## 2 Materials and Methods
### Analytical Methods  
A Flash 2000 elemental analyser (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a thermal conductivity detector was used to determine the content of nitrogen and oxygen present in all the biocrudes. This equipment was also used to measure the content of the main elements (carbon, hydrogen, nitrogen, oxygen and sulfur) in the microalgal biomass and in the biocrude obtained at the optimal operating conditions.  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Biological Composition** & \multicolumn{2}{c}{**Elemental Composition**} & \multicolumn{2}{c}{**Trace Element Composition**} \\ \hline Lipids (wt\%) & 35.5 \(\pm\) 1.2 & H (wt\%) & 7.1 \(\pm\) 0.2 & Na (mg/g) & 21.00 \(\pm\) 2.01 \\ Proteins (wt\%) & 43.8 \(\pm\) 3.5 & C (wt\%) & 48.7 \(\pm\) 0.1 & K (mg/g) & 3.53 \(\pm\) 0.09 \\ Carbohydrates (wt\%) & 15.7 \(\pm\) 3.6 & N (wt\%) & 6.80 \(\pm\) 0.04 & Mg (mg/g) & 0.90 \(\pm\) 0.03 \\ Ashes (wt\%) & 4.5 \(\pm\) 0.8 & S (wt\%) & 0.90 \(\pm\) 0.04 & Ca (mg/g) & 0.08 \(\pm\) 0.01 \\  & & O (wt\%) & 36.5 \(\pm\) 0.20 & P (mg/g) & 6.43 \(\pm\) 0.02 \\ \hline \hline \end{tabular}
\end{table}
Table 1: Composition of _N. gaditana_.  
The high heating value (HHV) of the optimal biocrude and the microalgal biomass was estimated using the [PERSON]'s model (Equation (1)) [18]:  
\[\text{HHV (MJ/kg)}=0.3516\times\text{C}+1.16225\times\text{H}-0.1109\times\text{O}+0.0628\times\text{N} \tag{1}\]  
The element recovery in this biocrude phase was determined and calculated using Equation (2) [19], whereas the energy recovery (ER) of the biocrude oil was defined as Equation (3) [16].  
\[\text{Element Recovery (\%)}=\text{(Element content in biocrude }\times\text{Mass of biocrude)}/ \tag{2}\] \[\text{(Element content in microalga }\times\text{Mass of dry microalga)}\times 100\] \[\text{ER (\%)}=\text{(HHV biocrude }\times\text{mass biocrude)}/\] (3) \[\text{(HHV microalgae }\times\text{mass dry matter of microalgae)}\times 100\]  
The composition of the biocrude at optimal operating conditions was determined by gas chromatography-mass spectrometry (GC-MS) (Bruker 450 GC, Bruker Corp., Billerica, MA, USA). The samples were diluted with carbon disulphide and filtered with a 0.45 um nylon filter. The GC-MS was fitted to a triple quadrupole mass spectrometer detector (Bruker 320 MS, Bruker Corp.,Billerica, MA, USA) operating in electronic impact mode. It was provided with a Rai-SSil MS 30 m 0.25 mm ID column (Restek, France). Data acquisition and processing were performed by using Bruker MS Workstation software v.7 (Bruker Corp., Billerica, MA, USA).  
For the phase of the water-soluble compounds, the water recovery was calculated considering the mass of water after the reaction with respect to the initial mass of water. In addition, total organic content (TOC) was analysed in a Shimadzu-V equipment (Shimadzu Corp., Kyoto, Japan), and the pH was measured in a Basic 30 pH meter (Crison Instruments, Barcelona, Spain). The composition of this phase was also measured by GC-MS.  
The biochemical composition of the microalgal biomass was characterised using the following methods: Bligh and Dyer [20] for lipids, [PERSON] [21] for carbohydrates and Lowry [22] for proteins.  
Trace elements were quantified by induced plasma atomic emission spectroscopy (ICP-AES), using a Vista AX CCD equipment (Varian Inc., Palo Alto, CA, USA). For its analysis, the sample was treated by acid digestion. For this, 0.1 g of sample was treated with 2 mL of 98% vol. sulfuric acid. and 10 mL of water. The sample was heated on a hot plate until both water and acid were removed, and the resulting solid was calcined using a ramp from 50 \({}^{\circ}\)C/min to 750 \({}^{\circ}\)C, keeping this temperature for 5 h in a CWF 1300 muffle (Carbolite, Hope, UK). The resulting ash was digested with 2 mL of 98% vol sulfuric acid and 10 mL of 35% vol hydrofluoric acid, heated until the latter was removed (appearance of white fumes), thus obtaining the sample ready for measurement.