ProFuture

ProFuture

The safety of novel protein sources such as microalgae is not fully deciphered. Therefore, one of ProFuture´s objectives is to assess potential risks such as acrylamide and polycyclic aromatic hydrocarbon (PAHs) in microalgae products. PAHs are toxic pollutants that can be bioaccumulated by aquatic organisms from contaminated water or can be formed during food processing at high temperature. Due to their toxicity, maximum contents for 4 target PAHs have been established in different foods within the EU (Commission Regulation (EC) No 1881/2006).

In the ProFuture project, four microalgae species (Chlorella vulgaris, Spirulina, Nannochloropsis oceanica, Tetraselmis chui) are cultivated in two locations under different conditions, and several drying technologies are applied to obtain the final single-cell ingredients.

Ensuring the safety of the consumer is primordial, therefore, we sought mandatory to accurately quantify the PAHs content of the microalgae products developed in ProFuture as well as the fulfilment of the legal limits for the four target PAHs following the Reg. /EC) nº1881/2006. 
Preliminary trials carried out with a protocol based on Quechers and Gas Chromatography Mass Spectrometry (GC-MS) showed that many interfering peaks in microalgae extracts made the correct quantitation of the target PAHs difficult. 

Therefore, a specific purification protocol was developed, including a two-step clean-up after Quechers extraction, allowing the elimination of the matrix effects. At this stage, the whole protocol to quantify the four regulated PAHs (i.e. Benz[a]anthracene, Chrysene, Benzo[b]fluoranthene and Benzo[a]pyrene) in the single-cell ingredients of the four microalgae species has been validated in-house (IRTA - Institute of Agrifood Research and Technology). The next step will be the assessment of the effects of the different drying technologies.

In the realm of the ProFuture project, in 2021, the Centre for Social Innovation (ZSI) organised and facilitated two multi-stakeholder workshops to co-create scenarios of desirable future microalgae scenarios. The results of this workshop have been analysed and recently published in the journal Frontiers in Aquaculture. The paper “Actor groups influencing and shaping sustainable microalgae value chains in Europe” describes three distinct yet interdependent groups of actors whose roles needs to be considered for supporting the evolution of sustainable microalgal based food value chains in Europe: (1) microalgae producers and processors, and researchers who work with microalgae and their development, (2) policy makers, and large industrial players, who influence the political and economic possibilities of the microalgae value chain and (3) consumers and civil society actors, who play a key role in order to root sustainable microalgae food products in the market. The paper shows how these groups interlink and draws conclusions about their roles for shaping the European microalgae value chain. The authors argue for the need of strengthened collaboration between these groups for rendering microalgae-based food products sustainable and relevant alternatives.

To communicate and disseminate the ProFuture work to diverse target audiences, the whole consortium cooperates and contributes. A good example is the communication towards consumers, showing that consumer research and communication efforts are closely related. 
Based on surveys and focus groups, the team from Ghent University found that consumers showed interest/ had questions about the taste of microalgae, nutritional benefits (in relation to sports and vegan diets) as well as food safety. Based on these outcomes, the communication team from the European Food Information Council (EUFIC), developed an infographic on the potential of microalgae on a sustainable and healthy diet, together with social media images: https://www.pro-future.eu/news/new-project-infographic-whats-the-potential-of-microalgae-on-a-sustainable 
Other examples for communication towards consumers include a lay article, translated into Spanish, Italian, French and German, on producing new food products that consumers accept: https://www.eufic.org/en/food-production/article/microalgae-producing-new-food-products-that-consumers-accept 
The article explains what microalgae are, showcases some of the products developed within the project and explains the different factors that influence how people perceive microalgae in food. The article was integrated in a campaign on alternative proteins, e.g. via interactive posts to find out what the general public thinks about microalgae.

The conventional method for drying liquid ingredients that are temperature-sensitive in the food industry is generally spray drying. This technique is often used for the drying of milk, coffee and spices and can also be used for microalgae. Spray drying is characterized by a high evaporation capacity and a low residence time, through vaporization of the product in a hot air vortex. On the downside, spray drying is an energy-intensive method that could increase the production cost of microalgae while also increasing its otherwise low carbon footprint. At ILVO, ProFuture’ researchers are trying to find alternative sustainable methods for the drying of microalgae that ensure a high-quality end product. A promising method is agitated thin film drying (ATFD). In essence, the ATFD is a heated tube under vacuum with a rotor inside to move the material along. In ATFD, the energy consumption is reduced since evaporation is accelerated by a vacuum. This allows for the use of lower drying temperatures, which might also help preserve the nutritional and functional properties of microalgae cells. In fact, the effect on the nutritional and functional characteristics will be the main criteria used to identify the most suitable drying technique for microalgae single cell protein. Studies will be conducted to determine the effect of ATFD on important nutritional properties such as the amino acid profile, fatty acid profile, vitamins and antioxidants of the single cell protein, as well as on the functional properties (solubility, foaming, gelling, interaction with oil and water). These data combined will provide the necessary insights to identify the most suitable method for drying microalgae protein rich ingredients.

ProFuture works on improving each step of the microalgae value chain. This abstract gives an overview of the first step, called “innovations in microalgae culturing and harvest”. And as part of ProFuture, this workpackage has 5 tasks aimed at achieving the goals of the project, making the production of microalgae more sustainable and more effective. As a first task, ProFuture partners worked on the Characterization and improvement of algae production strains for demonstration. Here, GreenColab (GCL) developed 6 colorless Chlorella vulgaris mutants (3 pale green, 2 yellow and 1 white). The second task focused on CO2 absorption and concentration from the air (Direct Air Capture/ DAC), was led by University of Twente (UT), who installed a pilot for direct CO2 capture at Allmicroalgae production site (Portugal). Allm has been operating their systems with CO2 directly provided from the air to produce microalgae without buying bottled CO2. In task number 3 partners worked on increasing production efficiency and cost reduction. In this task, Wageningen University (WU) scaled up an extremophile microalgae species (Galdieria sulphuraria) grown at 45 degrees and at pH 1-2; trials done at pilot scale (1300 litres). NORCE tested the use of insect production waste as culture medium for microalgae. Allmicroalgae scaled up the mutants grown on sugars (without sunlight), and the biomass was given to other profuture members for further trials. Task number four worked on energy reduction strategies, led by Necton, who is developing the following: Necton is testing different strategies to reduce costs: reactors running only on solar energy, expanding flatpanels reactors to compare production strategies, testing different nitrogen sources. Finally, in the task last task of this workpackage, ProFuture focused on the supply of biomass to other partners developing ingredients, a task led by Necton and Allmicroalgae.

Microalgae are considered the food/feed of the future for the high numbers of nutrients they contain: proteins, lipids, carbohydrates or vitamins. However, all of them are enclosed inside the cells, often protected by a tough cell wall. To be used in food and feed products, those nutrients must first be efficiently extracted from the microalgae cells. Several techniques exist to extract internal compounds from cells, some relying on chemicals with solvents or bases and others relying on mechanical disruption such as high-pressure homogenization or bead milling. Different factors must be considered when selecting a cell disruption technique such as its efficiency, its productivity, its operational costs, its scalability and its environmental impact. 
The bead milling is a well-known technique used industrially in ceramic, pharmaceutical, paint, paper and cosmetic industries and is now also used in biotechnology to destroy cells. The system consists of an agitated chamber filled with ceramic beads with a size usually between 0,3 to 2 mm. The collisions between beads break the cells that are between them and allow the nutrients to come out of the cells to the extract liquid. 
The disrupted cells are then separated from the liquid extract containing the nutrients by a solid/liquid separation technique. In ProFuture, proteins are the nutrient of interest. By selecting the optimal conditions, microalgal proteins could be recovered in the liquid phase after bead milling of the cells. Further purification steps could then increase the purity of the proteins and their functionality for food and feed applications.

A big-data analysis of the information available on algae on the Internet (Facebook, Twitter, Instagram, blogs, news, etc.) has been conducted. The analysis has been carried out not only on consumers' opinions, but to companies selling (producers, shops, restaurants, etc.) or using these products (spas, clinics, nutritionists), as they may influence consumer’s perception. Information has been obtained from the Internet using several tools available (awario.com, python libraries). State of the art machine learning techniques for natural language processing has been applied to process and classify the information obtained. BERT and Latent Dirichlet Analysis has been the algorithms selected for the analysis. Results suggests that the consumption of algae products is associated with a very strong positive sentiment. It is widely regarded as a sustainable vegan source of proteins, with lots of nutrients (vitamins, minerals, etc.) and properties (antioxidant, immunity boosting, etc.) and they are usually called “superfoods”. The consumption of these products is usually linked to a healthy lifestyle. However, some negative views can also be found (less than 5% of the posts analyzed). They are usually associated to side effects of consuming these products (iodine excess intake, health problems), false claims on product nutrition characteristics (not a source of omega3 or B12) or the bad taste and smell of these products. A lot of the information associated with positive sentiments is overly optimistic and are not supported by scientific evidence. Negative sentiment is closer to the current scientific evidence. The unrealistic optimism may constitute a long term risk for these products.

Carbon dioxide (CO2) is the main nutrient needed by microalgae to grow into a protein-rich biomass. CO2 can be obtained from the air around us, but only at very low concentrations (0.04%). During large scale production, microalgae need to be supplied with sufficient CO2, and preferably at higher concentrations, since they grow much faster when CO2 is more concentrated in the air.
To face this challenge, ProFuture’s researchers at the University of Twente are now developing a unique process to concentrate CO2 from ambient air in an efficient and sustainable way.
In this process, a large amount of air is blown through a system containing particles able to capture CO2 temporarily - also known as sorbent particle. After the capture, the system is heated at low pressure, causing the CO2 to be released from the particles. This causes our system to vent out highly concentrated CO2, which can be given directly to the algae. 
The ProFuture Direct Air Capture facility uses an efficient low pressure drop concept, meaning it consumes less energy than other similar technologies. The process also recycles the sorbent particles to maximize efficiency.
In order to make this process CO2 net negative – meaning that the process removes as much carbon as it emits - the energy consumption and the CO2 footprint (CO2 emission) must also be considered. ProFuture’s calculations showed that when ProFuture Direct Air Capture Facility uses renewable energy from the sun or wind, the CO2 emission is 0.1 kg CO2 per kg of CO2 captured from the air, thus resulting in a net removal of 0.9 kg CO2 per kg of CO2 captured. 
ProFuture will now test the cultivation of microalgae using CO2 from Direct Air Capture under the real-life conditions of a microalgae production facility. This system aims to be operational in the course of 2021.

In 2021, the Centre for Social Innovation (ZSI) organised and facilitated two multi-stakeholder workshops to co-create scenarios of desirable future microalgae scenarios. Using virtual whiteboard- and communication tools, the invited workshop participants active in the microalgae production, processing, retail, research, or policy, discussed the current state of microalgae production and identified existing bottlenecks and barriers for socially responsible and ecologically sustainable value chains. Having reached a common ground, participants started thinking beyond existing barriers and focused on desirable futures and solutions. The ZSI team led the participants when working on their future ideas, fleshing out the social, ecological, economic, and political factors and contexts. At the end of both workshops participants turned these factors into more concrete scenarios elaborating about the current state and its underlying reasons, future goals, related barriers and most importantly suggestions and measures to reach these ambitious goals. In total, workshop participants co-created four scenarios; Scenario I focussed on the recognition and acknowledgement of microalgae as part of a healthy diet, Scenario II focussed on the use of microalgae-based products in relation to other food products causing less environmental impact. Scenario III focussed on microalgae cultivation in a circular economy setting and Scenario IV focussed on microalgae being presented as common food to a wide public. The workshop results are currently under analysis and will be shared once analysis will have been concluded.

Sustainable food trends become step-by-step central for modern consumers. A growing number of people look today for products that can help them live in a more healthy and sustainable way.
Coop has decided to participate in the ProFuture project because they see a great potential in this type of ingredients. However, it is still too early to define how to integrate microalgae-based products into the Italian market. 
A challenge will be certainly represented by the cultural change of consumers.
One opportunity for Coop is to learn about the latest innovation before others (industries, retailers, etc…) by participating directly in the project. Coop’s participation in the project represents an important link in the supply chain, developing contacts with consumers through dissemination activities, product approval tests, etc…
ProFuture is corresponding to the expectations of modern consumers, but also is challenging the main barriers (the look, taste and price) to be roughly equal to regular products.

Based on the Means-End Chain approach, this study investigated Dutch (n= 10), Spanish (n= 10), and Hungarian (n= 10) consumers’ most important considerations (i.e. cognitive structures) when choosing between different types of algae food products. During so-called laddering interviews, participants were first asked to rank products from most to least preferred after which a systematic interview followed. The interviews aimed at exploring what kind of concrete product properties are linked to expected disadvantages or benefits in consumers’ mind, which in turn are related to values that one considers important in life. Dutch consumers’ most prevalent motive behind their product choice was health with a strong focus on quality of life. Another dominant aspect was attractiveness of food which was determined by colour and design. Enjoyment and positive feelings were central aspects of Spanish consumers’ choices. Additionally, they liked convenience to save time, and versatility to avoid getting bored with food. Hungarian consumers were health-oriented as well, with a focus on quality of life. They evaluated food based on its expected taste and ingredients which were more important than how appetizing food looked. Dutch consumers were open to novel food if it was perceived as innovative, versatile, and evoked positive feelings. Spanish consumers openness was affected by positive feelings as well, together with the opportunity to try something new and familiarity. Hungarian consumers were open to novel food with algae only if it was familiar and if they knew what to expect from the product. Our results show that reasons to accept algae food at least differ partially between European countries.

Consumer perception of food products with microalgae added will be extensively studied within ProFuture. Previous studies have already indicated that many factors play a role. First and foremost, sensory properties are important. Addition of microalgae can have a negative influence on the sensory properties of food products and changes in colour and taste are usually perceived as negative by consumers. Willingness to compromise on taste is a positive driver for microalgae-enriched food products. The higher the percentage of microalgae, the lower the sensory acceptance. The negative perception of the sensory properties has been mentioned as a reason for the fact that products with microalgae added are not yet commercially marketed in large numbers. Secondly, the product itself or the product category to which microalgae are added – which is also referred to as the ‘carrier product’ – is also important. It has been shown that when microalgae were added to milk, bread and supplements, the resulting products had a lower consumer acceptance while microalgae were much better accepted as an ingredient in pasta. Thirdly, familiarity with microalgae or food products with microalgae added, had a positive influence on the perception and liking of products in which microalgae were used. Also, when consumers are familiar with spirulina, one type of microalgae, they reported less disappointment with the taste. Fourth, knowledge about microalgae has a significant influence. Linked to this, nationality (as a proxy of knowledge) is a significant factor as in some countries the knowledge of microalgae is higher. For example, it has been shown that French consumers are more positive about food with microalgae added compared to German consumers, which has been attributed to a higher knowledge of microalgae among French consumers. Last, personal characteristics may also influence consumer acceptance of microalgae in food. Health-conscious individuals are more willing to adopt products with microalgae added. On the other hand, environmental concern has not been confirmed to influence the perception of microalgae in food products. A cross-sectional consumer survey within ProFuture and involving consumers in multiple EU countries is planned to verify these effects.

A research was conducted at Ghent University to distinguish different consumer segments and determine the most interesting ones for the adaptation of food products with microalgae proteins added. The study was held in five European countries: Germany, the Netherlands, Hungary, Spain and Italy (n = 3027). Based on participants’ willingness to try food products with microalgae-proteins added and their perception of these products, consumers were divided into four segments by means of Two-Way clustering. These segments were named based on their characteristics: ‘Enthusiast’, ‘Accepter’, ‘Indecisive’ and ‘Uninterested’.
The segments were characterized by amongst others sociodemographic characteristics, food related behavior and – attitudes. A general trend that could be observed over the segments was that when segments with a lower perception and lower willingness to try, showed a higher level of food (technology) neophobia and they thought that food products with microalgae-proteins added did not provide value for money. 
A second trend showed that when perception and willingness to try food products with microalgae-proteins added increased over the segments a number of other factors were increased too: general health interest, environmental concern, interest in related information, willingness to consume products derived from animals fed with feed with microalgae-proteins added and specific food products with those proteins added. 
To conclude, differences exist in consumer willingness to try and perception of food products with microalgae-proteins added. Therefore, it is important to focus on those with a generally higher willingness to try these food products and a better perception of these products for increasing adoption of these products.

This study investigated consumers’ associative thoughts on and affective reactions to different types of algae names and algae applications in food. Dutch participants (N = 20) were asked to complete a concept mapping task with different types of algae names (i.e., scientific name, name in the local language, Asian name) combined with a clip watching task with algae food products followed by a semi-structured interview. Algae names were predominantly associated with algae or plant properties, visual sensory properties, personal impressions, and food applications. After seeing food products with algae, participants’ associations about different aspects of food and sensory properties increased substantially. Additionally, associations generated by different algae names differed concerning the content, for instance, the scientific name “chlorella” evoked the most associations about biological-scientific terms. Participants showed interest and intentions to try the algae food products shown however, willingness to buy remained low. Participants expected the products to taste salty/savoury/briny and to be healthy. Our results indicate that consumers do not necessarily associate algae with food. Therefore, strategies focusing on stimulating algae consumption should provide consumers with concrete examples of possible applications. Additionally, curiosity evoked by the products stimulated willingness to taste, which might be a promising first step of introducing novel food containing algae.

ProFuture’s partner NORCE is researching the use of insect waste as a source of nutrients to grow microalgae. The main goal is to assess the potential of replacing chemical nutrients with organic insect waste and see how it affects the growth and nutritional quality of the final biomass. 
NORCE conducted preliminary experiments to study the growth of microalgae species (Nannochloropsis and Tetraselmis) using different side streams, including insect frass (from the excrements and the exoskeleton).
These preliminary studies showed that microalgae fed with insect waste could grow faster, due to the high bioavailability of their nutrients. In other words, insect waste provided a variety of important nutrients in a form that algae could easily take them up (absorb) and use them to grow and multiply.
Nevertheless, a few challenges that arise from this circular process - the three main ones being related to:
- Conversion efficiency - transforming insect fertilizer into microalgae feed 
How much insect fertilizer can be converted into microalgae feed? How much insect fertilizer is needed to grow high amounts of microalgae?
- Assessing the nutritional quality of the final biomass 
NORCE will conduct trials using a freshwater microalgal species (Chlorella vulgaris) and assess how the protein content and quality of the fully-grown microalgae is affected.
- Labelling and regulatory affairs. 
No detailed production rules have been specified for microalgae used for food and feed.
Read more here: https://www.pro-future.eu/blog/insects-and-microalgae

The production of protein isolate requires as a first step to extract proteins from the microalgae. Several techniques exist, some involving a mechanical disruption such as bead milling or high pressure homogenization, while others rely on a chemical disruption at high pH. These techniques are effectives to extract a large quantity of proteins, but they destroy entirely the cell, which is troublesome for the following steps of protein purification and concentration. Moreover, these techniques are often energy intensive and/or consume a large amount of chemicals. 
The Pulsed Electric Fields (PEF) is a physical technique which consumes a low amount of energy. It uses short electrical impulses to permeabilize the cell, without destroying it by electroporation. The soluble proteins can pass through the pores and be extracted. 
The cell isn’t totally destroyed, then the proteins are not mixed with cell debris and pigments, which facilitates their recovery and the purification. Moreover, this mild extraction allows the recovery of other compounds, such as polysaccharides, without altering their structure and functionality, making them valuable for cosmetic application for example. 
In ProFuture, this technique is applied to Tetraselmis, Chlorella and Spirulina and will be compared to a more traditional technique, bead milling. The comparison of the product obtained with both techniques will be done in a Life Cycle Analysis (LCA), aiming at calculating the lifetime environmental impact of both products, and in a Life Cycle Cost (LCC), analysing the direct monetary costs.

Decision support tools aim to facilitate the decision-making process of organisations in a structured manner, utilising specific IT software. In the ProFuture project, all the research, along with experimental results, from the utilisation of the microalgal proteins will be modelled by our project partner AXIA, in a complex set of rules and processes. The processes will use specific software and mathematical tools for the optimisation of particular outputs. RDC is preparing a Web-based platform where the final users will be able to access and visualise results. Particularly, this Decision Support serves as a host for the optimisation models, with a back-end administration system and custom database design. The database design makes it possible to represent all the stages of the value chain, via a back-office management system for the database input. The tool was prepared in an “Open” design form, that allows for the addition of more data in the future, apart from the ones studied throughout the project duration. The user interface wireframing, has two options of operation, and particularly: a) Step-by-Step Wizard, for users cognizant of the supply chain, and b) Results-driven optimal paths, for users non-cognizant of the supply chain. Accordingly, via a user-friendly interface and front-end screens, this tool will be ultimately used to boost the production and use of microalgae protein-rich ingredients in food and feed.

Despite the time that has been spent on research related to algae-based proteins there is a long way towards their production at industrial scale. Facing the obstacles of high cost and low efficiency it is essential to follow a more strategic approach in exploiting the advantages of microalgal proteins to stimulate further the implementation of a Bioeconomy in Europe. To exploit all the possibilities, a whole value chains review is required at the same time to maximize the value extracted. 
Within the ProFuture project, AXIA INNOVATION is involved in providing a particular Decision Support Tool to efficiently screen and assess this emerging value chain. The work includes the systematic integration of microalgae processing, cultivation harvesting, extraction, and drying technologies consideration simultaneously all the available types of microalgae and the uses of the proteins for foods and feeds. Based on the principals of process synthesis, the network of options develops a superstructure where the connections are translated to a mathematical model. The proposed approach provides a holistic overview of the value chain that is assessed based on single or multiple criteria. Alternative scenarios scope the impacts of the market uncertainties, the efficiencies of the processes, logistic restrictions, and sustainability in terms of environmental and economic feasibility of the value chain paths. 
The developments envision a step further than the creation of an appropriate framework of a User-Friendly Interface, providing the possibility of the use of the tool from all the relevant stakeholders in the area. The action is supported by RDC Informatics.

The microalgal strains chosen for the ProFuture project are Chlorella vulgaris (light green), Tetraselmis chui, Arthrospira platensis (known as Spirulina) and Nannochloropsis oceanica. The first three strains are currently accepted for food and feed use in the EU, while the last one is widely used for feed purposes and arouses the interest of food producers. Despite the general affiliation to the microalgae group the selected strains have quite different taxonomic classification making, for example, Chlorella and Nannochloropsis as different as an elephant and a shrimp (more details about the taxonomy at http://lifemap-ncbi.univ-lyon1.fr/#). This enormous difference among the microalgal strains is reflected in ad hoc culture conditions, harvesting and refinery treatments for protein extraction, which constitute a big challenge. 
Here a brief description of the different species. 

Chlorella vulgaris is a freshwater microalga with a spheric shape and very thick cell wall. The mean diameter is between 2 and 10 µm. It is consumed in food, especially in Japan. The strain used in ProFuture has a decreased chlorophyll content, to reduce the associated bitter taste.

Tetraselmis chuii is marine microalgae, characterized by an elliptic shape and surrounded by a theca composed of organic scales. It has a diameter between 12 and 14µm. It is mainly used to feed shellfish and shrimp larvae.

Arthrospira platensis is an alkaline brackish water cyanobacterium, characterized by a spiral shape and a diameter of 8 µm. It is the most produced microalgae in the world, it was historically consumed by the Aztecs and near Lake Chad. The cell wall is much more fragile than for the previous microalgae and its main soluble protein is a protein-pigment complex with a brilliant blue color, called phycocyanin. 
Nannochloropsis oceanica is a marine microalga which appear as small, non-motile sphere with a diameter of 2-3 µm. This strain was widely studied for biodiesel production, due to it high lipid content. Recent interest is paid to its EPA (eicosapentaenoic acid) content for food supplement purpose.

ProFuture partner German Institute of Food Technologies (DIL e.V.) investigates the effect of Chlorella vulgaris addition on pasta Lasagna sheets with the addition of 3 and 5% Chlorella vulgaris were prepared on a pasta machine (Pasta V 300, Häussler GmbH). Following the sheets were dried for 20 h at defined conditions in a cooking cabinet. The cooking time, colour and swelling capacity of the dry lasagna sheets was investigated. Furthermore, the colour, cooking-loss and cut resistance of 10 minutes cooked lasagna sheets were analyzed and scanning electron microscopy (SEM) pictures were taken. Results revealed that durum wheat semolina substitution with Chlorella vulgaris hadn’t any impact on the cooking time. However, swelling capacity of algae-containing lasagna was slightly reduced and algae-containing lasagna became much darker, more green and more yellowish (dry and cooked). Also, the cooking-loss of algae-containing lasagna sheets was little enhanced but without statistical significance. Lasagna sheets with 3% Chlorella vulgaris showed reduced and with 5% Chlorella vulgaris increased cut resistance, but again no statistically significant difference to the control was found. In the SEM pictures, no major differences could be found between the control and the pasta with 5% Chlorella vulgaris, too. In summary, the substitution of durum wheat semolina with 3 and 5% Chlorella vulgaris has only a minor influence on the pasta structure. Additional sensory analyses are required to determine whether the pasta is also acceptable in terms of colour as well as taste.

NextGenProteins, ProFuture, Smart Protein and Susinchain - four EU-funded projects working on alternative proteins, collaborate under the name Horizon4Proteins. These projects join forces to promote the transition towards a more sustainable and resilient food system in Europe.
Given the reality of climate change and natural resource scarcity, it will become increasingly challenging to ensure sufficient, nutritious, safe and affordable food to a growing population. The protein supply is critical – integration of a variety of new or alternative protein sources from both terrestrial and aquatic origin into new and/or existing processes or products needs to be explored in order to develop and ensure more sustainable and resilient food supply, featuring high levels of consumer acceptance by a clean labelling approach and attractive market opportunities.
Sharing this goal, Horizon4Proteins kicked off in 2021 with a webinar series exploring key aspects of alternative proteins such as consumer acceptance, safety and regulatory challenges, food applications, and sustainability. Horizon4Proteins invites researchers, farmers, producers, policymakers, and all those with an interest in sustainable food systems to join the conversation and contribute to the future of our food systems.
Supported by the Horizon Results Booster, the group will undertake further communication and dissemination activities together. 
https://www.pro-future.eu/news/alternative-proteins-eu-funded-projects-teaming-up-to-develop-sustainable

The microalgae market is nascent in the European Union. For it to scale-up, it is important to identify the different actors involved in the microalgae value chain. 

To understand the different stages of valuation/transformation and the types of actors and their distribution among the stages of transformation, 10 interviews were realized with actors from seven different countries. The main market for microalgae is human food with 74% of the production, followed by pet foods with 25% of the production and health and care with 1% of the production. Half of the microalgae consumed by humans is Spirulina sp. 

The value chain of microalgae is composed of four main stages: microalgae cultivation, harvesting, processing and distribution. Then, according to the targeted market, microalgae biomass undergoes different processes: it can be used as such for animal feed; it can be dried for human food and consumed as a whole or cracked to get biomolecules of interest such as proteins. The price of the microalgae is, thus, expensive due to high production costs, which can be an impediment to the development of the market. For the geographical layout, Germany, Spain, France and Portugal are the main producers of microalgae with plants generally placed on the coastal area. 

According to our study, the production part is the key point of value analysis with 3000 people hired in the production link. There are four models of microalgae production: photo-autotrophic, heterotrophic, photo-heterotrophic and mixotrophic using three main systems: open ponds, photo-bioreactors and fermenters. The latter, with a production of 500-1000 tons/hectares/year, has a yield 250 times superior to open basin production (5-10 tons/hectare/year). The harvest also represents a critical point that can represent 20-30% of the production cost of algal biomass. There is no standard process for this step, it depends on the strains of microalgae used (i.e. morphological, dimensional, and physiological properties) as well as the efficiency of the treatment, the final dryness, the impact on the quality of the biomass, the performance drifts, and the cost.

It was important to identify all the constraints of the microalgae value chain to scale up the production. Within our analysis of the microalgae value chain based on 10 interviews with actors from 7 countries, the distribution stage offers some room for improvement. 

For the distribution link, it is to be noted that the producers of microalgae often are also producers of macroalgae. Therefore, we suggest that macroalgae can be an example to follow in shaping the future of the microalgae market. Even though the first production of microalgae in Europe was 40 years ago, we found that the market is still not very mature and presents weak points such as the need for different technologies and equipment according to the customers’ requests and a weak sector ecosystem. 

To solve these issues and scale-up the market of microalgae, some of our recommendations are: 
• Develop interactions between links in the sectors 
• Develop visibility in the sectors, mainly with end consumers 
• Reduce the product price in different ways: developing production system based on the sector of application, developing intensive cultivation, developing automation, working on sustainable techniques, improving flow management 
• Develop know-how and the business dimension

Microalgae are normally cultivated as plants, harvesting the energy of sunlight, taking up CO2 and obtaining O2 as a by-product. This is called autotrophic growth. In addition, some species can also grow heterotrophically. That is, consuming sugar or other organic compounds, for which they require O2 and produce CO2 in turn. Both cultivation methods have advantages and disadvantages that limit their industrial application. In this part of the project, we combine the best of both worlds by developing a mixotrophic strategy in which light and an organic compound are provided simultaneously in a balanced manner. In this way, we are improving the low productivities of autotrophic cultivation, while maximizing substrate utilization (in this case, sugar), a weak point of heterotrophy. Because O2 and CO2 are both consumed and produced, we do not need to provide them externally, simplifying the construction of the system and saving energy costs. We aim to see this new concept applied in industry, and for that we need to translate it from the laboratory to industry. However, bringing the process to large scale is not an idle issue, as several aspects become more complex. For example, we have to adapt the way the organic carbon source is fed into the medium due to the difference in volume and size of an industrial system. We also need to consider external factors under outdoors conditions, such as day/night cycles and light and temperature changes. We are investigating separately these different elements under controlled conditions in the laboratory. With the data obtained, we are designing a suitable mixotrophic strategy that we will test and validate in a representative pilot-scale industrial setting.

The Profuture project wants to bring microalgae proteins into the market. Microalgae have many advantages for human and animal health, but above all they can be sustainable and carbon negative. Inside Profuture you can find a group of experts working closely together in WP3: Innovation in microalgae cultivation and harvesting. Here, 2 companies and 4 academic partners joined forces to solve some of the main bottlenecks of microalgae cultivation. Colab (PT) is using laboratory experiments to produce novel varieties of microalgae with improved production and without any genetic modifications, just exploring nature’s potential! Next, NORCE (NO) and Wageningen University (NL) are testing how to use waste streams from industry to grow microalgae, lowering costs and increasing sustainability. Twente University (NL) is designing and constructing a device to concentrate CO2 directly from the air, to be transported to our industrial partner Allmicrogalgae (PT). Normally, industries must acquire bottles of concentrated CO2 and here in WP3 we want to show it is possible to do it right there next to the algae farm. Finally, the industrial partners Allmicroalgae (PT) and NECTON (PT) will test new strategies and protocols to reduce water and electricity consumption, integrate solar energy, implement new processes and reactors design to grow microalgae. Finally, a very important task is providing other partners from Profuture with microalgal biomass, so they can run tests and make new formulations rich in protein. This task is done in WP3 by our industrial partners. All in all, WP3 works in the cooperative spirit of Profuture to move beyond the current limitations and deliver innovations in microalgae production for the European Market.

ProFuture worked on improving each step of the microalgae value chain. This abstract will focus on the results about “innovations in microalgae culturing and harvest”. This workpackage had 5 tasks aimed at making the production of microalgae more sustainable and more cost effective. As a first task, ProFuture partners worked on the Characterization and improvement of algae production strains for demonstration. Here, GreenColab (GCL) developed 6 colorless Chlorella vulgaris mutants (3 pale green, 2 yellow and 1 white); these strains were scaled up in heterotrophic mode (growing on glucose in fermenters) by the industrial partner Allmicroalgae; these biomasses were given to other industrial partner. The second task focused on CO2 absorption and concentration from the air (Direct Air Capture/ DAC), was led by University of Twente (UT), who installed a pilot for direct CO2 capture at Allmicroalgae production site (Portugal). Allm and UT operated their systems with CO2 directly provided from the air to produce microalgae without buying bottled CO2; this ran for >100 days with ~7kgCO2/day, close to the target of 10kg/day (although the weather conditions were suboptimal; high humidity). In task number 3, partners worked on increasing production efficiency and cost reduction. In this task, Wageningen University (WU) scaled up an extremophile microalgae species (Galdieria sulphuraria) grown at 45 degrees and at pH 1-2; trials done at pilot scale (1300 litres); data fed a model for further optimization; biomass was supplied to partner Algosource for protein extraction trials. NORCE tested the use of insect production waste as culture medium for microalgae; insect frass can be used as nutrient source to generate biomass with similar composition as the control. Task four worked on energy reduction strategies, led by Necton, who developed the following: reactors running only on solar energy (off-the-grid reactor; pumps turned off at night), flatpanels reactors installed to compare production strategies (energy consumption; input for LCA models), different nitrogen sources tested to control contaminants; ammonia effectively reduced contaminants, nitrate used as control (where contaminants remained). 

Vegetables creams are traditional and nutritious foods prepared using vegetables and legumes. Consumer awareness about the relatedness between health and nutrition is boosting the development of healthier vegetable cream products. Microalgae are seen as a forerunner resource to close the so-called “protein gap”. Reformulating vegetable creams using Arthrospira platensis (spirulina), Chlorella vulgaris, Tetraselmis chui, and Nannochloropsis oceanica enabled the production of new products that can be claimed “source of proteins” (in which protein provides more than 12% of the energy value) and “high protein” products (in which protein provides more than 20% Kcal) according to the EU Regulation (European Parliament and of the Council Regulation (EC) No 1924/2006 of the European Parliament and of the Council of 20 December 2006 on Nutrition and Health Claims Made on Foods. Available online: https://eur-lex.europa.eu/legal-content/en/ALL/?uri=CELEX%3A32006R1924 (accessed on 6 September 2021)). 

Beside proteins, all the new creams can be claimed “high fiber” (in which fiber is higher than 3 g/100g Kcal). The addition of microalgae did not affect the texture of the creams, while significant changes were observed in the color. Creams made with Chlorella vulgaris showed small differences compared to the conventional products. Based on advanced statistics analysis, we were able to conclude that vegetable creams made with 1.5% Chlorella vulgaris were the most similar to the standard formulation. Ongoing works are being planned to conduct sensory analysis on the creams to evaluate consumers perception and intention to purchase. 

More details about the results can be found here: https://www.mdpi.com/2304-8158/10/11/2550/htm

This abstract presents the results that Ghent University obtained based on a study on consumer interest in food products with microalgae-proteins added. The study investigated consumers’ familiarity, willingness to try and perception of those food products. The total sample consisted of Dutch, German, Hungarian, Italian and Spanish participants (n = 3027). To determine familiarity, participants were asked whether they heard of or tried food products with microalgae-proteins added. 48.1% of the total sample already heard of food products with microalgae-proteins added and 14.1% already tried these products. Significant differences between countries were observed as more participants from Spain, Italy and Germany heard of food products with microalgae-proteins added compared with participants from the Netherlands and Hungary. In addition, significantly more Spanish participants already tried or tasted them compared to participants from other countries. 
Following, consumers’ willingness to try food products with microalgae-proteins added was measured by willingness to eat them for the first time and again. Results showed an average willingness to try food products with microalgae-proteins added (i.e. mean = 3.54 on a five-point Likert scale). In addition, willingness to try is significantly higher for Spanish and Italian participants compared to Dutch, Hungarian and German participants. 
To summarize, results indicated that an average amount of participants already heard about food products with microalgae-proteins added but that these have only been tried by a small proportion. Willingness to try was above average. Therefore, specific strategies can be used to convince more consumers to try food products with microalgae-proteins added.

Life cycle assessment aims to investigate and compare the environmental impacts of products or services from cradle to grave. This means that the whole life cycle from resource extraction to final waste treatment is examined. In this project, it is accompanied by life cycle costing to also include economic aspects. Different technologies for cultivation and downstream processing are being compared on a pilot-scale level. Therefore, researchers from ESU-services are collecting data on the quantities of material used and energy consumed as well as on emissions and waste products from the project partners. A standardized impact assessment method is being used to quantify various environmental impacts (e.g. greenhouse gas emissions) based on the data collected and which will allow comparisons of the technologies tested. The expected outcome is the identification of optimum technologies for the different species under study. A reduction in greenhouse gas emissions, water consumption and production costs is expected. The results will be used to implement best-practice production chains for microalgal food and feed production on an industrial-scale. These production chains will be compared with conventional protein sources like soy- and meat-based products. Through this comparison, the ProFuture consortium will gain insights into the environmental competitiveness of microalgal products. Moreover, the project will generate knowledge on the effect of up-scaling on the environmental impact of a process. The results can be used to identify further research needs and to better model theoretical upscaling of emerging technologies, which allows a fairer comparison with mature technologies from an environmental point of view.

Sustainable food trends become step-by-step on the center of the plate with modern consumers. A growing number of consumers today look for products that can help them live a more healthy, sustainable and socially responsible life. Even though the demand for alternative proteins is growing steadily, the plant-based offerings health and sustainability credentials will come under increased scrutiny as it attempts to evolve from a movement that is trending to a trend firmly established in the mainstream. 

Businesses are recognizing increasing vulnerability and risks in sourcing of their key ingredients within their current supply chains, see the opportunities to build resilience by looking at new ingredients, uptake less energy-exhaustive and more sustainable production methods. Whilst there is only limited number of companies focusing on algae production today, large amounts of investment is going into this arena and expect much more innovation in years to come. There are 20,000 species of edible plants in the world, yet 75% of the global food supply comes from only five animal species and 12 plant, dominated by rice, maize and wheat. While meat producers still have 98% of the market, plant-based foods have been enjoying the attention as more flexitarians attempt to cut back on their meat consumption for health and sustainability reasons. 

The plant-based momentum is clearly relevant and food brands are extolling a vegan lifestyle as the pathway to good health as well as the answer to future food security, but questions are beginning from the supposed health and environmental benefits of a plant-based diet unless the sourcing of vegan products comes specifically from no-dig soil and do not contributing to soil change and actively participating in the destruction of soil and life. 

Profuture project is highly contributing to the sustainable microalgae-based production at industrial scale that corresponds to the expectations of modern consumers, but also challenging the main barriers - the look, taste and price to be roughly equal to regular products.

There are two types of algae, macroalgae and microalgae. Macroalgae have a long history of use in the EU market specially their deriving polysaccharides (e.g., alginate, carrageenan, and agar) providing relevant textural functionality (e.g., stabilizers, thickeners, and emulsifiers) in several processed foods. Over the last decade, the production of food products enriched with microalgae or compounds derived from microalgae has increasingly gained attention due to consumer awareness about their nutritious compositions (e.g., proteins, antioxidants, and vitamins). In this light, we conducted a market search focused on EU market to map the position of microalgae and macroalgae as a food ingredient. Results showed that 5720 new products containing algae ingredients were launched in EU market from 2015 to 2020. These ingredients were mostly used in formulating dairy and desserts and ice cream playing different roles such as thickeners or natural colorants or functional ingredients. In term of ingredients, carrageenan was the most used macroalgal ingredient with a total of 4350 food products. Macroalgae mainly nori, wakame and kombu were also incorporated in foods but to less extent. For microalgae, spirulina was included in different products at level from 0.1% to 62% addition level, while chlorella was less used, and the amount added was ranging from 0.1 to 0.5%. Under the umbrella of ProFuture, scientists, business experts and companies are boosting the application of microalgae in a wide range of products keeping in mind nutritional value, cost, and sustainability.

Green microalgae are comparable to higher plants except that they are single-celled and grow in fresh, brackish or marine water and therefore do not consume agricultural land or form a special habitus. In the EU, three species are approved for human consumption in the category microalgae, Chlorella vulgaris a 4-10 µm green microalgae that can grow in fresh and brackish water, Tetraselmis chuii a 10-16 µm green microalgae that can grow in marine water, and the multicellular filamentous cyanobacterium Arthrospira platensis, known as Spirulina and formerly called blue-green algae. The approved “microalgae” are nutritionally very valuable, are rich in vitamins and antioxidants and have a high protein content as well as a high proportion of essential ω-3 and -6 fatty acids. However, they are rarely found in food. The main reasons for this are their price, consumers' lack of knowledge about microalgae, the assumption that they taste strongly of fish, and their green color, which is undesirable in many food. To successfully integrate microalgae into food, a holistic approach is taken. Besides the goal of making microalgae more affordable (WP3 and 7), we are aiming to incorporate these valuable ingredients or improved protein isolates from them (WP4) into new formulations (WP5) while educating consumers about the benefits of microalgae in their diets (WP 8 and 10). To be more precise, the microalgae are to be incorporated into vegan recipes of bread, sausages, pasta, soups, vegetable cream and sports nutrition. The resulting changes will be analyzed and the products adapted according to the wishes of the companies that want to distribute them on the market.

Consumers within the EU are increasingly asking for natural and healthier food products, which are additives-free and environmentally friendly. Within this framework, microalgae have recently gained popularity in the nutraceutical and functional food-market, thanks to their high bioaccesible compounds content. The aim of this study was to assess dry biomass of Spirulina, Chlorella sp. and Tetraselmis sp. as innovative and healthier ingredients in baked goods (breadsticks, crackers, brioche and muffins) at flour substitution levels of 1.5-3.5%. Incorporation of microalgae led to increased protein and total phenolic content. Antioxidant activity was also higher in microalgae-enriched goods. A sensory analysis was carried out with 30 semi-trained panelists aiming to evaluate taste, texture and overall acceptance. According to results, Breadsticks (Grissini) and croutons with Chlorella at 3.5% showed a highest overall acceptance within the panelists, while crackers showed the highes global acceptance rates using 1.5% levels of Spirulina. Other baked goods studied were muffin and brioche, which obtained relative high scores when using Chlorella 1.5%. In spite of having a good nutritional contribution, Tetraselmis was rated with a “strong salty taste” and panelists found it unpleasant. Obtained Results show that Spirulina and Chlorella could be a sustainable ingredient to formulate baked goods with an enhanced nutrimental matrix without altering the acceptability for consumers. Even though, in order to apply other microalgae such as Tetraselmis in food matrixes, more studies must be carried out to provide solutions for that unpleasant salty taste, and that the nutrimental contribution can be utilized.

Galdieria sulphuraria is a species of microalgae that is naturally found in geothermal hot springs worldwide. Due to the adaptation to this extreme environment, this species thrives at low pH, high temperature and high salt concentration. Very few other organisms can grow under these conditions, making this microalga a perfect candidate for large-scale cultivation under conditions that are prone to contamination. At Wageningen University we cultivated this species with a novel non-aerated mixotrophic approach to improve biomass and protein productivity while reducing costs and avoiding contamination. The produced biomass had a protein content of more than 60%, which is one of the highest among all microalgae. 

Moreover, we studied the quality of the protein by analysing the essential amino acid content. The protein from G. sulphuraria was rich in all the essential amino acids, especially in the sulfur amino acids methionine and cysteine. These two amino acids are scarce in plant and algal biomasses, highlighting the potential of G. sulphuraria as a protein source. In addition, the biomass contained 10% of the blue phycobiliprotein C-phycocyanin, a high value protein used in diagnostic histochemistry, cosmetics and food industry. The C-phycocyanin from G. sulphuraria was acid and thermostable, increasing its application range compared to the current commercial C-phycocyanin from Spirulina.

Nowadays, the increasing demand of proteins, in particular of vegetal and sustainable proteins, is pushing the academic and commercial world to find new and original solutions. In this framework, microalgae seem to bring the desired solution: they are unicellular plants which can be “easily” cultivated in closed and controlled systems called photobioreactors, using CO2 and agri-food wastes as nutrients. Hundred thousand species have been identified in different environments, but only few species are exploited at industrial level and just a little part of them is allowed for food use. The potential of microalgae as protein sources is quite evident since their protein content ranges between 30 and 60% of their dry weight. Among vegetal, some species of soy reaches 45% of protein per dry weight while peas only have 23%, lentils 25%, wheat 12%. 

The microalgal strains selected for the production of single cell proteins and proteins isolates in the ProFuture project are: Chlorella vulgaris (light green), Tetraselmis chui, Arthrospira platensis (known as Spirulina) and Nannochloropsis oceanica. The microalgae were produced by the Portuguese companies Necton (Tetraselmis and Nannochloropsis) and Allmicrolgae (Chlorella and Spirulina). The nutritional composition of dried microalgal powder has been analyzed within the project. 

The results showed that:
• Chlorella vulgaris (light green) is composed by 40% proteins, 9% lipids, 20% fiber, 16% assimilable sugars, 7% ashes
• Tetraselmis chui is composed by 37% proteins, 7% lipids, 15% fiber, 8% assimilable sugars, 33% ashes
• Arthrospira platensis is composed by 67% proteins, 7% lipids, 9% fiber, 7% assimilable sugars, 9% ashes
• Nannochloropsis oceanica is composed by 46% proteins, 19% lipids, 16% fiber, 6% assimilable sugars, 13% ashes

Polycyclic aromatic hydrocarbons (PAHs) are a group of about 10,000 chemical compounds characterised by a structure composed of two or more fused aromatic rings. Many studies have demonstrated the genotoxicity and carcinogenicity of PAHs in treated foods. Food is the main source of exposure to PAHs, contributing to more than 90% of total PAH exposures. PAHs levels in foods can vary mainly based on the type and fat content of the food and on the cooking or drying processes operating conditions. Commission Regulation (EU) No. 836/2011 identifies the PAHs substances that produce both carcinogenic and genotoxic effects and establishes their maximum tolerated levels on foodstuffs.
Considering the previous discussion, there is a growing need for accurate detection of trace-level PAHs in food products. The challenge for analysts is to maximise recovery of analyte and minimize the interferences by proper extraction and clean-up procedures. The different methodologies of extraction, combine liquid-liquid, Soxhlet or QuEChERS techniques for the purification phase. Regarding detection, it is mainly carried out by Gas Chromatography Mass-Mass spectrometry (GC MS-MS), with also a few examples of GC-MS and High-Performance Liquid Chromatography with Fluorescence Detection. 
In the ProFuture project, researchers at the Institute of Agrifood Research and Technology (IRTA) are using GC-MS (which was revealed to be the most accurate technique in this case) to identify PAHs in the drying algae biomass and compare the results obtained by using different drying techniques to find the more suitable one in terms of costs but also quality and safeness of the product. The results show that all the techniques do not produce a detectable amount of PAHs.

The potential of marine microalgae such as Spirulina, Chlorella vulgaris or Tetraselmis chui as an innovative ingredient in the fortification of bakery products has been evaluated at Institute of Agrifood Research and Technology (IRTA) in the Fruitcentre from Lleida. The first part of the study focused on the interaction of different types of flours (wheat flour, manitoba, standard bakery flour, wholemeal flour, organic bakery flour) with Spirulina in bread formulations. 

Overall, adding Spirulina powder to the bread dough impacted some of the bread’s physical characteristics and nutritional properties. Regardless of the flour used, the breads became smaller and lighter due to the weakening of the gluten network by the proteins added by Spirulina. The final bread also had green tonalities, due to the intense green colour of Spirulina. Interestingly, adding between 1.5-2.5% of Spirulina in the bread formulations increased the total protein content, but not necessarily the amount of digested protein, compared to the regular version of each type of bread. This means that even though the enriched breads contained more protein, that surplus might not be digested and absorbed in human bodies. Plus, the enriched breads had a higher phenolic content and antioxidant capacity, meaning that they contained more antioxidants such as polyphenols, which are compounds known to benefit health. 

Therefore, Spirulina presents as a promising ingredient to improve the nutritional value of bread. In general, consumers had a positive reaction towards the different reformulated breads (with concentrations of Spirulina between 1.5% and 2.5%) and were not discouraged by the change in colour. This preliminary result let to test the reformulation of other bakery products including muffins, grissinis and crackers with the collaboration of the SME Cale from Portugal. Substitution level of flour with up to 3.5% Spirulina, C. vulgaris or T. chui led to products with higher antioxidant and protein content. No major differences in physical parameters (besides colour) were observed when compared to wheat-only controls. Purchase intention of the products as well as the acceptability index suggested that the fortified products would have a good acceptance.

In ProFuture, we are exploring several conventional and innovative drying technologies among which pulse combustion drying. This technology is a promising since it enables the transformation of a paste into a powder. Compared to a traditional spray drying, pulse combustion drying presents several advantages such as energy efficiency, low injection pressure, high thermal exchange, and versatility of applications from dairy products to microalgae biomass, producing a high-quality product. In ProFuture, we used it to dry Chlorella vulgaris, Tetraselmis chui, and Nannochloropsis oceanica. Results showed that the quality of the powders depended on the microalgae strains. In brief, the powder of Chlorella vulgaris has a low moisture content, a high yield and agglomeration level. On the other hand, powders of Tetraselmis chui and Nannochloropsis oceanica have low moisture contents, low agglomeration and high yields. We noticed some variability in the final moisture content and yield, which can be attributed to the structural variability of microalgae. 

Noteworthy, regardless of the microalgae used, this drying process was quite fast and this implies a lower use of energy compared to conventional drying processing. In depth life cycle assessment analysis are being conducted to evaluate the efficiency of the application of pulse combustion for drying microalgae in comparison to other technologies namely freeze dryer, spray dryer, solar dryer and agitated thin film evaporator.

Microalgae are the food of the future. These eukaryotic unicellular organisms are capable of living in the most diverse environments, growing faster than other photoautotrophs in non-conventional growth media that do not require potable water or arable land. Although food products based on microalgae already exist and are consumed as a high-quality food, there is an urgent need to improve existing microalgae-based products. Their organoleptic properties, such as a “grassy” taste, intense green color and fishy odor are not consensually accepted by consumers. 

This work aims to generate novel strains from microalgal species already registered as novel food, namely Chlorella vulgaris, "Spirulina", Tetraselmis and Nannochloropsis to improve the quality of the produced biomass and the overall consumer’s acceptance. While mutations may occur spontaneously in nature, they can also be incited experimentally using laboratory procedures. For this purpose, the Marbiotech/CCMAR Group and Greencolab at the University of Algarve, in Portugal, together with Allmicroalgae are carrying out random chemical mutagenesis to generate non-heterologous DNA-based mutants with higher protein contents and improved organoleptic characteristics without losing the high nutraceutical value of microalgae. 

Bombo, G. C.1,2; Schüler, L. M.1,2; Trovão, M.3, Barros, A.3; Machado, A.3; Cardoso, H.3; Silva, J.3; Pereira, H. G.2; Varela, J.1,2
Marine Biotechnology Group, Centre of Marine Sciences, University of Algarve, Campus de Gambelas, 8005-139 Faro, Portugal.
GreenColab - Associação Oceano Verde, University of Algarve, Campus de Gambelas, 8005-139 Faro, Portugal.
Allmicroalgae Natural Products S.A., Portugal; CCMAR, University of Algarve, Portugal

To generate novel strains of Chlorella vulgaris with improved nutritional profiles and organoleptic features, random mutagenesis was performed to isolate mutants with different traits. 
First, two C. vulgaris mutants were isolated, namely C3, which is able to grow significantly faster on solid medium as compared to the wildtype (WT), and a second mutant (GL3) obtained from the C3 strain. Interestingly, although C3 cells presented higher protein contents, GL3 displayed vestigial chlorophyll contents, lower carotenoid levels, and higher protein contents than the WT, together with a significantly different amino acid profile. Glutamic acid was present in similar amounts as in soy and Spirulina, which has not been observed in WT C. vulgaris, or in other Chlorella found in the literature. GL3 cells grown under heterotrophic conditions reached higher cell concentrations than the WT, strongly suggesting that this mutant might become a relevant source of protein, being suitable to be produced at a larger scale. 
More recently, two other promising yellow mutants have been isolated, 7Y and 8Y. However, 7Y exhibited a significantly lower biomass productivity (1.88 ± 0.03 g L-1 day-1) and growth rate (0.045 ± 0.001 h-1) compared to those of the WT (2.44 ± 0.12 g L-1 day-1; 0.057 ± 0.000 h-1). Similarly, 8Y displayed a lower biomass productivity (1.61 ± 0.073 g L-1 day-1) and a slower growth rate (0.042 ± 0.001 h-1), compared to those of the WT (1.83 ± 0.072 g/L/day and 0.052 ± 0.000 h-1). However, growth performance of the novel mutants might be improved by optimizing the growth media for the novel strains. Moreover, enhanced biochemical profiles and organoleptic features, yet to be analyzed, might make up for the slightly impaired growth performances.

Microalgae recognized as present and consumed to a significant degree in the EU before 15 May 1997 - i.e. Arthrospira platensis, Aphanizomenon flosaquae, Chlorella vulgaris, Chlorella pyrenoidosa and Chlorella Iuteoviridis - can be freely used in food and food supplements production (whereas the European Commission has not yet clarified the Chlorella protothecoides status). Otherwise, microalgae introduction in the internal market as food and food supplements ingredients must be previously authorized as Novel Foods, under Reg. EU No 2015/2283. The European Commission - with the help of the PAFF (Plants, Animals, Food and Feed) Committee - has so far authorized Odontella aurita, dried Tetraselmis chuii, Ulkenia sp., Schlzochtrium sp and, latterly, dried Euglena Gracilis as novel foods. Other microalgae - such as Phaeodactlyum tricornutum, Galdieria sulphuraria, Haematococcus pluvialis - are currently under approval. 
Within the ProFuture project, researchers from Food and Agricultural Requirements (FARE) identified the procedures to be followed in the application for novel food authorisations: in the pre-submission phase, studies notification, scientific review and, possibly, consultation with the Authority; then drafting of the dossier and its e-submission to the European Commission. 

On the basis of the Novel Food Regulation and its implementing acts, EFSA’s guidelines and practical arrangements, and the previous microalgae authorization dossiers. The aim is to support research centres and food business operators in studies design and dossiers fulfilment, by clarifying requirements, procedures and data requested in order to place microalgae into the European market. 

You can learn more in this blog article: https://www.pro-future.eu/blog/regulatory-work-on-microalgae-meet-profutures-partner-fare

In 2021, the Centre for Social Innovation (ZSI) organised and facilitated two multi-stakeholder workshops to co-create scenarios of desirable future microalgae scenarios, resulting in four scenarios. These scenarios were analysed with reference to the Sociology of Knowledge Approach to Discourse (SKAD) as formulated by Rainer Keller (2012). Three of the four created scenarios were formulated within the conditions of a growth-based capitalist economy. The participants tied specific values to their scenarios, such as e.g., ecological sustainability, profitability or affordability. According to the analysis risks and uncertainties are a dominant frame of reference across all actors of the microalgae value chain. Also, classifications such as approved microalgae strains for the use in food and feed and legal regulations influence potential futures. Participants related the current situation to its unoptimized and hence unsustainable production, its niche market position as well as inertial effects of existing food sectors. Responsibilities to act extend from consumers, to media, policy makers, retailers and other actors of the microalgae value chain. More political support, closing the persistent knowledge gaps in the realm of microalgae production, processing and consumption, increased funding to allow for upscaling of production and sustainable, cost-effective technologies, as well as enlarged cross-sectoral cooperation were deemed necessary.

Oxygen-balance mixotrophy (OBM) is a novel type of microalgae cultivation that removes the need for aerating the cultures while boosting productivity. This is achieved by adjusting organic substrate supply to photosynthetic oxygen production, which results in intracellular recirculation of O2 and CO2. In addition, due to this recirculation the substrate is used very efficiently and only around 10% of the CO2 is lost, obtaining a high biomass yield on substrate (0.9 C-gx·C-gs-1). After successfully establishing this process at lab scale, we adapted OBM to large scale systems, in particular tubular photobioreactors. This is not a straightforward procedure, as fluid behavior in these reactors resembles plug flow, affecting the dynamics of the process. In order to address this issue, we designed a scale-up approach consisting of 3 blocks: (1) scale-down of glucose and oxygen fluctuations in tubular photobioreactors, (2) model-based validation of substrate feeding control strategy and (3) experimental validation in our pilot facility. The results from (1) showed that oxygen limitation was a critical factor in tubular reactors, but based on (2) it could be avoided with a proper substrate feeding strategy. Finally, using these inputs OBM was implemented in a pilot scale tubular photobioreactor (3), obtaining equivalent results to the lab experiments. Overall, the results open the door for cost-effective production of high-quality protein with G. sulphuraria and potentially of other products at large scale via OBM.

Attitudes and preferences towards microalgae-protein enriched food products will be assessed using a cross-sectional quantitative survey in five countries across Europe, notably The Netherlands, Germany, Hungary, Spain and Italy, with sample sizes of around 600 participants per country. The survey was drawn up in the online survey program Qualtrics and an initial version of the survey was pretested in a sample of researchers and university students for comprehensibility, terminology, and length. The survey was adapted based on received comments. Participants for the final survey will be recruited by means of probabilistic sampling from the online access proprietary panel of a professional market research agency.
Validated psychometric scales will be employed to measure preferences for various product attributes, attitudes towards nutrition and health, attitudes towards food-related aspects such as sensory appeal, convenience, price and other quality aspects, food (technology) neophobia, environmental concerns and food purchasing and consumption habits. All other constructs and items used in the survey are based on literature review and previous research. A choice experiment using pasta as the focal carrier product will be implemented to compare the importance of attributes like price and labels but also to determine the consumer preference between general dry pasta and microalgae-protein enriched dry pasta. All participants will be asked to provide written informed consent before the study (cf. Information for the participants). In order to guarantee the anonymity and confidentiality of the data, a code or id number will be used as identifier in the database, thus all data will be coded and processed anonymously. The questionnaire has been developed and the application for ethics approval is pending. Meanwhile, translation agencies and market research companies are contacted for fieldwork. Following the granting of ethics approval, the questionnaire will be distributed until the quota for gender, age and region have been reached in all five countries. Data collection is scheduled for September 2021 and the first insights will be communicated beginning 2022.

Besides their nutritional value, microalgae can also contribute to other non-nutritional functions in foods such as providing and/or stabilizing the structure of foods and feeds. In food science, these non-nutritional functions are called functional properties and include foaming, gelling, emulsifying, water/oil holding and organoleptic properties (taste, smell, etc.). Many of these valuable properties result from the presence or addition of proteins or protein rich ingredients to foods. Since microalgae contain high levels of protein (often surpassing 50% in dry weight), they have the potential to be highly functional ingredients of particular interest to the food and feed industries. However, the presence of resistant cell walls in microalgae could reduce their functionality as food ingredients by keeping the proteins trapped inside the cell. To test this hypothesis, ProFuture’ researchers at ILVO, determined the functional properties of single cell proteins (SCP) from three microalgae species: Chlorella vulgaris, Nannochloropsis oceanica and Tetraselmis chui and obtained different results. C. vulgaris showed good water binding and foaming capacity; T. chui had good water/oil binding, foaming and emulsifying capacity and N. oceanica displayed good water/oil binding capacity but lacked foaming properties. In the future, ProFuture will also determine the effect of different drying methods on the functional properties of SCP, as well as the functional properties of microalgae protein extracts.

Low percentages of microalgae are recommended in the feed for young livestock animals. It has shown to have benefits on the immune system of some animal species as well as to improve lipid metabolism, gut function, stress resistance, appetite, weight, number of eggs and reproductive performance. The ruminants (as livestock species) are the most suitable species for microalgae supplementation because they can digest unprocessed algal biomass. Adding micoalgae to the diets of livestock and hen has also shown to improve meat and egg quality. 
The use of microalgae in the aquaculture has also several benefits. Microalgae contain antioxidants, peptides and fatty acids, which contribute to the improvement of health and the growth of individuals (especially in earlier stages). Additionally, microalgae provide prebiotic benefits which can improve the digestive and immune system of animals, which in turn can reduce the need for the use of pharmaceuticals, as this increases the tolerance to stress conditions. 
Due to the current position of microalgae, the market price is higher than that of other feed ingredients, such as soy oil meal for which the price is below 0.5 euro/kg. It is estimated that the current market price of microalgae production (oil and meal) is around 20-50 euro/kg. However, if microalgae production is increased, the market price could fall below 5 euro/kg. In this frame, ProFuture is working on finding cost-effective technologies to reduce the cost of producing microalgae ingredients to boost their use as a feed.

Efficiently extracting protein from microscopically small organisms like microalgae still presents a hurdle. Although microalgae contain relatively high amounts of protein, the precious nutrient is protected by a tough cell wall, making it difficult to purify. Today, microalgae protein is generally purified by techniques such as precipitation and ultrafiltration with often disappointing yields. In line with the goal of ProFuture to maximize the utilization of microalgae protein, we want to improve this segment of the microalgal refinery. To take on this challenge, researchers at Flanders Research Institute for Agriculture, Fisheries and Food (ILVO) are exploring a possible novel technique for protein purification: the spiral filter press. This press was recently successfully introduced in the production of fruit or vegetable juices. The spiral filter press contains a spiral that presses the pulp against a filter, aided by a vacuum, to separate the juice from the pulp. This system prevents product oxidation and preserve the bioactive compounds in their native form, resulting in nutritional rich fruit juice without discoloration (Kips et al., 2017). In the framework of ProFuture, spiral filter press was applied to produce a protein rich juice from microalgae or to at least enrich them. Tetraselmis chui and Arthrospira platensis (Spirulina) have been tested with the smallest filter element available at ILVO (60 µm) with little positive results so far. This could be explained by the size of the microalgae cells (< 60 µm) which are able to pass through the pores of the filter. We expect however, that this technique would produce promising results when the filter elements available will be adapted to the size of microalgal cells. 

Kips, L., De Paepe, D., Van Meulebroek, L., Van Poucke, C., Larbat, R., Bernaert, N., Van Pamel, E., De Loose, M., Raes, K., & Van Droogenbroeck, B. (2017). A novel spiral-filter press for tomato processing: process impact on phenolic compounds, carotenoids and ascorbic acid content. Journal of Food Engineering, 213, 27–37. https://doi.org/10.1016/J.JFOODENG.2017.06.010

Necton has acquired a Black Block® Hybrid Solar Dryer in May 2020 and has been ever since, developing methods to efficiently dry microalgae pastes. Necton has learned that several factors affect the drying process, including not only environmental factors, but also internal factors such as the biomass characteristics (microalgae species, paste concentration, etc.). The most efficient drying method identified so far was placing the biomass on top of an elevated grid, thus allowing the strong air flow on the system to pass above and below the biomass. With such approach, it is possible to dry frozen solid microalgal pastes in just over 24 hours during winter, which is approximately half of the duration time and energy costs of the freeze drying. Moreover, preliminary biochemical analysis indicate that the fatty acids are not affected by the solar drying, suggesting that this is a very promising drying method. Nevertheless, further analyses are needed to validate the use of solar drying as a high-quality, sustainable and efficient drying method.

The microalgae value chain is characterized by the creation of side-streams during processes such as extraction of high added value components (e.g. antioxidants, chlorophyll, proteins…). Within the ProFuture project, microalgae protein extraction is currently explored on different microalgae species by Algosource Technologies. The extraction of proteins from microalgae generally results in a “waste” stream that still contains other valuable components (lipids, insoluble proteins, antioxidants, etc.). This residual product may still prove to have value in other applications such as food, cosmetics, pharmaceuticals or agriculture. Finding a purpose for fractions that would otherwise be considered as waste enhances the circular economy aspect of the microalgae value chain, simultaneously reducing production costs as well as environmental impact and energy losses. 
ProFuture partners will analyze the protein extraction side streams for composition, functional properties and food safety aspects within the WP4. Based on the product characterization, bibliographic analysis will be conducted as well as consulting the project partners to recommend possible valorization pathways for the side products. Depending on the composition, possible use cases could include application in food and feed, biofuel production, fertilizer, plant bio-stimulants or high added value components for cosmetics and pharmaceutical applications.

Vegan diet is trending upward as a healthier and more sustainable lifestyle. Vegans and vegetarians greatly contributed into the expansion of vegan products market for ethical concerns over animal welfare and sustainability. Flexitarian consumers are also contributing in the growth of this market since these consumers are decreasing their animal deriving products to consumer more plant-based products for health motives. In 2020, the COVID-19 pandemic accelerated this process since it made consumers rethink their lifestyle and shift to a more plant-based diet as a healthier option. Microalgae is a valuable food ingredient with high nutritional value, and at the same time sustainable, hypoallergenic and versatile. Including microalgae in the vegan alternatives to dairy can be a promising solution to overcome the nutritional limitations of the marketed products in EU. Indeed, we conducted a market search of launches of the year 2020 and we compared the nutritional information of dairy cheese and yogurt and their vegan counterparts. We found that vegan alternatives provide more energy, total fats, and carbohydrates but less proteins than dairy products. In this light, food developers can consider microalgae as a clean label solution among other advantages to improve the nutritional values of vegan products while keeping in mind maintaining acceptable taste and texture. 

More details about the results can be found here: https://www.mdpi.com/2304-8158/10/11/2782/htm

As part of a larger consumer study in the Netherlands, Germany, Hungary and Spain (n = 3027), relative willingness to pay for several attributes of dry pasta with microalgae-proteins added, was researched. Analyses were done at Ghent University with the help of a discrete choice experiment and based on an unlabelled random parameter logit model. The attributes studied were colour, price, nutrition label and sustainability label. Colour was described by the levels "light to dark yellow" and "yellow to light green" which are the colour ranges linked to pasta with micro-algae proteins added. The four price levels used, were based on the average price for dry pasta and dry pasta with microalgae-proteins added and equal intervals were created by adding two more price levels. The Nutri-Score A label was chosen as nutrition label as it is upcoming in Europe and dry pasta mostly always receives Nutri-Score A. Lastly, the organic and vegan label were used for sustainability label. Next to two options per choice set, consumers were also able to select the opt-out option.
Results showed that the presence of an organic, vegan and Nutri-Score A label increased consumers’ willingness to pay for dry pasta with microalgae-proteins added. Colour did not influence willingness to pay in a significant way. Lastly, the main reason for choosing the opt-out option was that both types of pasta with microalgae-proteins were too expensive. This information is useful for producers of food products with microalgae-proteins added.