**3. Light-harvesting system of cyanobacteria**

In cyanobacteria, the antenna complexes for photosystem II (PS II) and to some extent for photosystem I (PS I) are extrinsic and formed as large multiprotein organelles, which are located on the stromal side of the thylakoid membranes. These supramolecular pigment-protein complexes, so-called phycobilisomes (PBSs), first described by Gantt [39], are the main light-harvesting antennae in cyanobacteria.

Phycobilisomes are primarily composed of phycobiliproteins, a colored family of water-soluble proteins. Their chemical and spectroscopic properties are determined by their structure and function that they perform in the photosynthetic process. The three classes of phycobiliproteins are allophycocyanin (APC), phycocyanin (PC), and phycoerythrin (PE). However, in some cyanobacteria phycoerythrin can be replaced by phycoerythrocyanin (PEC), or both pigments can be lacking; phycocyanin and allophycocyanin are constitutively present in all cyanobacteria. Actually, there are very slight species differences between detached phycobiliproteins, even between prokaryotic cyanobacteria and eukaryotic red algae [40].

Usually PBSs are assembled from 12 to 18 different types of polypeptides, which may be grouped into three classes: (1) phycobiliproteins, (2) linker polypeptides,

**13**

**Figure 3.**

*Self-Fluorescence of Photosynthetic System: A Powerful Tool for Investigation of Microalgal…*

and analysis of these data has revealed phylogenetic relationships [41].

promising fingerprints for practical and laboratory applications.

details about phycobilisome structure, see [18, 39, 47].

and (3) PBS-associated proteins. The amino acid sequences of all components constituting the phycobilisomes of some cyanobacterial strains have been determined,

The polypeptide composition of PBS varies widely among strains of cyanobacteria. It should be noted that the degree of PBS compositional variability, which reflects the ability of an organism to adapt to environmental changes, varies from strain to strain. Moreover, for a single strain it sometimes depends upon the environmental conditions such as nutrient availability, temperature, light quality, and

It is well-known that total biliprotein content of cyanobacterial cells is inversely related to the quality and quantity of irradiance. A comprehensive review given in Refs. [41–46] details the various degrees of such chromatic adaptation. However, for cyanobacteria cultured under white light of reasonable intensity and in the medium with habitual nutrient composition, no chromatic adaptation can occur, and PBS structure remains invariable within each strain. Thus the unique spectroscopic properties of different cyanobacterial strains, while analyzing in vivo, may become

Phycobilisomes are constructed from two main structural elements: a core substructure and peripheral rods that are arranged in a hemidiscoidal fashion around that core (**Figure 3**). Each core cylinder is made up of four disc-shaped phycobiliprotein trimers, allophycocyanin (APC), allophycocyanin B (APC-B), and APC core-membrane linker complex (APC-LCM). By the core-membrane linkers, PBSs are attached on thylakoids and structurally coupled with PSII. The peripheral cylindrical rods (six or eight) radiate from the lateral surfaces of the core substructure and are usually not in contact with the thylakoid membrane. The rods are made up of hexamers, disc-shaped phycobiliproteins (PE, PEC, and PC), and corresponding rod linker polypeptides [41–44]. Most linker polypeptides are colorless proteins, but some also contain phycobilin chromophores, endowing them with the ability to harvest light as well as aid in the assembly of the phycobilisomes [46]. For more

The phycobilisome is attached to the membrane by multiple weak charge-charge

Recently, it was established that phycobilisomes diffuse rapidly on the surface of the thylakoid membrane, while PS II reaction centers are normally almost immobile. Fluorescence recovery after photobleaching (FRAP) has been used to measure

interactions, either with proteins or with lipid head-groups. Binding is rather unstable. The core-membrane linker polypeptide provides a flexible surface, allowing interaction with a range of structurally distinct membrane complexes, including photosystem II (PSII) and photosystem I (PSI) (see **Figure 4**). The stability of each interaction may be modulated by covalent modification and/or the presence of

*Schematic drawing of phycobilisome and photosynthetic energy transfer to reaction center.*

*DOI: http://dx.doi.org/10.5772/intechopen.88785*

light intensity.

accessory subunits.

#### *Self-Fluorescence of Photosynthetic System: A Powerful Tool for Investigation of Microalgal… DOI: http://dx.doi.org/10.5772/intechopen.88785*

and (3) PBS-associated proteins. The amino acid sequences of all components constituting the phycobilisomes of some cyanobacterial strains have been determined, and analysis of these data has revealed phylogenetic relationships [41].

The polypeptide composition of PBS varies widely among strains of cyanobacteria. It should be noted that the degree of PBS compositional variability, which reflects the ability of an organism to adapt to environmental changes, varies from strain to strain. Moreover, for a single strain it sometimes depends upon the environmental conditions such as nutrient availability, temperature, light quality, and light intensity.

It is well-known that total biliprotein content of cyanobacterial cells is inversely related to the quality and quantity of irradiance. A comprehensive review given in Refs. [41–46] details the various degrees of such chromatic adaptation. However, for cyanobacteria cultured under white light of reasonable intensity and in the medium with habitual nutrient composition, no chromatic adaptation can occur, and PBS structure remains invariable within each strain. Thus the unique spectroscopic properties of different cyanobacterial strains, while analyzing in vivo, may become promising fingerprints for practical and laboratory applications.

Phycobilisomes are constructed from two main structural elements: a core substructure and peripheral rods that are arranged in a hemidiscoidal fashion around that core (**Figure 3**). Each core cylinder is made up of four disc-shaped phycobiliprotein trimers, allophycocyanin (APC), allophycocyanin B (APC-B), and APC core-membrane linker complex (APC-LCM). By the core-membrane linkers, PBSs are attached on thylakoids and structurally coupled with PSII. The peripheral cylindrical rods (six or eight) radiate from the lateral surfaces of the core substructure and are usually not in contact with the thylakoid membrane. The rods are made up of hexamers, disc-shaped phycobiliproteins (PE, PEC, and PC), and corresponding rod linker polypeptides [41–44]. Most linker polypeptides are colorless proteins, but some also contain phycobilin chromophores, endowing them with the ability to harvest light as well as aid in the assembly of the phycobilisomes [46]. For more details about phycobilisome structure, see [18, 39, 47].

The phycobilisome is attached to the membrane by multiple weak charge-charge interactions, either with proteins or with lipid head-groups. Binding is rather unstable. The core-membrane linker polypeptide provides a flexible surface, allowing interaction with a range of structurally distinct membrane complexes, including photosystem II (PSII) and photosystem I (PSI) (see **Figure 4**). The stability of each interaction may be modulated by covalent modification and/or the presence of accessory subunits.

Recently, it was established that phycobilisomes diffuse rapidly on the surface of the thylakoid membrane, while PS II reaction centers are normally almost immobile. Fluorescence recovery after photobleaching (FRAP) has been used to measure

**Figure 3.**

*Schematic drawing of phycobilisome and photosynthetic energy transfer to reaction center.*

*Microalgae - From Physiology to Application*

ties of the cells are not known beforehand.

epochs lays in the range from 300 to 800.

single-cell fluorescence spectra").

in this study, the training procedure has 500 iterations (epochs). After training, the network is tested. In this test phase, the characteristics of a number of cyanobacterial cells with known identities are fed to the network, and the solutions are compared with these known identities. In this study, after training the network was capable of recognizing about 96% of cyanobacterial cells in the test set. The analysis of generalization quality of ANN is identical to the test procedure; only the identi-

The ratio of training sample to the test sampling in this investigation was taken

70:30%. Other parameters of the selected training algorithm were as follows: acceptable error threshold is 0.01, the bandwidth parameter (size of error control window) is 20, the moment parameter is 0.1, and the regularization parameter is 0.001. The selected learning rate was chosen 0.01, and the number of training

The main criteria for assessing the quality of ANN operation is the value of classification accuracy. There are several approaches to evaluate the accuracy of classification. In the considered case, the classification accuracy is calculated for each class separately, as the ratio of the number of correctly classified class observations to the total number of observations in a given class. Then the average classification accuracy for all classes was obtained. In such case it is possible to build a matrix of errors with size N × N (N—number of classes) and present the results in a bar chart, on which a classification accuracy for each class can be visualized (see **Figure 8** in "Ataxonomic differentiation of cyanobacterial strains on the base of

On the base of the classification accuracy analysis, it is possible to evaluate the quality of ANN training as well as the quality of internal and external generalization. In our case, the evaluation of the quality of external generalization was obtained on the base a priori knowledge about new species, which was taken from an expert. To validate the correctness of the neural network operation, the results of

The ANN architecture presented in this paper, as well as the learning algorithm and its parameters were determined during the study of various configurations. The selected model after training consistently gives a classification accuracy of at least

In cyanobacteria, the antenna complexes for photosystem II (PS II) and to some

Usually PBSs are assembled from 12 to 18 different types of polypeptides, which may be grouped into three classes: (1) phycobiliproteins, (2) linker polypeptides,

extent for photosystem I (PS I) are extrinsic and formed as large multiprotein organelles, which are located on the stromal side of the thylakoid membranes. These supramolecular pigment-protein complexes, so-called phycobilisomes (PBSs), first described by Gantt [39], are the main light-harvesting antennae in cyanobacteria. Phycobilisomes are primarily composed of phycobiliproteins, a colored family of water-soluble proteins. Their chemical and spectroscopic properties are determined by their structure and function that they perform in the photosynthetic process. The three classes of phycobiliproteins are allophycocyanin (APC), phycocyanin (PC), and phycoerythrin (PE). However, in some cyanobacteria phycoerythrin can be replaced by phycoerythrocyanin (PEC), or both pigments can be lacking; phycocyanin and allophycocyanin are constitutively present in all cyanobacteria. Actually, there are very slight species differences between detached phycobiliproteins, even between prokaryotic cyanobacteria and eukaryotic red algae [40].

the NN classification were compared with the results of the LDA.

**3. Light-harvesting system of cyanobacteria**

95%. In this study, ANN was simulated using MATLAB software [33].

**12**

**Figure 4.**

*Model for the interaction between phycobilisomes and membrane components. (a) Phycobilisomes are shown as semicircles, with the core as a darker rectangle [48]. (b) A parallel section through the membrane with the terminal phycobilisome pigment in relation to photosystems I and II [51].*

the mobility of phycobilisomes in the intact cyanobacterial cells [48, 49], and it was clearly demonstrated that a significant proportion of phycobilisome-absorbed energy is delivered to PS I as well as to PS II [45, 49, 50].

The high mobility of phycobilisomes along the thylakoid membrane gives the opportunity of the occasional direct interaction of phycobilisome rods or core with PS I (**Figure 4**). Two ways that energy could be transferred from phycobilisomes to photosystem I are shown in **Figure 4**; "Spillover" from photosystem II with an attached phycobilisome (supposed by Su et al. [51]) (**Figure 4a**, left photosynthetic complex) and direct association of the phycobilisome core with photosystem I (**Figure 4a**, right photosynthetic complex).

Another possible variant of the interaction between phycobilisome and reaction centers of two photosystems was proposed by Gantt in the Chapter 6.3 of the book [40]. The author assumed that the special close arrangement of both photosystems around the base of the phycobilisome provides the partial transfer of the absorbed energy to PSII and PSI simultaneously (**Figure 4b**).

### **4. Fluorescence spectra of intact cyanobacterial cells**

The intrinsic fluorescence of photosynthetic organisms originates from excited states that were trapped by light-harvesting system and lost before photochemistry took place. Photoexcitation energy absorbed at the outer surface of phycobilisomes is transported sequentially through several rod chromoproteins to an inner core and then to core-membrane linker (the terminal pigment) that acts as the final energy transmitters from the phycobilisome to Chl a heterodimers of two photosystems (PSII and PSI), incorporated in the thylakoid membrane. This excitation transfer is recognized as due to the Förster dipole-dipole interaction with an extremely high efficiency, near unity.

The more distal parts of the antenna system, a peripheral antenna complex (phycobilisome), maximally absorb photons at shorter wavelengths (higher energies) than do the pigments in the antenna complexes that are proximal to the reaction center. Subsequent energy transfer processes are from these high-energy pigments physically distant from the reaction center to low-energy pigments that are physically closer to the reaction center (**Figure 5**). With each transfer, a small amount of energy is lost as heat, and the excitation is moved closer to the reaction center, where the energy is stored by photochemistry. Note that the probability of excitation energy escape from the trap in the form of fluorescence at all transfer steps is non-zero and depends on the intensity and wavelength of the excitation light.

During the energy transfer process, the occasional quenching of the absorbed light by fluorescence can occur, and this becomes the essential property for

**15**

cyanobacterial species.

synthetic systems.

**Figure 5.**

*complexes.*

*Self-Fluorescence of Photosynthetic System: A Powerful Tool for Investigation of Microalgal…*

fluorescent spectroscopy. It usually represents a small fraction of the excited states and diminishes in a functioning photosynthetic complex. Nevertheless, the fluorescence is an extremely informative quantity, because it reports on the energy transfer and trapping. Both steady-state and time-resolved fluorescence measurements are widely used methods for probing the organization and functional state of photo-

*Schematic illustration of the energy transfer in light-harvesting system of cyanobacteria (a). Panel (b) represents the example of normalized in vivo single-cell fluorescence emission spectra at excitation wavelength 488 nm. Dashed lines and letters over them indicate emission wavelengths of PE, PC, APC, and Chl a of PSII and PSI, correspondingly. Fine lines represent fluorescent spectra of corresponding detached pigment-protein* 

The fluorescence of intact living cyanobacterial cells is originated from the inefficiency of the energy transfer between all components of the energy transfer chain including the final step, the delivery to PSII or PSI (**Figure 5a**). Due to the occasional quenching by fluorescence each transfer step result in peak or shoulder on the corresponding spectrum (**Figure 5b**). This is due to the fact that when phycobilisomes are bound to the thylakoid membrane, most of the energy from phycobilisome is channeled to chlorophylls in the thylakoid membrane and thus did not shade the fluorescence of the previous steps in energy transfer chain. In the course of the energy transfer from the initially photoexcited phycobiliprotein to the reaction center of photosystems PSI and PSII, fluorescence is emitted from almost every type of pigment and can be used as a probe to examine the mechanism of

A convenient way to monitor this energy transfer process is to irradiate a sample with light that is selectively absorbed by one set of pigments and then monitor fluorescence that originates from a different set of pigments. Obviously, if the energy transfer is taken place between pigments, the light absorbed by one set of pigments is emitted by another set differently, depending on the excitation wavelength. This type of fluorescence excitation experiment can be used to measure quantitatively the efficiency of energy transfer from one set of pigments to another [43]. Moreover, different species of cyanobacteria contain different accessory pigment proteins and specific linker proteins between them; therefore a set of fluorescence emission spectra excited by different wavelengths have its own unique shape for the cells of one strain and are quite distinguishable from other species and strains. Such sets of fluorescence emission spectra can be used for automatic differentiation of

**Figure 6** shows several characteristic sets of single-cell fluorescence spectra corresponding to Microcystis CALU 398, Merismopedia CALU 666, Leptolyngbya CALU 1715, and Phormidium CALU 624, obtained by confocal laser scanning

energy transfer within the light-harvesting system [43, 44, 52].

*DOI: http://dx.doi.org/10.5772/intechopen.88785*

*Self-Fluorescence of Photosynthetic System: A Powerful Tool for Investigation of Microalgal… DOI: http://dx.doi.org/10.5772/intechopen.88785*

#### **Figure 5.**

*Microalgae - From Physiology to Application*

*Model for the interaction between phycobilisomes and membrane components. (a) Phycobilisomes are shown as semicircles, with the core as a darker rectangle [48]. (b) A parallel section through the membrane with the* 

the mobility of phycobilisomes in the intact cyanobacterial cells [48, 49], and it was clearly demonstrated that a significant proportion of phycobilisome-absorbed

The high mobility of phycobilisomes along the thylakoid membrane gives the opportunity of the occasional direct interaction of phycobilisome rods or core with PS I (**Figure 4**). Two ways that energy could be transferred from phycobilisomes to photosystem I are shown in **Figure 4**; "Spillover" from photosystem II with an attached phycobilisome (supposed by Su et al. [51]) (**Figure 4a**, left photosynthetic complex) and direct association of the phycobilisome core with photosystem I

Another possible variant of the interaction between phycobilisome and reaction centers of two photosystems was proposed by Gantt in the Chapter 6.3 of the book [40]. The author assumed that the special close arrangement of both photosystems around the base of the phycobilisome provides the partial transfer of the absorbed

The intrinsic fluorescence of photosynthetic organisms originates from excited states that were trapped by light-harvesting system and lost before photochemistry took place. Photoexcitation energy absorbed at the outer surface of phycobilisomes is transported sequentially through several rod chromoproteins to an inner core and then to core-membrane linker (the terminal pigment) that acts as the final energy transmitters from the phycobilisome to Chl a heterodimers of two photosystems (PSII and PSI), incorporated in the thylakoid membrane. This excitation transfer is recognized as due to the Förster dipole-dipole interaction with an extremely high

The more distal parts of the antenna system, a peripheral antenna complex (phycobilisome), maximally absorb photons at shorter wavelengths (higher energies) than do the pigments in the antenna complexes that are proximal to the reaction center. Subsequent energy transfer processes are from these high-energy pigments physically distant from the reaction center to low-energy pigments that are physically closer to the reaction center (**Figure 5**). With each transfer, a small amount of energy is lost as heat, and the excitation is moved closer to the reaction center, where the energy is stored by photochemistry. Note that the probability of excitation energy escape from the trap in the form of fluorescence at all transfer steps is non-zero and depends on the intensity and wavelength of the excitation light.

During the energy transfer process, the occasional quenching of the absorbed

light by fluorescence can occur, and this becomes the essential property for

*terminal phycobilisome pigment in relation to photosystems I and II [51].*

energy is delivered to PS I as well as to PS II [45, 49, 50].

(**Figure 4a**, right photosynthetic complex).

energy to PSII and PSI simultaneously (**Figure 4b**).

**4. Fluorescence spectra of intact cyanobacterial cells**

**Figure 4.**

**14**

efficiency, near unity.

*Schematic illustration of the energy transfer in light-harvesting system of cyanobacteria (a). Panel (b) represents the example of normalized in vivo single-cell fluorescence emission spectra at excitation wavelength 488 nm. Dashed lines and letters over them indicate emission wavelengths of PE, PC, APC, and Chl a of PSII and PSI, correspondingly. Fine lines represent fluorescent spectra of corresponding detached pigment-protein complexes.*

fluorescent spectroscopy. It usually represents a small fraction of the excited states and diminishes in a functioning photosynthetic complex. Nevertheless, the fluorescence is an extremely informative quantity, because it reports on the energy transfer and trapping. Both steady-state and time-resolved fluorescence measurements are widely used methods for probing the organization and functional state of photosynthetic systems.

The fluorescence of intact living cyanobacterial cells is originated from the inefficiency of the energy transfer between all components of the energy transfer chain including the final step, the delivery to PSII or PSI (**Figure 5a**). Due to the occasional quenching by fluorescence each transfer step result in peak or shoulder on the corresponding spectrum (**Figure 5b**). This is due to the fact that when phycobilisomes are bound to the thylakoid membrane, most of the energy from phycobilisome is channeled to chlorophylls in the thylakoid membrane and thus did not shade the fluorescence of the previous steps in energy transfer chain. In the course of the energy transfer from the initially photoexcited phycobiliprotein to the reaction center of photosystems PSI and PSII, fluorescence is emitted from almost every type of pigment and can be used as a probe to examine the mechanism of energy transfer within the light-harvesting system [43, 44, 52].

A convenient way to monitor this energy transfer process is to irradiate a sample with light that is selectively absorbed by one set of pigments and then monitor fluorescence that originates from a different set of pigments. Obviously, if the energy transfer is taken place between pigments, the light absorbed by one set of pigments is emitted by another set differently, depending on the excitation wavelength. This type of fluorescence excitation experiment can be used to measure quantitatively the efficiency of energy transfer from one set of pigments to another [43]. Moreover, different species of cyanobacteria contain different accessory pigment proteins and specific linker proteins between them; therefore a set of fluorescence emission spectra excited by different wavelengths have its own unique shape for the cells of one strain and are quite distinguishable from other species and strains. Such sets of fluorescence emission spectra can be used for automatic differentiation of cyanobacterial species.

**Figure 6** shows several characteristic sets of single-cell fluorescence spectra corresponding to Microcystis CALU 398, Merismopedia CALU 666, Leptolyngbya CALU 1715, and Phormidium CALU 624, obtained by confocal laser scanning

#### **Figure 6.**

*Four characteristic sets of single-cell fluorescence spectra. The excitation wavelengths (405, 458, 476, 488, 496, 514, 543, and 633 nm) are given over the curves. All spectra are normalized to the maximum intensity and shifted along x-axis for convenience of observation. The dashed lines indicate fluorescence maxima of the individual pigments (PE, 580 nm; PC, 656 nm; Chl a, 682 and 715 nm).*

microscope (CLSM) Leica TCS-SP5, which are placed near each set. Each spectrum in the set was obtained using different laser lines for excitation: 405, 458, 476, 488, 496, 514, 543, and 633 nm. Corresponding excitation wavelengths are given over each spectrum. All spectra are normalized to the maximum intensity and shifted along x-axis for convenience of observation. It can be easily noticed that laser line 458 nm excites mostly in vivo fluorescence of Chl a in both photosystems PSII and PSI around 682 and 715 nm, correspondingly, and the emission spectrum by cyanobacterial cells shows no appreciable emission of PC or APC. In cyanobacteria, the 458 nm excitation is preferentially absorbed by PSI that contains more Chl a than by PSII and is stoichiometrically more abundant than PSII. However, because reaction center of PSI turns over faster than the PSII, it has lower fluorescence intensity than the PSII antenna. This is indicated by PSI emission band at 715 nm which is much weaker than the PSII emission band at 682 nm. The excitation by intermediate (blue and green) wavelengths (405, 488, and 496 nm) reveals fluorescent maxima of all photosynthetic pigments, as the light in this range is absorbed by all pigmentprotein complexes almost in equal portions and fluorescence emits by all steps of energy transfer chain (**Figure 5**). The direct excitation of cells in the PE absorption region at 514 and 543 nm results in emission spectrum with two main peaks at 580 and 656 nm, which are due to PE, PC, and APC emission, and for species that lack PE, the emission accumulates mostly near 656 nm. Two chlorophyll fluorescence components can be resolved for some species in a number of spectra. The spectra of the 633 nm excitation directly give a prominent emission band at 656 nm that originates from C-PC, omitting band at 580 nm, which cannot be excited by 633 nm, even for species that have PE (see **Figure 6**). Other small emission bands, corresponding to fine pigment structure of antenna complex, are not resolved at the room temperature.

These in vivo fluorescence emission spectra reflect the structure of lightharvesting complex of corresponding species and correct or incorrect functioning of its energy transfer chain. Four characteristic wavelengths, corresponding to the fluorescence maximum or shoulder, can be easily distinguished: (1) peak near 580 nm corresponds to the fluorescence of phycoerythrin, (2) peak near 656– 560 nm corresponds to the fluorescence of phycocyanin and allophycocyanin in common (they are undistinguishable at room temperature), (3) peak near 682 nm

**17**

investigation.

*Self-Fluorescence of Photosynthetic System: A Powerful Tool for Investigation of Microalgal…*

corresponds to the fluorescence of chlorophyll a in photosystem II, and (4) peak or shoulder near 720 nm represents the fluorescence from photosystem I [10, 53]. Comparative analysis of the series of fluorescence spectra for different cyanobacterial species and strains reveals visible variations in their shape. If the fluorescence spectra were taken from live cells in normal physiological state, which are cultured in the same growth environmental conditions, then the interspecies variations in pigment/Chl a ratios are more pronounced than variations within the individual species. And species/strain differentiation could be carried out on the

**5. Ataxonomic differentiation of cyanobacterial strains on the base of** 

In the presented investigation, 307 sets of 8 single-cell fluorescent spectra for 23 cyanobacterial strains, belonging to 15 genera, were analyzed. An optimal set of classification parameters was considered that is sufficient for determining the generic membership of cyanobacterial cells by means of mathematical statistics. The results of this study show that LDA and ANN are able to recognize cyanobacteria up to species/strains according to the data recorded by means of CLSM. This implies that the classifier (LDA or ANN) is capable of defining a unique niche in a multiparameter space for each of 23 cyanobacterial strains, used in this

The results of LDA, evaluated over 63 parameters extracted from 307 single-cell fluorescence spectra, are presented in **Figure 7** as 3D-plots in the space of canonical discriminating functions. It is clear that the discrimination between species is sufficiently good. Moreover, the closely related species (e.g., *Spirulina* and *Oscillatoria*, *Synechococcus* and *Chlorogloea*, *Microcystis*, *Synechocystis* and *Myxosarcina*) appear close to each other. Such species as *Leptolyngbia*, *Geitleninema*, and *Oscillatoria*, which includes several strains, form big groups. However, inside these groups single strains also can be discriminated, which is demonstrated on the right panel, where the corresponding scaled region 1 is presented. This is confirmed by a classification diagram plotted in **Figure 7C**. The classification accuracy in the presented example was near 97.4%. The high classification accuracy is due to the fact that LDA works with distribution functions for classification parameters and their statistical charac-

In the legend all used cyanobacterial strains are named and enumerated according to CALU collection. Solid curves bounded the regions, occupied by seven strains

Fluorescence spectra have been used to classify phytoplankton populations since approximately the early 1970s [54, 55]. However, because of the generally low device precision and poor availabilities, the rate of species discrimination was relatively low. Recently new attempts to conduct the discrimination among microalga on the base of absorption or fluorescence spectra were reported [7, 10, 13, 15]. But again in published experiments only big algal groups with a considerable differences in pigment composition can be successfully separated (e.g., cryptophytes, chlorophytes, cyanobacteria, etc.). Moreover, all the authors pointed out that the discrimination among cyanobacterial species is quite complex and ambiguous. Actually, the correct discrimination of cyanobacterial species on the base of fluorescence signature is usually hampered by alterations in the pigment composition within one strain, which depends on the environmental conditions and physiological state of the culture. These difficulties can be overcome by using single-cell fluorescence spectra instead of bulk ones and by recording 7–8 spectra with different excitation wavelengths for

*DOI: http://dx.doi.org/10.5772/intechopen.88785*

basis of conventional multivariate analysis.

**single-cell fluorescence spectra**

each cell instead of one or two as usually is done.

teristics, which allows to build a good classification model.

#### *Self-Fluorescence of Photosynthetic System: A Powerful Tool for Investigation of Microalgal… DOI: http://dx.doi.org/10.5772/intechopen.88785*

corresponds to the fluorescence of chlorophyll a in photosystem II, and (4) peak or shoulder near 720 nm represents the fluorescence from photosystem I [10, 53].

Comparative analysis of the series of fluorescence spectra for different cyanobacterial species and strains reveals visible variations in their shape. If the fluorescence spectra were taken from live cells in normal physiological state, which are cultured in the same growth environmental conditions, then the interspecies variations in pigment/Chl a ratios are more pronounced than variations within the individual species. And species/strain differentiation could be carried out on the basis of conventional multivariate analysis.
