Bioreactor Design

The inception of the project involved a dream to engineer the oleaginous yeast Yarrowia lipolytica to produce bio-jet fuel in a sustainable and scalable way. To cater to the huge demands of the aviation sector, we need a huge infrastructure and financial investment to scale it up to that level. One of the necessary elements in this process is the bioreactor, where the engineered organism will grow on a very large scale and produce the desired product, which will be in a pool of other things and needs to be processed before usage.

This is an artistic representation of how our bioreactor would be. The scale taken here is 100 L but we will first start with smaller volumes like 5 L and keep optimizing conditions and increasing the capacity of the reactor.

Gif: Our Bioreactor Implementation

Before we came up with the design of our bioreactor, we sought advice from people who have more experience and expertise in the same.

• Prof. Catherine Madzak - INRAE French National Institute for Research on Agriculture Food and Environment
• She has decades of experience in research related to Yarrowia lipolytica and has been constantly guiding us through the entire process. She gave us tips on what parameters are the most important and the values we need to keep in mind. Y. lipolytica being an obligate aerobe, needs proper aeration as well agitation or else it can form a matty layer. She helped us with the dimensions of the bioreactor as well.
• Y. lipolytica can display dimorphism, so in starvation conditions, it can shift to its filamentous form. The media has to be rich in nutrients otherwise, it won’t grow properly or even die as it has an auxotrophic marker. She mentioned that our yeast after we have transformed it by selectable marker on YNB media, we should use normal YPD media as it increases the efficiency of our hp4d promoter.
• Dr. Anand Ghosalkar - Praj Industries
• Dr. Ghosalkar is the chief scientist at Praj R&D, and he suggested some important parameters like dissolved O₂ (DO), pH controls, agitation controls and temperature. He showed us around the plant where we saw many anaerobic and aerobic fermentors. He suggested that we could go for an airlift type of fermentor instead of a stirred tank to save on cost as well. He also said that we could use pre-treated sugars from agricultural wastes to reduce cost as well as make our product more sustainable.

We had to look through and consider the following constraints in order to come up with our own bioreacter design:

• Dissolved O₂: Yarrowia lipolytica is an obligate aerobic yeast and it has been seen that it produces the highest amount of lipids when the DO is around 30-35%.
• pH: We need mildly acidic conditions for Yarrowia lipolytica to grow. Even in lab experiments, we add citrate buffer to make the YPD media acidic around the range of 4.5-5 pH.
• Temperature: The yeast is capable of growing in a variety of different environmental conditions at different temperatures. However, its best activity is around 25-30 degrees Celsius. It cannot tolerate temperatures higher than 32 degrees, and this makes it even safe for human consumption (GRAS category yeast).
• Agitation: Generally, this is around the range of 180-250 rpm but can also go higher to 600 rpm. However, greater intensity and speed of agitation not only consume more energy but also cause oxidative and shearing stress to our organism.
• Media: We use YEPD media for Po1g strain of Yarrowia lipolytica (used for many other yeasts too). YEPD typically contains 1% yeast extract, 2% peptone, and 2% glucose in distilled water. It may be made as a broth or made into an agar gel by adding 1.5 to 2% agar. Yarrowia lipolytica Po1g strain has the capacity to sustain on various sources of carbon like glucose, dextrose, sucrose and xylan.
• Safety: Y. lipolytica is a GRAS (Generally Recognised As Safe) and doesn’t need elaborate safety measures. It is used as a food supplement as well. However, as we have engineered it to produce desired substances (hydrocarbons in our case), we will be taking utmost safety measures, hence UV sterilization, bleach and autoclave will all be a part of our safety procedures.
• Blue Light: This is required for activation of photodecarboxylase enzyme CvFAP that converts fatty acids into hydrocarbons (alkanes and alkenes) which can be used as drop-in jet fuels.

Further, some of the essential features of our bioreacter:

• Type: Our model is an airlift-type bioreactor that works by agitating the contents of the bioreactor pneumatically using gas. The gas used for agitation can act to either introduce new molecules to the mixture inside of the bioreactor or remove specific metabolic molecules produced by microorganisms. Airlift bioreactors have a built-in bubble column designed to release gas into the bioreactor. Gas is usually injected into the bubble column at the bottom of the bioreactor. Mixing occurs as the bubbles rise through the bubble column to the top of the bioreactor. This type is different from the traditional stirred tank bioreactor, which has a stirring rod inside to generate agitation and mix the broth. Airlift-type bioreactors are cheaper as well.
• Material: The bioreactor will be stainless steel which confers its strength and durability. The inner chamber of the reactor that will contain the media and yeast will be made of transparent material like glass or plastic. This is because there will be blue lights all around it to activate CvFAP enzymes. The outer layer would be made of a fairly cheap and reflective material like Aluminium to minimize the loss of light
• Blue Light between the Chambers: This is specific to our bioreactor, as light in the range of 460-470 nm is required to activate our photodecarboxylase to produce hydrocarbons. This is placed between the chambers such that it can penetrate the inner chamber, but due to the outer chamber being made of reflective material, the light is concentrated in the inner chamber. We will use LED lights to reduce power consumption and heating.
• Sensors and Controls: There will be sensors for pH, temperature and DO. This is to ensure that all of these parameters are at the correct range; if not it will be brought to that automatically via the sensors and control systems. We also have condensers to avoid overheating of the system.
• Media Input: The media will be YPD; however we will try to have different carbon sources like glucose, which is derived from pre-treated agricultural wastes (straw, rice, sugarcane etc as feedstocks) or sucrose from Cyanobacteria after carbon capture. This will help us reduce the carbon footprint of our product even further and make our fuel carbon-neutral or negative.^[1] Additionally, this will help us save on our sugar source as well.
• Biomass Management and Disposal: Once the batch is done, the slurry has to be disposed off to make space for the incoming batch. After the downstream processing to obtain our desired products, there will be a large amount of waste that needs to be disposed of. After the necessary safety protocols of UV and sterilization, this biomass can be utilised to produce Yeast extract, which is a constituent of media for many research purposes, including our media. This way we could try to reduce the waste, manage it and reuse it to minimise cost and environmental impact both.