Polyhydroxyalkanoates (PHAs) are polyesters synthesized and stored by various bacteria in their cytoplasm, inside water-insoluble inclusions known as granules 1. PHAs are usually produced when the microbes are cultured with nutrient-limiting concentrations of nitrogen, phosphorus, sulfur, or oxygen and excess carbon sources2. To optimize the cultivation of our bacteria and ensure the safety of the biosynthesis process, it is essential to follow specific steps as outlined in our implementation page. Among these steps, one of the most critical is the fermentation process.
To secure the optimal conditions for the cultivation of our bacteria a bioreactor is deemed essential. A bioreactor is a specialized vessel or container used in biotechnology, microbiology, and chemical engineering for the controlled cultivation and growth of microorganisms, cells, or biological processes. It offers a regulated environment where various biological reactions can take place under specific conditions, including temperature, pH, oxygen levels, and nutrient availability 3,4.

Selecting the right fermentation strategy is critical for optimizing bacterial cultivation, which, in turn, affects the detoxification of Olive Mill Wastewater (OMW) and the production of polyhydroxyalkanoates (PHAs). There are various fermentation strategies available, including batch, fed-batch, and continuous fermentation, all aimed at enhancing mcl-PHA production5. Batch fermentation is a straightforward and widely used method in biological fermentation research5. However, it is not ideal for PHA production due to its characteristics, which result in low cell density and PHA productivity6.
Continuous fermentation, although considered by many as having potential for PHA production, it is essential to note that PHAs are produced intracellularly by bacteria as a means of storing carbon and energy, particularly under specific stress conditions, as discussed earlier. In a continuous fermentation process, achieving these necessary stress conditions becomes challenging, resulting in reduced PHA accumulation7. Also, in a continuous fermentation process, there is a higher risk of infection6. Given that our synthetic consortium demands sterile conditions, it is imperative to minimize any significant risk of microbial contamination. Such contamination could pose a serious threat to the entire fermentation batch, potentially resulting in substantial economic losses. So, Following Dr. Muhammad's recommendation and a thorough review of the literature, we concluded that a fed-batch fermentation approach is the most suitable for our system.

Fed-batch cultures typically start as batch cultures, and then, nutritional components are gradually added to the culture to support growth and/or PHA synthesis. This method aims to extend the rapid growth phase, known as the exponential phase, and achieve maximum cell density quickly. High-density cultures like these have proven to be highly productive for mcl-PHA production.

As previously mentioned, we plan to utilize a Fed-batch fermentation process, recommended by Dr. Muhammad, as it appears to be the most fitting approach. This fermentation process will be conducted within a stirred tank configuration. Additionally, the selection of an appropriate fermentation strategy is closely tied to the effective control of essential bioreactor conditions, including dissolved oxygen (DO), impeller design, impeller agitation speed, aeration rate, pH, and temperature2. These controls are crucial for optimizing the utilization of our waste material in the production of PHAs. For this purpose: When we introduce microorganisms and OMW into the vessel, following Dr. Marras' advice, an impeller will initiate stirring at a speed ranging from 400 to 800 revolutions per minute (rpm)7. This speed is selected with precision to strike a balance: it provides the microorganisms with the necessary oxygen while avoiding excessive agitation that could potentially harm the cells. The agitator system plays a pivotal role in the bioreactor, primarily responsible for ensuring the proper mixing of the reactor's contents, thereby maintaining a homogeneous environment for the cells. This system comprises several components, with one of the most crucial being the impellers. Impellers serve multiple functions, including agitation, facilitating heat transfer, and aiding the spargers in aeration by breaking down and effectively mixing bubbles throughout the system7.
In addition, our system will incorporate three crucial sensors to ensure its proper operation: one for monitoring temperature, another for measuring pH levels, and a third for tracking dissolved oxygen levels. Concerning the determination of the optimal temperature, further experiments will be carried out to identify this critical parameter. We aim to find the temperature at which both bacteria species, Pseudomonas putida (with an optimal temperature of 30°C) and Escherichia coli (with an optimal temperature of 37°C), can coexist and function efficiently while achieving the highest levels of detoxification and PHA production. All in all, our team has developed straightforward Boolean software to ensure that none of these indicators exceed their specified limits. This software will be connected to the sensors, continuously monitoring their readings, and making adjustments if any values go beyond the established thresholds. By implementing this approach, we guarantee optimal conditions for the growth and productivity of our microorganisms.

fig.1. Within this boolean program, we've established a range of optimal values to maintain the ideal conditions within the bioreactor. Should any of these values exceed or fall below the specified limits, the system automatically readjusts them to stay within the predefined boundaries we've set.

Once the detoxification process is complete, we will proceed to the next step, which involves filtering the detoxified OMW to isolate the bacteria containing the PHA granules. Details of this process are explained in our implementation page.

Following our discussion with Dr. Marras, he suggested the use of stainless steel for constructing the bioreactor vessel. This recommendation is based on several advantages of stainless steel: it is easy to clean and sterilize, making it an excellent choice for bioreactors where maintaining a sterile environment is crucial for microorganism growth. Additionally, stainless steel can withstand high temperatures and the demands of industrial processes. Moreover, this material has a long service life, reducing the need for frequent replacements and minimizing overall maintenance costs8.

Also, our bioreactor will be a cylindrical shape, and its size was calculated according to the contribution guide provided by the iGEM team MIT MAHE 20219 To determine the appropriate dimensions for our cylindrical bioreactor, we began by considering the volume of OMW that needs detoxification. As indicated on our implementation page, the waste amounts to approximately 20 metric tons per truckload. Taking into account the density of OMW at 0.9 kilograms per cubic meter and a detoxification period of 24 hours, we used the formula for the volume of a cylinder (V = h * π * r^2) to calculate that our bioreactor should have a volume of approximately 23 cubic meters. With this data in mind, we estimated that the bioreactor should have a height of 3 meters, a diameter of approximately 3.1 meters, and a corresponding radius of approximately 1.6 meters. These calculations ensure that the bioreactor can effectively accommodate the specified volume of OMW for efficient detoxification. Internally, specific dimensions are critical. For instance, the impeller will have a diameter of 1.1 meters, positioned 1.1 meters above the tank bottom. The impeller blades themselves will measure 26 centimeters in length and 20 centimeters in width, while the impeller disk will possess a diameter of 78 centimeters. Furthermore, baffles will be incorporated into the design with a width of 0.31 meters and positioned at a distance of 0.62 meters from the bottom of the bioreactor. These specifications are essential to ensure the bioreactor's effective operation and optimize the detoxification process for OMW.

fig.2. A 3D design of our bioreactor.

Prior to fermentation, it's crucial to remove any solid residues to prepare for the bioremediation process. To achieve this, we will employ a 2-stage filtration system comprising two filters with pore sizes of 250 μm and 125 μm, respectively. These two meshes will be incorporated into a vibrating sieving machine, as recommended by Dr. Christakis. This machine is known for efficiently eliminating insoluble solid substances from diverse slurries, as evidenced by Xinxiang Dahan Vibrating Machinery Co., Ltd.
After the fermentation process in the bioreactor is completed, additional equipment is required to separate the PHA granules from the detoxified OMW. In line with Mr. Muhammad's recommendation, we have chosen to employ a filter press for this purpose. The filter press is a tool designed for liquid/solid separation using pressure filtration. We selected this method due to its proven efficacy and cost-effectiveness10.
Once the PHAs are successfully isolated from the OMW, the next step is to extract them from inside the bacterial cells. The specific protocols for this extraction depend on the type of PHAs produced by our system and their unique characteristics. Many of these extraction protocols involve the use of hardware equipment such as centrifuges and dryers11. The exact equipment needed can be determined once we identify the specific PHAs our system produces and verify the feasibility of our MEK-induced lysis system.
Finally, after isolating the PHAs and extracting them from the bacterial cells, an extruder is required to shape the PHAs into the desired form. Additionally, a pelletizer is used to transform the PHAs into pellet form. This entire procedure is detailed further in our implementation documentation.

Based on our literature review and Dr. Muhammad's recommendations, we have opted to incorporate a fed-batch bioreactor into our proposed implementation. However, it's worth noting that while this strategy is widely used in industrial PHA production, it does come with its own set of challenges. Many researchers are currently engaged in further research efforts aimed at modifying this strategy, primarily due to its substantial6. Additionally, to evaluate critical system parameters such as temperature, cultivation duration, pH, and others, we recognize the need for further research. As explained by Dr. Chatzidoukas, several steps should be followed before we can scale up to an industrial level, as indicated in the gaps on our Entrepreneurship page. To determine the most suitable bioreactor for our bacteria and process, our approach involves initially conducting experiments at the flask-scale level, followed by parameter measurements and subsequent pilot-scale testing, before ultimately making a well-informed decision on the industrial-scale bioreactor selection.