Bioreactor

We mainly proposed two systems in our project:

1. Lead recovery system
2. Lead quantifying biosensors

Our lead quantifying biosensor is based upon signals generated by a period modulating biosensor based on lead inducible expression of a quorum-sensing induced genetic circuit with the help of PbrR. Our lead recovery system is based on the surface expression of lead binding proteins such as PbrR, PbrR691, and PbrR MBD. Our goal from these systems is to achieve enhanced wastewater treatment for heavy metal ions, in present Lead(II), which is known to cause lead poisoning, which has life-threatening implications. We propose implementations for both aspects of our project in this section, also addressing the safety concerns surrounding these implementations along the way.

Bioreactors for Lead Recovery and Treatment

We propose a recovery of lead(II), an economically valuable metal ion. Here, we propose wastewater treatment for Lead(II) by a biological process instead of the well-known chemical process of chelation by EDTA because of its resistance to biodegradation and scarce absorbability. However, there are concerns with our process as well. Since we are using genetically engineered bacteria to serve our purpose, there is a concern about the possible exposure of these bacteria to the environment, so we keep this concern in mind while proposing our processes for wastewater treatment.

To scale the wastewater treatment for the recovery of lead with LEADer, we propose the use of MBBR (Moving Bed Biofilm Reactor). The MBBR consists of numerous free moving plastic rings on which engineered bacteria is allowed to grow and form a biofilm. These plastic structures are small and light enough to be aerated through influent water in the tank. The plastic structure mentioned is also known as carrier material for media which looks like a figure below. These plastic structures with grown biofilm on them are aerated to contact contaminated water in the tank and allow adsorption on the biofilm, thus helping lead to be removed from water since the biofilm formed by LEADer contains lead binding proteins on its surface.

This system has two advantages for us:

1. It keeps 90% of engineered cells within the reactor since the plastic structure traps it.
2. These plastic balls with lead adsorbed biofilm accumulated at one place are easier to process for recovery operation we are suggesting through acid leaching.

Point 1 is helpful for us since it helps with biocontainment of engineered bacteria, thus increasing the safety aspect of our implementation. Further, as we came to know through our visit to the wastewater treatment plant, we plant UV sterilised filtered water coming out of the reactor downstream. This eliminates the possibility of contamination of LEADer - bacteria engineered by us.

By mass transfer operation, it is known that processing large volumes is difficult and adsorption decreases with decrease in surface area of interface of adsorption.

Point 2 is helpful for us, since now we have lead accumulated on biofilm in a larger surface area with the help of small plastic ball-like structures making our bacteria easy to process for lead recovery operation and more efficient by larger surface area, as compared to sludge coming from a popularly used activated sludge process with more volume but less surface area, making it more difficult and inefficient to perform lead recovery operation. Here, we had mentioned activated sludge process because a thought may come in mind to aerate individual bacteria in water for treatment, since that will result in largest surface area of interface for adsorption, but the problem here is downstream operation of lead recovery we are proposing for economic value generation because these bacteria will accumulate to form bulky sludge making it difficult to process for lead recovery. Here, the advice from one of our technical stakeholders, Prof. Sanjay Ghosh, comes extremely handy. He stemmed the idea of integrating our quorum sensing circuit with lead recovery to promote biofilm formation for greater efficiency and this was mentioned by Nzila A et al. as well. Once the lead recovery system is developed, biofilm formation through quorum sensing would be a valuable addition to the engineered cells for being used in a MBBR.

Microfluidics

The lead sensing biosensor that we propose is a frequency-based biosensor instead of the traditional intensity-based biosensors. This allows it to quantify lead concentrations based on the time period of cumulative oscillations produced by a large number of colonies warding off any inconsistencies due to varied growth state of individual cells and differences in beam power and exposure time while handling.

To design such a biosensor the following objectives are required to be met:

1. The cells should be arranged such that the array is large enough for visualization to be possible but small enough that communication among colonies is possible for the oscillations to remain in phase.
2. The cells have access to nutritional requirements through their surroundings.
3. Excess cells and other metabolic wastes can be taken out of the environment to prevent any interference with the oscillations.

To achieve these objectives we devised two methods, one is through cell immobilization techniques making calcium alginate beads containing cells and arranging them in an array to visualize oscillations. Inputs from one of our stakeholders, Prof. Ravikrishna Elangovan helped us realise that this strategy can prove to be an easy and effective method for testing and providing a proof of concept, however, a large-scale implementation would require a more sophisticated and precise system like a microfluidic array. Microfluidic devices are considered tremendously efficient due to their miniaturized environment, ease of automation, low cost and disposability.

The above figure represents the schematic diagram of the microfluidic array that serves our goals. It consists of several small sections called traps where cells will be loaded. The trap size (100 μm x 85 μm x 1.65 μm) is optimized such that cells within the trap are able to communicate through strong but short range, quorum sensing via exchange of AHL. The traps are open to media channels from one end allowing take up nutritional requirements and removal of metabolic wastes and excess cells.

The number of traps in the array and the separation between the traps (25 μm) is such that weak but long-range gas-mediated communication via exchange of H2O2 produced through redox reactions is optimized. The material used for manufacturing the array is PDMS which is porous, thus allowing the exchange of gasses.

First of all, the media inlet is closed, and the engineered cells are loaded from the cell port keeping the waste ports open. The cell port channel is kept relatively narrow so that cells move under high pressure to make sure they are trapped inside the chamber. The channel towards the waste ports gets broader, reducing liquid speed, so that cells get enough time to be settled. Once the cells are loaded, the media port is opened which is used to feed the lead containing sample and constantly update the cells environment catering to its survival needs. The device will now be ready to be calibrated and utilized for lead quantification.

We intend to introduce this as a device that can be utilized by the general public as well as the industries as it does not require any scientific expertise and can be easily implemented as a point-of-care service. Especially people residing near industrial areas can use it to keep a check on their water supplies. The device can also be coupled with the lead recovery systems in wastewater treatment plants wherein recovery automatically occurs as a response to detection of excess lead in the sample. This device would decrease manual intervention in the lead detection process thus increasing its efficiency. The biggest challenge in implementing the device is that it requires the temperature to be maintained at 37° C which cannot be practically maintained at all times. Another concern lies with regards to the safety with which the device needs to be handled as we cannot afford to let these engineered microorganisms be released in the open environment and interfere with the ecosystem. Nevertheless, the introduction of such a device is a definite asset to the industry. Possible solutions to these problems could be to shift towards cell-free systems once we have established the proper functioning of our sensor. As highlighted by our stakeholder Prof. Arati Ramesh, cell-free systems would also require maintenance of optimal conditions for pH and temperature, but they would completely eliminate the issue of biocontainment.