Moving on to the legislation adopted on the matter at hand, Greece, despite being a prominent olive oil-producing and consuming country, has not managed to find a common and efficient way to solve the problem. Following the regulatory frameworks established by the EU (Directives 2008/98, Amending Directive 2018/851, Directive 91/271/EEC, and Commission Directive 98/15/EC) and Greece (4819/2021 and ΚΥΑ, which stands for Joint Ministerial Decision, 2017), the responsibility for waste management lies solely with the olive mill producers. This places a significant burden on their shoulders, as they are faced with additional costs and time-consuming tasks.
 As a result, many olive oil producers resort to haphazard waste disposal practices without proper strategic planning. Recent reports have brought attention to such actions. For instance, on November 20, 2022, it was reported in the municipality of Minoa, Crete, that OMW was improperly disposed of in the Anapodari River, which flows through several agricultural villages, making the local villagers express concerns about the environmental ramifications of this action 11. Another similar incident occurred on February 15, 2023, where OMW was discarded in the Euergetoula River in Lesvos, raising similar concerns among the locals. Not only have these actions raised environmental alarms but also have resulted in aesthetic degradation due to the pungent odor and dark color of the waste, as mentioned earlier 12. It is worth noting that both articles state that these incidents occur many times during the winter season when there is a significant increase in olive oil production, and thus, OMW.
 At the same time, the regulatory framework may become even more stringent due to the urgent need to achieve the UN's Sustainable Development Goals and the EU's 2030 Agenda for Sustainable Development. This will further encourage adopting improved waste management practices and environmental sustainability and thereby increasing the pressure on the producers.
  So, taking into account:

 We started working on oPHAelia. The name itself derives from the ancient Greek “ōphéleia” (ὠφέλεια), which means “aid” or “benefit”.

 ‘‘oPHAelia’’ represents a significant advancement in addressing the challenge of olive mill wastewater (OMW). To achieve this, leveraging the power of synthetic biology to design a synthetic consortium comprised of two bacteria: engineered Pseudomonas putida (KT2440) and Escherichia coli (BL21 DE3), based on a mutually beneficial symbiosis, for the efficient bioremediation of the OMW and its bioconversion into our high-value product.
 Firstly, E.coli will, under certain conditions, produce and secrete a white rot fungi laccase for the degradation of polymeric phenolic compounds (tannins, lignans, catechol melaninic polymers)13, a recalcitrant fraction found in OMW. Apart from that, E.coli will also preferentially utilize, among others, sugars like arabinose, xylose, and galactose, which P.putida cannot catabolize, and secrete free fatty acids. The expression of Acetyl-CoA carboxylase and acyl-ACP thioesterase from the plant Ricinus communis in E. coli will facilitate the provision of long-chain fatty acids to P. putida 14, 15, thereby promoting the production of medium-chain-length PHAs 16. To ensure the precise control of these genetic systems, we incorporated additional regulatory circuits, such as a negative feedback regulatory circuit based on a malonyl-CoA-based sensor-actuator 17. This circuit controls the expression of Acetyl-CoA carboxylase, preventing its uncontrolled expression, which could lead to reduced cell growth due to toxicity 18, 19.
 Secondly, P.putida, a robust bacteria with unique degradative metabolism 20, will convert monomeric phenolic compounds (phenolic acids), another serious obstacle for OMW treatment, sugars (mainly glucose), and free fatty acids from E.coli into our high-value product by specific substrate-induced systems. Lastly, in addition to that, product recovery is also very crucial. Hence, we will integrate a programmable lysozyme-based lysis system into P.putida for efficient and affordable final product collection 21.

Why Synthetic Consortium (SynCom)?

 Construction of a synthetic consortium between P. putida KT2440 and strains of E. coli has been suggested to enhance PHA accumulation 22, 23. Synthetic consortiums extend the scope of metabolic engineering and provide our system with many benefits both in the detoxification process and the PHA production, since the processes are divided among the two bacteria, resulting in less metabolic stress applied to them and enhancing PHA production. Also, the SynCom approach allows for the degradation of more complex biological compounds thus, increasing the output of the detoxification process, when occurred by only one bacterium 24. To further enhance those, we have included different aspects of previous work to control our system. Incorporating into our design control systems, such as promoters induced by compounds found in OMW or the negative feedback loop of malonyl-CoA sensor-actuator, the bacteria will be activated only when placed into OMW. This dual-bacterial approach allows for a comprehensive detoxification process, targeting multiple organic components of OMW simultaneously 14. In summary, by carefully combining the strengths of different systems and incorporating additional regulatory elements, we have developed a comprehensive approach to the valorization of OMW.

 The high-value product produced: Polyhydroxyalkanoates, or PHAs. PHAs are a group of biodegradable polyesters that bacteria and archaea can accumulate inside their cells under specific stress conditions, serving as a means of storing carbon and energy 25. Additionally, these biopolymers display similar mechanical and physical properties to various synthetic plastic like polypropylene and polystyrene. 26

 First of all, P. putida has the inherent capability to produce PHAs32. Moreover, compared to petrochemical polymers, PHAs offer numerous advantages, including their biodegradability and biocompatibility making them environmentally friendly 25. They pose a sustainable way to tackle plastic pollution, since PHA-based bioplastics, in contrast to traditional plastics, which have a life expectancy of between 100 and 1000 years, may degrade into water (H2O) and carbon dioxide (CO2) in 20 to 45 days if there is adequate humidity, oxygen, and a sufficient number of microbes in the open environment 27.
 Nonetheless, plastic pollution is a widely recognized global problem that demands immediate attention. Europe alone generates nearly 26 million tons of plastic waste annually, and approximately 80% of marine litter consists of plastic. According to WWF, in Greece, the production of plastic waste per capita reached 68 kilos in 2019, with a significant portion (84%) ending up in landfills and only a mere 8% of plastic waste being recycled. Consequently, a substantial amount of plastic waste finds its way into the sea, with an estimated 11.5 thousand tons leaking into Greek waters in 2016.
 

 This alarming situation has raised concerns among non-profit organizations, which emphasize the infiltration of plastic pollution into Greece's ecosystems. A recent study conducted by the Archipelagos Institute for Marine Conservation revealed the presence of microplastic contaminants in all 25 examined marine animals 28, while globally, the United Nations report that nearly 100 thousand marine animals are killed yearly due to plastic waste 29.
  These facts issue that plastic pollution poses a grave threat universally, including in Greece. The significant production and inadequate management of plastic waste have led to its infiltration into the environment, causing harm to marine life and ecosystems.
 On top of that, PHAs utilized to develop bioplastics can be the solution to some of the related sustainable development goals set forth by the United Nations, while they can help to reduce dependency on fossil fuels and uphold sustainability initiatives 3.

 PHA-based bioplastics find uses in other fields as well, including the biomedical sector in advanced drug delivery, electronics, construction, automotive, packaging, and agriculture 30. The main uses of PHA are in packaging and food services due to their degradable nature, non-solubility, non-permeable nature, and flexibility. Moreover, PHAs can serve as suitable feedstock for 3D printing due to their compatibility with 3D printing technology and inherent biodegradability. 26
 This sustainable solution has not yet taken over synthetic plastic production because of its cost of production primarily due to the cost of raw materials and the fermentation process involved. To make PHA production more economically viable, reducing costs is crucial 26. One approach is to utilize low-cost substrates such as olive mill wastewater (OMW), which can significantly decrease production expenses, typically ranging from $2 to $8 per kilogram (approximately 1.70€ to 6.80€). In addition, genetic and metabolic engineering tools offer a plausible way to enhance PHA productivity sustainably and economically 31. Addressing these challenges will contribute to the widespread adoption of PHA as a sustainable alternative to traditional plastics 32, 33 That is how ‘‘oPHAelia’’ aims to highlight the benefits and aid the transition to use PHAs as an alternative to petroleum-based plastics will not only help reduce the environmental impact of plastic pollution but also create new possibilities for industries to adopt more environmentally friendly materials.

 The first step of the process involves collecting olive mill wastewater (OMW) from the olive oil mills and transporting it to a bioremediation facility. At this facility, a specialized bioreactor will be installed to facilitate the detoxification process and optimize the growth and activity of a synthetic consortium. The primary objective of the bioreactor is to create a controlled environment that provides ideal growth conditions for P. putida and E. coli, the key microorganisms involved in the process. Parameters such as temperature, pH, nutrient availability, and oxygen levels will be carefully regulated to ensure the detoxification process operates at maximum efficiency 34. Once the detoxification process is completed, the resulting PHAs will be supplied to bioplastic companies. Additionally, the aqueous residue generated from the detoxification process will be integrated into our proposed implementation process. This integration serves the purpose of reducing additional operational costs, particularly for the production of PHAs. The residue can be utilized for tasks such as diluting OMW or cooling down the bioreactor, offering a more sustainable and realistic solution for the overall process.

 In closing, through the process of developing our project, we noticed that a clear, understandable way of explaining even the most complicated synbio parts of our project is key to attracting the attention of our stakeholders, as well as gaining the interest of the public. We need to highlight that synthetic biology is not something to fear and that, by taking the necessary precautions, it can offer solutions to multiple of the world’s most challenging problems. For that reason, we planned and organized a series of educational events, through our human practices department, while doing our best to include all age groups in the learning process. Our goal was to raise awareness about the issues raised by a local waste that is often being mistreated, and the vast problem of plastic pollution. At the same time, we mentioned to the different audiences of the events that our means of solving those prominent problems is synbio, whose meaning was explained through various interactive and engaging activities.