Environmental impact of phycocyanin recovery from Spirulina platensis cyanobacterium

Accepted Manuscript Environmental impact of phycocyanin recovery from Spirulina platensis cyanobacterium

Sofia Papadaki, Konstantina Kyriakopoulou, Ioannis Tzovenis, Magdalini Krokida PII: DOI: Reference:

S1466-8564(16)30526-4 doi: 10.1016/j.ifset.2017.02.014 INNFOO 1719

To appear in:

Innovative Food Science and Emerging Technologies

Received date: Revised date: Accepted date:

8 November 2016 6 February 2017 23 February 2017

Please cite this article as: Sofia Papadaki, Konstantina Kyriakopoulou, Ioannis Tzovenis, Magdalini Krokida , Environmental impact of phycocyanin recovery from Spirulina platensis cyanobacterium. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Innfoo(2017), doi: 10.1016/ j.ifset.2017.02.014

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ACCEPTED MANUSCRIPT Environmental impact of phycocyanin recovery from Spirulina platensis cyanobacterium Sofia Papadaki1, Konstantina Kyriakopoulou1, Ioannis Tzovenis2, Magdalini Krokida1 1. School of Chemical Engineering, National Technical University of Athens, Athens, Greece 2. Biology Department, National & Kapodistrian University of Athens, Athens, Greece

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Email: [email protected]

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Abstract

Multifunctional extracts from Spirulina platensis are suggested as food additives, due to their high

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content in in functional ingredients and specifically phycocyanin. The recovery of phycocyanin from the microalgal biomass is performed by using ultrasounds and polar solvents such water, ethanol or

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buffer. The application of drying pretreatment in combination with the use of different solvents presents variation in the yields, affecting the actual recovery of the protein and hence the

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environmental impact of the production of 1 kg phycocyanin. Life cycle analysis on the recovery

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techniques for the isolation of the desired phycocyanin was performed in order to evaluate the selected extraction processes’ sustainability. Drying exhibited increased environmental footprint due

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to the energy demand, while at the same time affecting not only the yielding but also the quality of the extracts. The use of aqueous solvents can lead to an environmental and efficient extraction,

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replacing organic solvent systems sufficiently.

Industrial relevance Phycocyanin is a pigment-protein complex which is used into various food products to enhance their nutritional qualities acting as food colorant, antioxidant and emulsifier, which can sufficiently replace or reduce the use of synthetic additives. For the effective recovery of phycocyanin, the nutrient should be extracted from the microalgae biomass of Spirulina platensis. The steps to

ACCEPTED MANUSCRIPT achieve that include the cultivation and harvesting of the microalgae, the drying of the biomass if necessary and the extraction process. However, these steps are resource and energy demanding processes which can affect the environmental footprint and the cost of the final product. Looking for more efficient practices combinations of materials (wet or dried biomass) and solvents (water, buffer and ethanol), which are currently used industrially, were examined in order to evaluate and

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suggest the most sustainable production line for phycocyanin.

Keywords: environmental footprint; extraction of bioactive compounds; microalgae cultivation

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process; life cycle assessment; pigments

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1. Introduction

Microalgae are microscopic photosynthetic organisms that are found in marine and freshwater

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environments (W. Chu, 2012). For centuries they have been exploited as food and animal feed,

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especially for aquaculture (Spolaore, Joannis-Cassan, Duran, & Isambert, 2006; Vigani, Parisi, Rodríguez-Cerezo, et al., 2015; Yamaguchi, 1997). However, recent achievements have renewed the interest in them, since studies have pointed out the ability of obtaining various high-value molecules

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from microalgae, such as carotenoids, chlorophylls, proteins (López et al., 2010; Pasquet et al., 2011;

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Spolaore et al., 2006; Vaz, Moreira, Morais, & Costa, 2016). Thus, the microalgal use in diversified sectors, including pharmaceutical, energy and food industries, has become especially attractive, with the latter sector benefiting greatly from this advancement (Chacon-Lee & Gonzalez-Marino, 2010; Enzing, Ploeg, Barbosa, & Sijtsma, 2014; Vigani, Parisi, Rodr??guez-Cerezo, et al., 2015). Microalgae are able to enhance the nutritional content of conventional food and hence, to positively affect the human health (Santos, Freitas, Moreira, Zanfonato, & Costa, 2015). Spirulina is a photosynthetic cyanobacterium that is produced commercially for alimentary use, as a dietary supplement and food additive. Furthermore, Spirulina possesses proven biological

ACCEPTED MANUSCRIPT functionality such as antiviral, anti-inflammatory and antioxidant activity (W.-L. Chu, Lim, Radhakrishnan, & Lim, 2010; Wu, Ho, Shieh, & Lu, 2005). Therefore, it is widely cultivated to produce biologically active food additives able to treat several diseases, including diabetes and obesity (Anitha & Chandralekha, 2010), arthritis (N. Kumar, Singh, Patro, & Patro, 2009), anaemia (Selmi et al., 2011), cardiovascular diseases (Deng & Chow, 2010), allergies (Vo, Ngo, & Kim, 2012), tumors

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and cancer (Singh, Kate, & Banerjee, 2005). Moreover, functional compounds from Spirulina are

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widely used in food industry as colorants (Martelli, Folli, Visai, Daglia, & Ferrari, 2014) and have been

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suggested also as emulsifiers (Batista, Raymundo, Sousa, & Empis, 2006).

Currently, the annual production of Spirulina exceeds 3000 tons on a dry weight basis and is realized

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by many companies in different countries. The extensive production of Spirulina is due to its original chemical composition (proteins, polyunsaturated fatty acids and vitamins). Dried spirulina contains

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about 60% proteins, as well as, many pigments which may be beneficial, including beta carotene, zeaxanthin, etc, plus phycobiliproteins, such as c-phycocyanin and allophycocyanin (Campanella,

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Crescentini, & Avino, 1999; E. G. Oliveira, Rosa, Moraes, & Pinto, 2009; Tang & Suter, 2011). Among

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those the most important functional ingredients are phycobiliproteins, a pigment-protein complex which is used into various food products to enhance their nutritional qualities acting as food

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colorant, antioxidant and emulsifier, and can sufficiently replace or reduce the use of synthetic additives. C-Phycocyanin is the main pigment and reaches 20% in dry weight of the cell protein,

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depending on cultivation conditions (Cuellar-Bermudez et al., 2015), and lately has been approved by the Food and Drug Administration as a natural blue food colorant (Fda, 2014). For the effective recovery of phycocyanin, the nutrient should be extracted from the microalgae biomass. Dehydration is considered an essential step pre-treatment step due to the high moisture content of the material, however it may lead to the decrease of its phycocyanin content. The drying of Spirulina constitutes approximately 30% of the total production cost (Elizangela G. Oliveira, Duarte, Moraes, Crexi, & Pinto, 2010). The most commonly used method is the spray drying

ACCEPTED MANUSCRIPT technique, in which the product is obtained in powder form, but usually, the powder does not satisfy all the criteria required for food use (Desmorieux & Decaen, 2005). Desmorieux et al., (2005) reported that apart from high operation cost, the dried product obtained by spray drying did not have the same aspect and color as the low-cost drying methods (Desmorieux & Decaen, 2005). Greenhouse drying and oven-drying have the potential to approach the advantages derived from

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spray drying in terms of quality and bioavailability at a lower cost (Tiburcio, Galvez, Cruz, & Gavino,

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2007). The development of an optimized drying process would therefore encourage farmers to grow Spirulina, as this will give them the assurance that they can produce dried Spirulina of marketable

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quality.

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The crucial step for the efficient phycocyanin recovery is the selection of the optimum combination of extraction technique and solvent system. The increasing legislative restrictions on the use of

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organic solvents in foods coupled to their negative effects on the functional properties of compounds are a challenge for an effective and eco-friendly recovery process (Chemat et al., 2017;

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Chemat, Vian, & Cravotto, 2012). Nowadays, Ultrasound Assisted Extraction (UAE) due to its high

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efficiency and low solvent consumption tends to replaced conventional extraction methods (Dey & Rathod, 2013; Patist & Bates, 2008). The higher yield obtained in UAE is of major interest from an

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industrial point of view, since the technology is an “add on” step to the existing process with minimum alteration, application in aqueous extraction where organic solvents can be replaced with

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generally recognised as safe solvents, shortening the extraction time (Vilkhu, Mawson, Simons, & Bates, 2008). The use of ultrasonic for extraction purposes in high-cost raw materials is an economical alternative to traditional extraction processes, which is an industry demand for a sustainable development. The environmental assessment of industrial processes, such as the phycocyanin recovery is based on Life Cycle Assessment (LCA) procedure. LCA is a tool that is being used to evaluate the environmental aspects associated with the entire life cycle of a product or process. It monitors the

ACCEPTED MANUSCRIPT materials, energy and wastes involved in each phase of the product’s life cycle, from raw materials extraction to final disposal, by compiling an inventory of elementary flows. These flows are then assigned to environmental impact categories according to the substances‘ ability to contribute to different key environmental issues (JRC European Commission, 2010). The results of the analysis will allow identifying the phases/processes and the relevant resource flows along the entire chain with

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the most significant environmental impacts (Andersson, Ohlsson, & Olsson, 1994; Schau, Fet, &

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Declarations, 2008). This will lead to the suggestion of effective alternative interventions (either technologies or management practices) for the upgrading of the food chain towards the reduction of

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its overall environmental footprint. These interventions may include, among others, (a) source

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material reduction, (b) process upgrading, (c) reuse of waste material and (d) recycling or composting practices (Kyriakopoulou, Papadaki, & Krokida, 2013, 2015; Schau et al., 2008). In

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addition, LCA as a tool can lead to the development of a cross-disciplinary agenda of research, linking together agricultural, environmental, social, and health concerns under the principle of

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sustainability and also applying them to daily consumption practices (Schau et al., 2008).

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In this study, the environmental performance of the isolation of phycocyanin from Spirulina platensis microalga using UAE and polar solvents such as water, ethanol or phosphate buffer was evaluated

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using the Life Cycle Assessment methodology. The analysis was carried out, using the Ecoinvent 2 database provided with the SimaPro 7. software (Pre’ Consultants, 2014; simapro manual PRe

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Consultants, 2008).The system examined included the open pond cultivation of the microalgae, the hot air drying step and their extraction processing for the recovery of extracts equivalent to 1 kg of phycocyanin.

2. Life cycle assessment methodology According to International Standards Organization (ISO) 14000 series, the technical framework for LCA methodology consists of four phases: (1) the goal and scope definition; (2) the inventory

ACCEPTED MANUSCRIPT analysis; (3) the impact assessment; and (4) the interpretation (ISO, 2006). Defining the goal and scope involves the description of the purpose, the functional unit (FU), the system boundaries, the data quality, assumptions and simplifications. The life cycle inventory involves collecting data for each unit process regarding all relevant inputs and outputs of energy and mass flows, as well as data on emissions to air, water and soil. The life cycle impact assessment phase evaluates the potential

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environmental impacts of the examined system, product and processes, while in the interpretation

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phase the results of the inventory analysis and the environmental impacts are combined together.

3. Goal and scope definition

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The objective of this study was to compare the environmental aspects and impacts associated with different extraction methods for the recovery of phycocyanin from the microalga Spirulina Platensis

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and to identify the ‘‘hot spots’’ of the evaluated technologies as a way to potentially improve their environmental performance. This study includes the analysis of different production stages, such as

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the cultivation, the harvesting, the drying treatment and the extraction of the microalgal biomass till

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the recovery of phycocyanin rich extracts. To evaluate the impact of the different processes, an inventory registration of the material and energy flows, as well as, the emissions that occur over

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these processes was established.

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3.1. Functional unit To remove performance variation and provide a fair comparison between the extracts recovered by the proposed production lines, the functional unit was defined as 1 kg of phycocyanin extracted from dried Spirulina platensis microalga to be used as a natural colorant and bioactive component in food, nutraceuticals and cosmetic applications. In general, the functional unit defines what the LCA study is measuring and provides a reference to which the inputs and outputs can be related. A direct comparison of different materials is not in accordance with ISO 14040 as their properties may vary and lead to unambiguous definition regarding their common function (Kyriakopoulou et al., 2015).

ACCEPTED MANUSCRIPT 3.2. Product system and system boundaries The system model is based on a fictional microalgae farming establishment using open pond cultivation systems. The open pond (raceway) infrastructure was selected over photo-bioreactors due to their simple technology and low energy, as well as, the fact that they are inexpensive and well researched on different strain cultivations (Jorquera, Kiperstok, Sales, Embiruçu, & Ghirardi, 2010).

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The simplicity of raceway ponds lays on their construction, since they are shallow open basins with a considerate length constructed using a concrete shell lined with polyvinyl chloride (PVC) with various

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dimensions (Aitken & Antizar-Ladislao, 2012). On the contrary to photo-bioreactors, which present

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high energy and cost intensity production and operation, their energy demand is limited, especially when utilizing natural sunlight, decreasing their environmental impact. Open pond systems have

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been used for the production of Spirulina biomass (Chanawongse, Lee, Bunnag, & Tanticharoen, 1994; Radmann, Reinehr, & Costa, 2007). Moreover, the warm and sunny climate of the Aegean and

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Mediterranean regions are ideal for outdoor cultivation, while algae can be also grown in

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Greek island of Chios was selected.

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greenhouses utilizing the sunlight. Therefore, to place the microalga cultivation for this study the

In this LCA analysis, cradle-to-gate systems of phycocyanin rich extract production were considered, broken down into two stages; the acquisition and treatment of the different raw material prior to

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extraction (cultivation, harvesting, drying, etc.) and the extraction process for the recovery of

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phycocyanin which can be used as additive in food, nutraceutical or cosmetic products. As in previous studies energy, water, carbon dioxide and nutrient requirements are treated as system inputs, while solar radiation provided for the cultivation stage is not modelled in the process as it is considered a natural energy source (Kyriakopoulou et al., 2015). Regarding the open pond installation, site leveling works for capital infrastructure, including plumbing, pumps, sheds, processing plant and machinery is not taken into consideration, due to the assumed low attribution of these elements, while the land occupation and the energy demands for the biomass production were incorporated in the analysis. The energy consumption of each component employed in the

ACCEPTED MANUSCRIPT production process (pumps, centrifuge, drier, ultrasound extractor) was calculated based on their specification (considering commercial equipment) and application time.

4. Life cycle inventory Spirulina platensis has been widely used as a food supplement due to its high protein content and

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nutritional value. In order to acquire the desired biomass several steps including cultivation of the

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treatment (drying temperature, storage conditions, etc.).

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microalgae in appropriate media, the harvesting of the biomass and the conditions of post-harvest

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4.1. Cultivation, harvest of microalgae Industrially applied microalgae cultivation systems include shallow big ponds, tanks, circular ponds and raceway ponds, however according to FAO the commercial cultivation of Spirulina platensis is

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done outdoors in open-air cultures (FAO, 2008). The microalga thrives in alkaline environment and this preference prevents external contamination, suggesting its suitability for environmental

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applications (Wuang, Khin, Chua, & Luo, 2016).

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For the cultivation phase of this study shallow ponds incorporated with low energy-consuming paddlewheels for gas/liquid mixing and circulation, called raceway ponds, are the available

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infrastructures. Guillard F/2 commercial media, is selected for the cultivation, diluted in the manufacturer's recommended dilutions (1:1000) in tap water with 30 g/L marine salt (Wuang et al.,

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2016). According to Jorquera et al. (2010), temperature regulation is performed during liquid evaporation, since the culture medium is directly exposed to the atmosphere (Jorquera et al., 2010). Even though open-air cultures are easy to construct and operate, they present several limitations such as large area requirement, contamination problems, impractical control of some environmental factors and the high running costs due to the large saltwater volumes (Prieto et al., 2011). The biomass productivity in such systems can reach up to 15 g m-2 d-1 (Jimenez et al., 2003; De Bhowmick et al., 2014), however it is difficult to maintaining optimal cultivation parameters (Guterman et al., 1990)(Zhang et al., 2015).

ACCEPTED MANUSCRIPT Following, the cultivation, a series of dewatering and drying processes are applied in order to concentrate the microalgae biomass. According to Grierson et al. (2013), a concentration of the microalgae cultivation by a factor of 18.4 can be achieved by flocculation is applied, followed by air floatation (Grierson et al., 2013). An additional concentration can be achieved by centrifugation leading to approximately 28.6% solids content and a recycling of the growth medium (Grierson et al.,

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2013). The moisture content of the Spirulina platensis biomass after centrifugation is around 88%

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(Olguín, Galicia, Camacho, Mercado, & Pérez, 1997; Stramarkou, Papadaki, Kyriakopoulou, & Krokida, 2016). Combining the cultivation and handling specifications of Spirulina with input and

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output data for open pond microalga cultivations provided by the literature (Campbell, Beer, &

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Batten, 2011; Collet et al., 2011), the mass and energy balances of the life cycle inventory of the cultivation and harvesting for the production of 1 kg wet biomass were constructed. This inventory

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analysis refers to a fictional open pond cultivation system utilizing sunlight and sea water, along with several nutrients for the cultivation of Spirulina biomass. Energy is required for pumping and

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recirculating the water, for pumping the carbon dioxide needed for the cultivation and for the

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dewatering of the biomass by centrifugation. In table 1 are presented the inputs and outputs of the inventory for the production of 1 kg wet Spirulina biomass.

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4.2. Pretreatment of Spirulina Platensis and Phycocyanin recovery Phycobiliprotein recovery involves the cell rupture of the microalga and the release of these proteins

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from within the cell (Moraes, Sala, Cerveira, & Kalil, 2011). Traditionally, the recovery is achieved with the use of polar solvents such as water, ethanol and buffer (Mahadev, 2005). The microalgal biomass is suspended and the pigments are leached in the selected solvent (Prabuthas, Majumdar, Srivastav, & Mishra, 2011). However, the wet biomass is immediately susceptible to bacterial decomposition and degradation due to of its nutritional composition. Therefore, dried biomass treatment gained popularity since it leads to effective storage and handling and at the same time retain the bioactive content (Elizangela G. Oliveira et al., 2010).

ACCEPTED MANUSCRIPT The microalgae paste collected is dried in an industrial rotary dryer, as the one describe in the study of Show et al. (2015), and the dried product at approximately 4% moisture is turned to powder ready for extraction. According to Show et al. (2015), wet slurry containing 30% solids of microalgae is treated at 120 °C for about 10 s in a pilot drum-dryer consuming about 52 kWh. In their assessment on energy requirement for drying algae with a water content of approximately 94% to

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4%, they report that for the production of 1 kg of dry algae product 18.2 kg of water need to be

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evaporated. The energy demand for this process was is 15.7 Mcal, leading to 1.15 Mcal/kg of evaporated water (or 4.815 MJ/kg water) (Show, Lee, Tay, Lee, & Chang, 2015). In the case of the

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centrifuged Spirulina biomass with an initial moisture content of 88% to produce 1 kg of dry material

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with 4% moisture about 8 kg of wet biomass need to be treated. By the end of the drying process 7 kg of water will be evaporated using 8.05 Mcal (34.704 MJ) and the final dry product of 1 kg will

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contain about 0.04 Kg of water.

As the water content is a determining factor on the energy consumption, the dewatering and

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centrifugation steps prior to drying are considered essential to reduce the amount of water entering

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the rotary drier. Adjusting not only the initial moisture content (to lower levels), but also the moisture content of the dried microalga biomass (to acceptable but not extremely low levels) can

performance.

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affect the energy requirement, the processes economic viability and the environmental

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Nowadays, for the disruption of Spirulina’s cell walls from both dried or wet biomass, sonication is suggested since it enhances the bioactive compound recover leading to shorter treatment times (Dey & Rathod, 2013; Hadiyanto, 2016). UAE is considered an inexpensive extraction techniques which involves the use of ultrasound of 20 kHz to 40 kHz; which increases the permeability of cell walls and produces cavitation (the formation, growth, and implosive collapse of bubbles in a liquid) (Drosou, Kyriakopoulou, Bimpilas, Tsimogiannis, & Krokida, 2015; Kyriakopoulou et al., 2015). The cavitation effect helps in the cell wall disruption allowing the solvent to penetrate into the biomass

ACCEPTED MANUSCRIPT and increasing the contact surface area between the solvent and compounds of interest, resulting into increased mass transfer also through good mixing (Naveena, Armshaw, & Tony Pembroke, 2015). Therefore, UAE provides increased yielding and leaching rate, along with the advantage of reduced solvent volumes and temperatures, which is crucial for the recovery of thermolabile compounds (Dey & Rathod, 2013).

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For the recovery of phycocyanin from wet and dried Spirulina platensis, the combination of

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ultrasound with solvents such as water, ethanol and phosphate buffer (pH 7) is suggested (Agustini,

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Suzery, Sutrisnanto, & Ma’ruf, 2015; D. Kumar, Dhar, Pabbi, Kumar, & Walia, 2014; Stramarkou et al., 2016). The selection of solvents is based on the nature of phycobilins, which are covalently bound to

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a water-soluble protein that aggregates on the surface of the photosynthetic membrane located in the cytoplasm or in the stroma of the chloroplast (Cuellar-Bermudez et al., 2015). The most efficient

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pigment extraction procedures combine mechanical and chemical methods that lead to protein release (Sobiechowska-Sasim, Stoń-Egiert, & Kosakowska, 2014). However, each combination of

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solvent and extraction conditions leads to different yielding as it was presented in our previous study

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(Stramarkou et al., 2016). The difference in the yield on the compounds of interest, which can be a result of the material selected or the recovery process examined, has shown among different studies

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than it can affect the environmental performance of the production process (Kyriakopoulou et al., 2015; S. G. Papadaki, Kyriakopoulou, & Krokida, 2016; Silva et al., 2015).

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In this study phycocyanin recovery from both wet and dried Spirulina biomass is examined and the specifications of the extraction processes are presented in Table 2. The selection of the conditions was made based on previous experimental work in which the comparison on the yield of phycocyanin was made after treating the samples in selected extraction time, ultrasound power and temperatures (Stramarkou et al., 2016). Taking into consideration the experimental yielding the mass and energy balances of each extraction line case study were prepared for the production of 1 kg of phycocyanin in the form of liquid extract (Table 3).

ACCEPTED MANUSCRIPT 5. Environmental Impact assessment and Interpretation The environmental impacts associated with the recovery of bioactive, phycobilin-rich extracts from Spirulina platensis were quantified using the problem-oriented approach, CML 2 baseline 2000 v2.04 (Center for Environmental Studies, University of Leiden) method provided by the SimaPro LCA tool (de Bruijn, van Duin, & Huijbregts, 2002; European Commission - Joint Research Centre - Institute for

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Environment and Sustainability, 2010; Guinée et al., 2002). The selected method includes a series of impact categories, namely Ozone layer depletion, Human toxicity, Fresh water aquatic ecotoxicity,

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Marine aquatic ecotoxicity, Terrestrial ecotoxicity, Photochemical oxidation, Global warming

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(indicator of Carbon footprint), Acidification, Abiotic depletion and Eutrophication, and has been previously used for the evaluation of microalgae cultivation and different extraction processes for

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the recovery of bioactive compounds (Collet et al., 2014; Lardon, Hélias, Sialve, Steyer, & Bernard, 2009; Rodríguez-Meizoso et al., 2012). The impacts in this method are quantified with appropriate

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physical units based on the life cycle inventory data according to physically based formulae (Guinée

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et al., 2002).

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In this study a life cycle assessment from cradle to gate was performed including the production of wet and dried Spirulina platensis biomass and the extraction of the biomass for the production of 1 kg of phycocyanin in the form of extract. Therefore, the environmental performance of the

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phycocyanin recovery process lines involves the footprints of all the necessary steps; the cultivation

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(raceways ponds and harvesting), the drying and the final biomass treatment using different solvents. Each step was found to contribute to the environmental footprint according to its energy and resources consumption. Specifically, for the cultivation step the most important contributions to the energy demand come from the electricity required to circulate the culture and the embodied energy in pond construction. These energy fractions were reported to be 22% to79% and 8% to 70%, respectively (Slade & Bauen, 2013). In addition, the embodied energy in the fertilisers, such as nitrogen and phosphorus, is reported that it may contribute to the energy demand by 6% to 40% (Slade & Bauen, 2013). Despite

ACCEPTED MANUSCRIPT these energy demands, the cultivation stage yields to some net benefit in relation to global warming impact due to the intake of CO2 from microalgae, presenting a slightly reduced overall carbon footprint. However, the electricity usage, also for the centrifugation, and fertilizer consumption contribute not only the global worming potential but also to the acidification and abiotic depletion potential.

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On the other hand, the recycling of harvest water during dewatering and centrifugation reduces the

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nutrient use and controls the fertilizer usage, which leads to the reduction of the natural resources

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depletion and the environmental impacts during nutrients production and use (Clarens, Resurreccion, White, & Colosi, 2010; Lardon et al., 2009). Eutrophication potential is reduced due to

oxides to the air (Kyriakopoulou et al., 2015).

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the avoidance of nitrate and phosphate leaching to the ground water, and ammonia and nitrous

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Although the dewatering steps together with drying have been reported as the most energy and cost intensive steps in algal biomass production due to the low concentration of microalgae in the culture

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medium (Soomro et al., 2016; Uduman, Qi, Danquah, Forde, & Hoadley, 2010), dewatering and

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centrifuge operation are considered an important step for condensing the microalga paste prior to extraction or drying (Grierson et al., 2013). Therefore, the selection of energy efficient systems is

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important to result to lower energy consumption and lower emissions (Weschler, Barr, Harper, & Landis, 2014). However, the selection of the drying processing can significantly affect the quality of

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the final dried microalga powder and thus the extractability of the targeted bioactive compounds (S. Papadaki, Kyriakopoulou, Stramarkou, Tzovenis, & Krokida, 2017). In this study an industrially applied hot air drying method was selected for all the production lines. Although the dried powder used in all extraction processes was considered of the same quality, the experimental extraction results for each process presented significant differences, affecting greatly the environmental performances of phycocyanin production.

ACCEPTED MANUSCRIPT As far as the extraction of both wet and dried biomass is concerned, the environmental impact is primarily affected by the energy demand of the ultrasound extractor and the nature of the solvent system used. The energy demand according to the experimental data was 54 MJ/kg dry biomass treated, while the corresponding energy demands for other ultrasound extractors can reach up to 148 MJ/kg biomass treated depended on the time of the treatment, the scale and the specifications

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of the machinery used (Ferreira, Dias, Silva, & Costa, 2016). However, focusing further on the

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recovery of bioactive compound the crucial factor for the overall environmental performance of the recovery process is the achieved yield. If the overall footprint of the process is allocated to the

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phycocyanin yield, pretreatment steps of high energy demand such as wet biomass drying influence

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insignificantly the overall environmental performance. Even though drying is considered an energy demanding step, especially due to the high moisture content of Spirulina platensis, the effect of the

the overall environmental performance.

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treatment on the phycocyanin content or its availability in the biomass showed opposite results on

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Examining the treatment processes for the extraction of phycocyanin from wet biomass using water,

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ethanol and buffer as solvent (Table 4), significant differences on several impact categories were observed. Among the three ultrasound assisted extraction processes, UAE 2 using ethanol as solvent

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exhibited the lowest environmental impact in categories of carbon footprint, ozon layer depletion and human toxicity even though an organic solvent was used. The positive effect of ethanol on the

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recovery of phycobiloproteins affects the environmental performance positively, since less biomass is needed to be cultivated, harvested and extracted for the recovery of the desired amount of phycocyanin. However, in some cases the effect of ethanol overcomes the positive effect of the high yielding, leading to slightly worsen impact, especially in the photochemical oxidation and the ozone layer depletion potential. The combination of buffer as solvent with dried Spirulina platensis biomass leads to significantly higher phycocyanin yields, contributing the lowest on the overall environmental performance,

ACCEPTED MANUSCRIPT despite the fact that an extremely energy consuming step is added prior to extraction. The treatment of dried biomass with ethanol exhibits the highest footprints due to the low yielding and the high material (biomass and solvent) consumption (Table 5). The carbon footprint of the two preferable extraction procedures for wet (UAE 2) and dried biomass (UAE 6) is 2.06E+03 and 1.18E+03 kg CO2 eq, respectively. This difference of the latter from the

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former is around 43% and shows once more that the yielding is the crucial factor for the

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environmental performance of phycocyanin recovery. The drying step prior to extraction permits the

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highest penetration of the solvent resulting to higher extraction rates, leading to lower biomass requirements and energy consumption for the extraction process (Drosou et al., 2015). If the daily

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consumption of phycobiloproteins is taken into consideration, functional food products containing small amounts of phycobilin will be burdened by the environmental footprint of the recovery

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process in an extent corresponding on the mass of the bioactive incorporated in the product. Therefore, with such low burdens a promising pathway for the use of cyanobacteria by industries to

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produce cell biomass for the production of high value products lies ahead (Mazard, Penesyan,

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Ostrowski, Paulsen, & Egan, 2016). Modifying cyanobacteria to utilize waste CO2 can yield industrialand food-grade sugars and a nutritional range of products, such as amino acids, nutrients and

6. Conclusions

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vitamins, making them ideal alternatives to plants for carbohydrate feedstocks (Hays & Ducat, 2015).

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Spirulina platensis is a natural rich source of bioactive compounds which can be recovered under several conditions and steps. The treatment steps employed involve the cultivation, harvesting, drying and extraction of the microalga cells. The environmental performance of each process is affected by the usage of vast amounts of biomass, consumables and energy for the recovery of 1 kg of phycocyanin thus extraction processes enhancing the yielding of the process such as UAE are considered essential. UAE’s low cost, short time and medium environmental impact and in combination with the right pretreatment and solvent can be even greener. Comparing wet and dried biomass treatment, wet biomass in combination with organic solvents such as ethanol exhibited high

ACCEPTED MANUSCRIPT selectivity leading to low environmental impact but still higher than that of dried biomass. In the case of dried biomass extraction, although drying is an energy consuming steps the combination with solvents such as buffer lead to extracts richer phycocyanin leading to unpredicted lower environmental impact. In both cases, the extraction techniques examined showed their potential for the recovery of phycocyanin rich extracts which can be used in nutraceutical, cosmetic and food

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industries.

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Acknowledgments

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The authors would like to thank Mrs. M. Stramarkou for her contribution and the information regarding the extraction of Spirulina platensis for the recovery of bioactive compounds and

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especially phycocyanin.

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Abbreviations

GWP: Global Warming Potential; LCA: Life Cycle Analysis/Assessment; LCI: Life Cycle Inventory; LCIA:

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Life Cycle Impact Assessment; UAE: Ultrasound Assisted Extraction

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ACCEPTED MANUSCRIPT Table 1. Life cycle inventory of the cultivation and harvest of the production of 1 kg wet Spirulina platensis biomass at an open pond installation occupying 1 ha.

1.0000

kg

0.0193 <0.0001

kg kg

397.6834 kg kg 0.6541

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MJ MJ MJ MJ MJ

(Collet et al., 2011) (Collet et al., 2011) (Collet et al., 2011) (Collet et al., 2011) (Campbell et al., 2011)

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0.4659 0.1031 0.6090 0.1279 0.0035

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kg kg kg kg kg ha

(Campbell et al., 2011) (Campbell et al., 2011)

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595.6757 1.4286 0.0069 0.0047 <0.0001 1

Reference (Campbell et al., 2011) (Campbell et al., 2011) (Campbell et al., 2011) (Campbell et al., 2011) (Campbell et al., 2011)

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Amount

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Inputs Water, salt, ocean Carbon dioxide Nitrogen Fertiliser Phosphorus Fertiliser Iron sulphate Occupation land open pond Electricity pumping water Electricity pumping CO2 Electricity paddlewheel Electricity centrifugation Traction Outputs Spirulina Platensis Emissions to air Carbon dioxide Nitrogen Emissions to water Water Salts

(Campbell et al., 2011) (Campbell et al., 2011)

ACCEPTED MANUSCRIPT Table 2. Specification of extraction conditions examined. UAE 1 wet paste

UAE 2 wet paste

UAE 3 wet paste

UAE 4 dry powder

UAE 5 dry powder

UAE 6 dry powder

water 1:20

ethanol 1:20

buffer 1:20

water 1:20

ethanol 1:20

buffer 1:20

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30°C, 5 min, 30°C, 5 min, 30°C, 5 min, 30°C, 5 min, 30°C, 5 min, 30°C, 5 min, 25 kHz, 25 kHz, 25 kHz, 25 kHz, 25 kHz, 25 kHz, 450 W 450 W 450 W 450 W 450 W 450 W

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*The extraction conditions are derived from the research of Stramarkou et al. 2016.

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Specs Spirulina platensis Solvent Solid liquid ratio (kg d.b.:L) Conditions*

ACCEPTED MANUSCRIPT Table 3. Life cycle inventory of the recovery processes for the extraction of phycocyanin in the form of liquid extract. UAE 3

UAE 4

UAE 5

UAE 6

0.46

2.71

0.94

3.22

0.02

3.41

217.39 1739.13

36.90 295.20

106.38 851.06

31.06 248.45

5000.00 40000.00

29.33 234.60

non

non

non

217.39

-

-

-

1046.74

168525.00

988.42

4347.83 11739.13

738.01 1992.62

2127.66 5744.68

621.12 1677.02

100000.00 270000.00

586.51 1583.58

1

1

1

1

1

1

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UAE 2

35000.00

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Experimental data Phycocyanin Yield (%)* Inputs LCI Material (kg d.b) Equivalent in wet biomass (kg) Water evaporated through drying (kg water) Energy for drying (MJ) Solvent (L) Electricity for extraction (MJ) Output LCI Phycocyanin content (kg)

UAE 1

205.28

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* The yield is referred to the consistency of the final extracts’ recovery of phycocyanin (Stramarkou et al., 2016) according to which the mass and energy balances are demonstrated in the Table.

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kg CFC-11 eq kg 1,4-DB eq kg 1,4-DB eq

UAE 2

UAE 3

5.49E+01 4.27E+01 1.51E+00

2.52E+01 9.92E+00 1.30E+00

2.69E+01 2.09E+01 7.37E-01

6.72E+03 4.68E-04 3.74E+03

2.06E+03 1.06E-04 7.95E+02

3.29E+03 2.29E-04 1.83E+03

9.52E+02

1.91E+02

4.66E+02

2.42E+06 3.49E+01

4.54E+05 6.85E+00

1.18E+06 1.71E+01

1.40E+00

9.44E-01

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kg Sb eq kg SO2 eq kg PO4-3 eq kg CO2 eq

UAE 1

kg 1,4-DB eq kg C2H4

1.93E+00

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kg 1,4-DB eq

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Impact Category Abiotic depletion Acidification Eutrophication Global warming (GWP100) Ozone layer depletion Human toxicity Fresh water aquatic ecotox. Marine aquatic ecotoxicity Terrestrial ecotoxicity Photochemical oxidation

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Table 4. Environmental footprint of processing wet Spirulina biomass for the recovery of 1 kg phycocyanin.

ACCEPTED MANUSCRIPT Table 5. Environmental footprint of processing dried Spirulina biomass for the recovery of 1 kg phycocyanin. UAE 5 3.86E+03 1.64E+03 1.86E+02 3.26E+05

UAE 6 1.00E+01 7.50E+00 2.61E-01 1.18E+03

kg CFC-11 eq kg 1,4-DB eq kg 1,4-DB eq

7.49E-05 6.31E+02 2.04E+02

1.57E-02 1.23E+05 3.69E+04

7.07E-05 5.96E+02 1.93E+02

kg 1,4-DB eq

5.20E+05

8.96E+07

4.91E+05

kg 1,4-DB eq kg C2H4

6.63E+00 3.48E-01

1.19E+03 2.02E+02

6.26E+00 3.29E-01

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kg Sb eq kg SO2 eq kg PO4-3 eq kg CO2 eq

UAE 4 1.06E+01 7.94E+00 2.76E-01 1.25E+03

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D

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Impact Category Abiotic depletion Acidification Eutrophication Global warming (GWP100) Ozone layer depletion Human toxicity Fresh water aquatic ecotox. Marine aquatic ecotoxicity Terrestrial ecotoxicity Photochemical oxidation

ACCEPTED MANUSCRIPT Highlights

PT RI SC NU MA D PT E CE

 

Solvent selection plays the most important role on phycocyanin recovery. Extraction with ethanol and buffer was considered the most sustainable choise for wet and dried biomass, respectively. Phycocyanin yield is the main factor affecting the environmental performance. Dried biomass extraction exhibited lower carbon foorprint for the recovery of phycocyanin.

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