Introduction
Overview
Through our Human Practices, we realized that addressing the problem of Olive Mill Wastewater (OMW) is essential for mitigating the environmental risks associated with its disposal, and relieving our local olive oil producers from the burden of dealing with OMW, which hampers their daily work. Determined to offer a solution to this problem, questions like: " Which approach is the most effective to tackle this waste? How can our approach benefit olive oil millers without disrupting their work? How we will ensure the safety of this approach and how should be implemented?"
These were only some of the questions we had at the beginning of our journey.
Also, engaging with stakeholders to create a viable business model was essential for the entrepreneurial development of our project. Simultaneously, connecting with the local community helped us refine our educational outreach efforts. Over the past few months, we gathered information, engaged with stakeholders and experts, reflected on our decisions, and identified areas for improvement, all to shape oPHAelia.
Our team envisioned oPHAelia as a solution to benefit our community and promote sustainability. To achieve this vision, we engaged with a diverse range of individuals, from olive oil millers to gain deep insights into the issue, to experts from various fields, and by integrating their feedback, we proposed oPHAelia and this is the story of how it was conceived!
The problem
Olive Mill Wastewater, OMW, is the toxic byproduct of olive oil production. Even though Greece is a small country, it ranks as the third-largest global producer of olive oil 1, and our local region, Thessaly, significantly contributes to this production, leading to the annual generation of vast quantities of OMW.
Locality and issue for the society
Engaging with local olive oil producers helped us understand the significance of the problem, and they informed us of the current situation regarding its management in our country. More specifically, they explained that this byproduct demands an immediate solution since the tons of OMW produced during olive oil season hampers their operation. Even though many methods have been suggested for its management, to this day, the main “solution” for OMW is its disposal in large evaporation ponds. However, in this approach, the organic content of OMW remains unutilized as it evaporates, while there is also a risk of leakage in the environment if the ponds are not adequately maintained. Also, the economic burden of OMW in conjunction with the applied legislation, leads many olive oil owners to resort to unsafe practices, including improper disposal into the environment.
Environmental problem
Improper disposal of OMW into the ecosystem triggers a cascade of problems. When disposed to an aquatic system, the high organic load in OMW induces anthropogenic eutrophication, disturbing the natural balance of water bodies 2,3. Similarly, when disposed into the soil, it disturbs microbial ecosystems and impacts plant life by affecting the soil's porosity 4,5.
Our approach
Through our Human Practices, we were able to recognize the severity of the issue; and drawing inspiration from the progress of synthetic biology in tackling similar challenges, our team embarked on a project aimed at detoxifying OMW. Its name? oPHAelia – an innovative synthetic biology approach addressing two significant challenges in Greece: OMW and the problem of plastic pollution.
Deepening our understanding of OMW's distinct composition and its potential to serve as a resource for Polyhydroxyalkanoate (PHA) production, we were determined to maximize its utility, promoting the concept of a circular economy. This approach represents a transformative shift in the treatment of industrial waste, a change sorely needed in our country.
oPHAelia represents a potent tool for upscaling PHA production, with far-reaching benefits. Foremost among these benefits is the reduction of the carbon footprint associated with synthetic plastic production. Our synbio design not only tackles OMW but also turns waste into a valuable resource.
OUR HUMAN PRACTICES METHODOLOGY
1. Identifying the problem
In the initial phase of our human practices, we reached out to our stakeholders to fully comprehend the problem of OMW. Our interactions began with olive oil millers who shared how OMW hampers their business, the current methods for its management in Greece, and how we could help them. Also, to ensure holistic understanding, we consulted experts who have been studying OMW’s composition, its environmental impact, and its potential effects on human well-being if not handled appropriately.
2. Searching for the best solution
After the first phase, we embarked on our journey for the best possible solution. The second stage of our human practices was very important since significant shifts in our project took place before concluding that the valorization of OMW for PHA production would be our approach. Dialogues with olive oil producers and experts, including microbiologists and engineers, played a pivotal role in shaping the evolution of oPHAelia.
3. Giving life to oPHAelia
At this stage we engaged with many experts from the academia to get personalized feedback for our project’s design, guiding both our wet lab and dry lab. We got in touch with microbiologists and experts in synthetic consortiums to decide which strains to use and how to make use of them, as well as experts in the PHA production field, to understand the steps of upstream and downstream procedures, meaning the PHA production and PHA recovery, respectively.
4. Implementing oPHAelia
Lastly,our team researched the possibility of designing a specialized bioreactor to facilitate the detoxification process and optimize the growth and activity of a synthetic consortium, while also ensuring its biocontainment. For this purpose, we consulted mechanics, and chemical engineers to understand the design of our bioreactor and the treatment of OMW before utilizing it in our bioremediation process. To close the loop, we sought to find the best alternative for PHA isolation, while consulting different bioplastic companies that would use PHA biopolymers for the production of bioplastics. Our proposed implementation is extensively presented on the implementation page.
Identifying the problem
Mr. Kantikos started by informing us of the severity of the problem. He noted that olive oil is an extremely important product for Greece since it exports 250-300 tons of olive oil annually from around 17 local producers. Moreover, he underlined the fact that our country has a lot of live oil mills- they estimate 2500- but they are scattered even in the most remote areas. Most of them produce OMW and have to deal with the severe issue it poses.
Additionally, he talked to us about the methods used to deal with OMW. To begin with, he informed us that they currently discharge OMW into large evaporation ponds outside the olive oil mill, where the liquid is vaporized, and the solids that remain are used as fertilizer. Moreover, Mr. Kantikos shared with us that the range of management techniques includes co-composting with plant or animal residues and thermal methods. Lately, he has been providing OMW to companies involved in biogas production. To elaborate, he described how these companies employ large tanks to collect OMW on a weekly basis, highlighting that this approach enables them to easily dispose of OMW without encountering any issues.
Mr. Kantikos explicitly told us how much olive oil mills like to get rid of OMW by making use of the most cost effective method, since it is nothing but a burden to them.
Moreover, the olive mill operator explained to us the distinction between two-phased and three-phased mills, which are the two main types of olive oil mills in Greece. His input helped us arrive at the conclusion that in the case of two-phase mills the issue of OMW is shifted towards the pomace biorefineries, which can also benefit from our project.
OMW originates directly from three-phased mills, whereas in the case of two-phased mills things are more complicated. Despite two-phased mills not generating OMW, they do yield Two-Phased Olive Oil Mill Wastewater (TPOMW) or wet pomace. TPOMW is also found in three-phase mills but typically contains less moisture because OMW is a separate by-product. This distinction arises from the fact that two-phase systems use less water in the olive processing, resulting in the mixing of this water with the solid wastes, producing one final by-product, TPOMW. Interestingly, when this by-product is transferred to pomace oil extraction plants and undergoes treatment, it results in the substantial production of OMW. Essentially, this implies that the issue of OMW is shifted from the two-phase mills to the pomace biorefineries, which can also benefit from our project.
The process is visualized in the accompanying diagram:
Since we learned that OMW also concerns olive pomace biorefineries, we contacted Mr. Mouzaki's pomace oil extraction plant in Almiros, Thessaly. They clarified that the by-product of the two-phase system (TPOMW) contains significant amounts of moisture that must be removed. After this process, they take a solid three-phase by-product, pomace olive oil, and OMW. Additionally, they informed us that they process approximately 17.5 thousand tons of TPOMW each year, a portion of which ultimately becomes OMW.
Mr. Zafeiropoulos' olive mill presents a unique scenario where the mill is close to residential areas. The owner must transport the OMW to the evaporation pond, situated away from the local community. This circumstance allowed us to engage in a conversation with Mr. Zafeiropoulos regarding the logistical considerations of such a situation. The expenses he had to go into for the transportation of OMW were, in his opinion, too high . After he heard about our project, he said that being able to get rid of OMW without any economic burden would be very helpful for his olive mill. Mr. Zafeiropoulos owns a small olive mill however, he told us that he produces an average of 40 tons of OMW every day during the olive oil production period. This is a huge quantity of liquid byproduct that is rather hard to manage. He employs two drivers that have the responsibility to transport the OMW to the evaporation pond.
In the case of Mr. Tzortzis, the olive oil mill is located in Lesvos, a Greek island. The olive miller told us that even there the current solution for dealing with the issue of OMW is not holistic.
“Many olive pomace mill owners are in desperation because they cannot manage the large volumes of OMW”, Mr. Tzortzis highlights specifically.
Since in Lesvos, most olive oil mills are two-phased, the problem of OMW is transferred from the olive oil mills to the pomace oil extraction plants, as Mr. Kantikos also suggested, where it is usually vaporized using heat, which really adds to the cost of OMW’s processing. As a result, many olive pomace mill owners might go for the easy solution and dispose of OMW at sea, with detrimental damages to aquatic life!
Furthermore, for Greek islands such as Lesvos and Crete, OMW can cause significant damage to the tourist sector, because of its repelling color and noxious odor.
“Personally, I would pay in order to remove that burden off my shoulders. The problem is that there are no entrepreneurs who understand the value and use of OMW yet”, Mr. Tzortzis said.
So, he praised our work for taking the initiative to deal with such a serious problem in our local community.
Dr. Kormas talked to us about the issues OMW can cause, when released into water bodies, like the sea or ponds. He mentioned that OMW contains groups of chemical compounds and elements that can harm organisms across different food chains living in aquatic ecosystems. The most studied group of compounds in OMW that can affect those ecosystems are polyphenols which have antimicrobial and phytotoxic properties . However, OMW also contains a significant amount of non-toxic organic load that can be used as a substrate for microbial growth. This phenomenon, the overload of an aquatic ecosystem with organic load can lead to quicker oxygen consumption, through increased bacterial metabolization, resulting in hypoxia (2mg of dissolved oxygen per 1L of water) or even anoxia, interrupting the ecosystem’s balance. This phenomenon is more intense in small, shallow ponds where water stagnation allows organic matter to persist longer, compared to the increased water circulation in the sea.
Moreover, he explained how aquatic organisms are affected when OMW is discharged in their natural habitat. The phytotoxicity of OMW can reduce the protogenic production, which in aquatic ecosystems happens primarily thanks to microscopic, photosynthetic organisms and secondly because of macro algae and macrophytes. These primary producers span a wide range of sizes, from single-celled organisms and cyanobacteria to larger marine plants like seagrasses in coastal ecosystems. Also, because of its dark color, OMW causes severe phenomena of turbidity in water bodies, obstructing light from reaching the organisms. In this case more affected are the robust plant photosynthetic organisms, for instance macrophytes and macroalgae. So, if there is prolonged discharge of OMW and thus, turbidity of the water, we expect the primary production , which is the basis of the food web, to be reduced.
Another question Dr.Kormas helped us answer was to what extent OMW can affect fishing areas. It appears that the most immediate way in which it can affect them is its toxic properties that threaten some organisms. In a simple food web energy flows from primary producers to superior organisms. If for some reason, this energy flow is perturbed, the organisms that will be more affected will be those on the upper layers. The organisms that concern the fishing industry belong to the upper levels of the food chain, which means that they depend on the well-being of the primary producers and consumers. This indirect impact on the foundations of the food chain can destabilize the entire ecosystem.
Last but not least, Dr.Kormas mentioned the different ways in which OMW affects coastal areas than other land areas. According to him,the issue is more severe in lakes with a broader land contact. A very enclosed sea bay, for example, can exhibit similar consequences to a lake. Dr. Kormas highlighted that OMW degrades slowly in nature, meaning that immediate discharge into a lake or a river can lead to long-term pollution.
Dr. Orfanoudakis, gave us valuable information on the severe consequences the discharge of OMW has to the soil. He noted that OMW can form organic- mineral complexes with different aromatic compounds.
Thus, OMW can change the quality and quantity of the soil’s organic load. Normally, agricultural grounds have very small content of organic compounds (less than 5%), while forest land for example has between 10 and 15%.
At the beginning, it is important to clarify what we mean with the term “terrestrial environment”. The soil is the surface layer of the earth, where all flora has its roots. Τherefore we could say that it is the basis of all ecosystems. Even though humans cannot perceive it, since it is an extremely slow procedure, the soil’s composition changes eternally. Primary rocks are created from the erosion of rocks that were formed in the bowels of the earth, under very high heat and pressure and through other mechanisms reach the earth’s surface. Later on, they weather off or they transform to yield secondary minerals, which are through time further reformed. The climate conditions are crucial for these processes and when they shift, the secondary rocks change again, reaching a thermodynamic state with high energy, meaning that they have to discharge. During this process happens the creation of colloids, which are, in essence, plant residues that form multiple complexes with organic compounds. In the same way, organic- mineral complexes of OMW can be formed with different aromatic compounds.
At the same time, OMW increases the soil’s electric conductance. Especially because of the dry climate on mediterranean ecosystems OMW is vaporized faster, leaving behind salts.
Like that, because of OMW, the soil’s pH changes, affecting particularly alkalic grounds, since OMW’s pH is acidic.
Moreover,
the rise in the soil’s phenolic content because of OMW, severely affects its health. In detail, the soil’s chemical oxygen demand (COD)- the oxygen needed for the oxidation of the phenolic compounds so that they are no longer toxic- is increased.
Lastly, there are some symbiotic fungi that grow in the roots of most plants, as they keep them healthy. They are responsible for the biodiversity in the plant ecosystems, while they are the reason why crops require less water and fertilizer, reducing the expenses needed for their growth. However,
the discharge of OMW in these ecosystems reduces biodiversity and causes the underdevelopment of plants, by affecting these fungi.
At the next olive oil mill, Mr. Spyroulis stressed the locality of the problem. OMW is an immense issue for Larissa and the areas close by (e.g. Agiokampos) where there are many olive oil mills.
Upon sharing our idea with him (regarding the collection, detoxification, and subsequent use of OMW-derived water for irrigating plants at the mill), he praised our project, but expressed reservations about its viability. He pointed out that such an approach might not prove advantageous and could lack support from the local community. His hesitation stemmed from the big quantity of the detoxified product that would be used for plant irrigation. It would necessitate additional investments in storage facilities by our stakeholders. Furthermore, the substantial volume of this detoxified byproduct would exceed the mill's irrigation needs.
Moreover, about the insufficiency of the use of evaporation ponds for dealing with OMW, he mentioned the potential leakage of the pond’s content into the soil. He clarified that, in an effort to prevent such occurrences, he utilizes materials such as Nylon to line the pond. However, even this measure does not guarantee 100% safety.On top of that its purchase is expensive for the olive oil mill. In general,
it would save the olive oil mill a lot of effort, time, and money if someone came and relieved them from the burden of OMW.
So, with all the valuable information we got from olive oil mills located both in a remote and a residential area in Thessaly, but also an island showed how hard the management of OMW is for their owners. Not only are they concerned about the time and resources they invest every year to deal with the specific waste, but also worried about the implications of its disposal into the environment. They know the harm it can cause to nature, which was also explained in detail to our team by microbial ecologists, soil scientists, and animal physiologists.
Searching for the best solution
To further research the composition of our substrate (OMW), we contacted Dr. George Zervakis. Having dealt with the detoxification of the specific waste as well, he gave us valuable information such as the fact that what makes OMW so toxic for aquatic and terrestrial ecosystems is its high phenolic content. Because of that, the conditions of OMW are challenging even for bacterial growth. He noted that if we wanted to use bacteria in its detoxification we would have to choose carefully the kind that would survive. On top of that, he highlighted the fact that OMW has a heterogeneous composition, which means that it can contain different quantities of phenolic compounds depending on the olive production period (fruit maturity), and the variety of the olives,
Moreover, he suggested that we construct a portable unit for the detoxification of OMW on-site, which would move from mill to mill. That would be extremely useful, since it would minimize the transportation costs and would add to the universality of our idea. However, we knew from the beginning that the detoxification of OMW must take place under specific conditions, in a bioreactor. Therefore, we thought that our portable unit would be a portable bioreactor. We looked into this idea by contacting chemical engineers.
Dr. Zervakis inspired our team by saying that OMW is not a waste but a by-product and that our team really “gives worth to waste” by viewing it as an opportunity and not a problem!
One of the most important things we learned from Dr. Zervakis was that to detoxify OMW we need to target its phenolic content. Therefore, our wet lab plan regarding the detoxification of the byproduct was designed based on this principle.
After looking into the composition of OMW, our wet lab team found out that its toxicity makes it lethal for many bacteria and set off to find the one resistant to its composition. At the same time, they took under consideration Dr. Zervakis’ advice and made sure that the bacterium had the ability to catabolize phenolic compounds. Based on the literature, we found out that P. putida could survive those conditions, so we took the initiative of contacting Dr. Mosialos, who has extensively researched different species of the genus Pseudomonas. He recommended the utilization of Pseudomonas putida KT2440 due to its renowned rich metabolism, which enables it to effectively degrade a wide range of phenolic compounds present in OMW. Additionally, he proposed that this particular strain, given its attributes, would likely exhibit a tolerance to the toxic compounds within OMW, thereby increasing its chances of survival in this mixture.
However, the feedback we got from Dr. Mosialos did not end there. When we asked him for further advice regarding our project, he suggested that we shift our focus towards valorizing OMW, rather than solely detoxifying it. So our new goal was to produce a high-value product from the olive oil mill’s toxic byproduct.
Dr. Marras informed us that such a portable bioreactor, theoretically proposed by Dr. Zervakis, would be rather impossible to implement, at least in such a short timetable. On top of that, he added that: “It is challenging to design a bioreactor, catering to the specific needs of your project, even experts need years to decide on the bioreactor best suited to their work as well.”
Despite this, he suggested we employ a bottom-up approach to calculate the required size of our bioreactor. The procedure he outlined begins with determining the amount of PHA that needs to be produced, subsequently working out the quantity of cell culture necessary to generate this desired amount, followed by determining the amount of nutrients needed to sustain this quantity of cell culture. By adhering to this systematic approach, we will be able to estimate the optimal size of the bioreactor, a step crucial for ensuring the efficiency of our proposed biological system. Nonetheless, he gave us valuable notes on the aspects we had to look into to begin our original research for the bioreactor.
First of all, it needs to have a good stirring with no blind spots, but not too because it could break the cells.
Oxygen bubbles created in the content of the bioreactor can enhance stirring, however, they should be small in diameter, to surface at a small speed.
Our bioreactor will need a cooling-down system, which may result in additional costs. Since P. putida KT2440, grows optimally at 30°C he noted that a cooling system will have to be implemented in our bioreactor.
At the beginning of our redefined journey, to valorize OMW, we explored the possibility of utilizing OMW for the production of electrical energy through Microbial Fuel Cell (MFC) technology. That is since we did not solely want to detoxify OMW, but also get an added-value product. The detoxification process, studied by our wet lab remained the same: with P. putida metabolizing the phenolic content of OMW. When we decided to integrate the MFC technology on our project, another bacterium was implicated, after extensive literature reviews: S. oneidensis. More information about this alternative was given to us by Dr. Ieropoulos (see below).
From the beginning, Dr. Ieropoulos seemed very keen on our project. However, after an extensive conversation with our team about the MFC technology, he noted that it has not yet been implemented in the real world, due to the lack of proper equipment to facilitate this technology. Therefore, we would not be able to bring our project to life in the near future and achieve a circular economy, while it would be rather impossible for us to gather the scientific data we need.
While the design for the detoxification of OMW remained the same, our quest for an added value product, went on.
Having worked with OMW himself, Dr. Topakas, noted that the choice of Pseudomonas was very accurate. Moreover, in his own experiments with OMW, he used the ¼ dilution method and added a buffer to reduce the pH. However, Dr. Topakas warned us that the dilution changes depending on the type of OMW and its chemical content.
Furthermore, since the beginning of our discussion, Dr. Topakas stressed the importance of finding a solution for liquid wastes and especially OMW. When we told him about the methods OMW is dealt with today, we mentioned that certain companies pick it up from olive oil mills- as Mr. Kantikos informed us- to use it in the production of biogas. Most importantly he remarked:
“New regulations proposed by the European Union, aim to minimize the use of biogas by 2050. Instead, I encourage your team to explore a more sustainable solution. In fact, the production of chemicals such as PHAs is part of the EU’s new agenda.”
Research about the possibility of our system producing PHAs had positive results. Beginning with the detoxification of OMW, P. putida, would catabolize the phenolic and other compounds of OMW and produce PHAs. In the next panel the evolution of our project is depicted.
Through trial and error, our project took its final form, oPHAelia.
Giving life to oPHAelia
Dr. Karpouzas, building upon Dr. Mosialos’ feedback, who agreed that P. putida is right for the degradation of the phenolic compounds in OMW since it is a resilient bacterium that can adapt to OMW. His opinion was very valuable since he has studied OMW himself. Moreover, he hinted that it would be a good idea to use another microorganism to comprise a complex bacterial community with many “helpers”, to increase its overall efficiency. His recommendation involved employing a bacterium like Pseudomonas putida, dedicated to producing PHAs, alongside another bacterium that would provide support to P. putida. This strategic approach would allow the metabolic workload to be shared within our engineered consortium, leading to improved detoxification and more efficient PHA production.
At this stage we made the decision to integrate a bacterial consortium to our wet lab’s design, consisting of two bacteria: P. putida and E. coli
Dr. Penoglou stressed the importance of a multi-stage pre-treatment of OMW involving pH adjustment and removal of solid compounds and proteins.
Afterward, he focused on the dynamic relationship between producing and degrading PHAs, according to the environment of the bacteria. He told us that when the bacteria are in a low-organic substrate, the expression of the enzyme depolymerase is triggered, resulting in the degradation of the PHAs. This information motivated us to consider knocking out this gene to reduce the chances of degradation, ensuring our product's vitality. Moreover, in PHAs, the molecular weight is of high importance because low molecular PHAs don't have enough stability, hampering further treatment procedures.
Regarding the fact that we utilize a variety of compounds found in OMW, he informed us that a mixture of PHAs will be obtained as our final product. Also, he mentioned that mixed PHAs often exhibit novel, valuable properties. But all in all, we need to characterize our final product, to be sure of its unique properties.
Lastly, Dr. Penoglou expressed confidence in our synthetic consortium approach, indicating that our approach is feasible.
At the beginning, she explained to us that the limited commercialization of PHAs versus other bioplastic materials such as PLA, is due to the expensive substrates (carbon sources) that must be provided to the bacteria. Thus, our approach of utilizing low-cost waste as a substrate is good enough to reduce upstream process expenses, indicating that our idea is feasible. After presenting our design to her she pointed out that free fatty acids (FFAs) are the best substrate for PHA production in P. putida. Additionally, phenolic compounds contribute to PHA production through the de novo synthesis of fatty acids. Later, we asked about the applications of mcl-PHA, and she highlighted that the specific type of mcl-PHA produced determines its distinct properties. For example, in contrast to short-chain PHAs, mcl-PHAs are more resistant to water and high temperatures.
In our conversations with Dr. Tsampika, we sought clarifications about the behavior of PHA synthases (phaC1 and phaC2), specifically for their reliance on different carbon sources. We were particularly interested in understanding whether the efficiency of these synthases is influenced by the carbon substrate, for example, whether phaC1 synthase might exhibit higher activity when free fatty acids (FFAs) serve as the primary substrate. Uncertainty also arose regarding whether we should incorporate both synthases into our system. In response, Dr. Tsampika responded that neither phaC1 nor phaC2 within P. putida exhibit differential recognition of distinct carbon sources. Instead, the only thing that changes is the way they degrade and connect to the PHA circle. She further explained that additional experiments would be necessary for us to discern the most suitable synthases to integrate into our system and to determine the appropriate regulatory controls.
The theoretical part of our project was ready to be implemented. The bacterial consortium that is the basis of our design needs ideal conditions to work with maximum efficiency. For that reason, our team consulted chemical engineers, to cater to the needs of the consortium and try to design the most suitable bioreactor. After consulting with the scientific community and finalizing our project, we came in touch with companies that specialize in waste management and bioplastic production to assess the feasibility of oPHAelia, from an entrepreneurial perspective.
Implementing oPHAelia
Dr. Muhammad Roil Bilad provided key insights for the design and construction of our bioreactor. He built on the feedback given to us by Dr. Marras. An initial recommendation from Dr. Bilad is to commence with a batch fermentation process, since in a Fed-Batch Culture, the new substrate is being continuously added until the maximum concentration of the biomass has been reached. This allows P. putida to produce the maximum quantity of PHAs, while a large volume of OMW has been processed. A critical goal lies in determining the zenith point at which P. putida, the bacterium responsible for PHA production, achieves its maximum intracellular biopolymer yield. In addition, Dr. Bilad concurred with Dr. Marras' view, affirming that, given our aerobic system, enhancing mixing through aeration is a prudent choice. Moreover, he noted that among various filtration methods, including centrifugation and membrane micro-filtration, the filter press stands out as a preferable option for bacterial separation due to its cost-effectiveness. Last but not least, we talked about the cell lysis that would have to occur in order to isolate the PHAs. That leaves a significant amount of dead biomass that can be further processed. Like that, we came up with the idea of transferring it to an anaerobic bioreactor in order to produce biogas.
Dr. Christakis is an expert in the field of treating OMW using specialized filtration methods. He mentioned that while there are numerous methods available for filtering OMW and extracting high-value phenols, these methods are often prohibitively expensive, making them impractical for implementing them in an olive oil mill.
In our discussion, we also explored the idea of using filtration methods for OMW pre-treatment before using it as a substrate for our bacteria. Dr. Christakis recommended employing a two-stage filtration process, where filters with pore sizes of 250 μm and 125 μm are used concurrently. The 250 μm filter would effectively capture solid residues present in OMW, such as pomace and olive leaves. The 125 μm filter would retain a small portion of the organic load, resulting in filtrated OMW with only 80% of its original organic content. These filters would be constructed from anoxic materials and require maintenance in the form of regular washing with water and soap.
“Furthermore, Dr. Christakis pointed out that after our detoxification process, the resulting detoxified by-product can be easily disposed of either in the city's biological wastewater facility or used for cleaning within our own facility.”
Firstly, Dr. Torri provided us with valuable insight into the techniques employed for PHA recovery. Then we proceeded to explain to him our idea of incorporating a lysis system in our bacteria to facilitate PHA recovery, induced by the solvent, butanone. He exhibited keen interest in our proposal, since butanone is quite a good solvent, resembling —a cheap and minimally toxic solvent used for PHA recovery. He also mentioned that because we will utilize small amounts of this solvent, costs regarding PHA recovery will be reduced, while also noting the environmental advantage of our approach, as high concentrations of certain solvents, such as chloroform are deemed to be toxic.
Lastly, he informed us that the type of PHA we aim to produce -middle-chain length PHAs like polyhydroxy-octanoate- are easier to extract than other PHA variants such as PHB, which is the most frequently commercialized PHA. This can be achieved by utilizing a well-defined nanofiltration system to separate the granules.
At the beginning of our engagement with Dr. Chatzidoukas, he placed a strong emphasis on the significance of sterilizing OMW. He also mentioned that subjecting OMW to thermal treatment at 120°C for 20 minutes could potentially induce physiological changes. Specifically, he highlighted the importance of conducting tests to measure phenolic concentration disparities between sterilized and unsterilized OMW. Furthermore, he recommended employing a toxicity assay to assess how our bacteria fare in the medium before and after sterilization, with and without dilution.
Transitioning to bioreactor design considerations, he advised that the task was considerably advanced in the timeline. For selecting an appropriate bioreactor, he advocated the use of laboratory-scale bioreactors, utilizing high-throughput systems. He explained to us that employing this approach entails utilizing numerous small lab bioreactors, each subjected to distinct conditions, to systematically screen and identify optimal parameters for achieving high-efficiency results. While acknowledging the cost-intensive nature of this strategy, he recommended a more budget-conscious approach for our case: conducting screening experiments in conical flasks within an incubator to identify the most suitable bioreactor configuration.
To get feedback on the feasibility of our idea we approached Qlab, a research and development lab in the agri-food and bioenergy sectors, to learn more about the methodologies they use for the treatment of OMW. At the beginning, they explained that they needed to assess the properties of olive oil’s byproduct to know how much yield they would have, before utilizing it for biogas production. They proposed that we do the same and estimate OMW’s composition before PHA production. Because we would use the same technology, we explored a potential partnership where Qlab's expertise would complement our project.
On top of that, Qlab expressed interest in the prospect of biogas production through our project. This idea was also proposed to us by Dr. Muhammad Roil Bilad, and as a team decided to further research after the iGEM competition.
Following our engagement with Qlab, they recommended connecting with Ergoplanning, a group of companies that provides complete technical and financial assistance. They suggested we communicate with them because one of the main challenges is transporting OMW from multiple olive oil mills to our
bioremediation facility . Ergoplanning, with years of experience in OMW transport to biogas facilities, provided insights into transportation costs. They informed us that the estimated cost of transporting 20 tons of OMW, depending on the distance can vary between 30-50 euros per ride. Regarding their policy, Ergopanning clarifies that they do not price the OMW to their owners and manage all the transportation costs with their budget, hinting that we should not be too concerned about it.
So, through those discussions, these companies found meaning in our endeavors and suggested a possible, future implementation of our idea-synergy. Sharing our future vision, Qlab is a company that believes in our idea, and as visible on the end of our page. To read their
letter of support provided to us by that company.
Mango Materials is a company specializing in producing PHA derived from methane. Since we share a similar vision, synthesizing PHA utilizing waste, we contacted the company to learn more. During our conversation, they couldn't answer all of our questions, as they needed to maintain secrecy and confidentiality about certain company information. One of the most significant insights we gained from this exchange is that PHA pellets can be manufactured using conventional plastic processing equipment like extruders, meaning that both producers of bioplastics and petroleum-based plastics utilize similar methods to create PHA pellets.
Furthermore, we learned that Mango Materials primarily serves customers in the fashion and beauty industries, demonstrating the potential for PHA to find applications in various sectors beyond the bioplastic industry. Additionally, blending PHA with other bioplastics, such as PLA and PBS, is a practice that is becoming more and more common. This insight suggests that our goal of expanding our customer segment to other bioplastic industries is feasible.
As we conclude our journey through Human Practices, we've successfully closed the loop by presenting the implementation of our idea. While we couldn't propose an on-site OMW detoxification solution within olive oil mills, we've come to understand that the process of transporting OMW to a bioremediation facility isn't as complex as initially thought. Upon transporting OMW to our facility, essential pre-treatment steps, including filtration, dilution, and sterilization, are necessary to prepare OMW as the substrate for our synthetic consortium. To facilitate detoxification and PHA production, we've meticulously designed a specialized bioreactor based on expert suggestions and an extensive literature review. Proceeding with the downstream process, our wet lab designed a specific MEK-induced lysis system for the efficient release of the granules (see more in Design ), which also according to literature review and experts seems to be a cost-efficient and environmentally friendly approach. After obtaining PHAs, additional treatment is required to produce our intended final product, PHA pellets, which aligns with our business plan. This pellet form is preferred by potential manufacturers for bioplastic production.
For all the steps needed for the bioremediation of OMW and pellet creation, we considered how to ensure safety for each step. From the transportation of OMW to the bacteria selected for our system, the MEK-induced lysis system for P.putida and establishing an auxotrophic relationship among the two bacteria to ensure their elimination after the end of our process. Additionally, through our proposed implementation, by-products are generated such as the detoxified OMW and the dead biomass. A plan for their safe management has also been developed, while taking into account advice from various experts. Each step of our proposed implementation is explained in detail on a dedicated implementation page, providing comprehensive information about the process and how to ensure its safety. We encourage you to visit our implementation page.
The Bottom Line
In summary, our project, oPHAelia, was born out of integrating feedback from key stakeholders, such as the local olive oil producers who highlighted the significant impact of OMW on the community. Additionally, learning about its environmental impact, especially on the well-being of aquatic life and health of soil, inspired our team to search for the best solution so as to efficiently detoxify OMW and be able to be implemented in the real world.
With these values in mind, initially, oPHAelia had a relatively narrow focus, mainly centered on a process for removing toxic substances from OMW. However, as we collaborated with key stakeholders and experts, and proceeded with our research, we realized we could expand our efforts to offer much more than originally envisioned! Polyhydroxyalkanoate emerged as our solution!
By repurposing this waste, to produce PHAs, an entire industry unfolded before us with our primary product, PHA pellets, finding potential end-users in the plastic and bioplastic industries, as outlined in our Business Model Canvas. (Entrepreneurship).
Yet, our project’s vision extends beyond the people who will employ our final product. oPHAelia strives to resolve a major environmental issue in Greece and other olive oil-producing countries such as Italy and Spain. By repurposing toxic waste from the olive oil industry, we also make a meaningful impact on tackling fossil fuel plastic pollution, aligning with efforts to scale up PHA production. Finally, we hope that our project will motivate 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, in our Entrepreneurship page).
References
- Bungaro, M. (2022, January 12). THE WORLD OF OLIVE OIL - International Olive Council. International Olive Council. Retrieved October 3, 2023, from https://www.internationaloliveoil.org/the-world-of-olive-oil/?fbclid=IwAR0Vqir-g3iX-fGfo-38jNHeAff76SpOVgEzIRPp5lsa7ntIsSpfBbrhDLo
- M. Della Greca, P. Monaco, G. Pinto, A. Pollio, L. Previtera, F. Temussi. Phytotoxicity of low-molecular-weight phenols from olive mill waste waters. Bull Environ Toxicol, 67 (2001), pp. 352-359. DOI: 10.1007/s001280132
- G. Manthos, D. Zagklis, C. Zafiri, M. Kornaros. Comparative life cycle assessment of anaerobic digestion, lagoon evaporation, and direct land application of olive mill wastewater. Bioresource Technology. Volume 388. 2023.
- M.J. Paredes, E. Moreno, A. Ramos-Cormenzana, J. Martinez. Characteristics of soil after pollution with wastewaters from olive oil extraction plants. Chemosphere, 16 (7) (1987), pp. 1557-1564. DOI: 10.1016/0045-6535(87)90096-8
- Comegna, A., Dragonetti, G., Kodesova, R. et al. Impact of olive mill wastewater (OMW) on the soil hydraulic and solute transport properties. Int. J. Environ. Sci. Technol. 19, 7079–7092 (2022). https://doi.org/10.1007/s13762-021-03630-6
