## 2 Materials and Methods
### Apparatus Used for This Investigation  
Commercial benchtop equipment was used to determine the optical and the morphological properties of the selected species. Absorption, fluorescence and single-cell flow cytometry were measured.  
Absorption spectra of each phytoplankton species were recorded with a setup that includes a 170 W tungsten light source (Newport NRC 6334 NS); a monochromator (Newport 74125); an optical fibre (Newport Standard Grade FS Fibre Optic), used to guide the light through the photodiode (Hamamatsu, S1336-5 BQ); and a picoammeter (Keithley 487) to measure the photodiode current. Absorption spectra were scanned from 400 nm to 750 nm at 1 nm intervals.  
Fluorescence spectra of species were acquired by a fluorimeter (Fluorolog of the brand [PERSON]).  
For the acquisition of different structural and functional parameters such as size (FS), relative complexity (SS) and fluorescent response (green (FL1), yellow (FL2), orange (FL3) and red (FL4)) of each of the phytoplankton species under study, a flow cytometer (COULTER(r) EPICS(r) XL-MCL(tm) Flow Cytometer, Beckman Coulter, Inc., Brea, California, USA) was used [22]. This flow cytometer uses a 488 nm excitation laser, directly measured in the forward scattering sensor. The side scattering (SS) sensor signal is separated using a 488 nm dichroic long-pass filter (488 DL) at a 45-degree angle to the light path (see Figure 1). This filter reflects the SS to the respective sensor but transmits fluorescent light of longer wavelengths. A 488 nm laser-blocking filter (488 BK) blocks any remaining laser light, transmitting only fluorescent light. The remaining optical filters separate the light for the four FL sensors. A 550 DL filter is at a 45-degree angle to the light path, and it reflects wavelengths lower than 550 nm to a 525 nm band-pass (525 BP) filter. In turn, this filter transmits light between 505 nm and 545 nm to the FL1 sensor. The 550 DL filter transmits between 555 nm and 725 nm to the next dichroic long-pass filter (600 DL), also positioned at a 45-degree angle to the light path, that reflects light between 555 nm and 600 nm to a 575 BP filter (560-590 nm) in front of the FL2 sensor. A dichroic long-pass filter, a 645 DL, reflects the light between 605 nm and 645 nm to a 620 BP filter (605-635 nm) in front of the FL3 sensor. The 645 DL filter also transmits light between 650 nm and 725 nm to a 675 BP filter (600-700 nm) in front of the FL4 sensor.  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Species** & **Family** & **Order** & **Class** & **Division** \\ \hline _Nannochloropsis_ & & & & \\ _gaditma_ & & & & \\ _Isochryss galbuna_ & & & & \\ _Tetraselmis_ & & & & \\ _sucecia_ & & & & \\ \hline \hline \end{tabular}
\end{table}
Table 1: Phytoplankton species in culture for the different studies.  
\begin{table}
\begin{tabular}{c c c c} \hline \hline
**Species** & **Size** & **Shape** & **Characteristic Secondary** \\  & & & **Pigments [15]** \\ \hline _Nannochloropsis gaditana_ & 3–5 μm & spherical & fucoxanthin \\ _Isochryss galbuna_ & 6–12 μm & ellipsoid & 19′-hexanoylylucoxanthin \\ _Tetraselmis suecia_ & 10–15 μm & oval & chlorophyll-b \\ \hline \hline \end{tabular}
\end{table}
Table 2: Characteristics of each of the phytoplankton species.