Week 1 HW: Final Project Ideas

(1) What you would like to biomanufacture and why?

I would like to biomanufacture an engineered anammox bacterium that is more efficient and able to survive in a wider range of environments than naturally occurring anammox bacteria.

Anammox bacteria can remove nitrogen by converting ammonium and nitrite into nitrogen gas, but one of their biggest limitations is that they grow slowly and can be sensitive to changes in things like temperature, oxygen, pH, and nutrient levels. My goal would be to develop a strain that can maintain high anammox activity even when environmental conditions are not ideal.

This could make anammox useful in more places, including wastewater treatment systems, agricultural runoff areas, and potentially nitrogen-polluted wetlands. Instead of having to carefully create the perfect environment for the bacteria, I would want to make the bacteria themselves more adaptable to the environment they are placed in.

If anammox bacteria could be made more effective and reliable in natural environments, they could potentially become another tool for restoring polluted wetlands without requiring the energy and infrastructure of a traditional wastewater treatment system. This could also be used in traditional waste water treatment sites saving costs where low temps are an issue

(2) What living system would you use?

My living system would be an anammox bacterium, such as a species related to Brocadia or Kuenenia. The bacterium itself would be both the manufacturing chassis and the final product I am trying to improve.

The main inputs would be ammonium and nitrite. Inside the cell, the anammox pathway would convert these nitrogen compounds through a series of reactions and eventually release nitrogen gas.

Rather than trying to completely redesign the pathway, I would focus on improving traits that limit where anammox bacteria can be used. For example, I would be interested in increasing their tolerance to temperature changes, small amounts of oxygen, changes in pH, and different concentrations of nitrogen compounds. I would also be interested in improving growth rate or the ability of the bacteria to form stable biofilms.

I am assuming that genetic tools could be developed or adapted to modify an anammox bacterium and that different engineered strains could then be compared to determine which changes improve its performance.

(3) How would you use automation? I would use automation to test a large number of engineered anammox strains under different environmental conditions.

An automated liquid-handling system such as an Opentrons robot could prepare cultures with different combinations of temperature, pH, ammonium concentration, nitrite concentration, and oxygen exposure. This would allow me to test how different engineered strains respond to conditions that they might experience in wastewater systems or natural environments.

The system could also automate sampling and preparation for measurements of ammonium, nitrite, nitrate, and bacterial growth. I could then compare the strains based on how much nitrogen they remove, how quickly they grow, and how well they recover after environmental stress.

(4) How much would you need to make, and how long would it take?

For a pilot-scale test, I would aim to produce about 20 kg of engineered anammox bacteria, enough to inoculate roughly 100,000 liters of water at about 0.2 g of biomass per liter.

The main goal of my project is to make anammox bacteria grow faster and remove nitrogen much more efficiently than they naturally do. For my engineered strain, I would aim for a doubling time of about 3 days while also improving how effectively it converts ammonium and nitrite into nitrogen gas.

Starting with about 1.25 kg of bacteria:

1.25 kg -3days- 2.5 kg -3days- 5 kg -3days- 10 kg -3days- 20 kg 4 doublings × 3 days = 12 days of growth

With extra time for setup, harvesting, and testing, I would estimate about 2–3 weeks total to produce enough bacteria for a pilot-scale test.

(5) What don’t you know yet?

Almost everything but thats how every engineering process begins. The biggest practical unknowns are how to make the bacteria both more stable and much faster growing without losing the traits that make it useful.

One major question is which genetic or metabolic pathways could be changed to increase stability, so the engineered bacteria can keep functioning reliably over many generations and under bioreactor conditions. Another major unknown is what pathway controls the very slow doubling time and whether it can be modified enough to reach our target of about a 3-day doubling time. A third concern is whether making the bacteria grow faster would reduce its efficiency at carrying out the desired anammox reactions. Finding the balance that allows for the optimal results would be the real obstacle.

Final Project