## 2 Materials and Methods
### Agronomic Trials on Microalgae Biomass  
The agronomic value of microalgal biomass produced in the photo-bioreactor was investigated through phytotoxicity tests and nutrients leaching tests in columns in comparison with liquid digestate and synthetic fertilizers. Plants trials with seeds of tomatoes (_Solanum Lycopersicum_) were performed in small pots with a volume of 0.5 dm\({}^{3}\), placed in a standard growth chamber (Fitotron, SGC 120 Weiss Gallenkamp, United Kingdom) under controlled conditions. During the trials, the environmental conditions were the following ones: day duration 16 h at 25 \({}^{\circ}\)C under 4600 lux (62.1 umol m\({}^{2}\) s\({}^{-1}\)), in 60% relative humidity, whereas night conditions were 8 h darkness at 18 \({}^{\circ}\)C in 80% relative humidity. Four conditions were tested: (1) soil alone (control), (2) soil + synthetic fertilizers, (3) soil + digestate and (4) soil + dried microalgal biomass. a dose of 170 kg N/ha was applied for the three last trials, according to the limits stablished by EU regulations for land application of fertilizers near watercourses (_EU Directive 91/676/EC_). For synthetic fertilizer, N sources was provided by ammonium nitrate. For P dosage of synthetic fertilizers, monopotassium phosphate was applied at 22 kg P/ha, same proportion than in liquid digestate. Such doses are within the range of N and P application rates for different crops systems [30]. The mixtures of soil and fertilizers sources were prepared into 10 cm diameter plastic pots and the mixture was brought to 70% of the water holding gravimetric capacity. Six tomato seeds were planted in each pot using four replicates for each condition. Each pot was manually watered every 24 h by weighing and adding water to achieve the initial weight. Once 70% of control seeds germination was obtained, four seeds of tomatoes were let in each pot, to make space for the plants to growth during the first vegetative stage. After 47 days, the leaves of each plant were counted before being harvested by cutting them at the soil level. Then, the biomass was dried for 48 h at 70 \({}^{\circ}\)C in a forced air oven, and finally weighted. For each condition, germination index (expressed in percent) of the initial seeds and biomass dry matter were measured and calculated as follows (Equations (4) and (5)):  
\[\textit{Germination Index}\ (\%)=\frac{\textit{Final number of seeds that germinated}}{\textit{Number of initial seeds}}\times 100 \tag{4}\]  
\[\textit{Dry weight}\ (g\textsl{TS}/100\ \textit{plants})=\frac{\textit{Dry weight of harvested plants}\ (70\ \textsl{C})}{\textit{Number of plants}}\times 100 \tag{5}\]  
Analysis of variance (ANOVA) was carried out to compare variables means. The results of plant growth were analyzed to classify the significant differences compared to the control (i.e., soil alone), the confidence level considered was 95%. Elementary analysis (C, H, N, S) have been determined using an elemental analyzer (varioMicro V4.0.2, Elementar(r), Germany). The method employed for determination of Chlorophyll a, Chlorophyll b and carotenoids was adapted from the one used by [31]. Accurately weighted 0.5 g of fresh plant leaf sample was homogenized with 10 mL of methanol. The mixture was centrifuged 8000 rpm for 20 min (Rotanta 460, Hettich Centrifugues, United Kingdom). The supernatant was separated and 0.5 mL of it was mixed with 4.5 mL of methanol. The solution was analyzed in a spectrophotometer (7315, Jenway, United Kingdom). Equations to determine concentrations (\(\mu\)g ml\({}^{-1}\)) of chlorophyll a (Ch a), chlorophyll b (Ch b) and total carotenoids (Ca) by methanol as solvent are as follows:  
\[\textit{Ch}\ a=16.72\ A_{665}-9.16\ A_{652} \tag{6}\]  
\[\textit{Ch}\ b=34.09\ A_{652}-15.28\ A_{665} \tag{7}\]  
\[\textit{Ca}=(1000\ A_{470}-1.63\ \textit{Ch}\ a-104.96\ \textit{Ch}\ b)/221 \tag{8}\]  
A method adapted from [32], was used to carry out leaching nutrients without plants.