Team:OLS Canmore/Demonstrate

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DEMONSTRATE

Last year the breaKERs asked how we could implement our project into the real world and this year our goals for implementation have become a reality. In the previous year we have brainstormed several ways to implement our construct into the wastewater and poultry industries, finding that bioreactors are used both in water treatment as well as in the poultry industry. We realized we could utilize a containment system in order to create an environment where the bacteria can express the keratinase, the enzyme can be nurtured, and the keratin can be broken down, all in a controlled setting that allows us to ensure safety. A bioreactor by definition is an apparatus in which a biological reaction or process is carried out, and was the perfect fit. We found that the bioreactor system is an adaptable solution to all of the problems that our implementation model posed, and we could run numerous trials to alter the design before planning for implementation.

Last year while working on our construct we brainstormed potential methods for implementation, but at the time didn’t really emphasize implementation, as it fell under “future goals.” This year, we completed a lot of brainstorming and designed a prototype model.

Brainstorming
As we considered designs for our own bioreactor we researched the bioreactors that already exist in industry and took visits to our local wastewater treatments plants in Canmore and Banff. There we observed their massive bioreactors that they use for the treatment of the water, these bioreactors were able to hold gallons of liquid, enzymes, and microbes. Further research into these reactors revealed that Canmore has a pressurized filter system and Banff has a large bubble reactor.

At most plants they use so many different kinds of bacteria in the treatment of water that it seems that we could safely implement a keratinase expressing bacteria into their systems. The Canmore treatment facility used bacteria with rock and sand filtration systems, so we could easily implement a bacteria into their system, but would have to alter where to house it in our plans. The sand filters use a porous rock called biolite to house the bacteria that filter the water. At the Banff Water Treatment Plant, they mostly use eukaryotes and not specifically bacteria, but there is nitrifying bacteria involved in their processes, so it could definitely be a possibility of integrating our bacteria into their system. Their bioreactor holds 3.4 million litres, and retention time in the bioreactor is 9 days, way longer than most literature states keratinase activity should take, which leads us to assume that our construct could potentially very well be implemented into their system. Before the water leaves the plant, it gets treated to remove all of this bacteria and eukaryotes by using sand and anthracite filtration, as well as UV radiation to ensure that any remaining “bugs” were dead.

Because we know that most plants utilize a bacterial treatment, as well as have additional treatment measures to get rid of the bacteria later, we know that it is definitely a possibility to add our construct into the existing treatment systems, but incorporating it in a place where keratin waste is an issue, as well as implementing it into numerous plants, each with its unique system would prove challenging. When we visited Banff’s treatment plant, they told us that our construct would be extremely useful to them, but would be hard to implement into the existing systems. From this we decided to design a stand alone bioreactor prototype for use in testing within our lab, as well as potential implementation into the poultry industry, where there is less variation of their systems, rather than trying to incorporate our bioreactor into a pre-existing system.

Because we know that most plants utilize a bacterial treatment, as well as have additional treatment measures to get rid of the bacteria later, we know that it is definitely a possibility to add our construct into the existing treatment systems, but incorporating it in a place where keratin waste is an issue, as well as implementing it into numerous plants, each with its unique system would prove challenging. When we visited Banff’s treatment plant, they told us that our construct would be extremely useful to them, but would be hard to implement into the existing systems. From this we decided to design a stand alone bioreactor prototype for use in testing within our lab, as well as potential implementation into the poultry industry, where there is less variation of their systems, rather than trying to incorporate our bioreactor into a pre-existing system.

A few months into our iGEM season we hosted a mentor workshop, with the objective of organizing the testing of our construct. The team split into two groups, with one researching lab assays and the other researching bioreactors to make our very own. We researched about the many different multitudes of bioreactor types, as well as specific parts that all bioreactors have. At the end of the day we came together and decided on a concept for our bioreactor which can be seen below. We decided to design a chemostat model bioreactor, with two media inflow pumps, and a stirring mechanism, with an aeration system through bubbling. ( Figures 1.2-1.3).

With the initial design of our bioreactor complete, when our team attended a Mindfuel lab workshop at the University of Lethbridge, we worked on our construct and solidified design , including specific parts for the bioreactor prototype so that we could begin the process of ordering and assembly. When this design was finalized we got a talented, artistic classmate and honorary team member to draw out our design that we had settled on, so we could start to visualize it.

Design
After some edits of our initial design, our design of the prototype bioreactor the breaKERs initially went with resembled a bubble column reactor mixed with a continuous stirred reactor as seen in figures 1.2 and 1.3. This design would have required some sort of container with different hatches for effluent media, hair/feathers, media dispensed in two separate IV bags, and air. We designed our bioreactor using 2 IV bags for media inflow, each with a different pH to prevent any back growth of biofilms up the tubes. We decided to use IV bags to allow for gravity to power the inflow of media, to save money and energy of getting another pump. Our design incorporated a air pump with a filter system and a bubble column with a blade attached for the physical degradation of the substance (hair/feathers).

After the Lethbridge mindfuel lab day we decided on an alternate design that would accommodate all the available parts that we had chosen, to be more cost efficient (Figure 1.4) . This new design utilized the concept of a spinner flask as the container with the idea to use bubble walls, used mainly in aquariums to aerate and stir the solution to avoid tangling of the hair/feathers around the blades. This design stayed mostly consistent when we started assembly, except that we purchased glass carboys, rather than the spinner flasks, to stay cost effective.

Assembly
For the bioreactor the breaKERs bought many parts to make our concept a reality. We bought parts from pet stores, local sellers, and hardware stores including: glass wine carboys, a fish tank aerator and pump, and a heat pad. Initially we ran into a few problems including but not limited to the LED backbone of the aerator not fitting inside the hole of the carboy, the tube from the aerator being too buoyant and floating when the carboy was filled with liquid, as well as the heating mat being an ineffective method of warming the liquid broth inside the carboy. We fixed these problems moderately quickly with simple solutions like separating the aerator tube from the a backbone, weighing down the tube with nuts and bolts, secured with elastic bands, and warming the growth medium before putting it into the carboy. The bioreactor took roughly a few months to plan, weeks to get the parts and a few weeks to assemble and it was a great opportunity to see how a few vague ideas about implementation could turn into a physical prototype of a bioreactor.

Testing
To test the bioreactor the breKERs still had to determine whether it could hold the construct at the optimal pH and temperature. This was done by assembling the bioreactor and filling it with water, a suitable placeholder for LB broth medium, and measuring the temperature and pH after a certain period of time. Through some initial problems we managed to keep the bioreactor at optimal growing conditions and then in theory convert to the optimal expression pH for the keratinaze enzyme at 8.5 for 50C. In theory the bioreactor will be able to create an environment for both the bacteria and the enzyme to perform to their maximum capacity.

Contact us at:
https://www.facebook.com/OLeSsence/
@igem_canmore
larvisais@redeemer.ab.ca