oPHAelia’s primary objective is to detoxify Olive Oil Mill Wastewater (OMW) while concurrently harnessing its potential for Polyhydroxyalkanoates (PHAs) production. To achieve this, we have addressed critical questions during the past months: How will we execute our idea in order to connect the detoxification process with the production of PHAs? Who will use it? How will we ensure the safety of our system? Engaging with the local community, key stakeholders, experts, and authorities, we developed a strategy for our project’s implementation.

Our proposed implementation can be divided into 9 distinctive steps:

Throughout the 9 months dedicated to building our project, we visited many olive oil mills within Thessaly’s prefecture. What became evident through these visits, is that these mills are not clustered together in proximity, making it impractical to construct a single large facility where the OMW from each mill could be centralized. Both our observations and feedback from the producers themselves underscored the importance of collecting and transporting OMW to our bioremediation facility, using trucks. To estimate the average cost of this procedure, we considered the following factors:

1. Through discussions with local olive oil producers, we learned that they process approximately 32 tons of olives daily.
2. For every 1 kg of olives processed, approximately 1.25 kg of OMW is generated [1]. In a typical three-phase olive oil mill, this amounts to 40 tons of OMW produced daily.
3. After consulting with experts from Ergo planning (see “integrated” for more details ), they informed us that their trucks can transport 20 tons of OMW, and the cost for one trip is around 30 euros (USD 31.97), with pricing varying based on the distance from the olive oil mill to our facility. So, to collect the OMW produced by an average olive oil mill, two trips are required every day.
4. Considering input from olive oil mill operators, who typically run their mills for 2 to 6 months depending on the olive season and mill location, we calculated that for 91 days (an average of 3 months) of continuous OMW collection from a single olive oil mill, the total cost would amount to 5,460 euros (USD 5,819).

This cost estimation provides valuable insights into the expenses associated with collecting OMW from olive oil mills daily for three months.

After its collection from the olive oil mill, OMW is stored in stainless steel tanks, bearing an external cooling cloak, to keep their storage temperature at a level below 4 ◦C. [2] This is considered necessary as it has been shown that close to this temperature, the concentration of carbohydrates is reduced (possibly due to microbial activity), thus affecting their performance during anaerobic fermentation. [3] In addition in this phase, samples will be collected for analysis, to estimate the PHA production as suggested by Qlab (see “integrated” for more details ).

Dilution

OMW is abundant in phenolic compounds, which confer antimicrobial properties to it [4]. This composition of OMW can potentially affect the bacteria we intend to utilize. To address this, several experts have recommended diluting OMW to create conditions conducive to the operation of our synthetic consortium. From the storage tanks, using a pump and stainless-steel ducts, the OMW is driven into a tank, where dilution with water takes place.

Filtration to remove large solid compounds.

OMW, as a bioproduct from olive oil production, even though it is liquid, contains some solid residues derived from the olive oil process, such as pomace, leaves, wood, etc. These solid contents need to be removed before beginning the bioremediation process. For this purpose, we will implement a 2-stage filtration system consisting of one filter with pore sizes of 250 μm and one with pore sizes of 125 μm. The two meshes will be integrated into a vibrating sieving machine, as Dr. Christakis Paraskeva suggested since it can quickly remove insoluble solid substances in various slurries (Xinxiang Dahan Vibrating Machinery Co., Ltd). This device will be made from stainless steel 316L which can withstand the harsh conditions of products derived from the agro-food industry, such as OMW (PALAMATIC PROCESS Inc.), and thus with the proper cleaning maintain the sieve for a lot of years.
  According to the company Xinxiang Dahan Vibrating Machinery Co., Ltd, the price for a circular vibrating sieve varies from $360 to $3,500.

A circular vibrating sieve from Xinxiang Dahan Vibrating Machinery Co., Ltd.

Following the filtration process, the solid contaminants of OMW can be further exploited. The extracted pomaces can be collected and transported to an olive pomace oil extraction plant for further processing. Meanwhile, the remaining solid materials can be utilized in the production of compost, as recommended by Dr. Christakis (see “integrated” for more details ). The filtered OMW will be transferred into a large bioreactor where additional treatment will be conducted before introducing the bacteria.

Sterilization

Despite the antimicrobial properties of OMW, it does host some microorganisms. According to a study by Ntougias, S., et al., (2013), proteobacteria, Firmicutes, and Actinobacteria were the most abundant bacterial phyla identified in OMW. Commonly found genera including Pseudomonas, Bacillus, and Lactobacillus are present in OMW [5]. To address this concern, in line with Dr. Chatzidoukas’ suggestion, we will do a sterilization of OMW involving heating to 121°C for 20 minutes. This treatment is effective in eliminating most of the microorganisms within OMW while not affecting its composition.

Following the filtration and sterilization of OMW, the prepared feedstock is directed to an aerobic bioreactor. At this point, the pre-treated OMW is ready for bacterial inoculation, marking the start of aerobic digestion for PHA production. In this stage, a large bioreactor capable of accommodating the detoxification and PHA synthesis process is needed.
  Dr. Marras informed us of the challenging task of designing a bioreactor tailored to the specific requirements of the project, whiles Dr. Chatzidoukas stressed the importance of employing multiple small laboratory-scale bioreactors to systematically assess the optimal conditions and parameters for achieving high-efficiency results. After an extensive literature review and with the experts' help, our proposed bioreactor will have the following characteristics:
-Bioreactor Material: The bioreactor will be constructed from stainless steel
-Fed-Batch Process: Dr. Muhammad Roil Bilad recommended implementing a fed-batch fermentation process. This approach is widely favored for both mcl-PHA and scl-PHA production and has demonstrated a high rate of PHA production per unit volume of culture broth per unit time [6]
-Bioreactor Size: To determine the bioreactor's size, we considered the volume of OMW transported to the bioremediation facility, which is 20 tons, as previously mentioned. Based on this data, we estimate that the bioreactor should have dimensions of approximately 3 meters in height, 3.1 meters in diameter, and a corresponding radius of approximately 1.6 meters.
-Mixing System: The bioreactor will be equipped with a mixing system to ensure uniform distribution of the organic load throughout the reactor.
-Operating Conditions: The bioreactor operates at ambient temperature (30-37°C, further experiments are needed to assess our synthetic’s consortium optimal temperature), and will be equipped with aeration, agitation, and exhaust systems, and operates periodically and automatically, completing each treatment cycle in 24 hours, as suggested by Dr. Tsampika.
  You can see more about the design of our bioreactor on our “hardware page” .

Separation of the detoxified OMW from the Bacteria-containing granules

Following the completion of the detoxification process and the production of PHA, it becomes necessary to separate the detoxified water from the bacteria that contain the granules. For this purpose, we will utilize a filter press as suggested by Dr. Muhammad Roil Bilad (see “integrated” for more details). An industrial filter press is a tool used in separation processes, specifically to separate solids and liquids. We decided to use a filter press because of its cost-effectiveness, as highlighted by Dr. Muhammad Roil Bilad. Filter presses are characterized by high CAPEX and lower OPEX, and for this reason, are the best option for larger-scale applications, when the scale factor decreases selectively the CAPEX-related cost. [7]

Filter press. (2023, September 29). In Wikipedia.

According to the company Shanghai Junyi Filter Equipment Co., Ltd., the price for an industrial filter press is around $4,800-$5,000

PHA recovery

As explained earlier, the biomass is separated from the detoxified OMW using a filter press and is transferred to a tank.
  PHAs recovery is considered particularly crucial as an integral part of the downstream process, impacting decisively the overall procedure cost. As Dr. Torri informed us, the downstream process seems to account for up to 30% of total PHA production. For this purpose, our wet lab department has proposed a programmable lysozyme-based lysis system for P. putida, tailored to the specific requirements of OPHAelia. With the aid of the solvent MEK, the PHAs are recovered from the biomass (see more details in design ).

Since we produce medium chain-length PHAs, we decided to follow the protocol proposed by T. de Vrije et al. (2023). Initially, they suggest adding ethanol before beginning solvent extraction to optimize final PHA purity by removing lipids and other ethanol-soluble non-PHA components and avoiding the cost and energy required to dry the biomass before solvent extraction. The next step is solvent extraction; the ethanol-treated biomass is mixed with small doses of MEK. Adding MEK triggers the lysozyme-based lysis system in P. putida, facilitating the extraction of the PHA granules. After adding the solvent, the extraction process occurs by incubating the mixture of ethanol-soaked biomass and solvent at 60°C in stirring conditions for 16 hours. After the end of this step, the PHA-solvent fraction can be separated by centrifugation from the biomass. After this, the solvent fraction is dried under an N2 flow, and the dried PHAs are finally collected. The biomass fraction undergoes more extraction cycles), to increase the yield of extracted mcl-PHAs.[8]

However, the downstream process is more complicated than it seems, and optimizing the best-fitting recovery of our project was beyond our capabilities. To ensure the most effective PHA recovery process, several critical factors should be considered, starting from the analysis of the PHA we produce and the bacteria we use [9]. Also, we will need to assess the lysozyme-induced lysis and how to achieve the best environment for the expression of this enzyme. We will need to contact more experts in the field of downstream processing to assess the best option for our PHAs.

As a team these concepts piqued our interest, aligning well with our vision of promoting a zero-waste solution for the problem of OMW. Regrettably, due to time constraints, we were unable to conduct further in-depth research on these promising ideas.

After acquiring the dried and purified PHAs from the previous step, a further process is needed to turn them into pellets which is the final form of our product [14]. This step is essential for melting the biopolymers and shaping them into the desired form.

Extrusion is a widely employed manufacturing process within the plastic industry, and it can also be effectively applied in the PHA industry, as indicated by Mango Materials (see “integrated” for more details). In this process, raw plastic material is exposed to elevated temperatures, causing it to melt and be molded continuously. In addition, to tailor the properties of the resulting material, various additives such as chain extenders, stabilizers, plasticizers, and colorants can be introduced and thoroughly mixed with the raw plastic in the machine's hopper.
  Extrusion stands out as a cost-effective and highly efficient process, particularly suited for large-scale production. It consistently delivers products of high quality, making it a preferred choice in both the traditional plastic and emerging PHA industries[15].

According to Nanjing Kairong Machinery Tech. Co., Ltd., large machines can cost anywhere between $50,000-$200,000. Sometimes a smaller machine may cost $100,000 because of its quality and/or capabilities.

Conical Twin Screw Extruder (Wuhu Kaijinhua New Material Technology Co., Ltd)

The blend of additives and raw plastic is transferred to the pelletizer, where it undergoes a transformation into pellets. We chose to process our polymers in a pellet form since most manufacturers seem to prefer it [16]. Once the pelletization process is complete, the resulting PHA pellets are discharged from the pelletizer and left to solidify. Subsequently, they are subjected to a quality assessment to ensure that all the required properties meet the specified standards.
  After passing the quality check, these PHA pellets are carefully measured and placed into plastic bags, in accordance with the desired quantity. These bags are then weighed, and the filled bags are stored in a designated storage area [15].

According to Nanjing Kairong Machinery Tech. Co., Ltd., a pelletizer costs USD 57,000.

PHA pellets, our final product, can find a variety of applications when used by manufacturers equipped with the proper machinery (e.g. extruders): They can be used for injection molding, a process used for large-scale plastic production [15]. Additionally, PHAs can be used in the 3D printing industry [17], which has evolved over the past few years and aims to become more diverse and sustainable. Other applications for pelleted PHAs are in the fashion industry for textiles, and polymer films [18]. Furthermore, they hold great potential in biomedical applications, shedding light on future commercial uses [19].

To sum up:

Conical Twin Screw Extruder (Wuhu Kaijinhua New Material Technology Co., Ltd)

How will we ensure the safety of our system?

Ensuring the safety of our system has been a top priority throughout the development of our project and to achieve this we had to consider several aspects of our project to ensure their safety.

1. Transportation of OMW: While consulting with Olive Oil Millers about OMW transportation, they informed us that the responsibility relies on the transportation company with which they synergize. As a result, to ensure the safety of this process we will work closely with these companies.

2. Bioremediation process: For the bioremediation process of OMW we utilize two bacteria harnessing the tools of synthetic biology. To begin with, the two bacteria we chose for our system, E. coli and P. putida, are generally recognized as safe (GRAS) and HV1 certified [20], respectively. After engaging with our Supervisors, they emphasized the need to enhance our biosecurity and prevent engineered bacteria from escaping into the environment. For this purpose, our wet lab department proposed the implementation of a MEK-induced lysis system in P. putida. Additionally, these bacteria have an auxotrophic relationship, meaning the elimination of P. putida will lead to the demise of E. Coli. Also, the detoxification process will take place inside a contained environment, a bioreactor, ensuring the utmost safety.

3. Downstream process: For the PHA recovery process, as explained earlier the most common methods use chlorinated solvents like chloroform. We propose a more environmentally friendly idea, aligning with the Green Chemistry Principles. We acknowledge the need for further experiments to confirm its feasibility and remain open to exploring sustainable alternatives.

4. By-products: After the bioremediation process is finished, there are two by-products generated, aside from the PHAs: detoxified OMW and dead biomass. As explained earlier, we envision a zero-waste facility, so we explored potential uses for these by-products. Using the detoxified water for the dilution of OMW sounds like a safe choice, since in case of any bacteria surviving inside, this by-product will return again to the OMW and undergo serialization prior to the bioremediation process. As for the dead biomass, we aspire to explore its potential for biogas generation. However, due to limited research on this process, we cannot guarantee its safety, especially considering that the dead biomass will mix with other bacteria. As a team, we recognize the need to thoroughly assess the risk of potential DNA contamination that may persist in the biomass. We will also need to consult with experts, which was not feasible during the competition due to time constraints. Nevertheless, as a team, we will always comply with the rules and safety will be our top priority.

Who will use it?

Once our bioremediation process for OMW is completed, our primary product will be PHA pellets. These versatile pellets have a broad range of potential end-users within the plastic industry, including Bioplastic producers, Plastic producers, Bioplastic and Plastic product Manufacturers, and 3D Printing Companies (you can see more in our Business Model Canvas, Entrepreneurship). In essence, our end-users are those who share our vision of advancing eco-friendliness and sustainability in their products. They can use our PHA pellets to obtain more environmentally conscious manufacturing practices.
  However, the vision of our project doesn't apply only to the people who will employ our final product. Considering Greece's extensive olive oil industry, the number of olive mills, and the annual OMW production per mill, we can infer that a significant number of people in the community are influenced by the challenges associated with OMW. By tackling the specific challenge of OMW faced by olive oil producers we contribute to the broader goals of waste reduction, environmental protection, and the sustainable utilization of valuable resources
  As a result, by utilizing the toxic waste of the olive oil industry, we manage to achieve additional impact on the environment by dealing with fossil fuel plastic pollution, contributing to the extended efforts of scaling up PHA production. We hope that our project will serve as a catalyst for various individuals, including the general public, children, scientists, and policymakers, to recognize the feasibility of zero-waste solutions, rather than viewing them as mere theoretical concepts (you can see more in our stakeholder matrix analysis, Entrepreneurship).