## 4 Results and Discussions
### Excitation/Fluorescence Spectrum Matrix  
Fluorometric measurements, using the commercial fluorimeter described in Section 2.2, were performed for each species. Excitation was scanned from 400 nm to 650 nm (steps of 20 nm) and emission scanned from 400 nm to 850 nm (steps of 1 nm) for each excitation wavelength. Fluorescence at the same wavelength of the excitation was removed, since it results mainly from dispersion. Here, 2-3 mL of monocultures was analysed. Figure 3 presents the fluorescence spectra (emission) of three of the presented phytoplankton species (_Nannochloropsis gaditana_ (A), _Isochrysis galbana_ (B) and _Tetraselmis suecia_ (C)) for each excitation wavelength. Fluorescence values were normalized to the maximum value measured in the spectrum of each studied species. \begin{table}
\begin{tabular}{c c} \hline \hline
**Species** & **A\({}_{530}\)/A\({}_{436}\)** \\ \hline _Nannochloropsis gaditana_ & 0.46 \\ _Isochrysis galbana_ & 0.80 \\ _Tetraselmis suecia_ & 0.56 \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Relationship between absorption at 530 nm and 436 nm for the phytoplankton species studied. Figure 2: Relative absorbance spectrum of each studied phytoplankton species. All curves were normalized to the same absorbance value at 436 nm. The maximum standard deviation (\(n=3\)) of 2% was obtained. The three species show a fluorescence peak at 685 nm, for several excitation wavelengths. This peak corresponds to the fluorescence emission of chlorophyll a. We then tried to find patterns in Figure 3 that are unique for each species. To this aim, we calculated the mean and standard deviation (\(\sigma\)), corresponding to the excitation/emission values for the three species using Equation (1), where \(F_{xy}\) is the fluorescence at each pair of excitation (\(x\)) and fluorescence (\(y\)) wavelengths, and \(\overline{F_{xy}}\) is the mean value from the three species. \[\sigma_{xy}\ =\ \frac{\sqrt{\frac{\sum_{1}^{3}\left(F_{xy}-\overline{F_{xy}} \right)^{2}}{3}}}{\overline{F_{xy}}}, \tag{1}\]  
Standard deviation between fluorescence in each excitation/emission wavelength from the three species is plotted in Figure 4. As depicted by Equation (1) of the standard deviation (\(\sigma_{xy}\)), a higher standard deviation means that the three species have different values of fluorescence (measured at wavelength represented in index \(y\) and when excited  
Figure 3: Fluorescence spectrum with excitation light scanned from 320 nm to 700 nm of (**a**) of _Nammochloropsis gaditana_, (**b**) _Isochrysis galbuna_ and (**c**) _Tetraselmis suecia_. at wavelength represented by index \(x\)). When the standard deviation has a value near zero, it means that all three species have equivalent fluorescence at that wavelength. The standard deviation is therefore used to understand which wavelengths can be used to better distinguish between species. The standard deviation of fluorescence for each pair of excitation/emission is higher when differences are found between the three species. Four main regions were found where standard deviation showed high (>30%) values (Figure 4). The respective excitation and emission wavelengths are presented in Table 4.  
The last region (excitation and emission at 680 nm) results from dispersion, since the same wavelength is used for excitation and emission and will not be considered for species differentiation. Emission in the 640 nm regions is almost equal in all species, at all excitation wavelengths (Figure 4). Figures 5 and 6 plot only these selected wavelengths, for the three species. Figure 5 represents fluorescence at 640 nm and 685 nm, when excitation is scanned from 320 nm to 700 nm, and Figure 6 shows the fluorescence emission spectrum, for excitation at 420 nm, 450 nm and 530 nm. \begin{table}
\begin{tabular}{c c c} \hline
**Excitation (nm)** & **Emission (nm)** & **Standard Deviation (\%)** \\ \hline
420 & 685 & 39 \\
450 & 670 & 33 \\
530 & 680 & 47 \\
680 & 680 & 39 \\ \hline \end{tabular}
\end{table}