Paula Perez-Lopez
email: paula.perez_lopez@mines-paristech.fr

Jeroen H De Vree

Gumersindo D Feijoo

Rouke Bosma

Maria J Barbosa

Maria Teresa Moreira

René H Wijffels

Anton J B Van Boxtel

Dorinde M M Kleinegris

Paula Pérez-López

María Teresa Moreira

Comparative life cycle assessment of real pilot reactors for microalgae cultivation in different seasons

Keywords: Microalgae cultivation, Life cycle assessment (LCA), tubular photobioreactor, open raceway pond, pilot plant, weather variations

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The scarcity of natural resources and particularly the exhaustion of fossil fuels are a global challenge to be addressed in forthcoming decades [START_REF] Draaisma | Food commodities from microalgae[END_REF][START_REF] Hallenbeck | Solar biofuels production with microalgae[END_REF][START_REF] Lam | Microalgae biofuels: A critical review of issues, problems and the way forward[END_REF]. The current production framework based on non-renewable energies poses several problems to society, including economic instability and political conflicts (due to raw material scarcity and related increasing prices), as well as environmental concerns [START_REF] Nienhueser | Economic and environmental impacts of providing renewable energy for electric vehicle charging -A choice experiment study[END_REF]. Alternative sources including biomass feedstocks such as vegetable oils, waste oils or algal lipids are currently under development to reduce harmful effects on environment and ecological threats such as global warming [START_REF] Hallenbeck | Solar biofuels production with microalgae[END_REF][START_REF] Clarens | Environmental life cycle comparison of algae to other bioenergy feedstocks[END_REF][START_REF] Zhang | Waste cooking oil-to-energy under incomplete information: Identifying policy options through an evolutionary game[END_REF].

Microalgae have shown a great potential for the production of numerous compounds with a wide variety of applications that include biofuels and other forms of energy as well as chemicals, food and feed, among others [START_REF] Draaisma | Food commodities from microalgae[END_REF][START_REF] Pérez-López | Life cycle assessment of the production of the red antioxidant carotenoid astaxanthin by microalgae: from lab to pilot scale[END_REF][START_REF] Van Boxtel | The potential of optimized process design to advance LCA performance of algae production systems[END_REF]. Some advantages of microalgae compared to other bioenergy feedstocks are their higher productivity per unit, the possibility to cultivate them on marginal land in fresh-or saltwater avoiding competition with food crops and the option of coupling their growth with the treatment of waste streams [START_REF] Hallenbeck | Solar biofuels production with microalgae[END_REF][START_REF] Clarens | Environmental life cycle comparison of algae to other bioenergy feedstocks[END_REF][START_REF] Brentner | Combinatorial life cycle assessment to inform process design of industrial production of algal biodiesel[END_REF][START_REF] Chisti | Energy from algae: Current status and future trends: Algal biofuels -A status report[END_REF][START_REF] Fortier | Life cycle assessment of bio-jet fuel from hydrothermal liquefaction of microalgae[END_REF][START_REF] Woertz | Life cycle GHG emissions from microalgal biodiesel -A CA-GREET model[END_REF]. Despite the advantages of microalgae and their lower requirements in categories such as land competition or eutrophication [START_REF] Clarens | Environmental life cycle comparison of algae to other bioenergy feedstocks[END_REF][START_REF] Razon | Net energy analysis of the production of biodiesel and biogas from the microalgae: Haematococcus pluvialis and Nannochloropsis[END_REF], some aspects of environmental sustainability, such as the energy balance or greenhouse gas emissions, are still liable to improve, especially for the use of microalgae for energy applications [START_REF] Lam | Microalgae biofuels: A critical review of issues, problems and the way forward[END_REF][START_REF] Clarens | Environmental life cycle comparison of algae to other bioenergy feedstocks[END_REF]. Life Cycle Assessment (LCA) has the potential to be used as a guiding tool for decision-making in process design [START_REF] Van Boxtel | The potential of optimized process design to advance LCA performance of algae production systems[END_REF][START_REF] Chiaramonti | Life Cycle Assessment of algal biofuels: Needs and challenges[END_REF]. LCA may contribute to identify the main bottlenecks to be addressed during the scale up towards sustainable industrial facilities.

Algae cultivation has been identified as a major contributor to the operational and embodied energy of microalgal processes [START_REF] Lam | Microalgae biofuels: A critical review of issues, problems and the way forward[END_REF][START_REF] Pérez-López | Life cycle assessment of the production of the red antioxidant carotenoid astaxanthin by microalgae: from lab to pilot scale[END_REF][START_REF] Stephenson | Life-cycle assessment of potential algal biodiesel production in the United Kingdom: A comparison of raceways and air-lift tubular bioreactors[END_REF]. The total energy demand for cultivation stage usually ranges from 0.1 up to 5 MJ energy input per MJ energy produced [START_REF] Brentner | Combinatorial life cycle assessment to inform process design of industrial production of algal biodiesel[END_REF][START_REF] Sills | Quantitative uncertainty analysis of Life Cycle 29 Assessment for algal biofuel production[END_REF]. This is mainly due to addition of nutrients and CO 2 [START_REF] Draaisma | Food commodities from microalgae[END_REF][START_REF] Clarens | Environmental life cycle comparison of algae to other bioenergy feedstocks[END_REF] and the specific requirements of the selected reactor configuration, such as mixing and temperature control [START_REF] Brentner | Combinatorial life cycle assessment to inform process design of industrial production of algal biodiesel[END_REF][START_REF] Stephenson | Life-cycle assessment of potential algal biodiesel production in the United Kingdom: A comparison of raceways and air-lift tubular bioreactors[END_REF][START_REF] Jorquera | Comparative energy lifecycle analyses of microalgal biomass production in open ponds and photobioreactors[END_REF].

The embodied energy for nutrients is related to manufacturing of synthetic fertilizers and the reactor materials as well as CO 2 production, whereas the operational energy consumption is linked to pumping for the delivery of culture medium and CO 2 [START_REF] Draaisma | Food commodities from microalgae[END_REF][START_REF] Clarens | Environmental life cycle comparison of algae to other bioenergy feedstocks[END_REF][START_REF] Brentner | Combinatorial life cycle assessment to inform process design of industrial production of algal biodiesel[END_REF]. Since algae are temperature sensitive, heating and cooling is required to operate relatively close to the optimal temperature of the algal species. Temperature regulation allows high productivities and prevents growth inhibition, but may increase the energy demand of the process [START_REF] Bosma | Design and construction of the microalgal pilot facility AlgaePARC[END_REF][START_REF] Slade | Micro-algae cultivation for biofuels: Cost, energy balance, environmental impacts and future prospects[END_REF]. The integration of options such as waste heat from power generation or cold water resources allows reducing the energy requirements for heating or cooling the water from room temperature to the set point temperature, and thus, contributes to the optimization of the cultivation stage [START_REF] Slade | Micro-algae cultivation for biofuels: Cost, energy balance, environmental impacts and future prospects[END_REF][START_REF] Taelman | The environmental sustainability of microalgae as feed for aquaculture: A life cycle perspective[END_REF]. Furthermore, climatic data including irradiation and temperature depend on geographic location. Therefore, the heating and cooling needs of the system vary between locations [START_REF] Moody | Global evaluation of biofuels potential from microalgae[END_REF]. The selection of an appropriate location according to available resources (energy, nutrients, waste heat, cooling water) and algal strains may serve to maintain the optimal temperature with low heating and cooling requirements so that the energy consumption is minimized.

The environmental performance of cultivation is also influenced by reactor selection.

Open raceway ponds (ORPs) and closed tubular photobioreactors (PBRs) are currently considered as the two most feasible existing configurations for large-scale cultivation of microalgae [START_REF] Chisti | Energy from algae: Current status and future trends: Algal biofuels -A status report[END_REF][START_REF] Stephenson | Life-cycle assessment of potential algal biodiesel production in the United Kingdom: A comparison of raceways and air-lift tubular bioreactors[END_REF].

Although simple reactors such as ORPs have fewer elements that consume energy than closed PBRs, the maximum biomass productivity is also lower and they are more sensitive to contamination risks. However, closed PBRs have higher costs of infrastructure and operation [START_REF] Stephenson | Life-cycle assessment of potential algal biodiesel production in the United Kingdom: A comparison of raceways and air-lift tubular bioreactors[END_REF][START_REF] Slegers | Scenario analysis of large scale algae production in tubular photobioreactors[END_REF]. These aspects should be considered when comparing the environmental performance of different configurations.

Numerous studies dealing with the environmental performance of different reactor designs for microalgae cultivation have been published [START_REF] Draaisma | Food commodities from microalgae[END_REF][START_REF] Clarens | Environmental life cycle comparison of algae to other bioenergy feedstocks[END_REF][START_REF] Brentner | Combinatorial life cycle assessment to inform process design of industrial production of algal biodiesel[END_REF][START_REF] Woertz | Life cycle GHG emissions from microalgal biodiesel -A CA-GREET model[END_REF][START_REF] Stephenson | Life-cycle assessment of potential algal biodiesel production in the United Kingdom: A comparison of raceways and air-lift tubular bioreactors[END_REF][START_REF] Jorquera | Comparative energy lifecycle analyses of microalgal biomass production in open ponds and photobioreactors[END_REF][START_REF] Taelman | The environmental sustainability of microalgae as feed for aquaculture: A life cycle perspective[END_REF][START_REF] Collet | Biodiesel from microalgae -Life cycle assessment and recommendations for potential improvements[END_REF][START_REF] Montazeri | Timedependent life cycle assessment of microalgal biorefinery co-products[END_REF].

However, most of this work considers hypothetical simulated scenarios or extrapolations from lab-scale data rather than existing pilot or commercial systems [START_REF] Draaisma | Food commodities from microalgae[END_REF][START_REF] Clarens | Environmental life cycle comparison of algae to other bioenergy feedstocks[END_REF][START_REF] Van Boxtel | The potential of optimized process design to advance LCA performance of algae production systems[END_REF][START_REF] Woertz | Life cycle GHG emissions from microalgal biodiesel -A CA-GREET model[END_REF][START_REF] Stephenson | Life-cycle assessment of potential algal biodiesel production in the United Kingdom: A comparison of raceways and air-lift tubular bioreactors[END_REF][START_REF] Jorquera | Comparative energy lifecycle analyses of microalgal biomass production in open ponds and photobioreactors[END_REF][START_REF] Montazeri | Timedependent life cycle assessment of microalgal biorefinery co-products[END_REF]. Few of them make a comparison between different reactor configurations, often restricted to a very limited set of indicators that only takes into account energy requirements and greenhouse gas emissions [START_REF] Draaisma | Food commodities from microalgae[END_REF][START_REF] Brentner | Combinatorial life cycle assessment to inform process design of industrial production of algal biodiesel[END_REF][START_REF] Stephenson | Life-cycle assessment of potential algal biodiesel production in the United Kingdom: A comparison of raceways and air-lift tubular bioreactors[END_REF][START_REF] Jorquera | Comparative energy lifecycle analyses of microalgal biomass production in open ponds and photobioreactors[END_REF]. Moreover, they are based on average growth parameters and omit the influence of weather fluctuations, affecting reactor stability, on the environmental results.

This work provides a comparative life cycle assessment (LCA) of the two most common reactor configurations (ORPs and tubular PBRs)

to evaluate the main environmental burdens of each option, to compare their performances and to identify bottlenecks for up-scaling. The evaluation considers the algal biomass production from the eustigmatophyte Nannochloropsis sp. due to its good biomass productivity and capability for high lipid content when stressed [START_REF] Razon | Net energy analysis of the production of biodiesel and biogas from the microalgae: Haematococcus pluvialis and Nannochloropsis[END_REF][START_REF] Rodolfi | Microalgae for oil: Strain selection, induction of lipid synthesis and outdoor mass cultivation in a low-cost photobioreactor[END_REF]. The evaluation is based on data from three real reactors operated in parallel at AlgaePARC pilot facility (Wageningen, The Netherlands) [START_REF] Vree | Comparison of four outdoor pilot-scale photobioreactors[END_REF]. The use of real pilot data is expected to overcome current concerns of microalgal LCAs related to the lack of large-scale information [START_REF] Chiaramonti | Life Cycle Assessment of algal biofuels: Needs and challenges[END_REF]. The data are obtained for comparable weather conditions for each reactor, during three seasons (summer, fall and winter). These systems have been designed and operated as a first step to facilitate the transition from laboratory research to outdoor production for industrial applications [START_REF] Bosma | Design and construction of the microalgal pilot facility AlgaePARC[END_REF].

Materials and methods

AlgaePARC cultivation systems

AlgaePARC is a research facility of Wageningen University and Research (The Netherlands) that was built with the aim of comparing different PBRs and optimizing process control strategies for microalgae cultivation and processing. The main objective of this facility is to develop systems with low production costs and energy requirements that can serve as a basis for the improvement of large-scale microalgae plants [START_REF] Bosma | Design and construction of the microalgal pilot facility AlgaePARC[END_REF].

AlgaePARC outdoor facilities comprise several pilot-scale photobioreactors, including horizontal and vertical tubular PBRs and an ORP.

Among the available systems, the operation of the The horizontal tubular PBR system (0.56 m 3 ) consists of three loops with transparent pipes that are placed in parallel at the same height, whereas the vertical system (1.06 m 3 ) is composed of seven loops with pipes "stacked" on top of each other. In both systems, the culture medium is divided over the loops by a distribution header.

The accumulation of excess oxygen is avoided by using an oxygen stripper. This system receives air, blown by a compressor via a sparger at the bottom. Oxygen is transferred from the liquid to the gas phase and leaves the stripper at the top.

The stripper contains three heat exchange spirals to keep the culture temperature between a minimum and a maximum value.

The operation of the three systems was performed under different Dutch weather conditions that can be classified as "summer", "fall" and "winter". However, the "winter" operation of the ORP was unfeasible as heavy rainfall in this period resulted in a too high dilution of the ORP in combination with the low solar radiation level and low temperatures.

Geometric and average operating parameters in each reactor and season are specified in Table 1.

Life cycle assessment methodology

Life Cycle Assessment (LCA) was used in the comparative evaluation of the environmental aspects and potential impacts of algae cultivation.

LCA allowed a systematic evaluation of the environmental performance of the studied systems throughout the whole process chain, from raw materials extraction to microalgal cultivation and waste disposal. Following the ISO 14040 standards [START_REF]ISO 14040. Environmental Management -Life Cycle Assessment -Principles and Framework[END_REF], four sequential stages were undertaken, namely i) goal and scope definition, ii) inventory analysis, iii) impact assessment and iv) interpretation of the results. The procedures and assumptions considered for each stage are detailed below.

Goal and scope of the study

The goal of the present study was to assess three different reactor configurations for the pilotscale production of Nannochloropsis sp in three periods of the year (summer, fall and winter). With this work, bottlenecks in environmental performance (referred to as hot spots) of the systems were identified.

Since the environmental performance of the different reactors is linked to the biomass production, the life cycle inventory and impact assessment are referred to 1 kg of produced biomass dry weight, contained in a 22% DW slurry, as the functional unit (FU).

The system boundaries were divided into foreground and background processes, referring to the steps that are directly affected by the study, and the processes that supply energy and materials via a homogeneous market, respectively [START_REF] Azapagic | Allocation of environmental burdens in multiple-function systems[END_REF]. The processes in the foreground systems were classified in four subsystems, shown in Figure 2: i) cleaning of the reactor, ii) preparation of the culture medium, iii) cultivation and iv) biomass concentration.

i) Cleaning of the reactor (S1)

In the first stage of the process, tap water was pumped to a silo (6 m 3 ) and sterilized with hypochlorite (2 mg/L) and passed through activated carbon filters to remove the hypochlorite before being supplied to the systems.

Before inoculation, each reactor was flushed with sterilized tap water to ensure the absence of competing algae and protozoa. In the case of the tubular PBRs, the system was rinsed with tap water three times. In the second rinse, 3% of a disinfection agent (containing hydrogen peroxide) was added, whereas in the third one, 0.5 g/L plastic beads for biofilm removal were used. For the ORP, tap water in a quantity equal to three times the usable volume of the reactor and a vacuum cleaning system was used for 1-2 h after the last washing step to remove all water.

ii) Preparation of the culture medium (S2)

The main source of nutrients for the

iii) Cultivation (S3)

This stage consisted of a semi-continuous process in which the biomass was operated with a fixed daily dilution rate and harvested according to the scheme explained by de Vree et al. [START_REF] Vree | Comparison of four outdoor pilot-scale photobioreactors[END_REF]. As no source of artificial light was provided, light intensity only depended on weather conditions. To maintain temperature close to the optimal temperature of the species, a central chiller and heater were used. To minimize the energy consumption, the culture temperature varied between 20 and 30ºC. Heating was applied to prevent temperatures below 20ºC and cooling when culture temperature rose above 30ºC. The set point was selected according to

Nannochloropsis optimal range of 20-30°C [START_REF] Sandnes | Combined influence of light and temperature on growth rates of Nannochloropsis oceanica, linking cellular responses to large-scale biomass production[END_REF].

No active cooling was needed for the ORP since cooling in this system occurs naturally by water evaporation [START_REF] Bosma | Design and construction of the microalgal pilot facility AlgaePARC[END_REF]. The purity of the culture was checked microscopically (Leica Laborlux S) three times per week to minimize contamination problems.

iv) Biomass concentration (S4)

Despite similar environmental conditions, the final biomass concentration varied for a given period depending on the cultivation system and seasonally due to the different weather conditions.

In order to make relevant comparisons between the performances of the different reactors, microfiltration and centrifugation were applied to obtain a defined biomass concentration (22% DW), regardless of the reactor system and season.

The life cycle inventory and determined environmental impact assessment are referred to 1 kg of produced biomass (dry weight), so 4.55 kg of slurry (22% DW).

Inventory analysis: Data acquisition and assumptions

The collection of inventory data related to the significant inputs and outputs to the system under study is an essential step of LCA methodology. In this study, the information for the foreground system mainly consisted of primary data collected in the facility.

The inputs and outputs for the cleaning stage (S1) were quantified with respect to the volume of water, by assuming a water consumption equal to three times the total volume of the corresponding reactor for each cleaning event. During the full monitoring period, the tubular PBRs were cleaned once after each of the six performed cultivations.

For the ORP the length of each cultivation period was shorter (due to several washouts resulting from unfavorable weather conditions and high dilution rates at some periods of the operations) and 10 cleaning cycles were applied in total. The total quantity of inputs for each evaluated period was estimated according to the ratio between the duration of the period and the total feasible operation time per year (approximately 10 months).

Similarly, the water and chemicals for the preparation of the culture medium (S2) were calculated by considering the average dilution rate of each period. The energy requirements were estimated with respect to the total seawater and medium, assuming that the equipment was operating at the maximum allowed capacity.

Regarding the cultivation stage (S3), the energy consumption for the different operations S1 in Supplementary Material).

The inputs for the biomass concentration (S4)

were the energy consumptions for the consecutive units of microfiltration and centrifuge, which were calculated according to the total volume of medium to separate from the biomass in order to achieve the final 22% DW concentration.

In all subsystems, the solid wastes were assumed to be disposed in either sanitary or inert landfills, whereas the resulting wastewater was collected in the general sewage system and treated in a conventional wastewater treatment plant. An average transport distance of 200 km was considered for chemicals and building materials and 50 km was estimated for wastes. No seawater transport was considered, since it was assumed that a commercial scale facility would be placed close to the coast. Materials for auxiliary equipment used in the process (filters, pumps, centrifuge, etc.) were neglected, since this equipment was shared between several systems and the corresponding quantities for each system after applying the appropriate allocation procedures would be very limited. Moreover, the equipment was common to the three analyzed reactors and thus, no additional information for the comparative purposes of the present work would be obtained.

The inventory data related to the background system were obtained from Ecoinvent database [START_REF] Frischknecht | Overview and methodology[END_REF]. These inputs include the production of the chemicals required for the cleaning and the nutrients for the culture medium, as well as the production of electricity used throughout the stages of the processes, the manufacture of the building materials for each reactor and the waste disposal. With regard to NaNO 3 production, this process is not defined in the Ecoinvent database.

Therefore, the considered inventory data correspond to the synthetic process as described by Pérez-López et al. [START_REF] Pérez-López | Life cycle assessment of the production of the red antioxidant carotenoid astaxanthin by microalgae: from lab to pilot scale[END_REF].

No allocation procedure was required, according to the goal of the study (the comparison of the different reactor configurations). Thus, all the environmental burdens were allocated to the total quantity of biomass harvested from each reactor.

The inventory data of the assessed scenarios are shown in Table 2. Consultants [START_REF] Frischknecht | Implementation of Life Cycle Impact Assessment methods[END_REF].

The impact categories were selected according to the most relevant environmental issues related to microalgal products and used in previous LCA studies [START_REF] Pérez-López | Life cycle assessment of the production of the red antioxidant carotenoid astaxanthin by microalgae: from lab to pilot scale[END_REF][START_REF] Collet | Biodiesel from microalgae -Life cycle assessment and recommendations for potential improvements[END_REF][START_REF] Collet | Recommendations for Life Cycle Assessment of algal fuels[END_REF][START_REF] Lardon | Life-cycle assessment of biodiesel production from microalgae[END_REF]. Eleven potential impact categories (specified in Table 3)

were evaluated according to the CML 2001 methodology. CED methodology was applied to obtain the total energy consumption throughout the whole process (Total CED), which included three categories of non-renewable sources of energy and three categories of renewable sources.

The inventory data were implemented in the software SimaPro 8 [START_REF] Goedkoop | Introduction to LCA with SimaPro[END_REF].

Results and discussion

Identification of hot spots for each reactor configuration

The LCA characterization results for the two applied methodologies (CML and CED) in each of the eight evaluated scenarios are listed in Table 3.

Figure 3a shows the average distribution of impacts for each reactor configuration, for the subsystems cleaning (S1), nutrient supply (S2), cultivation (S3) and concentration (S4). Figure 3 shows that, as expected, the cultivation stage is the main hot spot for all the reactors in nearly all the analyzed categories, with 80% or more impact.

This result confirms, for all operational systems, the findings of previous studies based on hypothetical scenarios with extrapolated data regarding the importance of cultivation stage [START_REF] Lam | Microalgae biofuels: A critical review of issues, problems and the way forward[END_REF][START_REF] Brentner | Combinatorial life cycle assessment to inform process design of industrial production of algal biodiesel[END_REF][START_REF] Stephenson | Life-cycle assessment of potential algal biodiesel production in the United Kingdom: A comparison of raceways and air-lift tubular bioreactors[END_REF]. These studies indicated that energy requirements and GWP of algal cultivation could represent more than 90% of the total environmental burdens for tubular PBRs and 55%

for the ORP [START_REF] Brentner | Combinatorial life cycle assessment to inform process design of industrial production of algal biodiesel[END_REF][START_REF] Stephenson | Life-cycle assessment of potential algal biodiesel production in the United Kingdom: A comparison of raceways and air-lift tubular bioreactors[END_REF]. For AlgaePARC facility, the main contributions to the total CED are those of NR-F (85% of total CED) and NR-N (10% total CED). These are two important categories that can be reduced by minimizing the energy requirements for algal products.

Concerning S2, only the CML categories of AP, EP, TEP and POFP, as well as the CED category of NR-B show significant contributions.

Most of these contributions are associated to the production of the nitrogen source. S1 and S4 have a very limited contribution to all the evaluated categories. S4 has for all categories more influence for the ORP than for the tubular PBRs. This is because the achieved biomass concentrations in the ORP are significantly lower than in the tubular PBRs. Both S1 and S4 have contributions above 5% for the categories of EP, TEP and R-HYD. For the tubular PBRs, the highest contribution from S1 is associated with the category of HTP, with only 4-6%, while the contribution from S4 only exceeds 1% for the summer and fall periods in the categories of EP, TEP and R-HYD. Figure 3b illustrates the breakdown of the contributions per involved production process.

The impact of the electricity production for cultivation constitutes the main environmental burden of S3. The figure shows that electricity for cultivation has contributions between 80-95% in most of the categories. The role of energy requirements with respect to the total environmental impacts is consistent with other studies in which the electricity for cultivation has been identified as the main hot spot, but with slightly lower relative contributions than that of AlgaePARC pilot systems [14-15, 20, 33]. For example, Stephenson et al. [START_REF] Stephenson | Life-cycle assessment of potential algal biodiesel production in the United Kingdom: A comparison of raceways and air-lift tubular bioreactors[END_REF] reported that electrical power during cultivation in ORPs has a contribution of 74% to fossil energy requirement and 65% to GWP. Similarly, energy was identified by Lardon et al. [START_REF] Lardon | Life-cycle assessment of biodiesel production from microalgae[END_REF] as one of the main causes of impact for a raceway pond (with contributions between 42-75% to CED and 18-36% to GWP), together with fuel combustion and use of fertilizers.

As further discussed in section 3.2., the main reason for the high energy consumption is the use of an electrical heater and chiller in this pilot plant. These units, used for temperature control, work fine for a pilot facility, but at large scale should be replace by much more efficient technologies, for example, using waste heat or a storage temperature buffer in the ground. It should be remarked that the absolute values in Table 3 are based on pilot-scale systems, and may change significantly after scaling up to commercial scale.

Electrical efficiencies of the equipment are expected to improve in commercial systems [START_REF] Ruiz | Towards industrial products from microalgae[END_REF].

This improvement jointly with optimized equipment dimensioning (to avoid oversizing) could reduce the energy consumption by approximately 66% when moving from pilot to full scale [START_REF] Liu | Pilot-scale data provide enhanced estimates of the life cycle energy and emissions profile of algae biofuels produced via hydrothermal liquefaction[END_REF]. The main trends for up-scaling, including the expected differences between the evaluated pilot-scale systems and hypothetical industrial-scale facilities, are presented in section 3.4.

The production of building materials for the reactors (plastics, steel and aluminum grouped as "infrastructure" in Figure 3b) or the compressed carbon dioxide, also included in S3, have low contributions. The infrastructure is responsible for more than 5% of the impacts in six of the eight assessed scenarios: HTP, POFP, LC and R-HYD.

The contribution of this production process only exceeds 16% for the category of R-HYD.

However, this category represented less than 1% of total CED in all scenarios. Most of the impacts from infrastructure are associated with the production of metals, specifically aluminum for the tubular PBRs (used for the supporting structure) and steel for the ORP (among others used for the shaft of the paddle wheel and the tubes of the heat exchanger).

Among the other processes, the production of nutrients has the highest impact, although it is restricted to the categories AP (between 14-30% depending on the season), NR-B (from 40% to 65%) and to a lesser extent to EP, TEP and POFP (from 4 to 10%). Sodium nitrate, which comprises more than 90% of the nutrients, has the highest contribution. Substituting the main nitrogen source by other alternatives would have a limited effect on impact reduction under the evaluated conditions. This finding differs with previous research that mentions a noticeable influence of the production of fertilizers in the environmental profile [START_REF] Draaisma | Food commodities from microalgae[END_REF][START_REF] Clarens | Environmental life cycle comparison of algae to other bioenergy feedstocks[END_REF][START_REF] Lardon | Life-cycle assessment of biodiesel production from microalgae[END_REF]. The low relative contribution of nutrients is caused by the higher contribution of electricity of the pilot-scale reactors, which attenuates the relative contribution of the other processes. In addition, the use of assumptions and extrapolated laboratory data for productivity used in life cycle and techno-economic studies of algal biofuels may lead to underestimation of the required raw materials due to the overestimation of the productivity potential [START_REF] Moody | Global evaluation of biofuels potential from microalgae[END_REF].

The relative contribution of nutrient production in the environmental profile depends on the season of cultivation. Summer period presented higher productivities (linked to higher dilution rates) and lower electricity requirements

Distribution of electricity requirements during the cultivation stage (S3)

The identification of the hot spots reveals that production of electricity for the cultivation of Nannochloropsis sp. is the major contributor to the environmental impact for all the reactor designs and operating conditions. These energy requirements arise from four activities: 1) temperature regulation (including heating and cooling of the culture medium), 2) aeration, 3) mixing and 4) base energy of monitoring system.

To determine the relevance of each activity, the distribution of electricity consumption is depicted in Figure 4.

. Electricity requirements for heating are moderate for the tubular reactors in summer (13% and 22% respectively for horizontal and vertical systems) but go above 62% for the ORP. Heating exceeds 50% of the total requirements for all the systems operated in fall and winter.

One of the most significant findings of this work is the high influence of temperature regulation system on the global environmental performance of microalgal cultivation. To the best of our knowledge, no previous LCA study discussed this key issue. Most studies exclude this activity from the system boundaries. In some cases, this seems a realistic assumption, since the operating conditions to estimate the inventory data are based on locations with warm temperatures and sunlight intensities [START_REF] Draaisma | Food commodities from microalgae[END_REF][START_REF] Collet | Recommendations for Life Cycle Assessment of algal fuels[END_REF], but for these locations the contribution of cooling will increase severely. Indeed, non-cooled closed reactors can reach temperatures above 60°C [START_REF] Ruiz | Towards industrial products from microalgae[END_REF], which would make Nannochloropsis cultivation unfeasible, according to its temperature growth range [START_REF] Sandnes | Combined influence of light and temperature on growth rates of Nannochloropsis oceanica, linking cellular responses to large-scale biomass production[END_REF].

For other studies that consider less favorable locations [START_REF] Clarens | Environmental life cycle comparison of algae to other bioenergy feedstocks[END_REF][START_REF] Stephenson | Life-cycle assessment of potential algal biodiesel production in the United Kingdom: A comparison of raceways and air-lift tubular bioreactors[END_REF], the effect of temperature regulation is expected to be relevant for the total impacts. Among the published LCA studies, only

Taelman et al. [START_REF] Taelman | The environmental sustainability of microalgae as feed for aquaculture: A life cycle perspective[END_REF] was found to specify the use of waste heat to maintain temperature in winter.

However, no contribution to the environmental burdens is reported for this input, being treated as a re-used waste flow, and thus having zero impact.

The applicability of Taelman's approach is restricted to scenarios in which a sufficient source of waste heat is available. Estimating the total energy needs for temperature regulation is essential to verify for each possible location whether this balance is met or not, and hence, guarantee the sustainability of a large-scale facility. Thus, this estimation may affect the decision-making process depending on the availability of natural cooling or waste heating sources. The quantification provided in the present paper highlights, for the first time, the importance of taking temperature regulation into account when conducting an LCA of a microalgal cultivation system.

When comparing both tubular systems, the electricity consumption for the heating in the vertical system is between 1.3 and 1.9 times higher than for the horizontal PBR. This is caused by the approximately two times larger tube area of the vertical system compared to the horizontal system, which involves a higher heating requirement to keep the temperature at the minimum set temperature of 20°C. In addition, during daytime, less light is absorbed per loop in the vertical system due to its design, light dilution effect and shading of the tubes. In fall/winter this effect is even more pronounced, because the lower tubes in the loops almost receive less light due to low inclination of the sun and shading.

Aeration and CO 2 addition has a relevant contribution to the impact of cultivation (associated with the blower) in the horizontal system (around 20% in the three scenarios), but is below 10% for all vertical PBR and ORP scenarios. The high impact of the aeration/CO 2 is due to the back pressure of the stripper on pilotscale. Small electrical blowers are not designed to overcome this pressure and for that an oversized blower was needed; on large scale, an air compressor would be used and electricity consumption would decrease largely. The impact of mixing (pumping and paddle wheel) is higher for the vertical PBR and ORP contributions than for the horizontal PBR due to higher volumes that needed to be mixed. For the ORP, the electricity consumption of the paddle wheel has already been pointed out as a relevant contributor by other authors [START_REF] Draaisma | Food commodities from microalgae[END_REF][START_REF] Stephenson | Life-cycle assessment of potential algal biodiesel production in the United Kingdom: A comparison of raceways and air-lift tubular bioreactors[END_REF]. Although the impact of the paddle wheel in fall is seven times higher than in summer (due to lower productivities), the relative contribution with respect to the total energy requirements is significantly higher for summer due to the lower consumption of other components (e.g. the heating system). The base energy used for monitoring had a minor contribution between 1% and 7%. In a hypothetical large-scale plant, this contribution may be even lower since such installation would have less equipment and fewer sensors (which are numerous in the case of a pilot plant to allow measuring a higher number of parameters).

Comparative environmental assessment of cultivation scenarios

The comparative evaluation of scenarios is represented in Figure 5, which reflects the environmental profiles, with the ORP operation under fall conditions as a reference.

For the operation in fall and winter, the difference between configurations is decisive.

While the environmental burdens of the horizontal PBR operated in fall approximately double compared to the summer period, the effects nearly triple for the vertical tubular system.

For the operation in fall, the horizontal PBR presents between 15% and 30% lower impacts than the vertical configuration;

Figure 1 .

 1 Figure 1. Pilot-scale systems at AlgaePARC facilities: a) Horizontal tubular PBR, b) vertically stacked tubular PBR c) ORP, d) simplified scheme of the main components of the tubular reactors and e) simplified scheme of the main components of the ORP (excluding, pumps, compressors and nutrient dosage tanks).

  cultivation of Nannochloropsis was natural seawater. To avoid contamination, this seawater was sterilized in an analogous manner as tap water for cleaning by adding hypochlorite (5 mg/L) and removing the chlorine with active carbon. Then, it was passed through a cascade filter (10 m, 5 m and 1 m) and supplied to the systems. Additionally, culture medium with NaNO 3 as the main nitrogen source was supplemented to the reactor. The nitrate solution consisted of 212 g/L NaNO 3 , 11.5 g/L KH 2 PO 4 , 3 g/mL Na 2 EDTA, 50 mL of trace mineral stock solution (see Supplementary material) and 17.5 mL NaOH 4 M to adjust pH. For the tubular PBRs, a dosage of 10 mL of nitrate solution per L of seawater was added in the final culture medium, whereas 2 mL/L were used in the medium of the ORP.

Figure 2 .

 2 Figure 2. Process value chain and system boundaries of the cultivation of Nannochloropsis sp. in pilot-scale reactors at AlgaePARC facilities.

(

  base energy of monitoring system, mixing, aeration and temperature control) was directly obtained from the on-line monitoring system. The energy consumption of the microscope for the purity check was considered negligible compared to the requirements of the reactors. The quantities of building materials for each reactor were calculated from measurements of the dimensions, which allowed determining the volume of each component and obtaining the weight by multiplying by the corresponding density. Life spans of 10 or 20 years were considered for the building materials depending on their properties and function (see Table

Figure 3 .

 3 Figure 3. Relative contributions to the environmental burdens of a) the subsystems and b) the production processes involved in the operation of the compared pilot-reactor configurations. For each category, 100% corresponds to the environmental impacts for each reactor (considering the average of the three operation periods) indicated in Table 2.

  than fall and winter operation due to high temperatures and light intensities. Higher dilution rates require large quantities of nutrients to replace the harvested culture volume. Electricity consumption is lower in summer due to low heating requirements compared to fall and winter, which largely compensate energy needs for cooling. The combined effect of higher dilution rates and lower electricity requirements lead to a higher relative contribution of nutrients to the environmental burdens in summer than in fall and winter.Waste treatment has a moderate contribution (around 10%) for ORP scenarios to EP and toxicity categories (FEP, MEP, TEP) and to R-HYD, while its influence is below 3% for the tubular PBRs in all categories and conditions. This impact was linked to the treatment of wastewater from S1 and S4. The difference is mainly due to the larger volume of water required by the ORP both for biomass production and cleaning, per functional unit. Although the tubular PBRs required the addition of chemicals (disinfectant) and materials (plastic beads) with higher impacts, they needed a lower number of cleanings and produced more biomass per year than ORP, according to the operation during the reference year. A minor difference in waste treatment between the ORP and tubular reactor results from S4. The harvested biomass from the ORP has a lower concentration than the biomass from the tubular PBRs, and therefore a higher volume of wastewater was generated to achieve the same biomass concentration.

Figure 4 .

 4 Figure 4. Distribution of electricity requirements during the cultivation stage for the evaluated pilot reactor configurations and operating conditions (100% corresponds to the electricity consumption in S3 per FU listed in

Figure 4

 4 Figure 4 shows that temperature regulation is the main consumer of electricity during cultivation in all evaluated scenarios, with total requirements ranging between 60% and 90%. The relative contributions of heating and cooling for the tubular systems depend to a large extent on the season. While cooling requires about 55% of the electricity consumption for cultivation in the summer for both tubular PBRs, it takes less than 10% of the requirements during fall and has no contribution for the winter period. Regardless of the weather conditions, the ORP needs no cooling

  mainly due to a 30% lower electricity consumption for cultivation. The difference between the summer and fall scenarios is much more pronounced in the case of ORP, for which the impacts in fall are between 12 and 17 times higher than for the summer operation. Hence, the fall performance of the ORP is 90% worse than any of the tubular systems under the same conditions and even exceeds the environmental profile of both PBRs operated in winter (with significantly colder conditions and less irradiation) with 40%.Although tubular PBRs were operated under winter conditions and present a better behavior than ORP in fall, the environmental burdens are significantly higher, compared to the relatively efficient performance during summer and fall periods. The contributions of the horizontal PBR during winter are about 5 times higher than those of fall, and up to 10 times above those of summer. Similarly, the vertical PBR in winter conditions has an average of 4 times the impacts of fall and more than 12 times the impacts of summer.The environmental impact of the horizontal PBR in fall is between 5-10% lower than that of the vertical PBR.

Figure 5 .

 5 Figure 5. Relative environmental profile of the compared pilot reactor configurations with respect to ORP in fall conditions for FU=1 kg DW microalgal biomass, according to the impact categories of a) CML methodology and b) CED methodology.

Figure 6

 6 Figure 6 shows a comparison of the best performing real and hypothetical scenarios

Figure 6 .

 6 Figure 6. Relative environmental profile of hypothetical pilot and large-scale reactors with respect to pilot ORP in summer conditions for FU=1 kg DW microalgal biomass, according to the impact categories of a) CML methodology and b) CED methodology.

Table 1 .

 1 Dimensions and operational average parameters for each reactor and period

	Reactor configuration	Horizontal PBR		Vertical PBR		ORP
	Season	Summer	Fall	Winter	Summer	Fall	Winter	Summer	Fall
	Reactor characteristics
	Volume (m 3 )		0.56			1.06		4.73
	Ground area (m 2 )		27.0			31.0		25.4
	Optical path (cm)		4.6			4.6		20
	Operational parameters
	Start-up date	04/07/13 29/08/13 04/11/13 04/07/13 27/08/13 07/11/13	08/07/13 22/08/13
	Shutdown date	22/08/13 31/10/13 16/12/13 27/08/13 04/11/13 17/12/13	22/08/13 18/09/13
	Dilution rate (%)	25.3	23.5	11.6	27.1	25.4	14.0	16.0	11.0
	Biomass concentration in the reactor (g/L)	2.5	1.3	0.6	1.9	1.0	0.4	0.5	0.5
	Ground areal
	productivity	12.1	4.6	1.2	19.4	8.3	2.7	10.5	2.1
	(g•m -2 •d -1 )
	Volumetric productivity (g•m -3 •d -1 )	654.8	250.2	66.7	568.5	241.7	79.2	56.5	11.3
	Recirculation flow (m 3 •h -1 )	8.1	8.1	8.0	18.7	18.8	18.7	0	0
	Air flow (m 3 •h -1 )	5.6	6.0	6.0	11.6	10.0	9.7	0	0
	CO 2 flow (L•h -1 )	40.1	21.3	8.8	53.4	26.1	14.0	30.3	30.3
	Weather conditions
	Temperature outside (ºC)	21.4	14.4	5.8	21.4	14.2	5.8	21.7	17.5
	Light intensity (mol photons•m -2 •d -1 )	36.9	18.6	5.0	36.9	18.6	4.9	38.2	23.9
	Photosynthetic efficiency, PE (%)	1.5	1.1	1.0	2.5	2.1	2.6	1.1	0.3
	Mean sunlight duration
	calculated from global	7.7	4.3	2.1	7.7	4.2	2.1	7.5	5.1
	radiation (h) 1
	Mean daily precipitation (mm) 1	1.2	4.3	2.9	1.1	4.5	2.0	1.3	3.9
	Mean precipitation duration (h)	0.6	2.3	2.2	0.6	2.4	1.6	0.7	2.2
	Mean daily cloud cover (in octans) 1	4.0	5.5	6.3	4.0	5.6	6.3	4.1	5.3
	Mean daily relative humidity (%) 1	76	85	89	76	84	89	76	81
	1 Calculated from data of the Royal Dutch Meteorological Institute (http://www.knmi.nl/index_en.html), for De Bilt
	weather station

Table 2 .

 2 Inventory

	Reactor configuration	Horizontal PBR	Vertical PBR		ORP
	Season	Summer Fall	Winter	Summer	Fall	Winter	Summer	Fall
		INPUTS from TECHNOSPHERE
	Materials
	S1. Cleaning
	Tap water (L)	128	398	994	163	439	977	2959	15984
	Chlorine solution (g)	4.49	13.9	34.8	5.70	15.4	34.2	104	559
	Disinfectant (kg)	1.56	4.84	12.1	1.98	5.34	11.9	0	0
	Plastic beads (g)	21.4	66.3	166	27.1	73.2	163	0	0
	S2. Nutrient supply
	Chlorine solution (g)	17.8	43.3	88.6	21.9	47.9	90.1	145	649
	Deionized water (kg)	3.85	9.33	19.1	4.71	10.3	19.4	6.27	28.0
	FeSO 4 •7H 2 O (g)	6.45	15.7	32.1	7.90	17.3	32.6	10.5	47.0
	MnCl 2 •2H 2 O (g)	0.368	0.893	1.83	0.451	0.988	1.86	0.600	2.68
	ZnSO 4 •7H 2 O (g)	0.142	0.344	0.705	0.174	0.381	0.717	0.231	1.03
	Co(NO 3 ) 2 •6H 2 O (g)	0.0151 0.0365 0.0748	0.0184	0.0405	0.0760	0.0245	0.110
	CuSO 4 •5H 2 O (g)	0.0051 0.0125 0.0256	0.0063	0.0139	0.0261	0.0084	0.0376
	Na 2 MoO 4 •2H 2 O (g)	0.0521	0.126	0.259	0.0638	0.140	0.263	0.0849	0.379
	Na 2 EDTA•2H 2 O (g)	22.6	54.8	112	27.7	60.7	114	36.8	164
	NaNO 3 (kg)	0.912	2.21	4.53	1.12	2.45	4.61	1.49	6.64
	KH 2 PO 4 (g)	49.5	120	246	60.6	133	250	80.7	360
	NaOH (g)	13.8	33.4	68.4	16.9	37.0	69.5	22.4	100
	S3. Cultivation
	Biomass (g)	84.1	197	210	100	189	206	399	3045

table for the cultivation of microalgae Nannochloropsis in pilot-scale systems (FU: 1 kg DW harvested biomass in 22% DW slurry).

Table 3 .

 3 Characterization results for the cultivation of microalgae Nannochloropsis in real pilot-scale systems (FU: 1 kg DW harvested biomass in 22% DW slurry).

	Impact category	Horizontal PBR Summer Fall Winter	Summer	Vertical PBR Fall	Winter	ORP Summer	Fall
		Abiotic depletion, ADP (kg Sb eq)	1.66	3.40	18.6	1.64	4.43	20.6	1.98	33.0
		Acidification, AP (kg SO 2 eq)	0.460	0.996	4.36	0.485	1.23	4.77	0.605	7.51
		Eutrophication, EP (kg PO 4 3-eq)	0.361	0.753	3.87	0.363	0.96	4.26	0.514	7.15
	METHODOLOGY 2001 CML	Global Warming Potential, GWP (kg CO 2 eq) Ozone layer depletion, ODP (kg CFC-11 eq) Human Toxicity, HTP (kg 1,4-DB eq) Freshwater Ecotoxicity, FEP(kg 1,4-DB eq) Marine Ecotoxicity, MEP(kg 1,4-DB eq) Terrestrial Ecotoxicity, TEP(kg 1,4-DB eq)	216 7.68•10 -6 1.58•10 -5 8.34•10 -5 443 2409 52.5 115 525 50.8 105 565 33.0 68.2 366 0.007 0.014 0.073	214 7.56•10 -6 2.01•10 -5 9.17•10 -5 574 2665 49.7 130 546 49.9 134 621 32.4 87.1 403 0.007 0.018 0.081	256 9.19•10 -6 1.47•10 -4 4256 55.2 836 66.8 1027 42.4 661 0.009 0.134
		Photochemical oxidants'
		formation, POFP (kg	0.016	0.035	0.169	0.016	0.043	0.185	0.020	0.295
		C 2 H 4 eq)
		Land competition, LC (m 2 year)	4.32	9.23	46.90	4.13	11.1	50.1	5.29	81.2
	CED METHODOLOGY	(MJ) Non-renewable fossil energy demand, NR-F (MJ) Non-renewable nuclear energy demand, NR-N (MJ) Non-renewable energy demand from biomass of primary forests, NR-B	3010 377 2.76•10 -3 6.38•10 -3 1.99•10 -2 6179 33692 771 4187	2983 370 3.10•10 -3 7.30•10 -3 2.09•10 -2 8033 37315 994 4622	3610 455 3.89•10 -3 3.13•10 -2 59727 7449

Table 2 ).

 2

Acknowledgements

The work presented in this paper has been developed within the framework of the BAMMBO Project (Project reference: FP7 KBBE-2010-4). The authors P. Pérez-López, G. Feijoo and M.T. Moreira belong to the Galician Competitive Research Group GRC 2013-032. P. Pérez-López would like to express her gratitude to the Spanish Ministry of Education for awarding a research scholarship (AP2012-1605). Furthermore, the authors would like to thank the Ministry of Economic Affairs, Agriculture and Innovation and Province of Gelderland, and BioSolar Cells, BASF, BioOils, Drie Wilgen Development, DSM, Exxon Mobil, GEA Westfalia Separator, Heliae, Neste Oil, Nijhuis, Paques, Cellulac, Proviron, Roquette, SABIC, Simris Alg, Staatsolie Suriname, Synthetic Genomics, TOTAL and Unilever for the financial support of the AlgaePARC research program.