Forschungs- & Entwicklungsinformationsdienst der Gemeinschaft - CORDIS

Final Report Summary - FUEL4ME (FUture European League 4 Microalgal Energy)

Executive Summary:
Executive summary
FUEL4ME: Future European league for microalgal energy
The EU funded FUEL4ME (FUture European League 4 Microalgal Energy) project was established to evaluate microalgae as potential sustainable source for second-generation biofuels that can compete with fossil fuels. To realise this an increase in the scale of microalgae production needed to be matched with a simultaneous decrease in production costs. The FUEL4ME project has achieved significant decreases in production costs of algal lipids, but the production costs for algal biodiesel are still (more than) an order of magnitude too high to make this process commercially attractive in the short term. However, microalgae as source for high-value components such as omega-3 fatty acids for application in food and feed products, has proven to be realistic and cost-effective using microalgae as production organism.
FUEL4ME aimed at exploiting algae's unique ability to produce high value biomass and lipids efficiently by photosynthesis. Such biomass and lipids could form an excellent starting material for the sustainable production of biofuels and other products such as animal feed. Moreover, microalgae, including the target algae of this project (Nannochloropsis oceanica and Phaeodactylum tricornutum), do not compete with food crops for land and freshwater as they are grown in sea, saline or other marginal water on unproductive dryland.
FUEL4ME investigated in detail the molecular and metabolic mechanisms governing lipid accumulation in two microalgae species and demonstrated enhanced lipid accumulation by metabolic engineering. Furthermore, we compared the current two-step batch nitrogen starvation production process for microalgal lipids with a newly developed continuous one-step nitrogen limitation process and optimised lipid production under different growth conditions. Researchers developed the various steps of the downstream processing chain, which involved harvesting, cell disruption, lipid extraction and fractionation and hydro-treatment of the lipids to create biofuel. In addition, biorefinery steps to valorise high value molecules such as omega-3 fatty acids, or high quality algae protein, were developed and demonstrated. They were shown to be successfully applicable to microalgae and are now ready to be used in commercial processes, especially for high-value applications in Food & Feed.
The consortium designed and set up three pilot plants for outdoor microalgae production based in Italy, the Netherlands and Israel, respectively, and one demonstration facility in Spain. At outdoor pilot scale it was shown that although with lower lipid content, the one-step N-limitation process had comparable lipid productivity to the traditional N-starvation batch process, but requires further testing for prolonged periods on a large scale.
To determine the actual state of the technology as well as study how key parameters influence the sustainability of the FUEL4ME integrated process a life-cycle assessment (LCA) study was performed. The main influences upon sustainability were cultivation and harvesting, electricity demand especially for PBR cooling, sources of freshwater and carbon dioxide, and suitable land. FUEL4ME has addressed some of these parameters by improving productivity of cultivation, high efficiency thermoregulation of the photobioreactors, and improved harvesting efficiency, including integrated water and resource re-use in cheap desert land. However, further major improvements, will be needed to make biofuel production with microalgae fully economic and environmentally sustainable. Currently the process is best suited for the production of high value products such as polyunsaturated fatty acids (PUFAs), and a promising biorefinery approach showed to have a strongly improved economic balance. We believe that FUEL4ME's long-term innovation strategy, with initial focus on high value products, will result in economically feasible and environmentally sustainable microalgae-based products. This will ensure a further decrease in production costs and an increase in the scale of production. However, microalgae as source for biofuels seems only viable when the majority of the microalgal biomass is commercialized as high-value chemicals/commodities and the biofuels are a co-product instead of the main product. Both for microalgae cultivation and downstream processing FUEL4ME has provided an excellent opportunity for industrial partners to conduct pilot tests of their technologies, improving them and demonstrating their use within the microalgae field. This has helped to achieve more reliable and scalable industrial solutions.. Additionally, FUEL4ME has generated highly skilled professionals with expertise in algal microbiology and microalgae cultivation and processing systems. By developing knowledge and skills as well as sustainable valorised products from microalgae, the project has made a valuable contribution to Europe's research capacity and bioeconomy. Furthermore, this eco-friendly process has the capacity to reduce dependence on fossil fuels.

Project Context and Objectives:
FUEL4ME: Future European league for microalgal energy
Project context and objectives
The aim of the 4-year FUEL4ME project was to develop a sustainable chain for continuous biofuel production using microalgae as a production platform, thereby making 2nd generation biofuels competitive alternatives to fossil fuels. Main focus points were:
• Transforming the current 2-step process for algal lipid production into a continuous 1-step process with high lipid content (production process);
• Development of a continuous downstream process using all components of the algal biomass (conversion process);
• Integration of production and conversion process.

FUEL4ME aimed to exploit one the unique strengths of algae: the ability to produce lipids using energy from photosynthesis. These lipids form excellent starting material for the production of bulk products; the largest fraction of the lipids will be used for the production of biofuel (NExBTL) and a smaller fraction will be used for food and feed components (ω3 fatty acids). This way optimal use of biomass results in simultaneous production of food and fuel.

Production process
In order to produce biofuels from microalgae, it is important to have significant algal production capacity. However, this capacity is not yet available. Two important reasons for the lack of significant algae production are:
• High costs; with the current knowledge and systems, it should be possible to produce biomass in a reactor of 100 hectare at a cost price of 5 € kg-1. To make microalgae truly interesting as a source of biofuels and able to compete with fossil fuels the scale of production needs to increase at least 3 orders of magnitude, with a concomitant decrease in the cost of production by a factor of 10.
• Negative energy balance; in the current situation, more than 24% of the production costs are energy costs. The energy balance is negative or neutral for most systems, with an energy input equal or larger than the energetic output. It is evident that, to make microalgae attractive as a source of biofuels, the energy balance should be positive. A net energy ratio (NER = energy output / fossil energy input) of over two is an ambitious yet achievable goal required to achieve the renewable fuels standards in terms of fossil fuels input, and GHG savings.
Both high costs, GHG balance and negative energy balance can be addressed by developing and improving the technology behind biomass and lipid production. Currently, production of lipids from microalgae is a two-step process. First, the microalgae are grown in batch cultures and, when a certain biomass concentration is obtained, stress in the form of nitrogen starvation is applied upon the algae to induce lipid accumulation. Consequently, the growth rate decreases resulting in low lipid productivity. Within FUEL4ME, we have developed a one-step continuous process. We know the basic principles to trigger lipid accumulation and to produce biomass continuously. However, to facilitate lipid production, higher biomass and lipid productivity may be achieved continuously in a single step process. First, we have worked on the in depth understanding of lipid metabolism in three microalgae species (Phaeodactylum tricornutum, Nannochloropsis oceanica, and Acutodesmus obliquus). Next, at lab scale a proof of principle concept for a one-step lipid production process was developed.
After proof of concept within controlled indoor conditions, the continuous process was tested outdoors under real production conditions in four different regions (NL, IL, IT – small scale test plants; ES – large scale demonstration plant) with different climates and two microalgae strains with different optimal growth temperatures (Phaeodactylum and Nannochloropsis) to investigate optimal growth conditions (temperature, sunlight, scale), the effect of optimal growth temperature on the energy balance and the robustness and reliability of the production process.

Conversion process
Simultaneous with research on biomass production, a continuous downstream process was developed. The conversion process consists of different steps (harvest, cell disruption, primary extraction, fractionation, conversion, hydrotreatment). For each step, the most promising technologies presently used in other industrial sectors, were applied to microalgal biomass. Where necessary, adaptations have been made. Important criteria for the selection of the technologies were low operational costs and low energy consumption. It is important to find the right balance between costs and energy consumption. In order to lower the operational costs of the total conversion process, we have designed a continuous conversion process that uses all components of the algal biomass. Besides biofuels, high-value lipids (PUFAs) are valorised. From the remaining biomass, hydrogen is produced which is used in the conversion process of lipids into biofuel.

Integration of production and conversion processes
In order to facilitate the continuous production of biofuels from microalgae, the continuous process for algal biomass production with high lipid content has been coupled to the continuous downstream process to convert the biomass into biofuel, high-value lipids and hydrogen that is used within the biofuel conversion process. We have established guidelines describing the process requirements and parameters (costs, energy consumption, characteristics of intermediate products, etc.), ensuring the right fit between the different process steps and providing the information required to design a fully integrated process chain. Finally the whole process (both biomass production and conversion into biofuel) was integrated and subjected to a sustainability assessment, including environmental, economic and social elements based on the whole value chain in a life cycle perspective. The results have been used to guide the technical development and future implementation in the desired direction and to be able to determine economic feasibility and environmental sustainability.

Partnership
In order to achieve the overall aim and the specific objectives, an excellent consortium has been established. The multidisciplinary team includes partners who are leading in their work field. Specialists from both algae production and algae conversion were brought together. The consortium consisted of a powerful mix of established research organisations and universities, small and medium enterprises (55% of total partners) and large-scale industry. These are: Wageningen Food & Biobased Research, Wageningen Plant Research, Wageningen University, Ben Gurion University of the Negev, Fotosintetica & Microbiologica S.r.l., Norsker Investigationes, Proviron, Evodos B.V., Cellulac, Feyecon Development and Implementation B.V., Neste, JOANNEUM RESEARCH Forschungsgesellschaft mbH and IDconsortium SL. (see also http://www.fuel4me.eu)

Final results and their potential impact and use
Understanding and improving lipid metabolism
~Omics analyses of Phaeodactylum tricornutum have identified genes, proteins and metabolites that are differentially expressed under nitrogen replete vs. nitrogen limited conditions. Key biological pathways include nitrogen and amino acid metabolism, fatty acid metabolism, membrane and neutral lipid metabolism and pigment biosynthesis. Proteomics analysis resulted in identification of novel lipid-droplet-associated proteins in P. tricornutum. Preliminary results suggest that overexpression of two of these proteins may result in increased lipid production in P. tricornutum. Finally, lipid droplet formation, fusion and movement seem to be controlled by the microtubule system, as indicated by inhibitor studies.
Next to insight in the lipid metabolism, the work in this project has brought forth Mapomics, a new tool for integrative transcriptomics, proteomics and metabolomics analysis. Mapomics has been implemented as a Galaxy server instance for the support of FUEL4ME and future research focussed on omics integration.
The insights and tools gathered in this task will be used in future projects aimed at targeted metabolic engineering for improved lipid productivity in microalgae.

Comparison of batch versus continuous process
Traditionally, two-step batch mode cultivation (Nitrogen starvation) is adopted for the production of microalgal lipids. In this cultivation mode, biomass is first grown under optimal conditions, after which the cells are induced to accumulate lipids by nitrogen starvation. For the first time, we have shown that production of microalgal lipids in continuous nitrogen limitation mode is, on average, in same range of the lipid productivities achieved in batch mode (both in lab and outdoor). Total fatty acid contents were generally higher in batch mode, especially when batch reactors were harvested well-beyond the moment at which maximum lipid productivity was achieved.
Varying results were obtained for robustness of either cultivation mode (i.e. in some cases batch was more robust, while in other instances continuous mode was more robust). Costs could not be properly assessed at the tested scale. However, in view of the comparable productivities achieved, costs (of entire process including downstream processing) should in the end be decisive for ultimately adopting one of the two cultivation modes. For this, further improvement and upscaling of both cultivation technologies is needed to improve, properly assess and finally compare the process economy. Key point for this analysis is also to take into account the compatibility of the cultivation technology with the downstream processing chain.

Conversion process
Researchers developed the various steps of the downstream processing chain, which involved harvesting, cell disruption, lipid extraction and fractionation and hydro-treatment of the lipids to create biofuel. They were shown to be successfully applicable to microalgae, have been demonstrated at industrial scale and are now ready to be used in commercial processes, which are not only limited to the production of biofuels.

Integration of production and conversion processes
A sustainability assessment, integrating environmental, economic and social aspects of the whole value chain, was carried out to determine the actual state of the technology as well as how key parameters influence the sustainability of the FUEL4ME integrated process. The assessment of the FUEL4ME integrated process includes the comparison to a reference system with the substituted conventional products. The conventional product for HVO (hydrotreated vegetable oil) is diesel, PUFAs would substitute high value lipids from fish oil, fertilizer would substitute synthetic mineral fertilizer; and for hydrogen, the conventional product is hydrogen from natural gas. The reference system provides the same services and products as the FUEL4ME integrated process with conventional products. Due to the state of technology and availability of data a modelling of a possible future full scale FUEL4ME integrated process with TRL 9 was developed to describe a possible future commercial HVO production from microalgae by giving guiding values and sustainability indicators for some key technological, economic and environmental data. The main influences upon sustainability were cultivation and harvesting, electricity demand, sources of freshwater and carbon dioxide, and suitable land. The research has addressed some of these parameters by improving productivity of cultivation, and harvesting efficiency, including water and resource re-use in cheap desert land. Further improvements, however, need to be made to make the process of biofuel production with microalgae fully economic and environmentally sustainable. The FUEL4ME integrated process could be economic viable and environmental sustainable because of the current TRL level of the FUEL4ME integrated process, and the future possible technology improvements in a long-term perspective. FUEL4ME believes that the opportunities and long term innovation strategy, with a stronger focus on high value products, will result in economically feasible and environmentally sustainable microalgae-based products.



Project Results:
3 Description of main science and technology results
WP 1 - Fundamental research and enabling technologies: genes, metabolism, biochemical aspects, bioprocess

Overall objectives of WP 1
1. Understand lipid metabolism in two microalgae species (Phaeodactylum tricornutum and Nannochloropsis oceanica). NB. After previous EU-review, Acutodesmus obliquus was added.
2. Develop proof of principle concept for a one-step lipid production process at lab scale

Key findings and conclusions:
1.1 Mapomics, a new tool for integrative transcriptomics, proteomics and metabolomics analysis was developed (figure 2).

1.2 P. tricornutum, cultivated in continuous mode in either nitrogen replete or limited condition, exhibits differential expression of genes, proteins and metabolites that relate to key biological pathways, such as nitrogen and amino acid metabolism, fatty acid metabolism, membrane and neutral lipid metabolism and pigment biosynthesis.

1.3 Three P. tricornutum lipid-droplet-associated proteins (identified by proteomics analysis of isolated lipid droplets) were overexpressed in P. tricornutum, fused to Green Fluorescent Protein, using a starvation induced promoter. All three fusion proteins were found associated to lipid droplets after nitrogen starvation. Preliminary results suggest that overexpression of LD7 and LDA may result in increased fatty acid production, while LDE might actually inhibit fatty acid accumulation under nitrogen starvation. Finally, lipid droplet formation, fusion and movement seem to be controlled by the microtubule system, as indicated by inhibitor studies. (Figure 3)

1.4 Overexpression of a fusion protein (of Green Fluorescent Protein and an oil globule protein from Haematococcus pluvialis) increased TAG accumulation in P. tricornutum by over 50%.

1.5 In contrast to what is generally acknowledged, synchronized cell growth does not require major transitory energy storage molecules in A. obliquus, but the presence of such molecules does improve photosynthetic efficiency (figure 4).

2.1 Technical feasibility shown for continuous (one-step) lipid production with P. tricornutum, A. obliquus and N. oceanica.

2.2 Optimal batch and continuous mode cultivation conditions were determined for lipid production with P. tricornutum, A. obliquus and N. oceanica, to ensure fair comparison of both cultivation modes.

2.3 It depends on the species tested whether batch starvation or continuous cultivation under nitrogen limitation has highest TAG production at lab scale. For A. obliquus lower TAG content and TAG yield on light were observed for the continuous mode (table 1), whereas for N. oceanica higher TAG content and TAG yield on light were observed for the continuous mode. For P. tricornutum, the TAG yield on light was highest for continuous mode, while the TAG content was highest in batch mode.


2.4 Regardless of the species used, the differences between both cultivation modes are in such a range that other factors, such as operational costs and compatibility with the downstream process, will likely become decisive in adopting either batch or continuous mode.


WP 2 - Translation to outdoors and production

Overall objectives of WP 2
• The overall objective of WP2 is to translate the developed (WP1) one-step process to outdoors, thereby achieving a robust and reliable production process, continuous year round lipid production under different climates and weather conditions, and oil production of constant quality. WP2 is closely linked to WP3 for the production and delivery of the biomass for downstream processing activity and to WP5 for providing data for LCA.

Key findings and conclusions:
1) Three pilot plants and a demonstration plant were designed, build and operated. The pilot plants were built in Italy, Israel and The Netherlands, whereas the demonstration plant was built in Spain (figure 6).

2) Research activity mainly focused on N. oceanica as it resulted the most promising strain in terms of TFA productivity and content with respect to P. tricornutum. P. tricornutum also presented recurring contamination and culture crashes that made difficult to cultivate it outdoors.

3) Outdoors, N. oceanica TFA productivity (g m-2 ground d-1) under a semi continuous N-limitation regime showed to be as productive as N-starvation and in some cases even better.

4) TFA productivity was inversely related with the amount of nitrogen supplied to the cultures. A clear effect of the available light on TFA productivity was observed, resulting the highest in Israel (IL) and the lowest in The Netherlands (NL). Having performed outdoor tests at different sites (NL, IT and IL) it was possible to evaluate which location better performed in terms of TFA productivities. By comparing TFA productivities, both per m2 of occupied ground and m2 of panel surface, obtained in the three pilots under comparable nitrogen supply regime (starvation and limitation) and period of the year, it is clear as solar radiation available played a fundamental role (figure 7, figure 8, table 2). The highest TFA content with N. oceanica (% d.w.) has been obtained under N-starvation, when N-limitation was applied TFA content was generally lower.

5) Continuous N-limitation was found to be much more stable and easy to maintain with respect to starvation in The Netherlands (WFBR, figure 9). This finding is in contrast to what obtained by F&M (Italy) when daily dilution (semi-continuous) was applied, in fact TFA productivity of daily diluted culture under N-limitation was not stable and decreased with time. A higher stability could be obtained by reducing nitrogen limitation at the expenses of TFA content and productivity.

6) Inputs (energy and materials) and outputs (biomass and TFA content and productivity) were collected in the pilots, and at F&M and WFBR the data were used to elaborate an energy and mass balance for a 1-ha and 100-ha scale plants. These data were then used by WP5 partner for the LCA analysis. Mass and energy balances results were reported in details in the following deliverables: D2.13; D2.15 and D2.17. In order to standardize as much as possible the energy inputs data for the LCA, a methodology for the extrapolation of the data, from the pilot plant scale to the 1-ha and 100-ha plant, was elaborated. This mainly focused to the identification of reference efficiency values, characteristic for each type of pumps and blowers. The activity was performed by all WP2 partners, particularly with the contribution from BIT/NIS and the outcome is reported in D2.7.

7) Biomass for downstream processing was produced. A first batch (48 kg) of frozen paste of N. oceanica was produced from 11.03.2016 to 10.04.2016 in 20 ProviAPT reactors (D2.14). During the FUEL4ME General Assembly held in Olhão in April 2016, the problem of the lack of large amount of lipid-enriched Nannochloropsis biomass for the downstream process was addressed. During the meeting, the quotations received for the production of the biomass outside the consortium were evaluated and considered too expensive to be afforded. Moreover the availability of good lipid-enriched biomass was not certain. As a possible solution, the partner F&M, proposed the use of the biomass produced during the EU FP7 project BIOFAT, in which F&M participated. In fact, despite the use of different strains, the two projects investigated the same algal species: Nannochloropsis oceanica sp.2 in the FUEL4ME project and Nannochloropsis oceanica F&M-M24 in the BIOFAT project. Thus, the lipid enriched Nannochloropsis oceanica F&M-M24 biomass produced in Camporosso Pilot Plant for the purposes of BIOFAT (collection of data on Pilot Plant performances) was harvested and stored in a frozen form and it was made available for FUEL4ME project.



WP 3 – Downstream processing and conversion of oils to biofuels
Overall objectives of WP 3
• The development and integration of an innovative and continuous downstream process for conversion of microalgae into biofuels with consistent volume and quality
Key findings and conclusions:
1) Harvesting of Nannochloropsis and Phaeodactylum was successfully demonstrated at F&M in Italy with the Evodos type 10. For both microalga the maximum separation efficiency was 95%, and the time required for reaching this separation efficiency was 45 min and 80 min for Phaeodactylum and Nannochloropsis, respectively. The harvested biomass paste contained 26% solids for Nannochloropsis and 22% for Phaeodactylum. The prototype centrifuge worked at a maximum input flow of 537 l.h-1 for Nannochloropsis and 580 l.h-1 for Phaeodactylum.

2) Improved Evodos 25 was developed with automated discharge system. Harvesting of Nannochloropsis was successfully demonstrated at NIS facility in Huelva-Spain (figure 10). Separation efficiencies above 92% in combination with capacity up to 1200 l.h-1 was achieved and an algae paste with concentration above 20% with less than 1% loss was obtained (figure 11).

3) A membrane filtration unit has been developed for the demo plant at Biotopic in Huelva-Spain for capacities up to 600 litres/ hour. The membrane system was used for re-cycling of effluent water. During the test with effluent water an average flow 600 litre/hour with effluent water from a Nannochloropsis harvesting process with Evodos algae harvester was obtained and no fouling observed. By using the re-cycling system a yield of 97.6% was achieved.

4) A continuous high throughput cell cracking system with capacity of 50-60L/min of algae biomass was built and the system was able to facilitate mechanical cell disruption before further downstream processing and the experience of using SoniqueFlo for microalgae provided data for sustainability assessment that identified options for further up-scaling, implementation and commercialization of the SoniqueFlo technology for cell disruption (figure 12).


5) FeyeCon developed analysis method for characterization of oils, fatty acids and type of lipids (phospholipids, glycolipids and triacylglycerol). The results showed the strain and method of cultivation has significant effect on types of lipids and fatty acid profile. The lipids were extracted by using supercritical carbon dioxide technology and yield up to 46% was achieved from TAG-rich biomass. The effect of starvation on extraction yield was studied and it showed that the yield of fatty acids extraction in starved cells was 25% lower which might be due to stronger cell wall.

6) A process for extraction of lipids by using ethanol as solvent was developed and optimized and the extract was successfully trans-esterified. The ethyl esters were fractionated by using supercritical carbon dioxide fractionation column and a separation up to 78% was achieved.

7) Several batches of biomass were extracted by using supercritical CO2. The effect of pressure, temperature, solvent to feed ratio, density and time was studied and extraction yield above 80% was achieved. For the TAG-rich Nannochloropsis more than 95% of the TAG was extracted by using only CO2 as solvent. The extracted oil has high free fatty acid content which is due to hydrolysis of lipids during storage and shipment. Free fatty acid content of oil was reduced to 1% by using CO2 refining column.

8) The fatty acid analysis of biomass and extracted oil showed the LC-PUFAs (EPA) remains mainly in biomass due to polarity of the molecule that EPA is attached to. The EPA content of extracted oil is below 8% which is too low to be separated by using fractionation technologies.

9) The esterified fatty acids including EPA were fractionated successfully by using supercritical carbon dioxide as solvent (figure 13). The fractionation process parameters including feeding rate, pressure, temperature, reflux ration and solvent to feed ratio were optimized and a fraction of LC-PUFA rich raffinate with concentration of above 70% and a low value fraction of short chain fatty acids in ester form was obtained. The process were performed in continuous mode and the robustness was tested in a 60 hours non-stop running. The process showed good stability during the process.

10) Neste used three algae oil samples including 2.7 L of extracted oil from TAG-rich Nannochloropsis. The analysis results showed all samples were suitable for NEXBTL process in terms of impurities. However, chlorine level in sample 3 was high. Oil sample 3 was pre-treated in the laboratory according to general procedure available at commercial NEXBTL plants. And pre-treated algae oil was tested in microreactor-scale at Neste to produce NEXBTL products. The microreactor testing simulated the process conditions in existing commercial NEXBTL-plants. Testing was done with blend of 20 % algae oil with 80 % of regular NEXBTL feedstock. Blending ratio was determined by chlorine content of the sample. The results showed measured properties of samples containing algae oil are similar with sample of typical NEXBTL feed.

11) De-oiled biomass was analysed. Components of value in the biomass residue were the carbohydrates (26%) and proteins (30%). The protein fraction of the biomass residue might have application as feed. Carbohydrates was used for the production of hydrogen (H2) which is subsequently applied in hydrotreatment of the algal lipids for the production of biofuels. For fermentative H2 production the biomass residue was enzymatically hydrolysed (by cellulase activity) thereby converting polysaccharides (mainly glucan) to soluble sugars. The fermentability of the hydrolysate with a sugar concentration of 18 g/l (a mixture of glucose, galactose, oligomeric galactose and mannitol) was tested using two strains of extreme thermophilic hydrogen producing bacteria. Next the H2 yield and productivity were determined using the same thermophiles in fermentations under controlled conditions in a bioreactor. The H2 potential of the algal biomass residue was 3.7 g of H2 per kg of dry matter. This is circa 50% of the maximal feasible amount. Further optimization of carbohydrate conversion efficiency and H2 fermentation performance is necessary.

12) In conclusion it is shown all the process steps to work: harvesting, re-use of water, cell disruption, extraction and further fractionation of lipids, conversion of low-value lipids into biofuel, quality of high-value lipid fraction after fractionation, fermentability of remaining biomass into hydrogen.



WP 4 – Demonstration at pilot scale
Overall objectives of WP 4
• Demonstrate the capability of the optimised process (cultivation and downstream processing) at pilot scale under representative industrial conditions, based on the technology and methodologies developed in the previous WPs.

Key findings and conclusions
1) The integration of all steps from biomass production in continuous mode to harvesting, re-use of effluent water, cell disruption, extraction of triglycerides, conversion of triglycerides to biofuel, fractionation of high value fatty acids from low value fatty acids and conversion of carbohydrates in biomass to H2 by fermentation were performed based on optimized conditions in WP2 and WP3.

2) Each process step has been performed with industrial-like equipment.

3) Continuous cultivation: 20 reactor units were used for cultivation of TAG-rich microalgae in continuous mode and total harvest from the system during the production period was 40.2 kg DW with an average biomass concentration of 2.3 g/L. Daily average dilution rate was 5 %.

4) Harvesting: Nannochloropsis was harvested by modified Evodos 25 with an efficiency of >90%, capacity > 6 m3/day, dry weight >20% and with minimized paste losses <1% of the total volume.

5) Re-use of effluent water: Average flow 600 litre/hour, effluent water from a Nannochloropsis harvesting process with Evodos algae harvester, No fouling observed and a yield of 97,6%, was obtained.

6) Cell cracking and extraction of TAGs in demonstration scale: by proper pre-treatment 40kg of lipid-rich Nannochloropsis (produced by EU project BIOFAT) was extracted by using CO2 as solvent and more than 90% of TAG was extracted. The free fatty acid content of extract was reduce to below 1% by using CO2 column and co-extracted water and solid particles were separated from the extracted oil.

7) Fractionation of fatty acids: LC-PUFAs were fractionated by using CO2 as solvent and purity of 72% was achieved. The process was performed in batch and continuous mode and a 60 hours run was carried out in order to evaluate the robustness.

8) Conversion of low value TAGs to fuel: the extract was further purified and tested in micro-reactors to simulate the NEXBTL process. The results showed the algal oil can be used for biofuel production.

9) Conversion of biomass to hydrogen (H2) for the hydro-treatment step: the sugars present in biomass after extraction of oils was converted to H2. A production of 7.6 g of H2 per kg should be feasible by an optimized carbohydrate conversion of 90% and a H2 yield of 75%.

WP 5 – Sustainability assessment of integrated process
Overall objectives of WP 5
• Assess the sustainability of a continuous production and conversion process based on the work performed in WPs 1-4, including environmental, economic and social assessment based on the whole value chain in a life cycle perspective. The results were used to guide the technical development and future implementation in the desired direction and to be able to determine economic feasibility and environmental sustainability.

Key findings and conclusions
The key findings and conclusions are on sustainability assessment of FUEL4ME integrated system, modelling of a full scale commercial plant; and technology assessment. The results are described below.

Sustainability assessment of FUEL4ME integrated process
The sustainability assessment integrates environmental, economic and social aspects of the whole value chain of the FUEL4ME integrated process and is assessed in comparison to the reference system (Figure 13). Within the life cycle sustainability assessment the methodologies Cost assessment, Life Cycle Analyses (LCA) and social Life Cycle Analysis (s-LCA) were used and specifically adapted to the considered algae concepts. A list of indicators was identified: 14 environmental indicators, 14 economic indicators and 11 social indicators. Due to the relevance of the indicators especially for the FUEL4ME integrated process and the availability of data for their assessment the most applicable indicators for the assessment were identified, e.g.:
• Economic: e.g. production costs, annual costs, value added, employment, market aspects;
• Environmental: e.g. global warming potential, primary energy demand;
• Social: e.g. employment, supplier relationships.
Social indicators were described in a qualitative way.
The sustainability assessment of the FUEL4ME integrated process includes the comparison to a reference system which supplies the substituted conventional products. The reference system provides the same services and products as the FUEL4ME integrated process with conventional products. In Figure 14 the whole value chain of the FUEL4ME integrated process in comparison with the reference system with conventional products is shown. The conventional product for HVO (hydrotreated vegetable oil) is diesel, PUFAs would substitute high value lipids from fish oil, fertilizer would substitute synthetic mineral fertilizer; and for hydrogen the conventional product is hydrogen from natural gas. All production steps of the value chain are included in the reference system as well as for the FUEL4ME integrated process, e.g. for transportation services the following process steps are included: extraction of crude oil, transport to a refinery, processing in a refinery, distribution and the use in passenger cars.

Modelling of a full scale commercial and integrated microalgal-based process
Due to the state of technology and availability of data a model of a full scale FUEL4ME integrated process with TRL 9 (Technology Readiness Level) was developed to describe a possible future commercial HVO production from microalgae. The modelling was done using the methodologies LCA, cost assessment, assessment of macro-economic aspects and s-LCA. Three cases with a production capacity of 100 kt/a HVO and coproducing up to 5 kt/a PUFA were modelled. For the modelling targets, characteristics, data and assumptions were defined among project partners (Figure 15). The three main targets are:
1) Revenue ≥ costs,
2) Reduction of GHG emissions ≥ 60 % for HVO biofuel instead of diesel based on LCA methodology according to Renewable Energy Directive (RED)
3) Cumulated fossil primary energy demand in relation to HVO energy content ≤ 30 % (≤ 0.3 MJfossil/MJbiofuel).
These targets give the possible framework conditions for a future full scale commercial plant. The results of the modelling of a full scale FUEL4ME integrated process are guiding values and sustainability indicators formulated as ranges for the most relevant technical, environmental and economic characteristics. These sustainability indicators show how the three targets are fulfilled simultaneously.

In Figure 16 the GHG emissions for a full scale integrated microalgal-based process (TRL 9) are shown on the left side. This figure provides information on the biochemical and chemical contribution to global warming by GHG emissions (process emissions). Biophysical impacts, displayed as equivalent GHG emissions are assessed within FUEL4ME as well. Biophysical impacts are due to the land use change and therefore the change in albedo, which is a coefficient measuring the reflection of a surface. Darkening the surface (lowering the albedo) causes more solar energy to be absorbed and so contributes to the global warming. The process emissions (biochemical GHG emissions) for the three cases were 18 – 25 CO2-eq/MJHVO. The three cases required an area for algae cultivation of 4,000 - 15,000 ha to produce the 100,000 tHVO per year. The equivalent GHG emissions due to albedo change are 5 – 19 g CO2-eq/MJHVO, then the combined effect on global warming in equal emissions are 23 – 44 g CO2-eq/MJHVO which are still less than emissions of diesel (84 g CO2-eq/MJ).
Figure 16 shows on the right side the results of the macro-economic assessment on employment. Expressed in absolute numbers the new algae-based technology leads to approx. 2,000 jobs at expenses of € 127 mio., whereas the reference system using conventional technologies – even if they can produce products cheaper – employment is approx. 1,500 to 2,000 jobs, which remains valid even at high price discrepancies between HVO and conventional fuels.
In FUEL4ME a methodology to assess the social aspects was developed to show at least in which categories and subcategories of the social dimension a company must be careful. Some of the most relevant social “hot spots” concerning the installation of a large algae cultivation system are identified in the category “society”. These “hot spots” include the engagement with local citizens, local employment and transparency to foster the acceptance of the new technology. In Table 3 the indicators in the category “Regional corporate citizenship” relevant for regional cooperations are listed. For each indicator four possible situations are described; green – this situation should be reached, red – this situation should not be reached at all.

Technology assessment
After this modelling of a commercial plant (TRL 9) the actual demonstrated state of technology (actual TRL) of the FUEL4ME integrated process was assessed with following characteristics:
• Location of the cultivation: South Europe;
• Microalgae species: Nannochloropsis;
• Capacity of biofuel production: 10,000 t/a, 100,000 t/a;
• Size of one algae cultivation site: 1 ha, 100 ha;
• Hydrogen (H2) surplus: “yes” or “no”;
• No protein separation; and
• Fertilizer production.
The assessment of the developed concepts shows that due to the actual state of technology the GHG emissions (biochemical only) and the primary energy demand of the FUEL4ME integrated process (current TRL) are higher as the values for the conventional reference system (TRL 9). The main influencing factors of the GHG emissions are the electricity demand especially needed for cultivation and harvesting, the yearly substitution of plastic films in PBRs, and the energy needed for drying the biomass. Furthermore the actual demonstrated state of technology is economically not feasible. The costs for personnel and for auxiliary energy contribute to a high share of the annual operational costs.
Due to actual demonstrated state of technology (current TRL) further technology development is needed. A comparison using the guiding values of the modelled full scale commercial and integrated FUEL4ME process was made to analyse the gap between the current TRL and the guiding values for TRL 9. An assessment of the actual state of technology, based on the developed concepts, in comparison to TRL 9 is provided. In Figure 17 the two important parameters for a future full scale commercial microalgal-based process are shown: algal biomass yield [tDM/(ha*a)] and electricity demand [kWh/tDM].

In comparison to the FUEL4ME integrated process cultivation parameters are listed for two other FP7 projects (BIOFAT and InTeSusAl) (Table 4). The data for the two other FP7 projects is based on information from project members of BIOFAT (BIOfuels From Algae Technologies) and InTeSusAl (Demonstration of integrated and sustainable microalgae cultivation with biodiesel validation). The parameters include extrapolated biomass production per cultivation ha, depending on the biomass productivity, operational days and irradiation per location of the cultivation.
Cellana, a company located in Hawaii, United States, intends to construct and operates commercial facilities to produce Omega-3 EPA and DHA nutritional oils, food/animal feed, and biofuels. Therefore they are cultivation marine microalgae in open ponds.
At the company website (Cellana, 2017) following basic data for microalgae can be found. But it is not stated if these data are general information or based and calculated on their own shown biomass productivities:
• Biomass productivity: 50 – 150 t/(ha*a);
• Oil content: 20 – 50% of dry mass;
• Biofuel productivity: 10 – 75 t/(ha*a).
A comparison with FUEL4ME data is difficult, due to the lack of additional data, e.g. biomass productivity per g/(m²*d), operational days, detailed information on the assumed cultivation area – with our without surroundings. Data on costs were not found. Furthermore the production system (open ponds vs. flat panels photobioreactors) and the location (Hawaii vs. Southern Europe) are completely different.
Comparison with other microalgae projects or processes is not very well possible as data is not simply available in the same units and with detailed background information. Furthermore production systems and products are not similar to the FUEL4ME process and calculation methods are not explicitly mentioned to get more detailed information.
FUEL4ME co-organised with the European Algae Biomass Association (EABA) and with the EU projects Miracles, Splash and the Algae Cluster (InteSusAl, BIOFAT and All-gas) the Conference “European Roadmap for an Algae-Based Industry” in April 2016 in Olhão, Portugal. As a result of the conference a Whitepaper (FUEL4ME partners are co-authors) will be published. In this Whitepaper essential needs of the sector will be identified and the direction of the European algae strategic research agenda for the upcoming years will be pointed out. Research areas that need to be addressed in the future are included in the fields of markets, production technology, industrial strains and biorefinery. This information was furthermore used as the basis for the roundtable discussion during the workshop "Algae Research Agenda for 2020 and 2030" held in Madrid 13-15 December 2016 (AlgaEurope 2016 Conference).
In future the FUEL4ME integrated process could become economic viable and environmental sustainable with future possible technology improvements in a long-term perspective. The possible future technology improvements in a large scale operation are expected in following aspects:
• Algae strain improvement:
o Higher yields
o More efficient land use and
o Reduction of electricity demand
• Innovation in cultivation technologies:
o Lower personnel demand due to possible automatization (it seems to be possible to get to 100 – 200 persons per 1,000 ha)
o Lower electricity demand of PBRs– a possible reduction to 10% of the current TRL could be reachable, more focus should be on reduction of electricity demand, decrease of electricity demand could be possible by optimization, higher productivities and bigger scale of cultivation
o Larger scale installations with continuous production
o Reduction of the high impacts from embodied energy in the cultivation system
The FUEL4ME partners will further work to realize the opportunities and long term innovation strategy with a stronger focus on high value products, to develop economic feasible and environmentally sustainable microalgae-based products in a long term perspective.
Potential Impact:
Dissemination of the foreground
Dissemination strategy
The main objectives of communication and dissemination activities have been:
• To enable an effective and fluent communication within the project and between project partners and other actors relevant to FUEL4ME purposes
• To ensure the widest dissemination of project results possible, so they reach their highest potential impact on society
• To raise awareness among stakeholders, general public, the scientific community, industry and public authorities
• To pave the way for the future commercialization and deployment of the technologies and processes being developed in FUEL4ME.

The dissemination and communication strategy intended to establish the proper channels, materials and activities to meet these objectives by:
• Setting up a Dissemination and Communication infrastructure: website, social networks, intranet
• Defining a graphical identity for FUEL4ME project
• Producing effective communication materials: project roll-up, brochure
• Keeping stakeholders informed about project activities and results: e-bulletin, publications on website and social networks.
• Organising and participating in activities to provide the project with opportunities and channels to share their results and interact with different audiences: participation in international conferences, organization of visits to the cultivation plants during the last year of the project
• Identifying relevant stakeholders and their interests in the processes and technologies being optimized in the project, and so better customize the dissemination activities and gather information for exploitation purposes.

More detailed information on the communication and dissemination strategy, channels, audiences and messages can be found on FUEL4ME deliverable D6.1Communication and dissemination plan.
Table 5 summarizes the main target groups per communication channel.

Dissemination materials and activities
Website
FUEL4ME website was designed considering the inputs of all partners regarding the sections to be included, the layout and the functionalities. It has received over 16000visits from all over the world, as shown in figure 17. The number of visits considered in the actual report are the visits that have stayed in the website longer than one minute.

The website presents a complete description of project concept, activities, objectives and consortium partners, as well as collection of FAQs and it’s updated with news on the progress of the project.
It also serves as a platform to keep stakeholders and FUEL4ME followers informed about the progress of the project through the regular publication of articles, posts and updates on the activities of the different WPs. Besides, dissemination materials and e-bulletins can be downloaded directly from the website. A calendar shows the most important upcoming events of the project.
The website contains a link to an intranet available for FUEL4ME partners, where all project documents from different WPs are shared and managed. The website is also linked to the Facebook and twitter profile of the project. It can be visited at www.fuel4me.eu.
After the end of the project the website will be kept and maintained by IDC for an additional 1 years (31st December 2017) to ensure visibility of the project results after the end of the project.

Brochure and roll-up
FUEL4ME brochure is a 3-folded leaflet containing an overall view on FUEL4ME project partners, activities and goals. 2000 copies have been distributed among the partners, so that they can provide their own contacts with some information about the project.
FUEL4ME roll-up has been designed to be taken to be taken to the congresses and conferences where consortium partners participate, as well as to be displayed in all the events where FUEL4ME is present. One roll-up has been provided to each partner. They are 2 metre high x 1 metre wide, made in polyester supported by an Aluminium structure.
In addition to the aforementioned brochure and roll up, a final brochure will be distributed at the end of the project with the goal of disseminating project results to stakeholders, the scientific community, the European commission and other private and public entities (figure 19). Future steps and recommendations to advance in the development of sustainable, scalable process for biofuels from microalgae and to valorise the by-products will be presented in the form of a several page booklet.
Stakeholders’ list
With the purpose of ensuring an effective flow of communication within the project, an internal contact lists with all project partners has been set.
FUEL4ME external contact list gathers more than 400 hundred stakeholders from academia, industry, organizations and public entities from sectors ranging from different sectors related to the project activities. All partners have contributed to create this list, providing the contacts from their local and international stakeholders.
FUEL4ME has contacted two related EU-funded projects, SPLASH and MIRACLES, both coordinated by Wageningen UR and has worked together with them to create common algae stakeholders’ list with over 1000 contacts. These contacts can be filtered according to:
• Project:
o FUEL4ME
o MIRACLES
o SPLASH
• Type of entity:
o Algae feedstock suppliers
o Industry
▪ Human food
▪ Feed
▪ Fuels
▪ Pharmaceutical
▪ Biotechnological
▪ Fertilizers
▪ Bioplastics
o University and R&D
o Public administration
▪ EU
▪ National
▪ Local
o NGOs and associations
o Press
o Consulting
o EU related projects

E-bulletin
FUEL4ME e-bulletin is distributed among FUEL4ME partners, stakeholders and external contacts every six months, informing them about the progress of each WP, the most recent project activities and the upcoming events where the project is participating.
All project Newsletters can be found on the FUEL4ME website. They have been sent to over 400 relevant external entities (public authorities, industrial stakeholders, universities and research centres, NGOs, etc.)

Social networks
FUEL4ME has opened a profile in three social networks: Twitter, Facebook and LinkedIn. The kind of information published is customized to the functionalities and audiences of these different networks.
Twitter and Facebook are used to publish short posts on FUEL4ME latest activities, conferences where the project is presented, links to interviews to project partners, related EU projects, videos on our microalgae production facilities and relevant news and findings in the fields of microalgae biomass production and/or conversion to biofuels, photobioreactors and new innovative cultivation sites.
LinkedIn group is meant to be a forum to invite partners and stakeholders to foster discussion on relevant topics, such as the prospective for the use of biofuels in different fields or its share in the energy mix, as well as to inform them about interesting project results.
Twitter has proven to be a very good channel to be in contact with the main actors in the field of renewable energies, algae biomass production and biofuels. So far, FUEL4ME follows 135 entities, has 145 relevant followers belonging to the mentioned fields. Publications are published on weekly basis.
After the end of the project these profiles will be kept public but with minimal maintenance until they are no longer relevant.

International Course on Microalgae Process Design
The Bioprocessing Engineering group from Wageningen University, in cooperation with the Graduate School VLAG and BioSolar Cells has organized in July 2013 and 2014 an International course on Microalgae Process Design. From cells to photobioreactors, which was also available for summer 2015.

The aim of the course is to provide the essential skills for designing optimal microalgae-based production processes, for both research and commercial purposes.

The course is targeted at PhD candidates, postgraduate and postdoctoral researchers, as well as professionals, that would like to acquire a thorough understanding of microalgal metabolism and photobioreactor design. An MSc level in bioprocess technology, or alike, is recommended.
Course motivation and background
The waters of the world, oceans, seas, rivers, creeks, lakes and even ice, house a tremendous variety of microorganisms able to use light as the only source of energy to fuel metabolism. These unicellular organisms, microalgae and cyanobacteria, have the potential to produce a variety of products.
Among them are therapeutic proteins, polyunsaturated fatty acids and pigments. In addition to these high-value products, microalgae are regarded as one of the most promising resources for the production of bulk products such as food and feed lipids, food and feed proteins, base chemicals and energy sources for the industry, and possibly even biofuels. To make economical large-scale production of such bulk products possible, optimal design of bioreactor and cultivation strategy are essential. This process design has specific demand because sunlight, or artificial light, is used. The figure above shows small scale, fully controlled, photobioreactors containing green microalgae that are cultivated either under optimal conditions (left) or under conditions that stimulate accumulation of an orange pigment (right).
Course contents
Through lectures, digital cases and a photobioreactor practical, the participants will learn: 1) how to describe microalgal metabolism quantitatively, 2) how to apply basic design principles and set up mass/energy balances for photobioreactors, 3) how to cultivate microalgae in fully controlled photobioreactors, and 4) how to integrate all acquired knowledge into optimal production strategies for microalgae biomass or secondary metabolites. The course is composed by lectures, digital classes and practical work and includes a visit to AlgaePARC (www.AlgaePARC.com).

The programme topics are the following ones:
• Fundamentals of photoautotrophic growth and light
• Quantifying light-limited microalgae growth
• Predicting productivity of photobioreactors and raceway ponds
• Microalgal metabolism and secondary product formation
• Metabolic (flux) modelling
• Metabolic engineering strategies
• Mass transfer in photobioreactors
• Photobioreactor cultivation strategies for optimal yield
• Going large scale: experiences and challenges

The last edition the course was coordinated by Dr. Packo Lamers (Bioprocess Engineering WUR, and FUEL4ME WP1 leader) and by Chantal Doeswijk, MSc - Graduate School VLAG.
Course lecturers are leading researchers in the fields of microalgae cultivation and give lectures in the course, such as:
• Dr Maria Barbosa,
• Prof Olaf Kruse, Bielefeld University, Germany
• Dr Luc Roef, Proviron, Belgium
• Prof Gabriel Acién-Fernandez, Almería University, Spain
• Dr Mark Michiels, Proviron, Belgium
• Prof René Wijffels, Bioprocess Engineering, Wageningen UR
• Dr Marcel Janssen, Bioprocess Engineering, Wageningen UR
• Dr Dirk Martens, Bioprocess Engineering, Wageningen UR
• Dr Arjen Rinzema, Bioprocess Engineering, Wageningen UR
• Dr Rouke Bosma, Bioprocess Engineering, Wageningen UR
• Dr Dorinde Kleinegris, Food & Biobased Research, Wageningen UR

Results, lessons learned and challenges for the future
As one may conclude from the previous section, no patents, trademarks, registered designs or other IP protection forms have been registered as a direct output of the research carried out, and therefore cannot be attributed directly to the FUEL4ME project. This is not unexpected as there is still a big gap between research and the commercial uptake of 2nd generation biofuels from microalgae that are competitive alternatives to fossil fuels, thus furtherresearch is required to bridge this gap.
As a result, the vast majority of the foreground generated during the project corresponds to general advancements in knowledge in the form of lessons learned and improved know-how leading to cost effective and energy efficient processes. And in the form of rigorous scientific research that can either be published or may serve as input to new R&D projects.
For the FUEL4ME industrial partners the project has served as an excellent opportunity to pilot test their technologies in order to obtain more reliable and scalable industrial solutions for microalgae cultivation and downstream processing.
In addition FUEL4ME has contributed to prepare highly skilled professionals with expertise in algal microbiology and microalgae cultivation and processing systems.
The following paragraphssummarize some of the most outstanding results, lessons learned and future challenges that can be extracted from the FUEL4ME project:
Result 1: Continuous lipid production vs. Batch production
Partners involved: DLO/WU/BGU/F&M
Conclusions: Batch and continuous mode are on average in same range of productivity (in lab and outdoor), while TAG contents were generally higher in batch mode. Varying results were obtained for robustness of either process (i.e. in some cases batch was more robust, while in other instances continuous mode was more robust). Costs could not be properly assessed at the tested scale. However, costs (of entire process including downstream processing) should in the end be decisive for ultimately adopting one of the two cultivation strategies.
Future Challenges: Further development and up scaling of both cultivation technologies is needed to improve, properly assess and finally compare the process economy. Key point for this analysis is to take into account the compatibility of the cultivation technology with the downstream processing chain.

Result 2: Evodos 25 harvesting and water recycling technology testing and optimization.
Partners involved: EVODOS
Conclusions: FUEL4ME project has served for the successful testing and optimization of the EVODOS 25 dynamic settler with Evodos proprietary spiral plate technology. The results of the project show that the integration of an improved splash screen in the fully automated harvester minimized paste losses. The overall system lead to an increase yield, top quality algae paste and high efficiencies (separation efficiency was higher than 90% with a harvesting capacity over 6m3/day for the selected microalgae strains). In addition a water recycling unit was successfully tested.
Future Challenges: More research must be carried out for optimizing the water recycling unit when using the recycled water for cultivation mainly in analyzing performance, yield and the need of adding nutrients. In addition future testing must be carried out to scale-up the process maintaining productivity and avoiding contamination.
Result 3: SoniqueFlo technology tested for Nannochloropsis cell disruption
Partners Involved: CELLULAC
Conclusions: The FUEL4ME project results indicates strongly that Cellulac´s proprietary SoniqueFlo technology has a role to play in improving the economics of commercial scale production using an enzyme based solvent free wet extraction process of algal oils. This process can lead to an estimated 25% decrease in operational costs, less capital expenditure and high energy efficiencies because the solution works directly in raw algae material while still in aqueous solution eliminating the need of de-watering steps and therefore improving energy balance and cost effectiveness. In addition, the solution tested has no organic solvents or toxic waste products and delivers high yields of target oils, sugars and proteins in a readily usable form.
Future Challenges: While the cell disruption technology is ready to be scaled up at commercial scales, the biggest bottleneck is to obtain large volumes and steady supplies of microalgae biomass to attract big industry players.

Result 4: Energy efficient and cost effective lipid extraction using supercritical fluid
Partners Involved: FEYECON
Conclusions: The FUEL4ME project has contributed to the extraction of TAG with very high efficiencies using supercritical fluid (>95%) obtaining a high value and stable biomass after extraction.In addition, very relevant data and cost calculations for scale-up were collected during the project. This acquired knowledge will prove to be useful for future development projects. Lessons learned regarding the effect of the physical properties of the dried biomass on extraction yield and the need for polishing of the extracted oils (drying, FFA removal, deodorization, bleaching, dewaxing) were gathered and shared for future R&D.
Future Challenges: A feasibility comparison must be carried out between dried microalgae and wet microalgae extraction to further understand the relative pros and cons of each method in future technology developments.
Result 5: Energy Efficient and cost effective high and low value fatty acid extraction using supercritical fluid
Partners Involved: FEYECON
Conclusions: As a result of FUEL4ME, novel reactive extraction and separation protocols based on advanced supercritical fluid technology (as opposed to chromatographic methods) have been developed. These protocols and technology application have lead to obtaining high purified PUFAs (>81%), a robust system with stable concentrations of PUFAs and a reduction in FFAs content of oil from 23% to 1% at 50C. In addition, lessons learned regarding the required pre treatments of oil before fractionation (de-waxing, solid removal and bleaching), the improved purification of PUFAs in esterified form at lower pressures (1110-150 Bar) and the possibility to separate FFA from TAG at low temperatures have been recorded and shared for future R&D.
Future Challenges: Scaling up with high productivity, cost and energy efficiency in order to meet market demands while ensuring production quality and minimizing product quality variability.
Result 6: Setting up and operation of a demonstration plant
Partners involved: BIT.
Conclusions: A demonstration plant of 1ha was set up and operated in Huelva (South region of Spain) using Proviron technology (ProviAPT). The results show promising potential for scalability and replication. FUEL4ME has lead to increased know-how on the installation, operation and maintenance requirements of ProviAPT technology that will prove to be important for future activities and product development for Proviron and BIT after the end of the project.
Future Challenges: Future research must focus not so much on scalability but on maintaining stable and high productivity and in preventing and control of contamination.

Sustainability assessment and inputs for strategic decision-making
JOAENNUM as part of the planned activities for WP5: Sustainability assessment of the integrated process carried out a detailed modelling of a full-scale integrated micro algal-based process with sustainability indicators of the whole value chain in comparison to a conventional reference system including the three dimension economic, environmental and social aspects.
A comparison of the modelled full-scale integrated micro algal-based process with the assessment of to actual state of technology reveals information on key critical parameters impacting the sustainability of the FUEL4ME integrated process.
The main factors that influence sustainability were identified to be cultivation and harvesting, electricity demand, source of CO2, source of water and suitable land.
Some of the conclusions extracted from this analysis clearly underline the need for further technology development to improve economic and environmental sustainability.
This analysis has provided valuable input to all project partners regarding the potential up-scaling of these processes, considering competition for price impacts on food, feed, costs of transportation, capital and operating costs for algal cultivation and conversion, expected final costs of products, and market prices for alternative products or means for supplying equivalent services.
This information will prove to be useful for the project partners to set the grounds for the generation of new business models and apply FUEL4ME project results for commercial exploitation in line with their business strategies. In addition to the sustainability assessment the partners have acquired input from external project stakeholder and the project industrial board and they have received advice from the exploitation team on best practices on IP commercialization strategies while ensuring proper protection of the intangible assets and the foreground generated during the project.


List of Websites:
Project public website
www.fuel4me.eu

Contact details
Project coordinator
Dr. Dorinde Kleinegris
Wageningen Food & Biobased Research
P.O. Box 17
6700 AA Wageningen
Tel: +31 317 480324
E-mail: dorinde.kleinegris@wur.nl
From 14 January 2017:
Dr. Lolke Sijtsma
Wageningen Food & Biobased Research
P.O. Box 17
6700 AA Wageningen
Tel: +31 317 480220
E-mail: lolke.sijtsma@wur.nl

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