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Ecoefficient Biodegradable Composite Advanced Packaging

Final Report Summary - ECOBIOCAP (Ecoefficient Biodegradable Composite Advanced Packaging)

Executive Summary:
EcoBioCAP aims to provide the EU food industry with customizable, eco-efficient, biodegradable packaging solutions with direct benefits both for the environment and EU consumers in terms of food quality, safety as well as food and packaging waste reduction. This next-generation of food packaging is developed using advanced composite structures based on constituents derived from food industry by-products only. Thanks to innovative processing strategies, the project enables the customisation of the packaging’s properties to fit the functional, cost, safety and environmental impact requirements of the targeted fresh perishable foods.
A strategy driven by food quality and safety requirements, environmental preservation as well as stakeholders’ preferences, acceptance and needs, was adopted. In addition to conventional detailed materials packaging specifications elaborated for each targeted foods, a decision support system (DSS) software was developed. The DSS, accessible to all actors along the food packaging supply chain, is constituted of two modules. The first one is based on virtual MAP model, databases and multi-criteria query. The second one aggregates preferences expressed by the stakeholders into consensual preferences using an argumentation approach.
Microbial polyesters (PHAs) with targeted functional properties have been successfully synthesized from liquid effluents such olive mill wastewaters (OMW) and cheese whey (CW), with similar PHA yield and no preliminary phenol recovery for OMW. Fibre-based fractions were obtained from solid by-products such as wheat straw (WS), brewing grains and olive pomace. Impact milling process offered the best compromise between particle size and energy consumption. Anti-oxidant nano-clays were developed from polyphenolic extracts of OMW and pomace as well as bio-adhesives based on zein and pullulan.
The best respiring bio-composite trays were obtained from PHBV and WS fibres (up to 30%) by thermo-moulding process, with increased permeability due to WS content. Barrier properties of PHBV monolayers film were improved by the developed cellulose nano-crystals and antioxidant nano-clays.
Multilayers lid films and trays were developed by combining PHBV and composites with others bio-polymers and using various processing technologies such as electro-spinning technology. The extrusion blowing of bi-layered films was the best prospective option and thermoforming of PHBV multilayer structures to trays was a good alternative to injection moulding.
The processes used to produce the constituents PHA and WS fibres and the composite materials, were demonstrated to eliminate the large range of initial contaminants (pesticides, side-products..) and result in a worst case migration in food of no safety concern. PHBV based materials showed to be stable during storage, in contact with micro-organisms and with all types of foods’ simulants except with ethanol 95% for which a significant swelling and an excess of overall migration (OM) were obtained. The addition of wheat straw fibres induced a detrimental effect by increasing OM and specific migration of additives.
Eco-toxicity test for both terrestrial and aquatic ecosystems revealed no inhibitory effects of the materials developed. The LCA concluded that the carbon source in PHA/PVB production should preferably be a waste fraction, that spray drying and transport of CW were hot points and as well as the total weight of the EcoBioCAP packaging.
Successful up-scaling of selected packaging production was achieved. No significant negative impact of the EcoBioCAP packaging when compared to the benchmark packaging was found both in shelf-life studies and global consumer acceptance studies performed on real distribution conditions.
Outreach turned out more than 50 A-level publications, more than 50 communications but also newsletters, 3 workshops open to the public and numerous promotional video clips, in close collaboration with the very active stakeholders’ advisory board.

Project Context and Objectives:
The specific objectives of WP1- Integrated analysis of packaging specifications, dimensioning and decision support system were to determine the permeability window for the packaging of the targeted respiring foods by simulating virtual MAP; to evaluate and analyse the requirements of the stakeholders (such as packaging producers, consumers and legislators) and summarise them in agreed specifications for packaging solutions; and to develop a Decision Support System as a tool to enable the stakeholders to select the right packaging solution to fit their needs. A procedure was developed by WP1 to elaborate packaging specifications for each targeted product. This methodology is based on preparing a packaging development brief, carrying out technical surveys, collecting knowledge for the project partners, food manufacturers, packaging material manufacturers and experts in the field. The method of building the packaging specifications was adapted from the industry practice. Even if the majority of these elements are relatively known in the industry, and the use of the product development brief for food product development was described in Campden’s product development guide, their specific combination for packaging material is innovative and no document was ever written gathering all this methodology.
One of the main expected innovative achievements of EcoBioCAP in the field of computer science, knowledge engineering and food engineering was the development of a Decision Support System (DSS) aiming at helping stakeholders in the choice of a packaging material for a given product according to a multi-criteria query considering constraints and wishes of the user.
With these integrated design of tailor-made packaging driven by food and stakeholder requirements, EcoBioCAP intends to bring new insights into the research and development of biodegradable packaging, which integrated chain concept not used earlier.

WP2-Development of packaging constituents by upgrading food industry by-products made it possible to check several food industry by-products and to make them usable elements in the constitution of novel food bio-packaging.
Polyhydroxyalkanoates (PHA) are a family of biodegradable and biocompatible polyesters predominantly composed of R-3-hydroxyalkanoic acid monomers ranging from 3 to 14 carbons in length and the enormous possible variations in the length and composition of the side chains make them suitable for an array of potential applications including packaging (Keshavarz and Roy, 2010). Many companies have been set up to commercialize PHA as biodegradable plastics, however still most processes for PHA commercial production are based on the use of pure cultures of particular microorganisms (including GMO organisms) grown on well-defined nutrient-deficient synthetic media, which cause high production costs. A promising alternative relies on the use of mixed microbial cultures (MMC) which also allow to easily employ non-costly by-products as feedstock (Reis et al., 2011) and, in this context, significant advances have been achieved in the EcoBioCAP project. In particular, the MMC PHA production has been tested in a multi-stage process with olive oil mill wastewater (OMW) and cheese whey (CW), the disposal of which, represents a critical environmental problem due to both high level of production and high COD (chemical oxygen demand) content. Specifically, OMW is the liquid waste deriving from the extraction process of olive oil and its production is mainly concentrated in the Mediterranean area (over 90%) (Ntaikou et al., 2009). CW is a major by-product in the manufacture of cheese, representing 80-90% of the volume of transformed milk (9 kg of whey per kg of cheese).
The main objectives achieved are:
- Production, optimization and characterization of the microbial biopolyester PHA (polyhydroxyalkanoate) from liquid effluents deriving from agro-industries.
- Production, optimization and characterization of fibre-based fractions from solid residues of beer, olive and wheat industry.
- Production, optimization and characterization of tailored and high performance additives and adhesives.

The objective of WP3 - Formulation and structuring of finalized materials was to develop finalized packaging materials by combining and structuring the different constituents (developed in an earlier step pf the project) according to their complementary roles, in order to achieve the intended targeted final properties (e.g. processability, barrier/permeability properties, mechanical strength, sealability) based on the requirements of processing and the selected applications. This included 3 specific material developments:
- flexible barrier film with a focus mainly on high oxygen and medium water vapour barrier;
- flexible respiring film with a focus on high permselectivity (carbon dioxide/oxygen permeability ratio) and high water vapour transmission rate;
- rigid/semi-rigid water resistant tray with a medium to high oxygen barrier.
The target was to primarily produce all new potential packaging materials based on PHBV in lab-scale as monolayer composites in order to select the most suitable one to achieve the targeted functionalities. After optimising the processability of the composites and blends, the compounding and extrusion were performed at larger lab scale and the permeability and mechanical properties of the materials were assessed.
Coated systems, generated by the electrospinning process, are produced with different layer combinations. The developed multilayer films must display excellent adhesion between the layers. The water vapour and oxygen permeabilities were tailored due to the requirements of the food. The technology of blown film extrusion turned out to be the most prospective for the development of thin PHBV lid films. Blown bi-layered films were considered as the best compromise in terms of barrier and mechanical properties as well as processing conditions. Regarding the studied options for the intended applications as a lid film or pouch, the most adequate film was determined to be a bi-layer of Tianan (40%-50%) and Ecoflex (60%-50%) without fibres to ensure best possible transparency.
Trays processed by injection moulding were recommended to use the formulation based on PHBV and 20wt% of wheat straw fibres. The production of the trays using an industrial plant was performed.
Proof of concept was performed by thermoforming of trays based on PHBV. First, a blending step of PHBV enabled an improved processability, which was then followed by the co-extrusion of the multilayer. The thermoforming process was carried out in a second process step.
The main objective was to demonstrate the feasibility to reach different permeation values by the combination of tray and lid film properties. Furthermore, the influences of the surface and the thickness of the packaging material on permeability were studied. Taking that into account, the requirements for a high barrier application (here: sandwich) were achieved by combining a tray and a lid. All tested materials had too high gas barrier properties to match the requirements of the respiring products (here: cheese, strawberries or mushrooms) as defined in WP1. A modulation of the film thickness in addition to micro-perforation of the lid film were studied.

In WP4 - Assessment of packaging physical-chemical stability and chemical safety, the objective was to assess the packaging materials developed in WP3 as food contact materials (FCM) in order to check their suitability as FCM. This investigation aimed to extend knowledge of stability and safety of biodegradable packaging by:
- Developing specific methodologies to simulate targeted usage conditions based on the development of representative solid model foods and to characterize the degradation of the initial polymeric network and the mass transfer of potential contaminants.
- Providing new insights and understanding into the structural and physico-chemical stability of PHBV-based materials with respect to the targeted food packaging application, i.e. by studying the influence of severe yet realistic conditions of storage and food contact on the evolution of their properties.
- Assessing the microbiological stability of some packaging materials by studying the influence of conditions of storage and food contact on the characteristics of bio-films formation in relation to the surface properties of the packaging materials.
- Investigating the chemical safety due to raw constituents by identifying migratable and toxic substances and evaluating the decontamination efficiency of production/extraction processes through challenge tests.
- Studying the chemical migration of such potentially toxic compounds through the final materials by studying the migration of a range of relevant molecules already present in raw materials as well as a range of representative surrogates and by considering mass transfer modelling approaches.
This approach is summarized in Figure 1.
The objective of WP5 - Assessing the environmental impact of the new biodegradable materials was to assess the environmental performance of the developed materials and packaging solutions. Life cycle analysis, organic (biodegradability in natural environment) and energy recovery (combusting ability) as well as (terrestrial and aquatic) ecotoxicity have been performed within the time-frame of the project.
The Life Cycle Analysis was performed: (i) on the developed PHA material in comparison to literature and database information from commercial materials, bio-based and petroleum based (ii) for fresh strawberries packed in a composite tray sealed in a film, both biodegradable PHA/PHBV materials developed within EcoBioCAP and compared with a commercial PET.
Biodegradability of the materials developed were characterized according to international standards: ISO14855 (Biodegradation in composting condition) and ISO15985 (Biodegradation in anaerobic conditions) and disintegration in a simulated composting plant tested at lab scale level according to standard EN14045 (ISO20200). Ecotoxicological tests for both terrestrial and aquatic ecosystems were employed.
The objectives were to demonstrate that PHA-based materials, being developed in the frame of the EcoBioCAP project, were biodegradable, compostable and had no ecotoxicity. The LCA intended to identify critical points to improve the environmental performance. Furthermore, combustion enthalpies of the PHBV materials were evaluated in order to gain knowledge about the incentive for potential energy recovery via combustion.

The objectives of WP6 – Industrial applicability were to up-scale and demonstrate at industrial scale the EcoBioCAP technologies:
- One respiring film, one non-respiring (barrier) film and rigid trays were produced at small pilot scale.
- Adequate characteristics of EcoBioCAP’s PHBV material were studied in order to be used in the production of the packaging materials (films and trays).
- In general, EcoBioCAP packaging solutions had to prove to be capable of ensuring similar quality of food products during the targeted shelf-life, compared to benchmark packaging materials. Therefore, EcoBioCAP materials can be used as more eco-friendly packaging solutions for food products.
- R&D efforts focus on improving the mechanical properties of the EcoBioCAP films and increasing the capability of these films to hold modified atmosphere. In any case, the objective was to demonstrate that mechanical resistance of the films produced was sufficient e.g. to withstand their use in a packflow machine.
- The production of EcoBioCAP film at large pilot scale was a crucial objective. It required the development of multilayers structure combining PHBV with aliphatic-aromatic biodegradable copolyester (e.g. MATER-Bi), where Tianan is located in the inner side of the film bubble.
- The production of trays at large pilot-scale was studied through injection molding of EcoBioCAP’s tray compound (consisting of PHBV and WSF).
- Impact of the EcoBioCAP packaging was compared to the benchmark packaging. Such a real scale study intends to reveal main gaps to fill to reach industrial applicability.

The objectives of WP7 – Outreach were to disseminate the project’s results through scientific publications in international journals and at key scientific conferences and to ensure the successful uptake of the project results by the food packaging industry. The outreach activities were also targeted at stakeholders notably the general public (consumers) and policy makers/local authorities. These activities were meant to raise awareness of the benefits of biodegradable packaging and how it should be disposed of to these stakeholders and also to create a demand for biodegradable packaging.
An extensive dissemination of EcoBioCAP results was successfully organised within the framework of EcoBioCAP. Several publications were submitted or accepted in renowned journals and partners communicated about the projects results in major national and international conferences. Interaction with the stakeholders’ advisory board was active throughout the project’s duration. To reach the general public and policy makers, several tools such as promotional videos, newsletters, brochures, the project’s website were successfully utilized.

Project Results:
• Key result n°1: Tools for the design of biodegradable multi-composite packaging materials

WP1: Integrated analysis of packaging specifications, dimensioning and decision support system

A systematic approach was applied for developing the packaging specifications, which was based on agreeing on packaging development briefs, collecting the available knowledge of the project partners on the relevant aspects and from the literature, carrying out experimental work and a consumer survey. Detailed information was defined in packaging specifications, which give the key characteristics of the packaging materials as clear as possible for each biodegradable packaging solution. (Figure 2)
At the beginning of the project, it was necessary to determine the different requirements of the packaging and conditions of the research and development work for the development of the new biodegradable packaging. Available knowledge and packaging data on the selected products were collected. Information about the requirements of the selected food products and about their quality and safety were collected and their characteristics were analysed. Information on functional properties of the packaging such as oxygen, moisture and carbon dioxide barrier properties were collected. These experiments were also performed to complement information from the literature. Information was gathered on current suppliers of biodegradable materials, additives and bio-packaging solutions and on the processing characteristics and physical properties of the constituents were gathered. Information about the amounts, composition, physical characteristics, seasonality etc. of the by-products was analysed. Their potential safety concerns were also considered. The knowledge of the different disciplines represented by the project partners were harmonized and integrated. Thus the first stage packaging specifications were prepared successfully. These completed packaging material specifications and the additional information specified in the packaging development briefs served as the basis for the further research and development work.
Among all aforementioned information, it was necessary to quantify mass transfer packaging properties requirements for maintaining quality and safety of the selected food products, one of the most important packaging properties as required food quality. First, the project partners agreed on the specified products for which they will develop the new packaging materials. Then they performed a market assessment for these specified selected products (package size and geometry, amount of products, density, types of packaging material). Project partners specified the optimal packaging and storage conditions (temperature, O2 and CO2 and RH%), quantified the respiration and transpiration rate and identified the critical quality and safety and shelf life requirements. All data collected on fresh produce were stored in a dedicated database called ‘Fresh produce database’, to be further use in the DSS developed at a later stage. Experimental design and data analyses was performed and project partners quantified the packaging barriers required for the selected products (OTR, CTR) and the water vapour transmission rates (WVTR) using dedicated virtual MAP modelling tools (PackInMap and Tailorpack,). Data analyses and MAP simulations were performed. An additional survey was carried out on some aspects of the quality and safety of the model food products and collected the knowledge and data available from the project partners related to the respiring products (soft cheeses and fresh produces) from which the gaps were identified and literature review was done according to the results. The collected information was built in to the relevant packaging specifications.
Another objective was to map and analyse the stakeholders’ preferences, acceptances and needs and to harmonize all the information received from all the stakeholders with the aim of the EcoBioCAP at the beginning of the project. First the food – packaging matrixes were specified for determination of the food products for which the new packaging materials should be developed. A questionnaire template was developed for packaging development briefs and was completed by project partners and SAB partners for each targeted packaging material and solutions based on their combination. A more detailed technical questionnaire was developed and completed by the project partners for all the specified products. Multilingual questionnaire templates were developed for survey the consumers’ preferences and their needs then the survey was carried out in 4 countries with 877 respondents. Reports were performed on 1) the analyses of the availability, volume, properties of by-products, potential safety concerns 2) verification of the needs, interest and acceptance of by-products, including regulatory constraints 3) collection of information on current suppliers of biodegradable materials, additives, bio-packaging solutions and on analysing processing and physical properties of the constituents. Packaging specifications were established for each biodegradable packaging solution. The specific values with the defined limits (chemical, microbiological and physical) of the packaging materials enabled the project partners to interpret them in a standardized way, to help the identification of the gaps in the information, to allow modification or further improvements where necessary in later phase of the research.

• Key result n°2: Decision Support System (DSS)

WP1: Integrated analysis of packaging specifications, dimensioning and decision support system

There is a growing trend for governmental efforts to promote health benefits of fresh foods, despite their short shelf life. Beyond respect of the chill chain, modified atmosphere packaging is an efficient way to delay senescence and spoilage without using controversial preservatives compounds. It can be achieved by matching the film permeation rate with the respiration rate of respiring products. In addition, the choice or design of packaging for fresh foods must take into account numerous other factors such as the cost, availability, potential contaminants of raw materials, process ability, preferences of consumer, waste management constraints, etc. This is especially important when developing biodegradable packaging. Within the project, knowledge engineering method and tools were designed to store, share and use information regarding packaging material and to solve the dilemma of multi-criteria demands. A specific methodology has been developed to query those databases when user’s preferences are bipolar (i.e. express both constraints and wishes about the desired result). Results are then completely ordered with respect to these bipolar preferences, giving priority to constraints over wishes. This approach has permitted to build a Decision Support System for designing biodegradable packaging for fresh produces.
As a first step of the development process, existing available mathematical mass transfer models were selected and the databases of these models were enlarged with the parameters resulted in the experiments related to cheeses and fresh produces (respiration rate and transpiration rate). Upgrading of the mathematical models has permitted to refine the window of permeability previously identified and to predict the permeability of each material used for tray and lid film respectively. A demonstration of the tool was performed and suggestions of improvement were collected from the project partners and stakeholders. The DSS combined two modules. The first module computes the list of the most relevant packaging taking into account the virtual MAP model previously presented and data stored in two databases (food and packaging databases). This ranking is obtained from a multi-criteria query, which takes into account food quality as a constraint but also others preferences or constraints of the user (price of the material, transparency, machinability, etc.). The second module of the DSS is able to aggregate preferences expressed by the stakeholders into consensual preferences using an argumentation approach. The tool can therefore perform an integrative engineering design for modified atmosphere and humidity packaging (MAHP) and can permit to quantify the window of OTR, CTR and WVTR, suitable for the packaging of the product selected. This decision making tool is useful for the development of new added-value materials for food packaging application from locally available and poorly valorised solid by-products and liquid effluents.

• Key result n°3: Development and optimization of a biotechnological process for PHA production by using agro-industrial by-products and mixed microbial cultures (MMC)

WP2: Development of packaging constituents by upgrading food industry by-products

The development and optimization of a biotechnological process for PHA production by mixed microbial cultures (MMC) have been accomplished by using both synthetic mixtures of volatile fatty acids (VFA) and liquid industrial effluents as feedstock. As for the latter, olive oil mill wastewaters (OMW) and cheese whey (CW) have been used as process feedstock. When dealing with MMC and organic by-products, the PHA production process is usually based on a combination of anaerobic acidogenic fermentation and aerobic stages, as depicted in Figure 3. After acidogenic fermentation of high-concentration biodegradable by-product (first stage of the process), PHA-producing mixed microbial cultures are selected and enriched from an activated sludge under aerobic periodic feeding in a sequencing batch reactor (SBR). The storage capacity of the enriched sludge is saturated in a PHA accumulation reactor and, finally, in the downstream processing the PHA-rich sludge is treated for extraction and purification of the final product.
As for OMW, a treatment for phenol removal and recovery and a subsequent acidogenic fermentation step were performed prior to being used for PHA production. Indeed, phenols removal and recovery allowed to decrease the wastewater toxicity in favour of the fermentation step and to obtain natural chemicals to be potentially exploited in the formulation of functional packaging material. UNIBO was in charge to operate the OMW pre-treatments. The removal of OMW polyphenols was carried out according to a solid phase extraction procedure with a non-polar resin as the adsorbent (Amberlite XAD16), while the adsorbed phenolic fraction was recovered with acidified ethanol. The dephenolized OMW was fed to an anaerobic mesophilic packed bed biofilm reactor filled with Vukopor S10® ceramic cubes, which was mainly aimed at promoting the bioconversion of the OMW COD into VFA.
The dephenolized and fermented OMW were used to feed the aerobic stages (from microbial culture selection to polymer accumulation) of the PHA production process. To accomplish this objective, a lab-scale reactors' configuration, consisting of a sequencing batch reactor (SBR) and a PHA accumulation reactor, was completely assembled and set-up. In order to identify the best operational conditions of the process, different reactors' operating conditions were analysed. More in detail, in the SBR a key parameter is the organic loading rate (OLR), which has been investigated at four different values (from 2.37 to 8.42 g COD/Ld). As for the accumulation stage, it was operated under nitrogen limiting conditions (i.e. with no further addition of N to that contained in the OMW) and pulse feeding strategy with undiluted OMW. Besides of PHA production, a PHA recovery procedure was also implemented. For this purpose, the PHA-rich biomass (from the accumulation reactor) was recovered through a centrifugation method with a subsequent chemical digestion with sodium hypochlorite, aiming at disrupting the cell wall and releasing the intracellularly accumulated PHA. Overall, the feasibility of using OMW as no cost feedstock of the process was demonstrated. Indeed, under the optimal operating conditions (SBR operated at 4.74 gCOD/Ld) between those investigated, it was found that around 2 grams of PHA can be produced per day corresponding to a productivity of about 1.5 g PHA/Ld (where the volume refers to the sum of volumes of both reactors where PHA is produced, i.e. both selection and accumulation reactors). This PHA productivity is quite good, especially considering that it has been obtained on a continuous-flow basis and with the true feedstock. The produced polymer was a poly (hydroxybutyrate-hydroxyvakerate) [P(HB-HV)] copolymer with an HV content of around 9% (ranging from 7 and 13%, w/w). After the extraction step with NaClO, around 66% of the produced polymer was recovered at a purity around 72%, a performance that would require to be improved for full exploitation of process potential.
As for CW, the effectiveness of this substrate as feedstock for PHA production has been evaluated also in combination with other by-products of food industries. Indeed, when dealing with PHA production with MMC from wastes/by-products, an important aspect to be considered is that most of feedstocks are seasonal and, in practice, PHA producing industries will have to comply with this seasonal availability. Therefore, two possible solutions may be proposed: (1) to store the feedstock for continuous operation, which might implicate huge storing buffer tanks together with the fact that feedstocks can be degraded during storage; or (2) alternate operation with different available feedstocks along the year, which may have implications in the final polymer composition. Particularly, the impact of feedstock shift on the final polymer quality through the individual effects on the acidogenic stage, the PHA-accumulating culture selection and the PHA production stages has been investigated. To accomplish this objective, the process was firstly fed with a synthetic mixture of volatile fatty acids, then shifted to sugar cane molasses (SCM) and then shifted again to CW. The response to the feedstock shift was measured for each stage of the process and the quality of the final polymer produced was evaluated in terms of composition and properties. The possibility of controlling the polymer composition by mixing the two fermented feedstocks in the accumulation stage was also investigated. It was observed that with all substrates a P(HB-HV) copolymer was obtained with different HV contents. In particular, PHA obtained from synthetic medium and fermented CW showed a similar composition in terms of HV content (about 19%, w/w) and had similar molecular weights (2.2 and 2.5 x 105, respectively); whereas the polymer obtained with fermented SCM showed a much higher HV content (52%) and presented a slightly higher molecular weight, 3.2x105.
Given that mixed cultures were utilised for the production of polymers, a wide chain length distribution would be expected. However, the chain length distribution was very narrow for the different polymers, shown by the low and consistent PDI values (between 1.3 and 1.7).
The experimental results obtained in the frame of WP2 demonstrated the feasibility to produce PHA by using food-industry by-products, with no any competition with the food chain. Both process innovation and product innovation were achieved with respect to the previous literature.

As for process innovation:
- The whole multi-stage PHA production process (from feedstock pretreatment to polymer accumulation) has been verified under long-term continuous operation, while also optimizing several process parameters and related conversion rates and yields. This also allowed to verify the robustness and reliability of the microbial mixed cultures to drive the multi-stage process (e.g. Figure 4, Villano et al. 2013).
- Three largely available feedstock have been tested such as olive oil mill wastewater (OMW), cheese whey (CW), and sugar cane molasses (M), the process has been shown an effective tool for their simultaneous treatment and valorization towards PHA production.
- As for OMW, an optimized flow sheet has been defined (Figure 5) to simultaneously obtain OMW treatment and valorization, the latter through both PHA production and phenol recovery
- As for CW and M, as main results it was shown that:
Acidogenic step: similar VFAs profile are obtained for long term operation but also quick response is obtained under feedstock variations and dynamic pH (e.g. see Figure 6). This means that production of different VFAs can be obtained by changing operating conditions (such as pH dynamic control).
Selection step: Highly enriched cultures in PHA-accumulating organisms (such as analyzed FISH & DGGE) are robust and quickly adapt under shift of different feedstocks.
Accumulation step: High volumetric PHA production rates are obtained and PHA cellular content increases up 65%. Polymer composition can be easily tailored mainly depending on VFA composition, which can be controlled through the 1st step.
- Based on bench scale results, the CW process has been scaled up and 1 kg of copolymer has been produced for extensive characterization and testing.

As for product innovation
- PHA of defined composition has been produced both from synthetic substrates and true feedstock, in a wide range of monomeric ratios (HV (HV+HB)) in the range 8-40%.
- Produced PHAs from different feedstock have been supplied for extensive characterization and testing (thermal, mechanical, rheological, and barrier properties and processability).
- In general, the performance of PHAs produced from by-products by means of MMC compares well with PHAs produced from ad hoc designed substrates by means with axenic cultures.
- Produced PHAs have been modified and/or blended towards obtaining desired properties and tested again.

• Key result n°4: Production, optimization and characterization of tailored and high performance additives and adhesives

WP2: Development of packaging constituents by upgrading food industry by-products

Cellulose nanowhiskers by the use of WS, BSG and OP were produced. The extraction process was optimized in terms of increased extraction yield. After subjecting the cellulose rich materials to a purification process for isolating cellulose, a stronger acid hydrolysis procedure was applied for digesting the amorphous domains of the material. By this method, it was possible to generate cellulose nanowhiskers with a good thermal stability and an acceptable crystallinity index and aspect ratio when using BSG and WS as the raw materials, whereas OP resulted in less crystalline nanowhiskers. In addition, the extraction yields were ca. 30% for BSG and WS and ca.5% for OP, which are considered to be very low if compared with different cellulosic resources such as bacterial cellulose and therefore the extraction of cellulose nanowhiskers from food by-products is not commercially viable.
Pure keratin with nanometric dimensions was extracted from chicken feathers. A process for the purification of chicken feathers, followed by nanokeratin extraction was developed and optimized. The extracted nano-keratin presented good thermal stability and preliminary results on its incorporation into PHAs by melt compounding showed water and oxygen permeability reductions with relatively low nano-filler loadings.
EcoBioCAP project also developed adhesives for multilayer structures based on zein, pullulan and WPI to stick to layers of Tianan PHBV plasticized with PEG. The multilayer systems in which zein and pullulan fibres were used as inner adhesive layers were selected as they also allowed reducing the oxygen and water permeability of the plasticized PHBV films while keeping almost unaltered their transparency and mechanical properties.

• Key result n°5: Development of a flexible barrier film

WP3 – Formulation and structuring of finalized materials

To produce the flexible barrier film with a focus mainly on high oxygen and medium water vapour barrier, different constituents developed in upstream WP2 were combined and structured according to their complementary roles. This was done in order to achieve the intended targeted final properties: processability, barrier/permeability properties, mechanical strength, sealability as defined in WP1 (packaging specifications). The potential packaging materials were produced in lab-scale as monolayer composites in order to select the most suitable one for the multilayer structure to achieve the targeted functionalities and enable the later upscaling.
In the case of the flexible barrier film development, the following strategies were adopted:
- Multilayers composed of electrospun protein or polysaccharide (wheat gluten, zein or pullulan) as functional adhesive between extruded PHBV films.
- Multilayers containing composites of PHBV and bacterial cellulose nanowiskers (BCNW) created by cast film extrusion.
- Multilayers containing composites of PHBV with no or low content of a filler gained by film blowing and cast film extrusion.
The fillers were by-products from food industry such as wheat straw fibres (WSF), brewers’ spent grain fibres (BSGF) or olive fibres (OF). The reason for incorporating fibres into the biopolymer matrix was on the one hand to reduce the costs of the composite material and on the other hand to increase the permeability.
As WP2 could only deliver small amounts of PHBV, a commercial grade of PHBV was used instead. The processing of the commercial PHBV grades was difficult because of the high brittleness of the material. To improve the processability of the most suitable PHBV (Tianan) further optimising strategies were evaluated:
- Plasticizing PHBV with specific plasticisers (ATBC, TEC, PEG and glycerol etc.)
- Blending PHBV with another biopolymer (MaterBi, Ecoflex, Ecovio, etc.)
After optimising the processability of the composites and blends, the compounding and extrusion was performed at larger lab scale and the permeability and mechanical properties of the materials were assessed.
The suitability of the resulting mono- and multilayers as well as an outlook regarding the possible applications for flexible barrier film either for lid or pouch application are summarised below:
- Trials showed that the incorporation of BCNW in the PHBV matrix did not lead to a significant decrease in permeability. Therefore this cost efficient approach was not followed in more detail.
- As coated systems, generated by the electrospinning process, multilayers with 2 possible setups were created: PHBV/ zein or pullulan/ PHBV (Figure 7) or PHBV fibre/ gluten/ PHBV fibre (Figure 8). These systems showed a hydrophobic surface (contact angle values > 70º) and in case of the second structure the effect to protect the thermoplastic wheat gluten from moisture, thus tailoring the water vapour and oxygen permeability. The developed multilayer films showed excellent adhesion between the layers which is, in fact, a key factor for the improved barrier properties attained. An upscaling of this method was not possible due to facility limitations.
- The technology of blown film extrusion turned out to be the most prospective for the development of thin films. Blown bi-layered films were considered as the best compromise in terms of barrier and mechanical properties as well as processing conditions. Regarding the studied options for the intended applications as a lid or pouch, the most adequate film was a bi-layer of Tianan (40%-50%) and Ecoflex (60%-50%) without fibres to ensure best possible transparency while keeping acceptable processability of materials by film blowing technology. This setup was recommended for further work within the frame of WP 6. (Figure 9)
- The characteristics of films produced with PBAT used as external layer and PHBV used as internal layer are gathered in Table 1, along with the blow up ratio (BUR) and the take up ratio (TUR) used during film blowing. These films showed the best mechanical and optical properties when compared to films with PHBV as external layer and PBAT as inner layer. In addition, larger variations of the film lay flat width were measured when PHBV is the outer film layer. This indicates that a more stable bubble is achieved when PBAT is the outer layer, possibly due to a higher, more consistent, melt resistance. Thicker PHBV layers were produced when using low BUR and high TUR. A range of processing parameters (namely TUR and BUR) was found to minimize the total thickness of the film, or to maximize/minimize the thickness of the PHBV layer, whereas all other film properties were less sensitive to BUR and TUR.

• Key result n°6: Development of a flexible respiring film

WP3 – Formulation and structuring of finalized materials

Production of a flexible respiring film with a focus on high permselectivity (carbon dioxide/oxygen permeability ratio) and high water vapour transmission rate, required the combination and structuring of different constituents developed in upstream WP2. It was important to take into consideration the complementary roles of the constituents with regards to the intended targeted final properties: processability, barrier/permeability properties, mechanical strength, sealability as defined in WP1 (packaging specifications). The potential packaging materials were produced in lab-scale as monolayer composites in order to select the most suitable one for the multilayer structure to achieve the targeted functionalities and enable the later upscaling.
In the case of the flexible respiring film development, the following strategies were adopted:
- Multilayers containing composite material composed of PHBV with high content of a filler created by cast film extrusion.
- Multilayer of PHBV and a biopolymer gained by film blowing extrusion and subsequent perforation.
Fillers were by-products from food industry such as wheat straw fibres (WSF), brewers’ spent grain fibres (BSGF) or olive fibres (OF). PHBV from Tianan was used and to improve its processability, further optimising strategies were evaluated:
- Plasticizing PHBV with specific plasticisers (ATBC, TEC, PEG and glycerol etc.)
- Blending PHBV with another biopolymer (MaterBi, Ecoflex, Ecovio, etc.)
After optimising the processability of the composites and blends, the compounding and extrusion was performed at larger lab scale and the permeability and mechanical properties of the materials were assessed. The following gives a summary of the suitability of the resulting mono- and multilayers and an outlook regarding their possible applications:
- In case of film blowing processing difficulties were predictable when more than 5 wt% fibres, e.g. BSGF, were added to Tianan. However, melt compounding could enhance the quality of the mixing when compared to powder mixing and thus impact the processability of the compound. Nevertheless, this low fibre content did not lead to a significant increase of permeability and thus this strategy was not further validated. (Figure 10)
- The incorporation of ultra-fine WSF fibres, obtained by ball milling, in the PHBV matrix was the best case for all tested fillers with respect to cast film extrusion. The highest achieved filler content was approximately 20wt%. Attention has to be put on a sufficient fibre length (lower than the film thickness) to avoid defects, especially relevant when being applied as thin lid film. The main results of the characterization of these composite materials revealed that mechanical properties were decreased which was ascribed to the poor adhesion between the hydrophobic PHBV matrix and the hydrophilic wheat straw fibres at the fibre/matrix interface and to a thermal degradation of the polymer. The degradation of the polymer during processing was enhanced when fibres were added, as revealed by the decrease of Mw. Interestingly it was shown that the O2, CO2 and water vapour permeability were strongly increased in the case of thin films but not for thick films due to a continuous PHBV layer coating the fibres. (Figure 11)

• Key result n°7: Development of a rigid/semi-rigid tray

WP3 – Formulation and structuring of finalized materials

The development of a rigid/semi rigid water resistant tray with a medium to high oxygen barrier required the combination and structuring of different constituents developed in upstream WP2. It was important to take into consideration the complementary roles of the constituents with regards to the intended targeted final properties. The potential packaging materials were produced in lab-scale as monolayer composites in order to select the most suitable one for the multilayer structure to achieve the targeted functionalities and enable the later upscaling.
In the case of the rigid/semi-rigid tray development, the following strategies were adopted:
- Composite material composed of PHBV and a filler, probably additivated, further processed by injection moulding.
- Multilayer structures composed of PHBV with and without fillers, partly blended with other biopolymers or plasticised, further processed by cast film extrusion and subsequent thermoforming
Composite material composed of PHBV and wheat straw fibers. Compounds were successfully produced, with a material process-ability affected by the presence of fibres. Indeed, it was highlighted that the macroscopic appearance of both extrusion rods and compression mould films became heterogeneous and lost their physical integrity while increasing fibre content, such phenomenon being intensified for increasing fibre size. This was ascribed to a favoured agglomeration of fibres while the molten matrix is flowing and to an increasing difficulty for the PHBV polymer to correctly wet the fibres. A significant decrease in thermal stability of the polymer was evidenced by thermogravimetric analysis. This was exacerbated in the presence of increasing content of fibres, but was not impacted by the nature of the fibre. The decrease in thermal stability of composite materials was related to the degradation of polymer chains, as revealed by GPC analysis, induced by mechanical shearing and hydrolysis reactions favoured by degradation products of lignocellulosic fibres (essentially water).
Mechanical properties, and especially ultimate properties, were negatively impacted in all cases, globally leading to an aggravation of the brittleness of PHBV-based composites (Figure 12). This was mainly due to a poor fibre/matrix affinity (best preservation being linked to best visual affinity and smallest fibres) and the occurrence of defects in the presence of fibres (Figure 13). Impact of PHBV matrix modification on mechanical properties was dismissed as, even after processing, PHBV chains were still long (around 170 000 g.mol-1) and crystallinity, measured by DSC was not much affected by fibres – due to an already high nucleating agents rate in the raw pellets.
The introduction of increasing fibre content also enhanced water vapour transfer rate, with an intensification of this enhancement for increasing fibre size (Figure 14). This phenomenon was explained by the hydrophilicity of the fibres as well as by structural changes induced by the presence of fibres, creating preferential pathways for the water vapour to permeate through the material, especially at the fibre/matrix interface. Based on all these results, it can be concluded that a new range of PHBV-based composites with tunable properties have been successfully produced, which could fulfil the requirements of respiring fresh food products such as strawberries, thus enabling to preserve them in a better way than currently used polyolefins. Furthermore, at the economical point of view, neglecting wheat straw price and considering only the cost of raw materials, the final cost of PHBV-based composites could be reduced by 30%.
Multilayer structures composed of PHBV with and without fillers. Flat film extrusion trials revealed a not sufficient film thickness to process films with an absolutely closed surfaced. Therefore a multilayer was processeed with the middle layer containing fibres. The results of the characterisation of these films are shown in Table 2 and Table 3.
The incorporation of fibres in the PHBV matrix led to a decrease in mechaical properties and an increase in permeability for the multilyer structures.
Fillers were by-products from food industry such as wheat straw fibres (WSF), brewers’ spent grain fibres (BSGF) or olive fibres (OF). PHBV from Tianan was used and to improve its processability, further optimising strategies were evaluated:
- Plasticizing PHBV with specific plasticisers (ATBC, TEC, PEG and glycerol etc.)
- Blending PHBV with another biopolymer (MaterBi, Ecoflex, Ecovio, etc.)

External plasticization of PHBV. Thermal and mechanical characterization of PHBV/plasticizer blends showed that a significant plasticizing effect was obtained using hydrophobic substances such as acetyltributyl citrate (ATBC) and glycerol triacetate (GTA), with a decrease in the glass transition temperature and an increase of the elongation at break from 1.8 % up to about 6 % for an additive content of 10 wt% (Figure 15). However, the incorporation of wheat straw fibres in plasticized PHBV led to a dramatic decrease in the elongation at break of composites, neutralizing the increase of this parameter by the addition of the plasticizers. The stress at break of plasticized films was also significantly decreased by the introduction of fibres. Such a loss of ductility was mainly explained by the occurrence of microscopic defects in the materials induced by the presence of fibres and to a poor adhesion at the fibre/matrix interface.
Blending PHBV with another biopolymer. In order to increase the flexibility and improve the processing of the PHBV an additional strategy to plasticize PHBV was performed by a blending step. Blend partners were MaterBi, PCL, Ecoflex, Ecovio and EVA. The results of the mechanical properties (see Table 4) state that by adding a blending partner the flexibility can only be slightly increased: The Yong’s Modulus (YM) is decreased from the pure PHBV to values of 2990 to 3420 N/mm² for the blend partners MaterBi, Ecoflex, Ecovio and PCL. The sample with EVA shows a greater effect by a significant reduction of the YM. The tensile strength (TS) is reduced by blending for all samples. Interestingly the sample with PCL increased the TS of the blend. The elongation at break (Eb) is increased for all samples. (Table 4)
The barrier properties (Table 5) reveal that both WVP and OP are significantly increased by the addition of a blend partner which is generally in favour of the requirements of packing respiring products like strawberries and mushrooms.
After optimising the process-ability of the composites and blends, the compounding and extrusion was performed at larger lab scale and the permeability and mechanical properties of the materials were assessed. The following gives a summary of the suitability of the resulting mono- and multilayers and an outlook regarding their possible applications:
The suitability of the resulting mono- and multilayers as well as an outlook regarding the possible applications for rigid/semi-rigid tray are summarised below:
- For the injection moulding of the trays formulations with a plasticiser or an impact modifier did not bring enough improvement regarding the processability and the mechanical properties to an extent which justifies their application. Therefore the formulation based only on PHBV and 20wt% WSF (obtained by impact milling) was considered as the most suitable formulation. The injection moulding process was recommended for the scale-up in WP 6. The studies performed before revealed that barrier properties (OTR and WVTR) of the PHBV-based trays were significantly decreased by the addition of plasticisers and WSF. (Figure 16)
- The approach of extruding a multilayer and further thermoforming it to a tray was successfully performed, a proof of concept was demonstrated. First, a blending step of PHBV enabled an improved processability which was then followed by the co-extrusion of the multilayer. Moreover, with this approach the potential issue of migration resulting from the ‘fibre containing layer’ could be overcome by incorporating the fibres in the middle layer.
The most adequate composition was plasticized and blended PHBV, the plasticiser and blend partner being also a biopolymer. In a first step different material combinations were produced by cast film extrusion, the thermoforming process was carried out in a second process step. Due to the poor mechanical properties it was only possible to form small trays (124 mm x 124 mm x 31 mm) in small amounts. (Figure 17) The values for the oxygen and water vapour transmission rates were within the range of high to medium applications. Anyhow, an upscaling of the thermoforming of trays to industrial scale in WP 6: Industrial applicability was not possible and not recommended at this stage.

Regarding the use of different materials to create multilayer structures, a tailored consensus between processability, mechanical and permeation properties had to be found. After the production of the different components: a flexible barrier film, a flexible respiring film and a rigid/semi-rigid tray, the concept which was recommended for further scale-up in WP 6 based on the results of WP3 was an injection moulded tray containing approx. 20% wheat straw fibres in combination with a sealed lid film or with a flow pack of bilayered blown film.
It can be stated that it is possible to reach different permeation values by the combination of tray and lid film properties. Furthermore, the permeation is strongly influenced by the surface and the thickness of the packaging material. Taking that into account, the requirements for a high barrier application (e.g. sandwich) can be achieved by combining a tray and lid film with the above-mentioned multilayer structure. To match the requirements of the respiring products (eg. cheese, strawberries or mushrooms) a modulation of the film thickness was not sufficient, so an additional micro-perforation of the lid film is necessary. This was due to the fact that all tested materials had too low permeabilities to match the requirements stated in WP 1.

• Key result n°8: Suitability of developed packaging materials as Food Contact Materials (FCM)

WP4 – Assessment of packaging physical-chemical stability and chemical safety

The results obtained are presented below:
Advances in methodology
New range of Food Simulating Solids (FSS). A new range of FSS based on agar gels, with a water activity ranging from 0.98 down to 0.72 has been developed in the frame of the EcoBioCAP project to extend the testing methods for water sensitive materials (Figure 18). They were appropriate to simulate non-fatty intermediate and low water activity products.
A non-destructive method allowing a fast analysis of mass transfer. This method is based on the combined use Raman spectroscopy and a mathematical solving procedure (Reference 1). Taking advantage of the high spatial resolution of Raman analysis, the diffusivity value is deducted from a local concentration profile i.e. the concentration gradient in the polymer thickness on the basis of a resolution of Fick’s law. The experimental design consists in to putting into contact two solid films at the desired temperature, the one containing a known concentration of the additive and other originally virgin. After a period of contact, which depends on the study design, the two media are separated and thin slices of film are prepared from the initially virgin polymer (Figure 19). Raman spectra are recorded between 95 and 3500 cm−1 and the profile in migrant concentration is analysed assuming that sorption/desorption phenomena follow Fickian kinetics.
Structural and physical-chemical stability. The physical-chemical stability of selected packaging materials, i.e. the evolution of the mechanical, thermal and mass transfer properties, has been evaluated by studying the influence of severe but yet realistic conditions of storage (water activity, storage time, light exposition), but also of food contact (using liquid and solid food simulants), which was completely original. The physical-chemical stability has been linked to the structural stability, which was mainly evaluated at the macroscopic scale by SEM observations, and at the molecular scale by focusing on the thermal transition temperatures (evaluated by DSC), the thermal degradation temperature (evaluated by TGA), the molecular weight of polymer chains (evaluated by GPC), and the formation of low molecular weight fractions (LMWFs) (using FTIR and screening analysis).
Microbiological stability. The microbiological stability of the packaging materials has been determined by adapting a methodology used by Kristo et al. (2008) for anti-microbial packaging materials (Reference 2). The methodology developed in the frame of EcoBioCAP consisted in enriching the surface of films with a bacteria and to evaluate its survival upon time and under different relative humidities. For that purpose, Listeria monocytogenes was chosen as a micro-organism model since it is one of the most relevant foodborne pathogenic bacteria.
Substitution of vegetal oil for migration tests. It was demonstrated that iso-octane could be considered as an appropriate substitute for fatty simulants (vegetal oil) for PHBV overall migration testing whereas ethanol 95% (v/v) induces an excessive overall migration value.
Chemical safety of PHBV films
Potential contaminants of PHBV produced from food wastes – Challenge tests. It was shown in the EcoBioCAP project that cheese whey could be used as feedstock for the production of PHBV (WP2). Literature study allowed concluding that the possible presence of a variety of low-molecular weight contaminants in raw milk is one of the key issues for milk safety due to the risk of direct toxic effects on consumers, allergic reaction in hypersensitive individuals, and the development of antibiotic-resistant pathogens (Reference 3). These contaminants belong to several chemical classes, such as sulfonamides and pyrimidines, β-lactams, benzimidazole, anabolic steroids, macrolides, mycotoxins, non-steroidal anti-inflammatory drug, pesticides, anticoccidials, triphenylmethane dyes, environmental hormones, glucocorticoids, quinoxalines, lincosamides, nitroimidazoles, quinolones, tetracyclines and sedatives (Reference 4). Due to their lipophilicity and resistance to biodegradation (Reference 5), these compounds accumulate in the biosphere and detectable levels may be found essentially on a global scale in many foods, especially milk and dairy products. Challenge tests have been carried out in WP4 to evaluate the decontamination efficiency of the process. It consists in voluntary enriching raw materials (cheese whey in the present study) with surrogates, extracting them at each step of the process and analysing the residual quantity (Figure 20).
β- hexachlorocyclohexane (HCH) has been chosen as surrogate as the representative of the wide family of organochlorines pesticides used in agriculture. The β-HCH isomer fate was investigated during different steps of the polyhydroxyalkanoates (PHA) production process by using mixed microbial cultures (MMCs) and cheese whey as carbon source. The cheese whey fermentation (first anaerobic stage) and the PHA accumulation (second aerobic stage) were considered as the key steps to evaluate the fate of the β-HCH during PHA production process. β-HCH was found not to substantially interfere with the microbial activity, with most of it (above 90.0%) being removed during the PHA production process. A further removal (up to 99.8%) was achieved during the final polymer purification. (Figure 21).
Physico-chemical stability of PHBV films upon storage conditions. As regards the pure PHBV matrix, it was demonstrated that the relative humidity (from 0 to 100% RH) had no significant impact on the crystallinity of PHBV (commercial grade of Tianan containing 3% of valerate), neither on the tensile properties, water vapour and oxygen permeability. It was shown that pure PHBV matrices become more brittle after 3 months of storage, which was ascribed to the increase in the degree of crystallinity of materials. An exposition under light (10 000 lux) during 15 days led to the decrease in both the strain and the stress at break of PHBV films, with no further change over 90 days, which was ascribed to the degradation of the polymer also revealed by a decrease in the melting temperature.
Microbiological stability of PHBV films. It was demonstrated that the relative humidity (from 0 to 100% RH) greatly influenced the L. monocytogenes survival. The results from microbiological stability are of great interest since this bacteria does not survive at low relative humidities, which could provide a safety application in foods stored at low RH. However, applications at high relative humidities could be compromised in the case of neat PHBV based films
Inertness of PHBV films upon food contact conditions. It was concluded from the overall migration tests that the tested PHBV (Tianan grade) films can be used as food contact materials for all types of food, i.e. high water activity, acidity, alcohol or fatty foods since overall migration values were all lower than the overall migration limit of 10 mg/dm2 of packaging surface in contact with food simulant (Figure 22). It was shown that iso-octane could be considered as an appropriate substitute for fatty simulants (vegetable oil) for PHBV overall migration testing whereas ethanol 95% (v/v) induces an excessive overall migration value. The functional properties of PHBV films (mechanical properties and water vapour permeability) were very stable after contact at 40°C during 10 days with all FSLs tested (water, acetic acid 3% (w/v), ethanol 20% (v/v), iso-octane and olive oil)), except with ethanol 95% (v/v) (Table 6), corroborating the possibility to use PHBV films as packaging material for a wide range of foodstuff. As previously evidenced for other polyesters such as PET, ethanol 95% (v/v) was clearly identified as the most severe food simulant for PHBV films, with a strong impact on their physical-chemical and chemical stability. In this case, a high sorption value together with a significant plasticizing effect and an increase in the water vapour permeability were noticed, which was mainly explained by a decrease in both the molecular weight and the crystallinity degree of PHBV films (Reference 6). As regards specific migration, boron nitride and crotonic acid were not detectable at a detection limit of 0.03 mg/6 dm2 in an extruded film of 100 % PHBV (Tianan).
Globally, a strong dependence between the structural, physical-chemical and chemical stability of polymers was demonstrated. However, it was shown that the occurrence of structural changes, intimately related to the affinity between the polymer and the FSL, was not the unique parameter governing the physical-chemical stability and the inertness of PHBV films. Indeed, in the case of water, a decrease in the polymer molecular weight and crystallinity were observed with no impact on functional properties and overall migration, meaning that such structural changes do not fully predict the physical-chemical stability and chemical safety of PHBV films. Thus, an in-depth interest should be also given to the affinity between potential migrants and FSLs and their migration behaviour.
Impact of fillers on the chemical safety of PHBV based materials – Study case of PHBV/wheat straw fibres biocomposites.
Based on the results of WP2 and WP3, wheat straw fibres obtained by impact milling (d50 of about 150μm) have been selected as fillers for the production of composite rigid trays and further up-scaling in WP6 (Figure 23).
Potential contaminants of wheat straw fibres – Challenge tests. The main contaminants of wheat straw fibres are pesticides, and especially fungicides such as epoxiconazole. The decontamination efficiency of the processes used for the production of wheat straw fibres (successive grinding of native straw and production of composite materials by extrusion) was studied through challenge tests and using a range of surrogates. Challenge tests have been carried out with molecules representative of food packaging additives (Irganox 1076, Uvitex OB, Chimassorb 81 and benzophenone), which also display similar chemical structures as pesticides, in such a way to couple results to the ones of specific migration tests (Figure 24). It was shown that the process used to obtain wheat straw fibres (successive grinding steps) allowed to remove 20% of the contaminants (surrogates) whereas the final step of preparation of composite films allowed removing up to 60% (Figure 25). Epoxiconazole, a fungicide largely used for the culture of wheat, was selected as study case for the prediction of toxicological risk. Based on previous results, it was shown that the remaining quantity of epoxiconazole in wheat straw, even if migrating integrally towards the packaging towards food, did not represent any danger for human health (since much more lower that the recommended ADI value).
Physico-chemical stability of PHBV films upon storage conditions. The stability of PHBV-based materials was globally affected by the introduction of fillers, either positively or negatively. For example, the introduction of wheat straw fibres results in unstable materials at high relative humidity, due to the swelling of such hydrophilic fibres. However, the presence of wheat straw fibres allowed stabilizing the materials towards light exposition since no significant change in thermal and mechanical properties was observed during 90 days of light exposition.
Microbiological stability of PHBV films. It is worth noting that the introduction of 20wt% of wheat straw fibres in PHBV results in a deep decline of Listeria monocytogenes titres along the storage time for materials stored at 53% RH. Indeed, in composite materials, Listeria monocytogenes only survives until day 15 whereas in virgin PHBV films, Listeria monocytogenes is still detected after 4 weeks of storage. This behaviour can be related, on the one hand, with the antimicrobial effect of polyphenols contained in the lignin of wheat straw fibres. On the other hand, the presence of residual phytochemical compounds inside the straw could also contribute to the sharp decrease in the listeria growth.
Chemical safety of PHBV/wheat straw fibres composites. The introduction of 20 wt% of wheat straw fibres in PHBV led to very high overall migration values in the case of hydrophilic FSLs including water, acetic acid 3 wt%, ethanol 20 wt% and ethanol 95% (v/v). The overall migration values of composite films in these latter FSLs were significantly influenced by the sample thicknesses, with much higher overall migration values for thicker films, probably due to edge effects. Such high overall migration values were attributed to the hydrophilicity of wheat straw fibres, leading to sorption values 2 to 8 folds higher than the neat matrix depending on the tested FSL. It also resulted in a decrease in the Young’s modulus of materials, which was ascribed to an alteration of the intrinsic rigidity of wheat straw fibres induced by swelling. However, these structural changes had no impact on the water vapour permeability.
Interestingly, low overall migration values were obtained under contact with iso-octane, olive oil, TenaxTM [modified poly(phenylene oxide)] and agar gel-based food simulating solids with a water activity (aw) lower than 0.90 demonstrating the possibility of using biocomposites as food contact materials for low or intermediate water activity products and/or fat products. (Figure 26)
The specific migration limit established by the European Plastics Regulation (EU) No 10/2011 for authorised boron species such as the used boron nitride (used as nucleating agent) was respected for the investigated PHBV/wheat straw fibres composite materials, whereas the residual limit for crotonic acid of 0.05 mg/6dm2 was largely exceeded. As crotonic acid is typically formed by thermal degradation of the PHBV, it can be concluded that the thermal stress during the tray production and favoured by the presence of wheat straw fibres (Reference 7) is responsible for these high concentrations of crotonic acid in the tray material. As regards specific migration of surrogates, i.e. molecules representative of packaging additives, the addition of wheat straw fibres in the material induced a detrimental effect by increasing slightly the migration level as well as the diffusivity of surrogates. (Figure 27) A possible explanation of this result lies in the lack of cohesion between hydrophilic wheat straw fibres and the hydrophobic PHBV matrix, which may result in the formation of a preferential transport pathway through channels at their interface with the continuous phase of PHBV. The resulting effect of fibre addition on the mechanism of transport of diffusing substances within the composite material remains to be clarified and requires more investigation.
Recommendations and perspectives
Based on all these results, it was assumed that rigid composite trays based on PHBV and 20 wt% wheat straw fibres may be used for the packaging of mushrooms and strawberries as well as of whole cheese with inedible rind and of sandwiches and toasted bread pizza and the likes (which contain any kind of foodstuff without fatty substances on the surface). This should be of course verified before placing them as food contact articles on the market.
Furthermore, considering the results of the screening analysis for the PHBV trays (where polymeric fragments / breakdown products of the PHBV polymer were detected), temperature control during all processing steps (compounding + film extrusion and especially injection moulding) is essential to produce PHBV-based food contact articles that are in compliance with the regulatory requirements and that can be safely used as food packaging materials. Thus, decomposition processes should be further investigated to show compliance of such materials with the general safety requirements for food contact materials according to the European Framework Regulation (EC) No 1935/2004, with a special attention to carry on the temperature conditions during material production.

• Key result n°9: Life Cycle Assessment of the EcoBioCAP packaging materials

WP5 –Assessing the environmental impact of the new biodegradable materials of packaging

A Life Cycle Analysis was used to quantify the values of the associate indicators of environmental impacts linked to the packaging materials. The Life Cycle Analysis was performed:
- on the developed PHA material (comparison to literature and database information from commercial materials, bio-based and petroleum-based);
- on a selected food product (fresh strawberries) packed in EcoBioCAP-developed biodegradable packaging (a composite PHA tray sealed with a biodegradable film).

LCA on the developed PHA material
The environmental assessment of the present PHA material developed in the EcoBioCAP was done based on information and data delivered from other consortium members. Since the production of final PHA material and packaging solutions still are under development, a lighter version of LCA was performed focusing on the environmental influence of different steps and parameters in the production of the PHA based composite material (Figure 28) from waste streams (whey and wheat straw). The effect of whey as a polymer feedstock, energy use and amount of filler composite present was discussed. This information was compared with information available on literature for commercial biopolymers and petroleum based polymers.
As mentioned before, it is not relevant at this stage to compare the production of PHA based composite packaging with conventional packaging since the PHA production and compounding to composite is a small scale lab/pilot production and hence still under development. Therefore, this study points out the important steps in the production, when it comes to environmental impact from the production of PHA and PHA composite material.
- The impact from spray drying of CW is significant. Options of using wet whey direct or evaporated concentrated wet whey instead of powder should be considered (Figure 29).
- If a higher yield of PHA polymer from CW content in the 3 stage lab-scale production is possible this should be prioritized.
- The electricity consumption per kg PHA is high especially connected to spray drying of wet whey to powder and therefore the impact of using renewable sourced electricity in the production of PHA has a significant effect on climate change.
- Optimization of energy use, input of nutrients and consumables and yield in every step in the PHA production should be prioritised.
- Reduce the loss occurring during compounding is important.
- The use of chemicals/consumables in the PHA production process should be optimised.
- The use of wheat straw filler in the composite PHA based packaging is positive from an environmental point of view. A use of renewable “green” electricity in the milling of the straws will have a significant positive effect on climate impact and is recommended.
LCA on packed strawberries in EcoBioCAP-developed biodegradable packaging
A life cycle assessment of fresh strawberries packed in a composite tray sealed in a film, both biodegradable PHA/PHBV materials developed within EcoBiocap was performed and compared with a commercial PET clam (Figure 30).
The highest climate impact from packaging of 0,5 kg strawberries is seen for the scenarios using packaging produced with PHA developed within EcoBioCap project (Figure 31). The four most influencing parameters are:
- the impact from the feedstock cheese whey, when considered to be a by-product and given a part of the climate burden from producing the cheese,
- the spray drying,
- the PHA production (in lab-scale)
- the transport of wet whey.
The impact from the feedstock itself (CW) can clearly be seen in the two scenarios without CW as FS (CW considered as waste). If CW is considered waste the climate impact from these packaging will be much lower.
The impact from spray drying is large, about 2 kgCO2 eq per packaging. The impact would be lower if renewable energy is used instead of UECT electricity and if less CWP is used in the PHA production process (i.e. increased yield). (Figure 31)
The impact from PHA production is also higher compared to commercial production (as seen for PHBV packaging). This is not surprising because the PHA production is NOT a commercial polymer production, rather a lab/small pilot scale production. No upscaling to commercial production has been able to do within the time frame of this project. In the upscaling process, usually efficiencies are made, both concerning yield from feedstock and use of energy.
The impact from the composite milled wheat straw filler is low and therefore it is the contribution from the filler here (approx. 2 gram/packaging) low. The present content of composite in the tray is 20% and from an environmental point of view a high content is good.
The transport resulting in the highest impact is the transport of the wet whey. Even though the transport isn’t that far (here assumed to 350 km) the volume transported is high and therefore also the impact.
Even though the PHBV processing data not are from Tianan, the transport from China to France/Italy have been added to the PHBV packaging (also for PET actually). This transport is only contributing with about 10 g of CO2eq/FU or 3% of the total climate impact from this scenario.
The climate impact from 1 kg of field cultivated strawberry is 0,14 kg CO2eq. This is a low carbon footprint comparing to other food produce. One liter of milk has a carbon footprint of around 1 kg CO2 eq and 1 kg of beef 25-30kg CO2 eq. Therefor often the relative impact from the packaging is low for food products.
The impact to climate change of in particular the CWP and transport of the wet whey is significant. To reduce the environmental impact the best case scenario would be:
- Cheese whey is considered a waste fraction and is not given any burden from the cheese making process.
- The wet whey should be used directly, no need of spray-drying.
- The production of PHA is done close to the cheese dairy, so no (or small) transport is needed.
- The production process have been optimised to increase yield.
The environmental impact of the best case scenario is comparable to the Benchmark PET clam. Considering PHA being a renewable material, and the composite packaging completely biodegradable this packaging is a sustainable packaging solution.

• Key result n°10: Biodegradability of the EcoBioCAP packaging materials

WP5 –Assessing the environmental impact of the new biodegradable materials of packaging

Biodegradability of the materials developed has been characterized according to international standards: ISO14855 (Biodegradation in composting condition) and ISO15985 (Biodegradation in anaerobic conditions). Disintegration in a simulated composting plant is tested at lab scale level according to standard EN14045 (ISO20200).
The goal is to fulfil European harmonized norm for packaging waste EN13432. The standard EN13432 explains the characteristics of a material to be defined “compostable” and recycled through composting of organic solid waste: biodegradability, disintegration during biological treatment; absence of negative effects on the composting process and low levels of heavy metals and absence of negative effects on the quality of the resulting compost.
The materials tested were:
1) Tray of PHBV Tianan Enmat Y1000P
2) Tray of a Blend PHBV with 20 % wheat straw fibers (PHBV + 20WSF).
3) Flexible film based on Tianan Enmat Y1000P and Mater-Bi used to pack the strawberries.
The methodology used was:
- The disintegration was tested in composting conditions according to ISO20200:2004 “Plastics Determination of the degree of disintegration of plastic materials under simulated composting conditions in a laboratory-scale test”.
- The biodegradation in aerobic conditions is performed according to ISO14855-1:2005 “Determination of the Ultimate Aerobic Biodegradability and Disintegration of Plastics under Controlled Composting Conditions”.
- The biodegradation in anaerobic conditions is performed according to ISO15985:2004 “Plastics –Determination of the ultimate anaerobic biodegradation and disintegration under high-solids anaerobic-digestion conditions -- Method by analysis of released biogas”.

Disintegration test showed that, after 70 days of test at 58°C, both samples of PHBV Y1000P and PHBV+20WSF are completely degraded (Figure 32) and no residual was recovered in the mass of compost.
Regarding aerobic biodegradation, both samples reached the 90% indicated in the harmonized norm EN13432 (Table 7).
Taking into account both aerobic biodegradation and disintegration test it can be concluded that the samples of PHBV Y1000P and PHBV+20WSF are biodegradable and compostable according to EN13432 (assuming absence of negative effects on composting process).
Regarding anaerobic biodegradation, after 55 days of test at 52°C, the sample of PHBV film reached a biodegradation of 78% (Table 8). It should be kept in mind that in the EN13432 it is indicated that anaerobic biodegradation should reach at least 50%, so PHBV Enmat Y1000P complied with this condition. In the standard, this relatively low value, is justified because in most of the bio-gasification plants the process provides a second composting phase in which the biodegradation can further continue.
In conclusion, the positive results obtained in terms of biodegradation and disintegration showed that the EcoBioCAP tray composition is biodegradable and compostable.
Disintegration test of the flexible film based on Tianan Enmat Y1000P and Mater-Bi used to pack the strawberries, was successful according to ISO20200. In fact at the end of the test, just a few residue (fragile and small) were recovered after sieving. The disintegration calculated is 96%. The disintegration of the Test Material has far exceeded the limit of 90% required by the standard. This sample can be considered biodegradable since both components of the film are totally biodegradable according to ISO14855 test.
The calorific potential of the EcoBioCAP material was determined by a bomb calorimeter. As a step in the LCA analysis, the purpose was to investigate whether the materials had calorific potential exceeding 5 MJ/Kg in order to be considered for energy recovery via combustion. The measurements also include a comparison with the developed PHBV-based packaging material EcoFlex and the more common packaging material Cellophane. Values for the obtained combustion enthalpies of the PHBV samples are given in Table 9.
The results show rather strong incentive for potential energy recovery via combustion based on the threshold value of 5 MJ /Kg and a comparison with Cellophane.

• Key result n°11: Ecotoxicity of the EcoBioCAP packaging materials

WP5 –Assessing the environmental impact of the new biodegradable materials of packaging

The conventional environmental chemical monitoring does not allow the determination of possible detrimental effects of chemicals, which derive from (bio) degradation of monitored compounds; furthermore, synergic effects are not considered by chemical analyses. Thus, the application of ecotoxicity tests has been performed in order to study the actual toxicity associated to target products.
A battery of ecotoxicological tests were exploited to assess the actual environmental impact associated to the persistence and transformation of the new developed packaging materials. In particular, already recognized ecotoxicological tests for both terrestrial and aquatic ecosystems, based on the use of both prokaryotic and eukaryotic biomarkers, i.e. the luminescent bacterium Vibrio fischeri, the water cress Lepidium sativum and the animal Folsomia candida, were employed.
The feasibility of the application of all mentioned tests on PHA-based films was verified by preliminary experiments, which were performed on six films developed within EcoBioCAP.
Main features of such films are reported in Table 10.
The application of ecotoxicicity tests on PHA-based materials did not result in relevant inhibitory effects on employed biomarkers. In particular, no EC50 values could be calculated, since inhibition of monitored parameters never reached 50%. Furthermore, after about 6 months of monitoring, inhibition percentages were restrained among 20 and 30%. L. sativum appeared as the most sensible biomarker, in agreement with the test nature.
Importantly, no significant differences were observed when developed material included or excluded 20% of wheat straw fibers. All this considered, no significant concerns about the toxicological risks associated to the release of tested materials into the environment were detected.
In conclusion, the environmental performances of PHA-based materials, which are being developed in the framework of the EcoBioCAP project, seem compatible with their employment for packaging solutions.

• Key result n°12: Implementation of production strategies for constituents of EcoBioCAP packaging materials

WP6 – Industrial applicability

The performed essays resulted in production at small pilot scale of several constituents of packaging materials (in upstream WP), namely polyhydroxyalkanoates (PHA) and fibre fractions from wastes of the food industry. The upscaling of a process to obtain a multilayer structure by the application of a zein-based adhesive through electrospinning was also done. This work is summarized below:
- The scaling up process, from lab to small pilot scale (20-100L), of the three stage PHA production system was accomplished using cheese whey as feedstock. Roughly 1 kg of polymer with an average valerate (HV) content of 22.4 wt% was produced from 6 - 8 kg of cheese whey powder. Process optimization required the assessment of operational conditions in order to improve polymer yield from the substrate during PHA production; and the development of an economic and a large-scale efficient extraction process. Wheat straw fibers-based fractions were prepared by the succession of two grinding processes: Cut Milling and Impact Milling (yielding a sample which will be referred to as IM-WSF). Several kilograms of IM-WSF, with a particle size of 100-150 μm, were produced. A further step of fibres’ sorting is suggested, in order to decrease IM-WSF heterogeneity.
- A small pilot scale production scheme was developed for cellulose-rich material from brewer’s spent grains. It was found that the amounts produced are directly scalable, being only dependent on the size of the vessels. Using 150 L-vessels, it was possible to produce 0.5 kg of cellulose-rich material in each batch (in total, ca. 2.5 kg were produced). The up-scaled process did not affect the average bulk yield (20%) and the properties of cellulose and lignin-rich extracts.
- An integrated pilot scale scheme was developed for olive pomace processing, obtaining a phenolic extract as well as a fibre-rich fraction. The extraction process, using ethanol-water as solvent, was able to retrieve a phenolic extract with antioxidant potential. The fiber fraction was mostly composed of lignin and cellulose.
- Zein solutions were electrospun onto multilayer structures developed using extruded blown polyhydroxybutyrate-valerate (PHBV), PHBV/Ecoflex and PHBV/Mater-Bi. A scaled-up multilayer structure was obtained by covering another similar layer on the extruded blown films containing the zein electrospun interlayer. It was found that the zein interlayer significantly reduced the water and oxygen permeability of the systems.
After that it was important to succeed on implementation of production strategies for packaging materials, namely the production at small pilot scale of several packaging materials: one respiring film, one barrier film and one rigid tray.
Film production
Fifty meters of a functional non-respiring film, specifically a bi-layered thin film obtained by co-extrusion of PHBV with Ecoflex were produced (Figure 33). For this processing conditions suitable for a further upscaling were applied. The values obtained for the permeability to water vapor and oxygen were within the requirements set for sandwiches.
Also, a small pilot scale production of films with fibres was possible. The incorporation of up to 20 % (w/w) fibres resulted in a film with a significant increase of both WVTR and OTR in comparison to pure PHBV. However, these values were not sufficient to cover the requirements for the targeted fresh produce and cheese, while the incorporation of higher amount of fibres was not applicable due to technical constraints during extrusion. Perforation was mentioned as the most feasible alternative to obtain respiring packaging films with the required O2 and CO2 permeability values.
Tray production
The production of trays by injection moulding at pilot scale was possible for four different formulations: PHBV, plasticized PHBV, PHBV/WSF (wheat straw fibres obtained by impact milling) composite and plasticized PHBV/WSF composite. All the tested formulations were processable, whereas plasticized formulations displayed a behaviour similar to that of polypropylene. Thermal degradation of PHBV induced by injection moulding at pilot scale was equivalent to the one noticed at lab scale. Tensile tests showed an increase in the strain-at-break for plasticized trays. Contrary to results obtained on thin films, the addition of fibres into the tray composition did not significantly increase the OTR of the packaging. Respiration properties could not be achieved by the tray and must be brought by the lid film. However, addition of fibres to the tray composition increased WVTR. As expected, addition of plasticizer significantly increased OTR and WVTR values of the trays to a larger extent than the addition of fibres. In all cases, the obtained values of OTR and WVTR were much lower in both cases than the values suggested in the specifications defined in WP1 for fresh produce. These values were nevertheless suitable for modified atmosphere packaging (MAP) of sandwiches.

• Key result n°13: Pilot scale production of EcoBioCAP packaging materials and trials with food products

WP6 – Industrial applicability

Three types of EcoBioCAP packaging materials were produced at pilot scale for the trials: one barrier film; one respiring film (obtained through perforation of the barrier film) and one rigid tray. The EcoBioCAP packaging materials were tested in quality, safety and shelf-life studies of three different types of food products; cheese, ready-to-eat meals (sandwiches), and fresh packaged fruit & vegetables (strawberries and mushrooms).
The barrier film, without further treatment, was used for packaging of sandwiches. Rigid trays were used for packaging of cheese and fruits & vegetables. In the case of fruit & vegetables, that are respiring products, the barrier film was perforated to improve gas permeation and was used as a pouch within the tray containing produce was put. For cheese, that is also a respiring product, dotted-sealing of the lid film on the tray was attempted to ensure sufficient OTR and CTR.
The samples were packaged and stored in real distribution conditions. The quality, safety and shelf-life were quantified in terms of: internal gas composition, weight loss, visual appearance, texture (compression), microbiological analysis, pH and total soluble solids, depending on the product. As a reference the results were compared to those of samples packaged with benchmark packaging solutions such as micro-perforated OPP (cheese), Cellophane (sandwiches) and Naturflex NVS (strawberries and mushrooms).
Although the desired internal atmosphere was hard to achieve and maintain, the EcoBioCAP solutions proved in general to be capable of ensuring the quality of the food products during the targeted shelf-life. In the case of cheese EcoBioCAP solution, with a dotted sealing, showed to be on pair with the benchmark solution. No anoxia was noticed and favourable low CO2 content was achieved.
Results
- For sandwiches, the EcoBioCAP could not maintain the desired modified atmosphere which was mostly likely due to macroscopic deficiencies in the film (uneven thickness, holes). However, the study showed no significant difference in quality, safety and shelf-life of the product between EcoBioCAP and the benchmark packaging as both films proved to be a good moisture barrier.
- The study on strawberries and mushrooms was limited by the initial quality of the fresh produce. The study showed that the sealability of EcoBioCAP film needs to be improved in order to maintain the desired atmosphere, a crucial parameter for an increased shelf-life of fresh fruits and vegetables.
Conclusion
In general, EcoBioCAP packaging solutions proved to be capable of ensuring similar quality of food products during the targeted shelf-life, compared to benchmark packaging materials. Therefore, EcoBioCAP materials can be used as more eco-friendly packaging solutions for food products. R&D efforts are still needed to improve the mechanical properties of the EcoBioCAP films and to increase the capability of these films to hold modified atmosphere. In any case, it is clear that mechanical resistance of the films produced is sufficient e.g. to withstand their use in a packflow machine.

• Key result n°14: Large scale production of EcoBioCAP packaging materials and testing in real conditions with packed strawberries

WP6 – Industrial applicability

Large scale production
Strawberries were chosen as the product to be tested further, packaged in EcoBioCAP trays and respiring film. Large pilot–scale production of high performance packaging materials was thus required. The scale-up activities comprised both film blowing and extrusion techniques.
The materials produced at this stage (trays and respiring film) were used by producers of the selected food product (strawberries) for packaging tests.
The film structure consisted of a bilayer film based on PHBV Tianan Enmat Y1000P (53 %) and an aliphatic-aromatic biodegradable copolyester (PExy) 8MATER-Bi) (47 %), where the Tianan is located in the inner side of the film bubble. (Figure 34)
In fact, the previous studies done in the EcoBioCAP project have demonstrated that PHBV alone cannot be transformed using film-blowing technologies, thus needing the contribution of another polymer that can confer some melt strength and elasticity.
Trays were obtained at large pilot-scale through injection moulding of EcoBioCAP’s tray compound (consisting of 80 % PHBV and 20 % WSF) and were used as containers for strawberries. (Figure 35)
‘Real conditions’ testing with strawberries
High performance biodegradable packaging materials which were produced at large pilot-scale by Novamont (flexible film) and Furstplast (trays) and were sent to Alterbio to be used for packing strawberries. Alterbio sent the packed strawberry samples to the partners CBHU (via their usual distribution chain) and UCC (directly, for shelf-life evaluation). (Figure 36)
The main objective of this work was to demonstrate the efficiency and weaknesses of EcoBioCAP packaging solution under realistic distribution conditions by:
(1) testing produce overall quality in shelf life assessment (shelf life study carried out by UCC)
(2) the global consumer acceptance of the biodegradable packaging, which was tested against the benchmark reference packaging through a consumer survey using a large panel of consumers (carried out by CBHU in Hungary). (Figure 37)
Shelf-life assessment
There were no significant negative impacts of the EcoBioCAP packaging when compared to the benchmark packaging was found. However, this real scale study revealed some gaps to fill to reach industrial applicability. Perforations used to counterbalance the insufficient permeability of the lidding film were not adequate. This underlines the importance of developing tailored high permeability materials for fresh fruits and vegetables preservation.
Consumer acceptance
The consumer survey also included tasting sessions in order to gain information about the impact of packaging variations in terms of sensorial attributes of fresh strawberries. For this purpose, the consumers evaluated sensorial properties of strawberries’ samples, in both packaging variations. In order to evaluate the packaging condition efficiency, a trained panel assessed the samples as well. It was concluded that the proposed EcoBioCAP packaging did not have any significant negative impact on sensorial attributes of strawberries in comparison to product in benchmark packaging. However, for further improvement it would be beneficial to consider the fact that consumers’ most important expectation for fresh strawberry product is a transparent packaging, or a transparent lidding foil at least. Transparency of the lidding film was indeed demonstrated to be important for consumer acceptability.
Therefore during future developments of biodegradable packaging for food applications an early emphasis should be put on obtaining adequate optical, gas permeability and mechanical properties values of the lidding film.
The results of this report are a basis for further improvements both at lab scale and industrial level.

Potential Impact:
• WP1 - Integrated analysis of packaging specifications, dimensioning and decision support system

Specification development process:
For the development of new biodegradable packaging materials and decision support tools, expectations, requirements and knowledge of different disciplines need to be harmonized and integrated. In the frame of a systematic approach for setting the targets of the research and considering technical requirements and consumers’ needs, the use of these management tools is essential in the early phase of the research and development projects. This is necessary for projects within a company, but integration is also important in collaborative European research projects where the knowledge of different disciplines is represented by different organisations from different countries. The physical distance between the workplaces of the project partners prevents frequent physical meeting between the representatives of the different disciplines. Therefore the adaptation of the methods applied at industry product development activities may provide benefits for international research projects.
The objective of the specification development process is to apply a structured approach for defining the requirements for new packaging solutions with specific, tailor-made properties. The structured approach is a management tool to consider, harmonise and integrate the expectations, requirements and knowledge of different disciplines and stakeholders in an EU project (several organisations) and/or in a business (several departments, a few organisations) and to set up an agreed reference base for the research and development process.
The main output of the systematic approach is the development of packaging material/solution specifications. Specifications are clear establishments of all important packaging material properties (for producers, for consumers, for traders) and all requirements of the production (quality, food safety, technical, labelling to consumer information, waste handling). It contains harmonized and integrated requirements and knowledge, so it is a basis for the further research and development work. It is important to use the packaging specifications to prevent misunderstandings, to provide precise description of all essential properties, requirements and limitations in a written form to help the identification of the gaps in the information and to deliver information to the customers, to the food manufacturers and to the packaging material manufacturers.
Decision Support System (DSS):
DSS is one of the main achievements of EcoBioCAP in the field of computer science and results are very positive (TRL 9). The specifications of the DSS are provided in D1.3. The first prototype of this software is available at http://pfl.grignon.inra.fr/EcoBioCapQuerying/.
DSS exists for a lot of application, it generates high activities about it. An obvious need for such a tool exists, but a part of this need is hidden, the market has to be improved by workshops, training etc. This kind of product depends on the involvement of the different actors and the need will evolve with improvement in packaging. One of the main inputs required to favour the exploitation of such tools by stakeholders is the filling of associated databases as regard ‘fresh produce’ and ‘packaging’ characteristics. The reliability of the DSS predictions relies on the quality and diversity of data stored in the databases. This requires a huge effort from the scientific and technical community in that field with the launching of dedicated programs to capitalize data (creation of European network on that topic).
The first prototype of DSS is available right now for the EcoBioCAP consortium and demonstration purpose by technical centres such as CBHU. This DSS should be licensed. The current version is in open access on an INRA server.
The main future goal is to enhance the DSS prototype to become a commercial tool.
Tool enhancement: graphical interface, new packaging database connexion, addition of new options (e.g. prediction of microbial quality, modified atmosphere packaging for non-respiring products...)
Database management enhancement: workflow creation to fuse packaging characteristics (permeability, mechanical and processing characteristics...) database querying module enhancement, database promotion targeted towards journal editors to become a reference repository associated with scientific publications.
In parallel, a market study will be performed in order to determine key actors and potential valorisation career (in technical centers or start-ups), market analysis, etc.
Work performed in WP1 has been well disseminated towards the industrial and scientific community with open workshops, trainings, papers and conferences.

• WP2 - Development of packaging constituents by upgrading food industry by-products
The main results obtained in the frame of the EcoBioCAP project, for the production and optimization of MMC PHA (Campanari et al., 2014; Duque et al., 2014) as well as at the characterization of produced PHA (Martinez et al., 2014), have been mainly exploited to assess mass balances of the overall process and to scale up the process from lab to small pilot scale. In particular, the proposed process is an efficient tool for simultaneous wastewater treatment (COD removal efficiency up to 85%) and valorization towards PHA production (yield of COD conversion into PHA between 10 and 20%).
It has been demonstrated the feasibility to produce PHA with mixed microbial cultures by using food industry by-products through a multi-stage process from feedstock pre-treatment to final polymer extraction and purification with no competition with the food chain. Olive oil mill wastewater, cheese whey, and sugar cane molasses have been tested as feedstock and the proposed process is an effective tool for their simultaneous treatment and valorisation towards PHA production.
Both process innovation and product innovation were achieved with respect to the previous literature.
The results show that the process for PHA production by MMC is reliable and good performances can be achieved in a large set of experimental conditions. In particular, the product composition can be controlled by adjusting the acidogenic reactor conditions and type of feedstock, thus opening the possibility to scale up this process and apply it at industrial scale with lower associated operation and investment costs. Furthermore, results from this project will contribute to the issue of transforming WWTPs into factories which integrate production of high value products with the treatment of the effluent, thus having positive environmental impacts (e.g. coupling PHA production to sludge reduction and/or biogas recovery).
As further steps in this direction, pilot-scale plants are still needed, in particular to produce large PHA samples for industry and market tests, including novel, advanced and tailored applications. Any improvements of extraction and downstream processing can also be a decisive factor of towards further exploitation of PHA potential. The main achievements have also been used to publish scientific papers for a wide useful dissemination in the scientific community.

• WP3 - Formulation and structuring of finalized materials
The impact of the work performed in this project is based on the broad knowledge, which was gained in the different optimising steps of processing PHBV, plasticising PHBV and blending it with other biopolymers. Additionally many processing techniques were performed which enabled the consortium to evaluate the options and drawbacks of this biopolymer.
It was possible to process this material in an injection moulding but also flat and blown film extrusion line. A special attribute of this project was the realisation of a bilayered PHBV film by film blowing as well as the thermoforming process of PHBV multilayers.
The developments of the tray by injection moulding and the thin films by cast film extrusion are the two major achievements of this WP and the project. Indeed, these two techniques are relevant to exploit with the industry. By the good processability in the injection moulding process, a range of applications from different forms of trays or cups to transport containers or disposable tableware can be offered. Additionally the possibility to create flexible thin cast films enables more converters of the packaging industry to introduce this biopolymer on their facilities and consequently in their product portfolio. Thereby the choice of bio-based and biodegradable alternatives in the market is enhanced. On base of the project results tailor-made solutions can be developed together with the industry for different applications.
There was also extensive dissemination to the scientific community via the publication of scientific papers and other actions in scientific seminars and conferences.

• WP4 - Assessment of packaging physical-chemical stability and chemical safety
The impact of the work performed in WP4 of EcoBioCAP is based on the gained knowledge on the approach followed to evaluate the chemical safety of materials produced from food wastes, including the consideration of potential contaminants of raw materials (via challenge tests), the study of the relationships between the physical-chemical stability and the chemical safety of PHBV-based materials, as well as the development of specific methodologies to simulate targeted usage conditions and to characterize the degradation of the initial polymeric network and the mass transfer of unwanted molecules.
One potential impact is to introduce a new methodology in the guidelines/regulations published by national and/or European authorities to evaluate the chemical safety of liquid water sensitive packaging materials such as fibre-based biodegradable composites.
INRA has developed solid food simulants to extend the possibilities of solid food testing to different types of products (intermediate or high water activity, and/or intermediate or low pH), displaying textures more representative of food products than the Tenax powder (poly (2, 6-diphenyl-p-phenylene oxide)), which is currently recommended by the European regulation to simulate solid products). It allowed demonstrating the inertness of fibre-based composite materials when put in contact with solid products characterized by a water activity below 0.90. Integration of such methodologies in the national and/or European guidelines/regulations published by EFSA should be done since none of the current methodologies is adequate for such materials (Reference 8). Peer reviewed and scientific publication of these EcoBioCAP results should allow national and European expert to know about EcoBioCAP methodology.
Furthermore, the use of food wastes as raw resources for the production for packaging materials can give rise to problems of acceptability by the consumers, mainly due to potential contaminants from wastes streams. The “challenge tests” approach developed in the project EcoBioCAP allowed to demonstrate the ability of the processing chain to eliminate potential contaminants and therefore to restore the confidence of the consumer. Presenting EcoBioCAP results in large public dissemination about using wastes for materials, energy and chemicals should make consumers and all stakeholders of the food chain more familiar with such raw materials.

• WP5 - Assessing the environmental impact of the new biodegradable materials
The materials and packing solutions developed contribute to the development of environmental friendly packaging systems. The LCA studies showed that the best-case scenario will be comparable to the Benchmark PET clam. Considering PHA being a renewable material, and the composite packaging completely biodegradable, this packaging is a sustainable packaging solution.
Valorising organic waste fractions by using the waste as an input and carbon source in biopolymer production as done within the EcoBioCAP project is a sustainable approach when it comes to developing new innovative food packaging solutions for the future. The challenge is the time and resources needed to invest in the research and developing stages to come all the way to commercial packaging solutions. The physical properties: brittleness, transparency, barriers, etc. and the input resource needed must be comparable to commercial fossil based packaging today. EcoBioCAP has promising started this work. The EcoBioCAP PHA biopolymer material also contains a filler (milled wheat straw fibres) that both facilitates the biodegradability and reduce the material cost. To continue the work, increased resource efficiency in combination with improving the yield in the PHA production process is most important; both will have positive impact on the final environmental footprint of the material.
The environmental performances of PHA-based materials, which have been developed in the frame of the EcoBioCAP project, seem compatible with their employment for packaging solutions.
The biodegradability and ecotoxicity tests also showed that the EcoBioCAP tray composition is biodegradable and compostable without inducing relevant ecotoxicological effects to employed biomarkers.
These results can therefore be used to support the results from the technical performance of waste-based eco-friendly packaging solutions.

• WP6 – Industrial applicability
The industrial applicability of three types of EcoBioCAP packaging materials produced in pilot scale (one barrier film; one respiring film and one rigid tray) were tested for cheese, ready-to-eat meals (sandwiches), and fresh packaged fruit and vegetables (strawberries and mushrooms) and benchmarked with reference packaging materials. Data on quality, safety and shelf-life obtained are: internal gas composition, weight loss, visual appearance, texture, microbiological analysis, pH and total soluble solids. These results permitted to evaluate the performance of the EcoBioCAP materials developed and identify bottlenecks/gaps and future needs of research. Despite the need for improvement regarding mechanical properties of the EcoBioCAP films, generally, the EcoBioCAP packaging solutions proved to be capable of ensuring similar quality of food products during the targeted shelf-life tests, when compared to benchmark packaging materials. For that reason EcoBioCAP materials showed to be a more eco-friendly packaging solution for food products. We must have in mind that economic as well as industrial feasibility of EcoBioCAP solutions must be investigated further in order to minimize some of the existing gaps, namely the ones related with films’ transport and optical properties. In any case, industrial upscalability was demonstrated and therefore it is expected that the know-how generated in the framework of EcoBioCAP can be of use to the pertinent stakeholders.
It is expected that producers of packaging material and food producers will take advantage of the very significant progresses made in the framework of EcoBioCAP as a clear pathway has been established towards the creation of innovative biodegradable food packaging materials from food industry by-products, including scale-up data and procedures, while providing an answer to society’s concerns regarding food safety and environmental issues both of food industry and food packaging.

• WP7 - Outreach
EcoBioCAP dissemination strategy was adopted at the early stages of the project to ensure the widest impact. It included (1) creation and update of specific tools such as the project’s website, brochure and poster; (2) communication with stakeholders, especially with Stakeholder Advisory Board members and (3) ensuring visibility during conferences in the field of packaging and related sectors.
Among the dissemination tools developed within EcoBioCAP, the website provided the general public with a window on the project’s progress. The website was regularly updated with key information related to the project such as newsletters, conferences/workshops in the packaging or related sector. Specific promotional videos were made to present the project: they are available on the website as well as on a specific EcoBioCAP channel on youtube (more than 1000 views combined). Scientists and industrialists have contacted the consortium through this website. The website has regular visitors – 70% of the visitors are new visitors and after the public seminar, the website registered 395 connections (from mostly EU countries). It should be noted that the project’s website (www.ecobiocap.eu) will be kept online after the project ends. Contacts of different consortium members can be found there; they can thus be reached for future collaborations. Six newsletters have been issued (each 6 months) within the project’s duration. Apart from focuses on advances from the project, these newsletters also contained information about upcoming conferences and workshops to which consortium members could participate. They were sent to a dissemination list comprising of contacts from the academic and industry fields and also published on the website for a wider dissemination. A project brochure and a customizable poster were also elaborated as part of the dissemination plan. EcoBioCAP partners distributed the brochure when they participated to conferences and workshops and also used the poster for presentations related to the project. This allowed the project to be known.
The Stakeholder Advisory Board was constituted with members from the academic field and industry. Their main role was to ensure that the project met its objectives and became a success. Members from the scientific field provided input on the scientific content of the project and those from the industry gave feedback on how to meet end users’ expectations. Several meetings between the consortium and the SAB were organised; special sessions were planned at Annual meetings to foster discussion on the abovementioned points.
Dissemination to the scientific community was mainly done through the organisation of the following workshops: (1) a specific workshop on the occasion of the European Symposium on Biopolymers - ESBP2013 in Lisbon, Portugal on the 9 of October, 2013; 2) a workshop dedicated to eco-efficient packaging on the 25th of November 2014 in Uppsala, Sweden. This workshop was organised as a satellite event of the EFFoST 2014 and participants (20) were mostly from the academic field and were also participating to the EFFoST conference.
On top of these specific workshops, to which mostly students participated, EcoBioCAP was present at important conferences of EcoBioCAP field such as Effost 2012 and 2014, FUZZIEEE 2013 (7-10 July 2013), Hyderabad, India.
Three specific events dedicated to the general public were organized within the project framework.
(1) EcoBioCAP had a stand at the Salon International de l’Agriculture (SIA) in Paris in 2012 and 2013. The SIA is an international event (more than 800K visitors from France and elsewhere) dedicated to agriculture and it was an excellent opportunity for members to interact with the participants of this event who are the potential end-users of materials developed within the project.
(2) A public session dedicated to "Training and job opportunities in the frame of Bio-based economy and the best practices from European research projects" on the 21st of March 2014 in Roma, Italy. This workshop was organised after the 3rd AM of the project; there were around 50 participants to this event.
(2) A public seminar was organized on the 26th of February 2015 during the final meeting 25-27 February 2015 in Montpellier, France. This seminar was dedicated to the building of the next generation of sustainable food packaging in Europe. The main themes of this event were devoted to Resource efficiency, Innovations and Safety in food packaging with discussions and debates involving invited guest speakers (from the academic field, other project members and PhD students), EcoBioCAP partners and outside participants (from the industrial sector and students). The objectives were to define together R&D priorities and to lay down the basis of future collaborations and projects. The seminar was a success with about 70 participants.
Partners participating in EcoBioCAP have published 38 scientific papers (peer reviewed and proceedings); 13 more are in preparation or have been submitted for publication. Results from the project have been widely disseminated with over 90 oral presentations done worldwide by partners.

List of Websites:
The website address is the following: www.ecobiocap.eu
EcoBioCAP Coordinator:
Prof. Nathalie Gontard
UMR 1208 IATE
2 place Pierre Viala, Bat. 31
34060 Montpellier cedex 01, France
Tel:+33 4 99 61 30 02
Fax: +33 4 99 61 29 00
E-mail: Nathalie.Gontard@univ-montp2.fr

EcoBioCAP Project Manager:
Yoan Emritloll
INRA Transfert (IT)
3 rue de Pondichéry
75015 Paris, France
Tel: + 33 1 76 21 61 97
Fax: + 33 1 45 77 63 90
E-mail: yoan.emritloll@paris.inra.Fr