Final Report Summary - OLI-PHA (A novel and efficient method for the production of polyhydroxyalkanoate polymer-based packaging from olive oil waste water)
Executive Summary:
Oli-PHA was a three year applied research project, which commenced in June 2012 and finished in May 2015, and was funded under the European Commission’s Seventh Framework Programme (FP7) and specifically under the “NMP - Nanosciences, Nanotechnologies, Materials and New Production Technologies” programme. Following a circular economy approach, the overall goal of the project was to valorise the olive mill wastewater (OMWW)tosimultaneously clean such water and produce high value added products such as polyhydroxyalkanoate (PHA) biopolymers using photosynthetic microorganisms, but also to recover polyphenols that could be used as additives in cosmetic and in active packaging. PHAs is a family of biopolyesters produced by number of microorganisms, generally heterotrophic bacteria grown in refined feedstock, that present suitable properties for food and cosmetic packaging (good gas and moisture barrier, thermo-mechanical properties) and excellent biodegradability in number of environments. New research is needed to enhance PHA market uptake using more sustainable/non food competing substrates than commercially available PHA, especially obtained from wastes, reduce its cost, as well as to fine tune its formulation to different rigid and flexible functional packaging applications.
In terms of main results, the project delivered:
• A pretreatment process for OMWW was developed to recover polyphenols of very high antioxidant effect and obtain a suitable media for cultivating cyanobacteria. An interesting biogas potential was observed with left solids from OMWW.
• 7 different cyanobacteria and 4 heterotrophic bacteria PHA-producers were tested at lab scale. The optimal culture conditions (pH, temperature, CO2/air, light intensity and cycles, tolerance to dephenolized OMWW)were determined both on lab and pilot scale
• A novel method for genetic modification and characterization of a cyanobacteria with enhanced capacity of producing PHA was developed to allow increasing the PHA yield very significantly vs. wild strains (patent filled).
• Trials were made to upscale the microalgae culture using a 6 m3 PBR pilot plant with innovative design that was delivered in the project. In spite of challenges met to reach a stable production due to the microalgae acclimatation to large scales and tolerance to non axenic conditions, a proper 3-stage culture approach to maximise PHA production was identified.
• A new environmentally friendly extraction process was developed using non-chlorinated solvent after biomass concentration by flocculation and cell disruption by surfactants. The polymer extracted from the biomass was characterized as poly-hydroxybutyrate (PHB) of equivalent properties to commercial one.
• Optimal compounds reducing issues linked with crystallization kinetics & processing conditions were developed based on commercial PHA and PHB. Films were obtained by blow moulding, jars were injected and sheets were compressed and extruded before thermoforming trials to obtain trays.
• Active packaging with improved oxygen barrier and antimicrobial properties were developed using the polyphenols extracted from OMWW and validated showing a very significant shelf life extension of packed food products. PHA films had compliant migration levels to be used as food packaging. PHB jars were validated as suitable packaging solution for most cosmetic products. The polyphenols extracts also showed excellent potential as preservative for cosmetics.
• Biodegradation and organic recycling were tested on developed compounds meeting requirements for industrial composting, home compostability, freshwater and anaerobic degradation. The PHA produced within the project, is biodegradable in these environments. Moreover it is a suitable material to produce compostable products. Recyclability was also proved without affecting the properties of injected parts. An LCA was performed vs. standard packaging and further required optimizations were suggested.
Project Context and Objectives:
The bioplastics market in Europe is going through a truly exciting time with more and more bio based materials produced at a commercial scale. In this context, PHAs are gaining increasing attention in the biodegradable polymer market due to their high biodegradability and versatility. Indeed biogenic PHAs polyesters represent a potentially ‘‘sustainable’’ replacement to fossil fuel-based thermoplastics. However, current commercial practices that produce PHA with pure microbial cultures grown on renewable, but refined, feedstock (i.e. up to 5 kg of glucose are needed to produce 1kg of PHA) under sterile conditions do not result in a sustainable commodity. The simultaneous cleaning and valorization of waste as culture media, appears, on the one hand, as a much more sustainable alternative reducing the material’s environmental impact of PHAs and avoiding food competition, and, on the other hand, presents potential for reducing the cost of derived products.
The European and Latino America industries generate huge volumes of effluents. Olive mill wastewater (OMWW) generated by the olive oil industry is an extremely hazardous pollutant. Indeed, OMWW is characterised by a dark colour, a strong smell and a high degree of organic and inorganic species (sugars, fats, proteins, organic acids, phenols, pigments, potassium and phosphate salts). Among them, polyphenols exhibit antimicrobial, ecotoxic and phytotoxic properties which limit the OMWW disposal and in fact also the culture of microorganisms.
Cyanobacteria are the largest and most diverse group of oxygenic photosynthetic microorganisms that can live in water and produce their own food based in nutrients and light. Several families exist varying greatly in shape and living as solitary cells or as colonies of many shapes, possibly forming filaments. Those organisms can live under extreme environmental conditions of light, temperature and nutrients. Several strains of cyanobacteria can be used in the control of eutrophication, due to their ability to grow under high nutrient concentration and capacity to remove nutrients or toxic compounds from waste water. This group of microorganisms can produce organic compounds which can be used in several applications in biotechnology, cosmetology or pharmaceutical.
In such context, the Oli-PHA project developed a holistic environmental approach, referred to as Maxi-Use, based on the circular economy for the recovery and re-use of OMWW for the purpose of obtaining PHA biopolymers. The developed technologies and materials not only hold significant benefits for the packaging and plastics industries and a whole host of end-user applications, but it also has a major impact for the sustainability of the olive oil sector, which faces huge challenges in the disposal of olive oil wastewater.
The Oli-PHA project aimed at building on promising preliminary results into the growth of photosynthetic microorganisms in wastewaters to produce PHAs whereby the yield and cost effectiveness was optimized by engineering optimized photobioreactors (PBR), genetically modifying the cyanobacteria, but also by developing tailored compound formulations. In addition, the Oli-PHA project developed versatile functional packaging solutions (flexible, rigid, active) using the new PHA polymer, natural fibres and the antioxidants extracted from the OMWW. Indeed, the most abundant polyphenol in OMWW (1-2 g/L), hydroxy-tyrosol, is one of the strongest natural antioxidant with recognised therapeutic properties. Therefore, their extraction and application in active packaging for food and cosmetic preservation also represents an interesting advancement. The left-over biomass obtained during the OMWW treatment process, was used as source for biogas.
The overall aim of the Oli-PHA project was to produce promising PHAs biopolymers (of which the specific type produced in the selected microorganisms was determined to be PHB, the most valued in the PHAs family) using cyanobacteria cultured in wastewaters of the olive oil industry to replace conventional fossil fuel-based plastics in packaging materials.
To this end, the specific objectives can be summarised as follows:
1. To carry out an analysis and treatment of OMWW for the effective removal of phenolic fractions
The presence of the phenolic fraction inhibits the growth of microorganisms, and as such their removal was necessary for PHA production. To this end, the project developed an environmentally friendly process for the removal of polyphenols from OMWW based on either active carbon process (patented and owned by SME partner AFT) or ion exchange resins.
2. To optimise the production of PHAs by cyanobacteria and bacteria in OMWW
The organic matter in the OMWW can be transformed into PHB by selected microorganisms. First, a bio-prospection was carried out with previously reported PHA-producing cyanobacteria and heterotrophic bacteria in order to select the most promising microorganisms in terms of phenolic tolerance and PHA production yield.
3. To produce PHA from cyanobacteria cultures in OMWW at pilot scale and to carry out research into cyanobacteria selection, as well as the growth and optimisation of PHA
A pilot photobioreactor (PBR) was designed and built for the production of PHA by the culture of cyanobacteria in OMWW in non axenic culture conditions. This PBR is made of modules to be readily scalable and integrated in the facilities of the olive oil processing SMEs.
4. To optimise the PHA extraction process in terms of environmental performance and cost- effectiveness via the use of thermal, mechanical and improved chemical pathways, and to obtain biogas from the remaining biomass
The PHA recovery/extraction process from the biomass synthesised in the photobioreactor was developed by investigating several environmentally friendly options- extraction by cell degradation or cell disruption - as well as the use of modified chemical pathways with for example bio-sourced solvents.
5. To present the complete PHA production process from OMWW to the olive oil industry in order to showcase its functionality and performance, as well as its benefits and the business opportunity it presents to olive oil producers for valorising their residues and obtaining an additional source of income. Presentations were made in San Javier and AFT.
6. To process plastic at sample scale with the resulting Oli-PHA polymers and to validate its ability to replace fossil fuel-based thermoplastics as packaging material by carrying out processability (injection, formability, printability tests, etc.) and performance tests (mechanical, thermal, etc.)
The PHB samples extracted from the cyanobacteria were tested vs. commercially available PHAs. Processability and performance tests to validate the usability of the new Oli-PHA as packaging material were performed vs. other plastics used in the packaging industry. Formulation work was dedicated to the optimization of PHAs, and in particular PHB, for packaging requirements. Indeed it is well known that PHB can be brittle due to partial crystallinity and this can be overcome through blending with other polymers, or addition of nucleating agents or natural plasticizer.
7. To develop Oli-PHA active packaging solutions and validate their effectiveness for safeguarding the quality and safety of the packed foodstuffs and cosmetics, and to carry out migration tests to enable widespread approval for the use of the Oli-PHA material in the food and cosmetic packaging sectors
Polyphenolic antioxidants recovered from OMWW were used to develop active packaging solutions. To this end, an application technique for the polyphenolic substances on the surface of packaging items was developed selecting suitable binder. The suitability of PHA-based materials with and without added polyphenols was tested as packaging of selected foodstuffs.
8. To carry out trials with the Oli-PHA (PHB) items in order to characterise and heighten their biodegradability/recyclability and composting
Depending on the structure and polymerisation route of plastic during processing and norm considered, packaging derived from renewable resources may be non-biodegradable. To this end, a comprehensive series of trials including biodegradability under controlled composting conditions, recycling (i.e. to detect any loss of properties of the PHB during reprocessing) disintegration tests and ecotoxicity tests were carried out.
9. To carry out a complete eco-efficiency analysis on the Oli-PHA products in order to demonstrate the sustainability and viability of the Oli-PHA results
Following recognised rules and EC regulations, LCA and LLC analyses were performed on the Oli-PHA products, including inventory analysis, impact assessment and interpretation of the results, taking into account every stage in the Oli-PHA product’s life cycle: extraction of the raw materials, processing, manufacturing, transportation phases, ending with use and disposal or recycling. Comparisons were made with currently available PHA and with other plastics used in the packaging sector.
10. To scale up the process to obtain packaging items with the new Oli-PHA material, to compare them to existing solutions
A series of packaging items (films, pouches and jars) were processed at the industrial facilities of the packaging processing companies from the consortium, PEMU and SELLO, in order to validate their processability within their installations with their existing equipment and compare them to the conventional packaging being produced by these companies. Tentative to thermoform trays were also made at CONICET.
11. To characterize the Oli-PHA material developed during the project in order to evaluate its suitability for its use in wider applications and to draw up corresponding recommendations for future development work
The Oli-PHA material developed during the industrial trials was characterised in terms of thermo-mechanical properties, barrier, compostability in order to evaluate the possibility of its use in packaging but also in different markets, such as biodegradable medical devices with lower raw material cost, and automotive components, and to draw up recommendations for future development work. The cost of the new material was also evaluated also.
12. To carry out industrial validation work to prove the viability of new packaging
The food and cosmetic producers LAGRANA and COSMETIC respectively, used the resulting packaging to pack their products and validate the performance and suitability of the resulting Oli-PHA packaging items in comparison with the materials they currently use.
13. To present the new material to industry in order to facilitate knowledge transfer and uptake by industry
Presentation sessions were hosted at the industrial sites from PEMU and IRIS so that other industry representative and stakeholders were able to see the material being processed, the resulting products and understand its benefits.
14. To carry out knowledge management and innovation related activities to ensure the project results are widely disseminated without jeopardising their commercial exploitation and to plan for their swift uptake by industry
In order to complement and support the overall goals of the project and contribute to the realisation of its expected impacts, the above scientific and technological objectives were complemented by a series of innovation-related objectives. Firstly, in order to ensure a swift route to market for the results of the research, the protection of the knowledge generated has been of paramount importance, as well as activities to ensure knowledge transfer and training to the industry value chain, and to ensure compliance with food contact packaging and environmental regulations, as well as to effectively raise awareness about the research among industry and the general public in order to stimulate market demand. The issue of communication was given careful attention
Project Results:
The first target of the project focused on the selection of the most suitable cyanobacteria and heterotrophic bacteria strains with tolerance to the phenolic compounds found in OMWW and ability to produce PHAs. Beforehand, an OMWW treatment was developed to avoid the microorganism growth inhibition due mainly to phenols. Suitable strategies for treatment and dephenolisation of OMWW, but also phenol recovery processes to allow their reuse as antioxidant in packaging and cosmetic application were developed. This process was tested on OMWW from Europe and Latin America and an OMWW simulant was formulated to ensure the reproducibility of the results for production of PHAs by cyanobacteria. Stabilisation and concentration of OMWW were necessary steps to enable efficient storage of the OMWW produced during the milling season. The polyphenol removal process carried out at the facilities of AFT was improved and scaled up so the 2000T OMWW currently produced per year could be treated.
After the OMWW treatment, the recovered olive pulp was valorised by the anaerobic production of biogas with co-substrates to maximise the yield. The assessment of biogas and methane yield was carried out by means of batch tests of wet anaerobic digestion according to ISO FDIS 15985, ASTM D.5511-94 and DIN 38414. In a second step the biogas production was evaluated in a continuous digestion test over long-term. The biogas potential of such substrate appeared very interesting within the range of 190 Nm³ biogas produced from olive pulp per ton olive pulp (with 29.2% dry matter) derived from OMWW treatment.
The dephenolised media was used for lab scale culture of 7 different cyanobacteria, including the wild type strain selected by CICESE to make the genetic modification, and 4 heterotrophic bacteria strains selected on the basis of the literature review reported in the DoW. In particular, these microorganisms can reduce the organic matter in the OMWW while accumulating PHA in their cytoplasm with a yield depending upon a large number of factors. As such, a wide campaign of tests was run with different origins and compositions of wastewater and culture conditions. Selected strains were tested at lab level to find the effect of various bio-chemical parameters (pH, dilution of OMWW, nutrients addition, light, polyphenols residual amount) on the cell growth (g/L), PHA yield (w/w) and productivity (g/L/h). In addition to optimization by manipulating culture conditions, another workline was the genetic modification of a promising cyanobacteria strain which allowed to significantly increasing PHA yield of the wild type strain (patent application CICESE (PCT/IB2014/002412) on the method used for genetic modification and characterization of cyanobacteria with enhanced capacity of producing PHA).
The resulting GMO able to grow on high content of OMWW even without nutrient supplementation, which could reduce the fresh water consumption during the process exploitation. In addition, parallel tests were performed at laboratory level to determine the conditions for scaling up the process.
In parallel, the Oli-PHA precompetitive photobioreactor (PBR) was designed and built for the production of PHA from cyanobacteria. Taking as reference the state of the art search, a novel design was proposed to maximise the cyanobacteria growth and also to allow performing a design of experiments, critical for the ongoing work of inducing and optimizing the PHA accumulation in cyanobacteria. The protototype PBR, including a control system to monitor different aspects of the culture in real time, was built at incremental scales and tested leading to iterative improvements. A pre-competitive 6 m3 pilot plant was delivered for further optimization of the PHA accumulation conditions. The bioreactor allows variations of the bio-chemical parameters determined in WP1 and additional process parameters, such as the addition of CO2, specific lighting, etc. As cyanobacteria use CO2 as a carbon source converting it to organic carbon and incorporating it to their biomass, a system to facilitate gas exchange with the culture medium (agitation, bubbling) or enrichment of the gas in the gas mixture for a biomass increase, was included. The use of CO2 emissions from combustion industries as carbon providers, as well as heating sources to maintain the culture in an optimum temperature range could be considered to further enhance the process eco-efficiency. Extensive trials were performed to identify the conditions for upscaling the culture of the microalgae in the pilot PBR leading to the identification of a 3 phases approach:
• 1° biomass growth in photoautotrophic conditions in PBR
• 2° biomass growth in heterotrophic static conditions with addition of dephenolized waste water
• Polymer accumulation in heterotrophic conditions with addition of sodium acetate to maximize yield
Nevertheless, significant challenges were met due to the microalgae acclimating to large scales and tolerance to non axenic conditions preventing reaching a stable production of large amount of PHB during the project.
The cyanobacteria harvesting and downstream process for extracting PHA were optimized. The harvested biomass was concentrated by flocculation and a more environmentally friendly PHA extraction process (vs. the current industrial approach) using non-chlorinated solvent was developed after cyanobacteria cell disruption by surfactants. The harvested biomass was subsequently concentrated in order to obtain a biomass cake (20-30 % dw) from the liquid culture by filtration by centrifugation or microfiltration increasing concentration from 0.7-1 g/L to 200-300 g/L.
To study the most promising extraction process considered three approaches:
• Direct extraction with solvent;
• Biomass degradation, with surfactants and with a specific biphasic process;
• Biomass disruption, in hypotonic medium and by ultrafiltration.
The optimal process developed consisted in:
✓ 1° Biomass degradation in aqueous solution of surfactant and chaotropic agent to solubilize non-PHA material;
✓ 2° Biomass degradation in non-chlorinated non-PHA solvent to remove further non-PHA material;
✓ Polymer recovery by centrifugation; and drying (purity 94-95%, recovery 93-96%);
✓ Solvent recovery by distillation.
In terms of polymer extraction, the investigation was particularly challenging because of the low amount of polymer in the biomass, ranging from 3 to 4 % (on weight of dry biomass). A complete assessment of the most performing method was made on a GMMO biomass. The proposed solution is an attractive alternative to the common chloroform extraction, which is a carcinogenic product and requires a further biomass drying step, and could be used also with other PHAs which are easier to extract than PHB. According to the experimental activities, the PHB was obtained with a purity of 94-95% with a recovery yield of 93-96%.
The PHA polymer extracted from both wild and genetically modified strains was characterized showing it was equivalent to commercial poly-hydroxybutyrate (PHB) with average molecular weight of 328000 Da and a melting point of 176°C. An overall production cost analysis carried out on the lab scale process estimated that the PHB from the project could be cost competitive vs. commercial products already on the market.
The specifications for the different packaging items to be obtained from the project were defined based on the properties of the conventional packaging materials currently used by the partners. Among PHAs, Poly (3-hydroxybutyrate) (PHB) is a homopolymer of 3-hydroxybutyrate which offers good resistance to moisture and aroma barrier properties therefore of specific interest in the Oli-PHA project focusing on functional packaging. Plasticizers must be added during extrusion of PHAs and in particular of PHB in order to modulate both processability and mechanical properties. The most suitable plasticizers allowing stabilising the kinetics of PHB crystallisation were selected and allowed obtaining stable materials in terms of mechanical properties after about two weeks from production. Different compounds of commercially available PHA, PHBV and PHB and blends were formulated for further converting into flexible and rigid packaging and the suitability of their mechanical properties for such purposes were checked. Natural fibres were also tested for production of biocomposites with lighter weight and reduced cost versus fully PHA based material.
Polyphenols have excellent antioxidant, anti-allergic, anti-inflammatory, anticancer, antihypertensive, and antimicrobial properties. In the spirit of circular economy (MaxiUse), the polyphenols extracts (80 % Hydroxityrosol) recovered from OMWW were either used in cosmetic formulations or applied as coatings on different plastic films to develop active packaging solutions. Active packagings are intended to extend the shelf-life or to improve the condition of the packaged product, here taking benefits of the polyphenols inherent antioxidant and antimicrobial properties. They are of specific interest for minimally processed food or when additives are not authorised as it is often the case for organic food.
Several foodstuffs and cosmetics were selected and the suitability of PHA-based materials with and without added polyphenols to be used as packaging of selected foodstuffs was tested. A comprehensive series of tests were carried out in order to validate the effectiveness of the Oli-PHA based packaging for safeguarding the quality and safety of the packed goods, such as microbial assessment, sensory analysis, shelflife determination. In addition, migration tests were performed in order to gather results for enabling the wide approval of the Oli-PHA materials for use in the food and cosmetic packaging sectors. Furthermore PHA/PHA films resisted to most cosmetic simulants.Finally the barrier properties to oxygen, light and humidity were tested to specify the type of product that can be packed with different formulations of PHA materials. While PHAs present interesting barrier properties even vs. other standard and bio-sourced polyesters to be used as passive packaging, the use of active coating with polyphenols allowed decreasing the oxygen permeability by a factor 4.
In terms of food packaging validation, significant reductions in lipooxidation in minced meat and in microbial growth in Gouda cheese were observed with active packaging solutions. In terms of use in cosmetics, the polyphenolic extract also displayed high antimicrobial activity and potential to be used as alternative to standard preservatives. In particular, the use of the extract as preservative in a cream and in a shampoo allowed reducing the microbial activity vs. standard packaging. Biodegradation and organic recycling were tested on developed compounds based on commercial PHAs meeting requirements for industrial composting, home compostability, soil, freshwater marine and anaerobic degradation. The marine biodegradability was also validated on the PHB extracted from the microalgae. Recyclability was tested through the partial substitution of virgin material by production scrap without affecting the properties of injected parts. The LCA showed no improvement vs. standard packaging, at least using lab scale data for the Oli-PHA production, and indicated the most intensive steps in terms of environmental impact being the biomass growth whereby further optimization were suggested.
In terms of industrial upscaling to obtain packaging items, previously obtained compounds were processed via extrusion, thermoforming and injection moulding. Several formulations appeared suitable for injection, and the process conditions and moulds were optimised to produce cosmetic jars. Although challenges were met to obtain sufficiently flexible formulations over time for thermoforming and blow moulding, several results were obtained. On the one hand, compression moulding and sheet extrusion were possible as prior stage to the thermoforming of lids or trays which was so far limited to low deformation ratio. On the other hand, blown films were successfully obtained at pilot scale, and those could be further converted into pouches. Further improvements needed in terms of processing were highlighted.
In terms of demonstration, the obtained packages (films and jars) were used for further validation of their suitability for packing additional foodstuffs and cosmetics. Thermoformed PLA trays were also coated with polyphenol to validate the active packaging solution in the industry. Those were validated with strawberry cream truffles, and grounded nuts showing in general shelf life increase. PHB jars were suitable for packing over 98% of cosmetic and personal care products (shampoos, (o/w) & (w/o) creams, lotions and massage oils) and appeared comparable to standard materials in terms of shelf-life. No detrimental interaction of the product was observed with the PHB containers both in terms of cream emulsion stability (colour and smell) as well as in terms of maintaining the preservative activity.
The commercial PHA/PHB packaging (as well as when extrapolating PHB cost from the project) resulted economically viable solution for their applications into organic food sector and cosmetics.
The knowledge generated during the project has been continuously evaluated in terms of its novelty and exploitation potential. A patent review has been carried out throughout the duration of the project in order to position the Oli-PHA technologies relative to existing IPR, leading to a patent application and a possible future filling. Besides the scientific knowledge generated in the project in terms of PHA production by microalgae, the foreground elements have been further analysed in terms of market exploitability. Indeed, developments such as Oli-PHA offer enormous opportunity to create added value, novel offering and market differentiation, to contribute to increasing the competitiveness of companies operating along the olive, plastic and packaging industries. Making such industries more competitive will have impacts for the European economy, growth and jobs.
Potential Impact:
The precompetitive results generated in the Oli-PHA project consist in different products (PHA and its compounds, biogas, polyphenols, fibers) and required processes to obtain them (waste water treatment, photobioreactor, anaerobic digestion, extraction, compounding). It gives exploitation potential to all participating partners by uptaking them in their own processing plants, and possibly further licensing them to external users. The subsequent material converting and use as packaging for food and cosmetics by end-user partners, essentially acting as early adopters or first clients of the new material, will extend the benefits derived by the consortium.
There are numerous socio-economic impacts that will be derived from the results of this Oli-PHA research project beyond the consortium in Europe, Latino America and beyond.
On a first place, the project contributes with a solution to the environmental problems associated to OMWW disposal, which is particularly important in the leading oil producing nations in EU, namely Spain, Italy, Greece, Turkey, Portugal and France. The industry in these countries is characterised by a high territorial scattering of mills (in Italy, for example, there are some 6,000 oil mills), over 90 % of which are SMEs. Production is seasonal, with the typical press season lasting between 3 and 4 months. Over this short timeframe, each mill can generate on average over 30 million m3 of liquid effluents per press season, the disposal of which poses an enormous challenge to mills. Beyond the sheer volume of waste being created, if one considers that OMWW contains long chain fatty acid compounds and polyphenols of up to several grams per litre (150-200 g/l), which exhibit antimicrobial, and phytotoxic properties, the staggering intensity of the problem becomes really critical. The negative effects on the environment render this waste as “ecotoxic”, in the meaning of in Annex III to Directive 91/689/EEC and to Directive 2008/98/EC. As such, if the olive oil producer do not treat or use himself the OMWW, a third party should collect and treat all wastes with the same CER code only if authorized. Traditionally OMWW are treated with enzymes or organic acids. The Oli-PHA developed process is not only more economically competitive but also better performing, safer and environmentally sustainable than conventional treatments.
Central to the expected socio-economic impacts, is the boosting of the competitiveness of companies operating along the olive, plastic and packaging industries. The Oli-PHA results will be introduced into 3 very large markets. Making such industries more competitive will have impacts for the European economy, growth and jobs.
OMWW possesses a double nature. While being a strong pollutant it is at the same time a possible source of valuable components, such as polyphenols, flavonoids, anthocyanins, inorganic trace elements, etc., that can be extracted and economically exploited. Thus, the vision of OMWW is now evolving from a waste to a raw material.Hence the market of the reuse of OMWW to produce valuable products is in expansion, because it is revenue and the resolution of a problem at the same time.Oli-PHA developed a novel process for the enrichment of low molecular weight phenols, which can be used as antioxidants in different sectors such as cosmetic, pharmaceutical and food industry. Growing health awareness is the primary factor responsible for the large growth of the antioxidant market in the last decade. The superior antioxidant property of polyphenols and their application in the treatment of cancer, diabetes and cardiovascular diseases has further contributed to the growth of the polyphenols market. Functional beverages were the largest application segment of polyphenols and accounted for over 44% of the total polyphenols demand in 2011 followed by functional foods which accounted for over 33% of the total consumption in 2011. In terms of volume, global consumption is expected to grow at a CAGR of 8.2% from 2012 to 2018 driven by the growing popularity of antioxidant potential and the shifting trend in consumer health from being reactive to proactive.
However, the limited supply of raw materials due to other overlapping commercial applications such as wine manufacturing, preparation of juices, jams and jellies is expected to inhibit the market growth over the next six years. As such, there is market space for new offer such as polyphenol recovered from OMWW as developed in Oli-PHA, for example as natural preservative in cosmetics and in active packaging as tested in the project (under the condition of complying with legislative aspects set in the EC 450/2009 requiring further characterization of the polyphenol extracts for the latter application).
Besides the reuse of food waste in their formulation, Oli-PHA active packaging have the potential extend the shelf-life of the packaged product and therefore could be a key to SAVE FOOD (one third of all food is either wasted or lost, often at the consumer level)contributing therefore on one of the current greatest global socio-economic challenge to feed a growing population. Several factors affect the quality, safety and shelf life of food and cosmetic products such as oxygen, light, humidity or the growth of microorganisms, among others. Oxidation is one of the most frequent mechanisms of food deterioration along with microbial spoilage. Antioxidants can be incorporated into or coated onto food packaging materials to control the oxidation of fatty components and pigments, and thus can contribute to the quality preservation of foods. The release of antioxidant agents in active food packaging causes consumer concerns regarding their safety. For this reason, there is a growing consumer preference for natural antioxidants to synthetic antioxidants, such as butylated hydroxytoluene (BHT). Preservatives such parabens which are currently under revision in Europe are used in a high number of cosmetic products. As such Oli-PHA answers to the consumers demand for more natural cosmetics and food packaging solutions.
Another key socioeconomic impact is the development of bio-based and biodegradable packaging materials with suitable thermo-mechanical properties that can reuse waste and prevent the use of depleting fossil fuel derived raw materials and resources. The Bioplastics European association predicts that the global bioplastics production capacity is set to grow 500 % by 2016 (definition including under bioplastics partly or fully biobased, biodegradable, or both). The main drivers of this growth are twofold: consumer demand for environmentally-friendly products and volatile price of petroleum. In spite of announced production increase, PHA is still a minor production whereas drop in solutions such as biobased PE and PET have taken a major place. PHAs are currently is produced in Europe at small scale by the companies Biomer and Bio-On, in the USA by Metabolix, in China by Tianan and in Brazil by Biocycle among others. Commercial PHAs are generally extracted with chlorinated solvents from heterotrophic bacteriagrown on refined sugars (up to 5 kg per kg of PHA) or oils. PHAs are more expensive than most other (bio)plastics (eg. 5-19€ per kg of pure PHAs). Nevertheless, due to good mechanical and barrier properties, its biodegradability and recyclability, PHAs have potential to substitute some of the 250 MT of plastics used worldwide per year in packaging (market consuming over 45% of all plastic produced worldwide). The share is even bigger in case bioplastics (>75%) and biodegradability can be seen as a real asset for certain types of packaging which are very short term uses or of difficult recycling due to residual packed product contained at end of life. Among all PHA polymers existing, PHB, its copolymers, and PHBV are today the most widely commercially available. Depending on the composition of the copolymers, grafting co-monomers or tailoring of the formulations (using plasticizers and others additives), PHAs’ properties are very versatile can be used in applications ranging from surgical sutures, tissue engineering and agricultural foils to packaging for the storage of food products. PHA also exhibits good barrier properties, and can be used in the packaging industry as a biodegradable plastic for contributing to solving environmental pollution problems.
In order to improve PHA biopolymers sustainability, the use of waste as feedstock or of genetically engineered bacteria should all lead to reductions in production cost. Oli-PHA research effort focused on the valorisation of wastes (OMWW) as non-food competing sources for cultivating PHA-producers. In addition to studies of heterotrophic bacteria as PHA producers, PHA production using photosynthetic organisms as production hosts have been also explored as a novel approach in Oli-PHA. Cyanobacteria are oxygen-evolving photoautotrophs with the added advantage that some of them naturally possess the key enzyme in PHA biosynthesis. They can grow on high content of OMWW even without nutrient supplementation, which could reduce the fresh water consumption during the process exploitation. In addition, due to their minimal nutrient requirements in comparison with heterotrophic bacteria and fast growth in comparison with transgenic plants, cyanobacteria are attractive hosts for the production of PHA. However, in comparison with other heterotrophic bacterial strains, literature prior to Oli-PHA showed that cyanobacteria can only produce very small amounts of PHAs. Therefore, strategies to improve PHA production yield, such as those studied in Oli-PHA, hold an enormous potential for the industrial production of this biopolymer. In the Oli-PHA project, cyanobacteria were further optimised via genetic modifications allowing to significantly increase PHA yield and tolerance to some residual phenols. In addition, the incubation conditions, photobioreactor (PBR) designs and harvesting operations were optimized for large-scale industrial production of the cyanobacterial products to increase productivity and to decrease costs of PHA production. Despite difficulties in the scaling up of the cultures, great advances were made in the Oli-PHA project, showing that in spite of the lower accumulation in PHA in the GM cyanobacteria than in the currently used heterotrophic bacteria, the use of waste feedstock and optimised downstream process could still lead to market competitive PHB products.
Besides the potential of microalgae for producing PHA, the biodiesels generation represents the biggest volume application and potential for microalgae. In the past decade, the biodiesel industry has seen massive growth globally, more than doubling the production every 2 years. Indeed, algae can serve as a feedstock for first generation biodiesel and ethanol plants, aviation fuels market and biocrude for biofuel. Nowadays, 50% of the cars in EU are running with diesel. The problem is that the EU cannot produce enough rapeseed and sunflower to cope with this demand so this can be seen as a potential opportunity for algae. The USA is the first consumer of diesel fuel for military use in the world, their industrial Diesel market represents a 25% of the petrol consumption; also more than 95% of private cars use gasoline, while a 3% runs on diesel. The problem is the same as in the EU. The USA cannot produce enough corn and soybeans to meet the demand, even with cellulosic corn, so algae could assume an important role. Within the Oli-PHA project biogas productivity was evaluated using the two types of waste streams as substrates for anaerobic digestion: the solid fraction of OMWW (olive mill wastewater) after the pretreatment step to extract polyphenols, and the algae biomass - harvested from the photobioreactor - after extraction of PHA. It was concluded that olive pulp (solid fraction of OMWW) is a very usable substrate for anaerobic digestion which together with algae biomass as a nitrogen source has the potential to be a successful input mixture for anaerobic digestion. Nevertheless this latter has lower biogas potential and is not recovered as a solid from the extraction process, as such alternative co-substrate such as manure are preferred.
Furthermore, Oli-PHA assists European packaging producers to adhere to the various EC legislations that exist in the plastic industry, such as the packaging waste legislation in force in Europe. Moreover, by the CO2 consumption via microorganisms to grow, reducing the energy, as well as raw materials consumption and waste generation, the results of this project will also contribute to assisting the EU in meeting its CO2 cutting objectives under the Kyoto Protocol. All in all, microalgae have much faster growth than other crops and a huge potential still to be fully disclosed. The biorefinery and bioplastics sectors are growing at a very fast pace and, and while Oli-PHA met interesting properties, shall further research allow scaling up its production and therefore adjust its price to a suitable range, solutions like Oli-PHA would match different current technical needs opening up an interesting market application.
In terms of exploitation, the project developed significant know-how exploitable by the partners and their novelty was scanned in terms of patentability leading to a patent application on a method for genetic modification of bacteria, and a possible future filling. The key exploitable results (KER) were identified and characterized in terms of commercial significance, market potential, IPR status and the conditions needed to enable the results to be exploited. In addition to the number of foreground elements of scientific relevance dealing with the “Selection of microorganisms with highest yield, of suitable culture conditions and culture media”, achieved by cooperation of partners involved in WP1 and WP2 activities respectively at lab and large scale, the Oli-PHA exploitable foreground elements are as follows:
• Novel process for the enrichment of low molecular weight phenols in OMWW
• Improvement of polyphenol removal process from OMWW and of their recovery
• Method for genetic modification and characterization of cyanobacteria with enhanced capacity of producing PHA. Patent pending CICESE (PCT/IB2014/002412)
• PBR with its control system (draft patent in process)
• Optimal conditions for biogas production of solid fraction of OMWW and algae residue after extraction
• Formulation and processing parameters for production of PHA based material by injection moulding, thermoforming and blow moulding.
• Active packaging based on polyphenols from OMWW
An economic analysis was carried out for the individual process steps as well as for the integrated Oli-PHA plant taking into account the profits and costs of feedstock, energy, current and expected waste treatments costs, etc. This led to different balances depending on the commercialisation scenario but overall highlighted the potential cost competitiveness of the resulting products like PHB and polyphenols especially when valorising the biogas in closed loop as energy source. Further potential exploitation incomes arise from the licensing of the IP/knowhow, provision of services, etc. besides the use of the foreground generated by each partner in its own business, improvement of its operations.
The exploitation intentions of the Consortium members were discussed during project meetings and the exploitation strategy further elaborated with the support of the NMP Exploitation support services (escic2). A Joint exploitation agreement of the foreground was laid out based on the above described elements but also on the scientific know-how generated in the project, benefitting from the synergistic role of the partners covering the whole supply and value chain. In addition, for specific cases where the foreground was owned by several members, Joint Ownership agreements were signed to define shares, rights and obligations in order to avoid competition between co-owners.
In parallel, the consortium organized a comprehensive series of training and demonstration sessions for external stakeholders in order to maximize market uptake as well as to gather valuable feedback from researchers, end users and technology providers in terms of sustainable polymer trends, OMWW managing initiatives and key limiting factors for introduction of new solutions in the marketplace. The Consortium plans to undertake future development efforts to bring Oli-PHA technology from pre-industrial to a commercial system, and to perform practical evaluations of the technology and further demonstration work that will be bespoke to industry. Further funding to mitigate the cost of the additional development work will be applied for.
Additionally, a number of dissemination activities have been carried out to on the principles of the Oli-PHA technology and products in preparation for the future exploitation of the same. These include the development of a project website (http://www.oli-pha.eu) which informs the public on the basics of the technology, latest news and the progress of the project insofar, as well as various press releases for raising the awareness of Oli-PHA both in industry and in the public domain. The project partners also took part in several scientific conferences, trade fairs and TV programmes. In terms of scientific dissemination, the project allowed the preparation of 4 peer-reviewed publications (some are in revision) and 5 book chapters. 4 public demonstration events including a scientific workshop were organised, as well as training sessions.
List of Websites:
www.olipha.eu
Dr. Elodie Bugnicourt
IRIS SL.
Avda Carl Friedrich Gauss 11
08860 Castelldefels
Spain
Tel: 00 34 935542503
Fax: 00 34 935542511
E-mail: ebugnicourt@iris.cat
Oli-PHA was a three year applied research project, which commenced in June 2012 and finished in May 2015, and was funded under the European Commission’s Seventh Framework Programme (FP7) and specifically under the “NMP - Nanosciences, Nanotechnologies, Materials and New Production Technologies” programme. Following a circular economy approach, the overall goal of the project was to valorise the olive mill wastewater (OMWW)tosimultaneously clean such water and produce high value added products such as polyhydroxyalkanoate (PHA) biopolymers using photosynthetic microorganisms, but also to recover polyphenols that could be used as additives in cosmetic and in active packaging. PHAs is a family of biopolyesters produced by number of microorganisms, generally heterotrophic bacteria grown in refined feedstock, that present suitable properties for food and cosmetic packaging (good gas and moisture barrier, thermo-mechanical properties) and excellent biodegradability in number of environments. New research is needed to enhance PHA market uptake using more sustainable/non food competing substrates than commercially available PHA, especially obtained from wastes, reduce its cost, as well as to fine tune its formulation to different rigid and flexible functional packaging applications.
In terms of main results, the project delivered:
• A pretreatment process for OMWW was developed to recover polyphenols of very high antioxidant effect and obtain a suitable media for cultivating cyanobacteria. An interesting biogas potential was observed with left solids from OMWW.
• 7 different cyanobacteria and 4 heterotrophic bacteria PHA-producers were tested at lab scale. The optimal culture conditions (pH, temperature, CO2/air, light intensity and cycles, tolerance to dephenolized OMWW)were determined both on lab and pilot scale
• A novel method for genetic modification and characterization of a cyanobacteria with enhanced capacity of producing PHA was developed to allow increasing the PHA yield very significantly vs. wild strains (patent filled).
• Trials were made to upscale the microalgae culture using a 6 m3 PBR pilot plant with innovative design that was delivered in the project. In spite of challenges met to reach a stable production due to the microalgae acclimatation to large scales and tolerance to non axenic conditions, a proper 3-stage culture approach to maximise PHA production was identified.
• A new environmentally friendly extraction process was developed using non-chlorinated solvent after biomass concentration by flocculation and cell disruption by surfactants. The polymer extracted from the biomass was characterized as poly-hydroxybutyrate (PHB) of equivalent properties to commercial one.
• Optimal compounds reducing issues linked with crystallization kinetics & processing conditions were developed based on commercial PHA and PHB. Films were obtained by blow moulding, jars were injected and sheets were compressed and extruded before thermoforming trials to obtain trays.
• Active packaging with improved oxygen barrier and antimicrobial properties were developed using the polyphenols extracted from OMWW and validated showing a very significant shelf life extension of packed food products. PHA films had compliant migration levels to be used as food packaging. PHB jars were validated as suitable packaging solution for most cosmetic products. The polyphenols extracts also showed excellent potential as preservative for cosmetics.
• Biodegradation and organic recycling were tested on developed compounds meeting requirements for industrial composting, home compostability, freshwater and anaerobic degradation. The PHA produced within the project, is biodegradable in these environments. Moreover it is a suitable material to produce compostable products. Recyclability was also proved without affecting the properties of injected parts. An LCA was performed vs. standard packaging and further required optimizations were suggested.
Project Context and Objectives:
The bioplastics market in Europe is going through a truly exciting time with more and more bio based materials produced at a commercial scale. In this context, PHAs are gaining increasing attention in the biodegradable polymer market due to their high biodegradability and versatility. Indeed biogenic PHAs polyesters represent a potentially ‘‘sustainable’’ replacement to fossil fuel-based thermoplastics. However, current commercial practices that produce PHA with pure microbial cultures grown on renewable, but refined, feedstock (i.e. up to 5 kg of glucose are needed to produce 1kg of PHA) under sterile conditions do not result in a sustainable commodity. The simultaneous cleaning and valorization of waste as culture media, appears, on the one hand, as a much more sustainable alternative reducing the material’s environmental impact of PHAs and avoiding food competition, and, on the other hand, presents potential for reducing the cost of derived products.
The European and Latino America industries generate huge volumes of effluents. Olive mill wastewater (OMWW) generated by the olive oil industry is an extremely hazardous pollutant. Indeed, OMWW is characterised by a dark colour, a strong smell and a high degree of organic and inorganic species (sugars, fats, proteins, organic acids, phenols, pigments, potassium and phosphate salts). Among them, polyphenols exhibit antimicrobial, ecotoxic and phytotoxic properties which limit the OMWW disposal and in fact also the culture of microorganisms.
Cyanobacteria are the largest and most diverse group of oxygenic photosynthetic microorganisms that can live in water and produce their own food based in nutrients and light. Several families exist varying greatly in shape and living as solitary cells or as colonies of many shapes, possibly forming filaments. Those organisms can live under extreme environmental conditions of light, temperature and nutrients. Several strains of cyanobacteria can be used in the control of eutrophication, due to their ability to grow under high nutrient concentration and capacity to remove nutrients or toxic compounds from waste water. This group of microorganisms can produce organic compounds which can be used in several applications in biotechnology, cosmetology or pharmaceutical.
In such context, the Oli-PHA project developed a holistic environmental approach, referred to as Maxi-Use, based on the circular economy for the recovery and re-use of OMWW for the purpose of obtaining PHA biopolymers. The developed technologies and materials not only hold significant benefits for the packaging and plastics industries and a whole host of end-user applications, but it also has a major impact for the sustainability of the olive oil sector, which faces huge challenges in the disposal of olive oil wastewater.
The Oli-PHA project aimed at building on promising preliminary results into the growth of photosynthetic microorganisms in wastewaters to produce PHAs whereby the yield and cost effectiveness was optimized by engineering optimized photobioreactors (PBR), genetically modifying the cyanobacteria, but also by developing tailored compound formulations. In addition, the Oli-PHA project developed versatile functional packaging solutions (flexible, rigid, active) using the new PHA polymer, natural fibres and the antioxidants extracted from the OMWW. Indeed, the most abundant polyphenol in OMWW (1-2 g/L), hydroxy-tyrosol, is one of the strongest natural antioxidant with recognised therapeutic properties. Therefore, their extraction and application in active packaging for food and cosmetic preservation also represents an interesting advancement. The left-over biomass obtained during the OMWW treatment process, was used as source for biogas.
The overall aim of the Oli-PHA project was to produce promising PHAs biopolymers (of which the specific type produced in the selected microorganisms was determined to be PHB, the most valued in the PHAs family) using cyanobacteria cultured in wastewaters of the olive oil industry to replace conventional fossil fuel-based plastics in packaging materials.
To this end, the specific objectives can be summarised as follows:
1. To carry out an analysis and treatment of OMWW for the effective removal of phenolic fractions
The presence of the phenolic fraction inhibits the growth of microorganisms, and as such their removal was necessary for PHA production. To this end, the project developed an environmentally friendly process for the removal of polyphenols from OMWW based on either active carbon process (patented and owned by SME partner AFT) or ion exchange resins.
2. To optimise the production of PHAs by cyanobacteria and bacteria in OMWW
The organic matter in the OMWW can be transformed into PHB by selected microorganisms. First, a bio-prospection was carried out with previously reported PHA-producing cyanobacteria and heterotrophic bacteria in order to select the most promising microorganisms in terms of phenolic tolerance and PHA production yield.
3. To produce PHA from cyanobacteria cultures in OMWW at pilot scale and to carry out research into cyanobacteria selection, as well as the growth and optimisation of PHA
A pilot photobioreactor (PBR) was designed and built for the production of PHA by the culture of cyanobacteria in OMWW in non axenic culture conditions. This PBR is made of modules to be readily scalable and integrated in the facilities of the olive oil processing SMEs.
4. To optimise the PHA extraction process in terms of environmental performance and cost- effectiveness via the use of thermal, mechanical and improved chemical pathways, and to obtain biogas from the remaining biomass
The PHA recovery/extraction process from the biomass synthesised in the photobioreactor was developed by investigating several environmentally friendly options- extraction by cell degradation or cell disruption - as well as the use of modified chemical pathways with for example bio-sourced solvents.
5. To present the complete PHA production process from OMWW to the olive oil industry in order to showcase its functionality and performance, as well as its benefits and the business opportunity it presents to olive oil producers for valorising their residues and obtaining an additional source of income. Presentations were made in San Javier and AFT.
6. To process plastic at sample scale with the resulting Oli-PHA polymers and to validate its ability to replace fossil fuel-based thermoplastics as packaging material by carrying out processability (injection, formability, printability tests, etc.) and performance tests (mechanical, thermal, etc.)
The PHB samples extracted from the cyanobacteria were tested vs. commercially available PHAs. Processability and performance tests to validate the usability of the new Oli-PHA as packaging material were performed vs. other plastics used in the packaging industry. Formulation work was dedicated to the optimization of PHAs, and in particular PHB, for packaging requirements. Indeed it is well known that PHB can be brittle due to partial crystallinity and this can be overcome through blending with other polymers, or addition of nucleating agents or natural plasticizer.
7. To develop Oli-PHA active packaging solutions and validate their effectiveness for safeguarding the quality and safety of the packed foodstuffs and cosmetics, and to carry out migration tests to enable widespread approval for the use of the Oli-PHA material in the food and cosmetic packaging sectors
Polyphenolic antioxidants recovered from OMWW were used to develop active packaging solutions. To this end, an application technique for the polyphenolic substances on the surface of packaging items was developed selecting suitable binder. The suitability of PHA-based materials with and without added polyphenols was tested as packaging of selected foodstuffs.
8. To carry out trials with the Oli-PHA (PHB) items in order to characterise and heighten their biodegradability/recyclability and composting
Depending on the structure and polymerisation route of plastic during processing and norm considered, packaging derived from renewable resources may be non-biodegradable. To this end, a comprehensive series of trials including biodegradability under controlled composting conditions, recycling (i.e. to detect any loss of properties of the PHB during reprocessing) disintegration tests and ecotoxicity tests were carried out.
9. To carry out a complete eco-efficiency analysis on the Oli-PHA products in order to demonstrate the sustainability and viability of the Oli-PHA results
Following recognised rules and EC regulations, LCA and LLC analyses were performed on the Oli-PHA products, including inventory analysis, impact assessment and interpretation of the results, taking into account every stage in the Oli-PHA product’s life cycle: extraction of the raw materials, processing, manufacturing, transportation phases, ending with use and disposal or recycling. Comparisons were made with currently available PHA and with other plastics used in the packaging sector.
10. To scale up the process to obtain packaging items with the new Oli-PHA material, to compare them to existing solutions
A series of packaging items (films, pouches and jars) were processed at the industrial facilities of the packaging processing companies from the consortium, PEMU and SELLO, in order to validate their processability within their installations with their existing equipment and compare them to the conventional packaging being produced by these companies. Tentative to thermoform trays were also made at CONICET.
11. To characterize the Oli-PHA material developed during the project in order to evaluate its suitability for its use in wider applications and to draw up corresponding recommendations for future development work
The Oli-PHA material developed during the industrial trials was characterised in terms of thermo-mechanical properties, barrier, compostability in order to evaluate the possibility of its use in packaging but also in different markets, such as biodegradable medical devices with lower raw material cost, and automotive components, and to draw up recommendations for future development work. The cost of the new material was also evaluated also.
12. To carry out industrial validation work to prove the viability of new packaging
The food and cosmetic producers LAGRANA and COSMETIC respectively, used the resulting packaging to pack their products and validate the performance and suitability of the resulting Oli-PHA packaging items in comparison with the materials they currently use.
13. To present the new material to industry in order to facilitate knowledge transfer and uptake by industry
Presentation sessions were hosted at the industrial sites from PEMU and IRIS so that other industry representative and stakeholders were able to see the material being processed, the resulting products and understand its benefits.
14. To carry out knowledge management and innovation related activities to ensure the project results are widely disseminated without jeopardising their commercial exploitation and to plan for their swift uptake by industry
In order to complement and support the overall goals of the project and contribute to the realisation of its expected impacts, the above scientific and technological objectives were complemented by a series of innovation-related objectives. Firstly, in order to ensure a swift route to market for the results of the research, the protection of the knowledge generated has been of paramount importance, as well as activities to ensure knowledge transfer and training to the industry value chain, and to ensure compliance with food contact packaging and environmental regulations, as well as to effectively raise awareness about the research among industry and the general public in order to stimulate market demand. The issue of communication was given careful attention
Project Results:
The first target of the project focused on the selection of the most suitable cyanobacteria and heterotrophic bacteria strains with tolerance to the phenolic compounds found in OMWW and ability to produce PHAs. Beforehand, an OMWW treatment was developed to avoid the microorganism growth inhibition due mainly to phenols. Suitable strategies for treatment and dephenolisation of OMWW, but also phenol recovery processes to allow their reuse as antioxidant in packaging and cosmetic application were developed. This process was tested on OMWW from Europe and Latin America and an OMWW simulant was formulated to ensure the reproducibility of the results for production of PHAs by cyanobacteria. Stabilisation and concentration of OMWW were necessary steps to enable efficient storage of the OMWW produced during the milling season. The polyphenol removal process carried out at the facilities of AFT was improved and scaled up so the 2000T OMWW currently produced per year could be treated.
After the OMWW treatment, the recovered olive pulp was valorised by the anaerobic production of biogas with co-substrates to maximise the yield. The assessment of biogas and methane yield was carried out by means of batch tests of wet anaerobic digestion according to ISO FDIS 15985, ASTM D.5511-94 and DIN 38414. In a second step the biogas production was evaluated in a continuous digestion test over long-term. The biogas potential of such substrate appeared very interesting within the range of 190 Nm³ biogas produced from olive pulp per ton olive pulp (with 29.2% dry matter) derived from OMWW treatment.
The dephenolised media was used for lab scale culture of 7 different cyanobacteria, including the wild type strain selected by CICESE to make the genetic modification, and 4 heterotrophic bacteria strains selected on the basis of the literature review reported in the DoW. In particular, these microorganisms can reduce the organic matter in the OMWW while accumulating PHA in their cytoplasm with a yield depending upon a large number of factors. As such, a wide campaign of tests was run with different origins and compositions of wastewater and culture conditions. Selected strains were tested at lab level to find the effect of various bio-chemical parameters (pH, dilution of OMWW, nutrients addition, light, polyphenols residual amount) on the cell growth (g/L), PHA yield (w/w) and productivity (g/L/h). In addition to optimization by manipulating culture conditions, another workline was the genetic modification of a promising cyanobacteria strain which allowed to significantly increasing PHA yield of the wild type strain (patent application CICESE (PCT/IB2014/002412) on the method used for genetic modification and characterization of cyanobacteria with enhanced capacity of producing PHA).
The resulting GMO able to grow on high content of OMWW even without nutrient supplementation, which could reduce the fresh water consumption during the process exploitation. In addition, parallel tests were performed at laboratory level to determine the conditions for scaling up the process.
In parallel, the Oli-PHA precompetitive photobioreactor (PBR) was designed and built for the production of PHA from cyanobacteria. Taking as reference the state of the art search, a novel design was proposed to maximise the cyanobacteria growth and also to allow performing a design of experiments, critical for the ongoing work of inducing and optimizing the PHA accumulation in cyanobacteria. The protototype PBR, including a control system to monitor different aspects of the culture in real time, was built at incremental scales and tested leading to iterative improvements. A pre-competitive 6 m3 pilot plant was delivered for further optimization of the PHA accumulation conditions. The bioreactor allows variations of the bio-chemical parameters determined in WP1 and additional process parameters, such as the addition of CO2, specific lighting, etc. As cyanobacteria use CO2 as a carbon source converting it to organic carbon and incorporating it to their biomass, a system to facilitate gas exchange with the culture medium (agitation, bubbling) or enrichment of the gas in the gas mixture for a biomass increase, was included. The use of CO2 emissions from combustion industries as carbon providers, as well as heating sources to maintain the culture in an optimum temperature range could be considered to further enhance the process eco-efficiency. Extensive trials were performed to identify the conditions for upscaling the culture of the microalgae in the pilot PBR leading to the identification of a 3 phases approach:
• 1° biomass growth in photoautotrophic conditions in PBR
• 2° biomass growth in heterotrophic static conditions with addition of dephenolized waste water
• Polymer accumulation in heterotrophic conditions with addition of sodium acetate to maximize yield
Nevertheless, significant challenges were met due to the microalgae acclimating to large scales and tolerance to non axenic conditions preventing reaching a stable production of large amount of PHB during the project.
The cyanobacteria harvesting and downstream process for extracting PHA were optimized. The harvested biomass was concentrated by flocculation and a more environmentally friendly PHA extraction process (vs. the current industrial approach) using non-chlorinated solvent was developed after cyanobacteria cell disruption by surfactants. The harvested biomass was subsequently concentrated in order to obtain a biomass cake (20-30 % dw) from the liquid culture by filtration by centrifugation or microfiltration increasing concentration from 0.7-1 g/L to 200-300 g/L.
To study the most promising extraction process considered three approaches:
• Direct extraction with solvent;
• Biomass degradation, with surfactants and with a specific biphasic process;
• Biomass disruption, in hypotonic medium and by ultrafiltration.
The optimal process developed consisted in:
✓ 1° Biomass degradation in aqueous solution of surfactant and chaotropic agent to solubilize non-PHA material;
✓ 2° Biomass degradation in non-chlorinated non-PHA solvent to remove further non-PHA material;
✓ Polymer recovery by centrifugation; and drying (purity 94-95%, recovery 93-96%);
✓ Solvent recovery by distillation.
In terms of polymer extraction, the investigation was particularly challenging because of the low amount of polymer in the biomass, ranging from 3 to 4 % (on weight of dry biomass). A complete assessment of the most performing method was made on a GMMO biomass. The proposed solution is an attractive alternative to the common chloroform extraction, which is a carcinogenic product and requires a further biomass drying step, and could be used also with other PHAs which are easier to extract than PHB. According to the experimental activities, the PHB was obtained with a purity of 94-95% with a recovery yield of 93-96%.
The PHA polymer extracted from both wild and genetically modified strains was characterized showing it was equivalent to commercial poly-hydroxybutyrate (PHB) with average molecular weight of 328000 Da and a melting point of 176°C. An overall production cost analysis carried out on the lab scale process estimated that the PHB from the project could be cost competitive vs. commercial products already on the market.
The specifications for the different packaging items to be obtained from the project were defined based on the properties of the conventional packaging materials currently used by the partners. Among PHAs, Poly (3-hydroxybutyrate) (PHB) is a homopolymer of 3-hydroxybutyrate which offers good resistance to moisture and aroma barrier properties therefore of specific interest in the Oli-PHA project focusing on functional packaging. Plasticizers must be added during extrusion of PHAs and in particular of PHB in order to modulate both processability and mechanical properties. The most suitable plasticizers allowing stabilising the kinetics of PHB crystallisation were selected and allowed obtaining stable materials in terms of mechanical properties after about two weeks from production. Different compounds of commercially available PHA, PHBV and PHB and blends were formulated for further converting into flexible and rigid packaging and the suitability of their mechanical properties for such purposes were checked. Natural fibres were also tested for production of biocomposites with lighter weight and reduced cost versus fully PHA based material.
Polyphenols have excellent antioxidant, anti-allergic, anti-inflammatory, anticancer, antihypertensive, and antimicrobial properties. In the spirit of circular economy (MaxiUse), the polyphenols extracts (80 % Hydroxityrosol) recovered from OMWW were either used in cosmetic formulations or applied as coatings on different plastic films to develop active packaging solutions. Active packagings are intended to extend the shelf-life or to improve the condition of the packaged product, here taking benefits of the polyphenols inherent antioxidant and antimicrobial properties. They are of specific interest for minimally processed food or when additives are not authorised as it is often the case for organic food.
Several foodstuffs and cosmetics were selected and the suitability of PHA-based materials with and without added polyphenols to be used as packaging of selected foodstuffs was tested. A comprehensive series of tests were carried out in order to validate the effectiveness of the Oli-PHA based packaging for safeguarding the quality and safety of the packed goods, such as microbial assessment, sensory analysis, shelflife determination. In addition, migration tests were performed in order to gather results for enabling the wide approval of the Oli-PHA materials for use in the food and cosmetic packaging sectors. Furthermore PHA/PHA films resisted to most cosmetic simulants.Finally the barrier properties to oxygen, light and humidity were tested to specify the type of product that can be packed with different formulations of PHA materials. While PHAs present interesting barrier properties even vs. other standard and bio-sourced polyesters to be used as passive packaging, the use of active coating with polyphenols allowed decreasing the oxygen permeability by a factor 4.
In terms of food packaging validation, significant reductions in lipooxidation in minced meat and in microbial growth in Gouda cheese were observed with active packaging solutions. In terms of use in cosmetics, the polyphenolic extract also displayed high antimicrobial activity and potential to be used as alternative to standard preservatives. In particular, the use of the extract as preservative in a cream and in a shampoo allowed reducing the microbial activity vs. standard packaging. Biodegradation and organic recycling were tested on developed compounds based on commercial PHAs meeting requirements for industrial composting, home compostability, soil, freshwater marine and anaerobic degradation. The marine biodegradability was also validated on the PHB extracted from the microalgae. Recyclability was tested through the partial substitution of virgin material by production scrap without affecting the properties of injected parts. The LCA showed no improvement vs. standard packaging, at least using lab scale data for the Oli-PHA production, and indicated the most intensive steps in terms of environmental impact being the biomass growth whereby further optimization were suggested.
In terms of industrial upscaling to obtain packaging items, previously obtained compounds were processed via extrusion, thermoforming and injection moulding. Several formulations appeared suitable for injection, and the process conditions and moulds were optimised to produce cosmetic jars. Although challenges were met to obtain sufficiently flexible formulations over time for thermoforming and blow moulding, several results were obtained. On the one hand, compression moulding and sheet extrusion were possible as prior stage to the thermoforming of lids or trays which was so far limited to low deformation ratio. On the other hand, blown films were successfully obtained at pilot scale, and those could be further converted into pouches. Further improvements needed in terms of processing were highlighted.
In terms of demonstration, the obtained packages (films and jars) were used for further validation of their suitability for packing additional foodstuffs and cosmetics. Thermoformed PLA trays were also coated with polyphenol to validate the active packaging solution in the industry. Those were validated with strawberry cream truffles, and grounded nuts showing in general shelf life increase. PHB jars were suitable for packing over 98% of cosmetic and personal care products (shampoos, (o/w) & (w/o) creams, lotions and massage oils) and appeared comparable to standard materials in terms of shelf-life. No detrimental interaction of the product was observed with the PHB containers both in terms of cream emulsion stability (colour and smell) as well as in terms of maintaining the preservative activity.
The commercial PHA/PHB packaging (as well as when extrapolating PHB cost from the project) resulted economically viable solution for their applications into organic food sector and cosmetics.
The knowledge generated during the project has been continuously evaluated in terms of its novelty and exploitation potential. A patent review has been carried out throughout the duration of the project in order to position the Oli-PHA technologies relative to existing IPR, leading to a patent application and a possible future filling. Besides the scientific knowledge generated in the project in terms of PHA production by microalgae, the foreground elements have been further analysed in terms of market exploitability. Indeed, developments such as Oli-PHA offer enormous opportunity to create added value, novel offering and market differentiation, to contribute to increasing the competitiveness of companies operating along the olive, plastic and packaging industries. Making such industries more competitive will have impacts for the European economy, growth and jobs.
Potential Impact:
The precompetitive results generated in the Oli-PHA project consist in different products (PHA and its compounds, biogas, polyphenols, fibers) and required processes to obtain them (waste water treatment, photobioreactor, anaerobic digestion, extraction, compounding). It gives exploitation potential to all participating partners by uptaking them in their own processing plants, and possibly further licensing them to external users. The subsequent material converting and use as packaging for food and cosmetics by end-user partners, essentially acting as early adopters or first clients of the new material, will extend the benefits derived by the consortium.
There are numerous socio-economic impacts that will be derived from the results of this Oli-PHA research project beyond the consortium in Europe, Latino America and beyond.
On a first place, the project contributes with a solution to the environmental problems associated to OMWW disposal, which is particularly important in the leading oil producing nations in EU, namely Spain, Italy, Greece, Turkey, Portugal and France. The industry in these countries is characterised by a high territorial scattering of mills (in Italy, for example, there are some 6,000 oil mills), over 90 % of which are SMEs. Production is seasonal, with the typical press season lasting between 3 and 4 months. Over this short timeframe, each mill can generate on average over 30 million m3 of liquid effluents per press season, the disposal of which poses an enormous challenge to mills. Beyond the sheer volume of waste being created, if one considers that OMWW contains long chain fatty acid compounds and polyphenols of up to several grams per litre (150-200 g/l), which exhibit antimicrobial, and phytotoxic properties, the staggering intensity of the problem becomes really critical. The negative effects on the environment render this waste as “ecotoxic”, in the meaning of in Annex III to Directive 91/689/EEC and to Directive 2008/98/EC. As such, if the olive oil producer do not treat or use himself the OMWW, a third party should collect and treat all wastes with the same CER code only if authorized. Traditionally OMWW are treated with enzymes or organic acids. The Oli-PHA developed process is not only more economically competitive but also better performing, safer and environmentally sustainable than conventional treatments.
Central to the expected socio-economic impacts, is the boosting of the competitiveness of companies operating along the olive, plastic and packaging industries. The Oli-PHA results will be introduced into 3 very large markets. Making such industries more competitive will have impacts for the European economy, growth and jobs.
OMWW possesses a double nature. While being a strong pollutant it is at the same time a possible source of valuable components, such as polyphenols, flavonoids, anthocyanins, inorganic trace elements, etc., that can be extracted and economically exploited. Thus, the vision of OMWW is now evolving from a waste to a raw material.Hence the market of the reuse of OMWW to produce valuable products is in expansion, because it is revenue and the resolution of a problem at the same time.Oli-PHA developed a novel process for the enrichment of low molecular weight phenols, which can be used as antioxidants in different sectors such as cosmetic, pharmaceutical and food industry. Growing health awareness is the primary factor responsible for the large growth of the antioxidant market in the last decade. The superior antioxidant property of polyphenols and their application in the treatment of cancer, diabetes and cardiovascular diseases has further contributed to the growth of the polyphenols market. Functional beverages were the largest application segment of polyphenols and accounted for over 44% of the total polyphenols demand in 2011 followed by functional foods which accounted for over 33% of the total consumption in 2011. In terms of volume, global consumption is expected to grow at a CAGR of 8.2% from 2012 to 2018 driven by the growing popularity of antioxidant potential and the shifting trend in consumer health from being reactive to proactive.
However, the limited supply of raw materials due to other overlapping commercial applications such as wine manufacturing, preparation of juices, jams and jellies is expected to inhibit the market growth over the next six years. As such, there is market space for new offer such as polyphenol recovered from OMWW as developed in Oli-PHA, for example as natural preservative in cosmetics and in active packaging as tested in the project (under the condition of complying with legislative aspects set in the EC 450/2009 requiring further characterization of the polyphenol extracts for the latter application).
Besides the reuse of food waste in their formulation, Oli-PHA active packaging have the potential extend the shelf-life of the packaged product and therefore could be a key to SAVE FOOD (one third of all food is either wasted or lost, often at the consumer level)contributing therefore on one of the current greatest global socio-economic challenge to feed a growing population. Several factors affect the quality, safety and shelf life of food and cosmetic products such as oxygen, light, humidity or the growth of microorganisms, among others. Oxidation is one of the most frequent mechanisms of food deterioration along with microbial spoilage. Antioxidants can be incorporated into or coated onto food packaging materials to control the oxidation of fatty components and pigments, and thus can contribute to the quality preservation of foods. The release of antioxidant agents in active food packaging causes consumer concerns regarding their safety. For this reason, there is a growing consumer preference for natural antioxidants to synthetic antioxidants, such as butylated hydroxytoluene (BHT). Preservatives such parabens which are currently under revision in Europe are used in a high number of cosmetic products. As such Oli-PHA answers to the consumers demand for more natural cosmetics and food packaging solutions.
Another key socioeconomic impact is the development of bio-based and biodegradable packaging materials with suitable thermo-mechanical properties that can reuse waste and prevent the use of depleting fossil fuel derived raw materials and resources. The Bioplastics European association predicts that the global bioplastics production capacity is set to grow 500 % by 2016 (definition including under bioplastics partly or fully biobased, biodegradable, or both). The main drivers of this growth are twofold: consumer demand for environmentally-friendly products and volatile price of petroleum. In spite of announced production increase, PHA is still a minor production whereas drop in solutions such as biobased PE and PET have taken a major place. PHAs are currently is produced in Europe at small scale by the companies Biomer and Bio-On, in the USA by Metabolix, in China by Tianan and in Brazil by Biocycle among others. Commercial PHAs are generally extracted with chlorinated solvents from heterotrophic bacteriagrown on refined sugars (up to 5 kg per kg of PHA) or oils. PHAs are more expensive than most other (bio)plastics (eg. 5-19€ per kg of pure PHAs). Nevertheless, due to good mechanical and barrier properties, its biodegradability and recyclability, PHAs have potential to substitute some of the 250 MT of plastics used worldwide per year in packaging (market consuming over 45% of all plastic produced worldwide). The share is even bigger in case bioplastics (>75%) and biodegradability can be seen as a real asset for certain types of packaging which are very short term uses or of difficult recycling due to residual packed product contained at end of life. Among all PHA polymers existing, PHB, its copolymers, and PHBV are today the most widely commercially available. Depending on the composition of the copolymers, grafting co-monomers or tailoring of the formulations (using plasticizers and others additives), PHAs’ properties are very versatile can be used in applications ranging from surgical sutures, tissue engineering and agricultural foils to packaging for the storage of food products. PHA also exhibits good barrier properties, and can be used in the packaging industry as a biodegradable plastic for contributing to solving environmental pollution problems.
In order to improve PHA biopolymers sustainability, the use of waste as feedstock or of genetically engineered bacteria should all lead to reductions in production cost. Oli-PHA research effort focused on the valorisation of wastes (OMWW) as non-food competing sources for cultivating PHA-producers. In addition to studies of heterotrophic bacteria as PHA producers, PHA production using photosynthetic organisms as production hosts have been also explored as a novel approach in Oli-PHA. Cyanobacteria are oxygen-evolving photoautotrophs with the added advantage that some of them naturally possess the key enzyme in PHA biosynthesis. They can grow on high content of OMWW even without nutrient supplementation, which could reduce the fresh water consumption during the process exploitation. In addition, due to their minimal nutrient requirements in comparison with heterotrophic bacteria and fast growth in comparison with transgenic plants, cyanobacteria are attractive hosts for the production of PHA. However, in comparison with other heterotrophic bacterial strains, literature prior to Oli-PHA showed that cyanobacteria can only produce very small amounts of PHAs. Therefore, strategies to improve PHA production yield, such as those studied in Oli-PHA, hold an enormous potential for the industrial production of this biopolymer. In the Oli-PHA project, cyanobacteria were further optimised via genetic modifications allowing to significantly increase PHA yield and tolerance to some residual phenols. In addition, the incubation conditions, photobioreactor (PBR) designs and harvesting operations were optimized for large-scale industrial production of the cyanobacterial products to increase productivity and to decrease costs of PHA production. Despite difficulties in the scaling up of the cultures, great advances were made in the Oli-PHA project, showing that in spite of the lower accumulation in PHA in the GM cyanobacteria than in the currently used heterotrophic bacteria, the use of waste feedstock and optimised downstream process could still lead to market competitive PHB products.
Besides the potential of microalgae for producing PHA, the biodiesels generation represents the biggest volume application and potential for microalgae. In the past decade, the biodiesel industry has seen massive growth globally, more than doubling the production every 2 years. Indeed, algae can serve as a feedstock for first generation biodiesel and ethanol plants, aviation fuels market and biocrude for biofuel. Nowadays, 50% of the cars in EU are running with diesel. The problem is that the EU cannot produce enough rapeseed and sunflower to cope with this demand so this can be seen as a potential opportunity for algae. The USA is the first consumer of diesel fuel for military use in the world, their industrial Diesel market represents a 25% of the petrol consumption; also more than 95% of private cars use gasoline, while a 3% runs on diesel. The problem is the same as in the EU. The USA cannot produce enough corn and soybeans to meet the demand, even with cellulosic corn, so algae could assume an important role. Within the Oli-PHA project biogas productivity was evaluated using the two types of waste streams as substrates for anaerobic digestion: the solid fraction of OMWW (olive mill wastewater) after the pretreatment step to extract polyphenols, and the algae biomass - harvested from the photobioreactor - after extraction of PHA. It was concluded that olive pulp (solid fraction of OMWW) is a very usable substrate for anaerobic digestion which together with algae biomass as a nitrogen source has the potential to be a successful input mixture for anaerobic digestion. Nevertheless this latter has lower biogas potential and is not recovered as a solid from the extraction process, as such alternative co-substrate such as manure are preferred.
Furthermore, Oli-PHA assists European packaging producers to adhere to the various EC legislations that exist in the plastic industry, such as the packaging waste legislation in force in Europe. Moreover, by the CO2 consumption via microorganisms to grow, reducing the energy, as well as raw materials consumption and waste generation, the results of this project will also contribute to assisting the EU in meeting its CO2 cutting objectives under the Kyoto Protocol. All in all, microalgae have much faster growth than other crops and a huge potential still to be fully disclosed. The biorefinery and bioplastics sectors are growing at a very fast pace and, and while Oli-PHA met interesting properties, shall further research allow scaling up its production and therefore adjust its price to a suitable range, solutions like Oli-PHA would match different current technical needs opening up an interesting market application.
In terms of exploitation, the project developed significant know-how exploitable by the partners and their novelty was scanned in terms of patentability leading to a patent application on a method for genetic modification of bacteria, and a possible future filling. The key exploitable results (KER) were identified and characterized in terms of commercial significance, market potential, IPR status and the conditions needed to enable the results to be exploited. In addition to the number of foreground elements of scientific relevance dealing with the “Selection of microorganisms with highest yield, of suitable culture conditions and culture media”, achieved by cooperation of partners involved in WP1 and WP2 activities respectively at lab and large scale, the Oli-PHA exploitable foreground elements are as follows:
• Novel process for the enrichment of low molecular weight phenols in OMWW
• Improvement of polyphenol removal process from OMWW and of their recovery
• Method for genetic modification and characterization of cyanobacteria with enhanced capacity of producing PHA. Patent pending CICESE (PCT/IB2014/002412)
• PBR with its control system (draft patent in process)
• Optimal conditions for biogas production of solid fraction of OMWW and algae residue after extraction
• Formulation and processing parameters for production of PHA based material by injection moulding, thermoforming and blow moulding.
• Active packaging based on polyphenols from OMWW
An economic analysis was carried out for the individual process steps as well as for the integrated Oli-PHA plant taking into account the profits and costs of feedstock, energy, current and expected waste treatments costs, etc. This led to different balances depending on the commercialisation scenario but overall highlighted the potential cost competitiveness of the resulting products like PHB and polyphenols especially when valorising the biogas in closed loop as energy source. Further potential exploitation incomes arise from the licensing of the IP/knowhow, provision of services, etc. besides the use of the foreground generated by each partner in its own business, improvement of its operations.
The exploitation intentions of the Consortium members were discussed during project meetings and the exploitation strategy further elaborated with the support of the NMP Exploitation support services (escic2). A Joint exploitation agreement of the foreground was laid out based on the above described elements but also on the scientific know-how generated in the project, benefitting from the synergistic role of the partners covering the whole supply and value chain. In addition, for specific cases where the foreground was owned by several members, Joint Ownership agreements were signed to define shares, rights and obligations in order to avoid competition between co-owners.
In parallel, the consortium organized a comprehensive series of training and demonstration sessions for external stakeholders in order to maximize market uptake as well as to gather valuable feedback from researchers, end users and technology providers in terms of sustainable polymer trends, OMWW managing initiatives and key limiting factors for introduction of new solutions in the marketplace. The Consortium plans to undertake future development efforts to bring Oli-PHA technology from pre-industrial to a commercial system, and to perform practical evaluations of the technology and further demonstration work that will be bespoke to industry. Further funding to mitigate the cost of the additional development work will be applied for.
Additionally, a number of dissemination activities have been carried out to on the principles of the Oli-PHA technology and products in preparation for the future exploitation of the same. These include the development of a project website (http://www.oli-pha.eu) which informs the public on the basics of the technology, latest news and the progress of the project insofar, as well as various press releases for raising the awareness of Oli-PHA both in industry and in the public domain. The project partners also took part in several scientific conferences, trade fairs and TV programmes. In terms of scientific dissemination, the project allowed the preparation of 4 peer-reviewed publications (some are in revision) and 5 book chapters. 4 public demonstration events including a scientific workshop were organised, as well as training sessions.
List of Websites:
www.olipha.eu
Dr. Elodie Bugnicourt
IRIS SL.
Avda Carl Friedrich Gauss 11
08860 Castelldefels
Spain
Tel: 00 34 935542503
Fax: 00 34 935542511
E-mail: ebugnicourt@iris.cat
