**3.2. Ultrasonic treatments**

**Figure 4.** The illustration to the process of the culture viability estimation. (a–c) Averaged SFS for cyanobacterial strain Synechocystis CALU 1336, excited by three laser lines 458, 488, and 514 nm. *Blue* lines show spectra of alive cells, *red* lines are for the cells in the depressed physiological state. (d–f) Fitted and integral normalized spectra for the whole culture under consideration. *Black* lines show a linear combination of CLSM spectra of alive and depressed cells summed in ratio 2:1. *Green* lines show the integral spectra of the whole culture obtained using Cary Eclipse fluorimeter (Varian Cary) at corresponding excitation wavelengths. At all plots dashed lines indicate the fluorescence maximum of chlorophyll a

eight obtained spectra are presented in **Figure 4**. Then, the integral spectra of the whole culture were obtained at corresponding excitation wavelengths using Cary Eclipse fluorimeter (Varian Cary) (**Figure 4d–f**; green lines). Two sets of eight fluorescence spectra for cells in normal and depressed physiological state were taken to obtain a linear combination for fitting procedure a set of integral spectra. Each spectrum in two sets was averaged over 10–15 cells. It should be noted that to raise accuracy of calculations, the curve fitting process was done over the whole set of the eight spectra simultaneously, so that to exclude any ambiguity. The fitting coefficients for alive and depressed cells were about 2 and 1, correspondingly. Thus, the whole culture can

(680 nm) and phycocyanin (656 nm), respectively. For SFS averaging was carried out over 10–15 cells.

be considered as a healthy and being in the exponential or logarithmic growth phase.

significantly for the healthy and depressed cells.

24 Cyanobacteria

As it is following from the plots of **Figure 4a–c**, the degree of cell damage is determined mostly by the relative fluorescence intensity of phycobilins at 656 nm and chlorophyll-binding proteins at 680 nm. It is especially clear from the excitation wavelength 488 nm (**Figure 4b**), where the fluorescence spectra for healthy and depressed cells have the mirror shape. For the 458 nm excitation, the shape and the intensity of fluorescence spectra for this two states differ not so much because this wavelength excites better the peak of chlorophyll-binding proteins in PS II (680 nm), which is not affected. On the other hand, the 514 nm wavelength strongly excites the fluorescence of the antenna pigments and the fluorescence spectra intensity differs In recent years, several environmentally friendly methods for preventing of toxic cyanobacterial "blooms" of water bodies have appeared. One of them is a weak ultrasound treatment. Unfortunately, due to the low intensity of the applied sonication and its constant but weak influence on the biophysical parameters of the cyanobacteria most of the results of the previous investigations are quite ambiguous [32–34, 84–89].

Despite the significance of the problem of cyanobacterial blooms and a variety of applied methods to control them, the sufficient principles of investigation and monitoring the results of various external actions on cyanobacteria are not developed. Currently, the most of all studies are based mainly on traditional visual methods of obtaining results or on the analysis of fixed or dissociated environmental samples. However, standard methods can only record the presence of the bloom in reservoir, but cannot determine at what evolution stage it is situated, or, all the more, to predict the possibility of further cyanobacterial bloom. This owes to the use of rather crude methods of monitoring of the physiological state of the culture during the experiment. All previously elaborated monitoring methods either destroy cells or change significantly their physiological state just before the measurements, so it is impossible to determine the initial physiological state of the treated culture.

For example, in the paper [87], the ultrasonic inhibition of *Microcystis aeruginosa* cell growth and extracellular microcystins release was examined. The authors reported the decrease of antenna complexes like cyanobacterial chlorophyll a and phycocyanins (PC), and the oxygen evolution rate. The conclusion about slowing down of the photoactivity and damaging of the antenna complexes was made according to the measuring of the growth rate of the whole culture. This is not the case because there is no any confirmation that the single-cell physiological state really changes. May be the whole culture died due to other reasons or simply several cells were destroyed during sonication. Moreover, the pointed sonication power settings (about 0.32W/mL) cannot be considered as a weak and environmentally friendly treatment. It cannot be applied to the natural reservoirs, so that to carry out a real control on cyanobacterial blooms in open water. Obviously, this high intensity of sonication was used to obtain any possible results because used methods are not precise enough. Thus, this example shows that the standard methods do not give the correct results, and new precise, nondestructive *in vivo* methods for monitoring of the physiological state of cyanobacterial cultures are required for such investigations.

chosen following the literature reports [32, 86] and based on our preliminary tests. During sonication, the cyanobacteria solution was carefully shuffled and taken for analysis just in the process of sonication. Control sample without any treatment was kept under exactly the same

Fluorescence Microscopic Spectroscopy for Investigation and Monitoring of Biological Diversity…

http://dx.doi.org/10.5772/intechopen.78044

27

In **Figure 6**, single-cell fluorescence spectra for two emission wavelengths (488 and 543 nm), obtained by standard lambda-scanning using Leica TCS-SP5 CLSM, are shown for control and treated samples. Blue lines demonstrate the spectra of the control suspension, whereas red lines show the spectra of the experimental one. It is clear that the fluorescence intensity in the control group differ from the experimental one. According to the preliminary studies, enhancement of the fluorescence at 660 nm in the experimental culture indicates that this culture is in a depressed physiological state in comparison with the control group. Each spectrum in **Figure 6** was obtained by averaging over 15 cells. Spectral analysis on the cell level using CLSM makes it possible to obtain more comprehensive information on the small variations in physiological state of both single cells and the entire culture exposed to ultrasonic treatment. During ultrasonic treatment, single cyanobacterial cells were studied at the first day to obtain the reference spectra of the initial cells in a good physiological state. Then, after 24–30 h, both control and treated specimens were studied and their fluorescence spectra

Let us mention here again that the treated and control probe initially were taken from one cultural sample and were placed at the same environmental conditions. Else, all the obtained results were double controlled via conventional fluorometric methods such as pulse amplitude modulation (PAM) and absorption and fluorescent characteristics of the whole culture [90]. These measurements were conducted using the light curves method on the PAM 2500 pulse spectrofluorimeter (WALZ, Germany) and a standard spectroscopic technique using a Cary Eclipse fluorimeter (Varian Cary), correspondingly. The dependences of the electron transport rate (ETR) and the quantum yield of photosystem PS II (Y(II)) on photosynthetically active radiation (PAR) under blue actinic light, obtained via WALZ PAM, show that ETR and

**Figure 6.** Single-sell fluorescence spectra for cyanobacterial strain Synechocystis CALU 1336 obtained at two excitation wavelengths (a) 488 nm, (b) 543 nm. Red lines—the culture exposed to ultrasound; blue lines—the control culture. Each

conditions during the whole period of the experiment.

were compared (**Figure 6**).

spectrum was obtained by averaging over 15 cells.

In the previous section, we present a novel noninvasive technique for estimation of the physiological state of single living cyanobacterial cells. Let us demonstrate the results of the application of this technique to the ultrasonic treatment experiments with cyanobacterial cultures.

In this investigation, the photosynthetic activity of living cyanobacterial cells treated by ultrasonic radiation was studied. A strain *Synechocystis CALU 1336 aquatilis* from CALU collection of core facility Center for Culture Collection of Microorganisms of St. Petersburg State University was used in the experiment. After 8 days incubation, the tested culture was divided into two parts and placed in the same light, temperature and nutrient conditions. One part was a control sample and another part was sonicated via original, specially designed, ultrasonic laboratory device shown in **Figure 5**.

The sonication was performed in a 35-mm plastic Petri dish placed on the ultrasound emitter. Ultrasound emitter consists of a ceramic resonator connected with handheld pocket frequency generator HPG1 (Velleman Instruments Inc.), it has an emitting surface area about 13 cm<sup>2</sup> . The ultrasonic field inside the dish was measured out with a standard calibration ultrasound-needle-hydrophone connected to a TDS 3000 oscillograph (Velleman Instruments Inc.). For each experiment, 10 mL cyanobacteria solution was filled in a dish and kept at 25 ± 2°C. The ultrasound frequency and power density were about 60 kHz and 5.85 mW/mL, correspondingly, and the sonication time was near 24–30 h. Such sonication conditions were

**Figure 5.** The developed device for laboratory ultrasonic treatment of cyanobacterial culture. (1) Plastic petri dish, (2) ultrasonic emitter, (3) generator of ultrasonic frequencies and (4) sample culture.

chosen following the literature reports [32, 86] and based on our preliminary tests. During sonication, the cyanobacteria solution was carefully shuffled and taken for analysis just in the process of sonication. Control sample without any treatment was kept under exactly the same conditions during the whole period of the experiment.

In **Figure 6**, single-cell fluorescence spectra for two emission wavelengths (488 and 543 nm), obtained by standard lambda-scanning using Leica TCS-SP5 CLSM, are shown for control and treated samples. Blue lines demonstrate the spectra of the control suspension, whereas red lines show the spectra of the experimental one. It is clear that the fluorescence intensity in the control group differ from the experimental one. According to the preliminary studies, enhancement of the fluorescence at 660 nm in the experimental culture indicates that this culture is in a depressed physiological state in comparison with the control group. Each spectrum in **Figure 6** was obtained by averaging over 15 cells. Spectral analysis on the cell level using CLSM makes it possible to obtain more comprehensive information on the small variations in physiological state of both single cells and the entire culture exposed to ultrasonic treatment. During ultrasonic treatment, single cyanobacterial cells were studied at the first day to obtain the reference spectra of the initial cells in a good physiological state. Then, after 24–30 h, both control and treated specimens were studied and their fluorescence spectra were compared (**Figure 6**).

Let us mention here again that the treated and control probe initially were taken from one cultural sample and were placed at the same environmental conditions. Else, all the obtained results were double controlled via conventional fluorometric methods such as pulse amplitude modulation (PAM) and absorption and fluorescent characteristics of the whole culture [90]. These measurements were conducted using the light curves method on the PAM 2500 pulse spectrofluorimeter (WALZ, Germany) and a standard spectroscopic technique using a Cary Eclipse fluorimeter (Varian Cary), correspondingly. The dependences of the electron transport rate (ETR) and the quantum yield of photosystem PS II (Y(II)) on photosynthetically active radiation (PAR) under blue actinic light, obtained via WALZ PAM, show that ETR and

**Figure 6.** Single-sell fluorescence spectra for cyanobacterial strain Synechocystis CALU 1336 obtained at two excitation wavelengths (a) 488 nm, (b) 543 nm. Red lines—the culture exposed to ultrasound; blue lines—the control culture. Each spectrum was obtained by averaging over 15 cells.

**Figure 5.** The developed device for laboratory ultrasonic treatment of cyanobacterial culture. (1) Plastic petri dish, (2)

changes. May be the whole culture died due to other reasons or simply several cells were destroyed during sonication. Moreover, the pointed sonication power settings (about 0.32W/mL) cannot be considered as a weak and environmentally friendly treatment. It cannot be applied to the natural reservoirs, so that to carry out a real control on cyanobacterial blooms in open water. Obviously, this high intensity of sonication was used to obtain any possible results because used methods are not precise enough. Thus, this example shows that the standard methods do not give the correct results, and new precise, nondestructive *in vivo* methods for monitoring of the

In the previous section, we present a novel noninvasive technique for estimation of the physiological state of single living cyanobacterial cells. Let us demonstrate the results of the application of this technique to the ultrasonic treatment experiments with cyanobacterial cultures. In this investigation, the photosynthetic activity of living cyanobacterial cells treated by ultrasonic radiation was studied. A strain *Synechocystis CALU 1336 aquatilis* from CALU collection of core facility Center for Culture Collection of Microorganisms of St. Petersburg State University was used in the experiment. After 8 days incubation, the tested culture was divided into two parts and placed in the same light, temperature and nutrient conditions. One part was a control sample and another part was sonicated via original, specially designed,

The sonication was performed in a 35-mm plastic Petri dish placed on the ultrasound emitter. Ultrasound emitter consists of a ceramic resonator connected with handheld pocket frequency generator HPG1 (Velleman Instruments Inc.), it has an emitting surface area about

. The ultrasonic field inside the dish was measured out with a standard calibration ultrasound-needle-hydrophone connected to a TDS 3000 oscillograph (Velleman Instruments Inc.). For each experiment, 10 mL cyanobacteria solution was filled in a dish and kept at 25 ± 2°C. The ultrasound frequency and power density were about 60 kHz and 5.85 mW/mL, correspondingly, and the sonication time was near 24–30 h. Such sonication conditions were

physiological state of cyanobacterial cultures are required for such investigations.

ultrasonic laboratory device shown in **Figure 5**.

13 cm<sup>2</sup>

26 Cyanobacteria

ultrasonic emitter, (3) generator of ultrasonic frequencies and (4) sample culture.

Y(II) decrease in the sonicated culture, which indicates that the physiological state of the culture under sonication is depressed. At the same time, the nonphotochemical quenching of the absorbed light by the fluorescence rises considerably for the treated culture. Comparison of the results of the CLSM spectroscopic measurements with those obtained using conventional fluorimeter and pulse-amplitude modulation approaches confirmed the inhibitory effect of low ultrasonic frequencies (~60 kHz) on the physiological state of cyanobacterial cells and whole cyanobacterial cultures.

for unicellular species and is useless for numerous industrially cultured filamentous strains. HPLC is the only method, of the three, that is based on the chemical constituents in the sample. The problem is that during the chemical sample preparation, most of the information about the peculiarities of individual species is lost and the residual part of the information is not enough for species/strain classification inside cyanobacterial genera, and is suitable only for the rude differentiation of big classes of phytoplankton. As it was mentioned earlier, several factors contribute to the spectroscopic properties of the phycobilins: the number and chemical nature of the bilins attached to the polypeptide chains; the effects of protein conformation or aggregation state; and interaction between the bilins. Any of this feature can be unpredictably changed during the extraction and purification procedure [96]. Thus, only spectroscopic properties of the intact living cells can give pure unspoiled information about distinctive fea-

Fluorescence Microscopic Spectroscopy for Investigation and Monitoring of Biological Diversity…

http://dx.doi.org/10.5772/intechopen.78044

29

Analysis of the *in-vivo* absorption and fluorescence spectra is an alternative way of obtaining qualitative information about the phytoplankton abundance and composition, which is continuously demonstrated by various publications [21, 23–27, 36, 97–100]. The relative phytoplankton abundance can be calculated once initial assumptions about the phytoplankton classes present and their pigment compositions have been made [10, 24, 25, 36, 100]. However, the correct classification of cyanobacterial species on the base of their fluorescence signature was hampered by alterations in pigment composition within one strain, which depends on the environmental conditions [93]. On the other hand, several researchers show that the nutrient and light limitations do not significantly change the initial fluorescence spectra and can-

May be the first attempt to use phycoerythrins as chemotaxonomic markers was done by Glazer et al. [96] for red algae in 1982, but until now fluorescence spectra of phycobilins do not appear to be useful at familial, ordinal and class levels in taxonomic studies. Although the investigation in [96] concerns only purified high molecular weight phycoerythrin from red algae this work clearly demonstrates the possibility of the correct taxonomic analysis on the base of phycobiliproteins structural differences, which can serve as intrinsical fingerprints for taxons and genera in phytoplankton diversity. Later the correlation between the distribution of the biliproteins and the genera of *Cryptophyceae* was discussed in [102]. In 1985, Yentsch and Phinney [19] proposed an ataxonomic technique that utilized the spectral fluorescence signatures of major ocean phytoplankton. Seppälä and Olli [97] used spectral fluorescence signals to detect changes in the phytoplankton community. In 2002, Beutler et al. reported a reduced model of the fluorescence from the cyanobacterial photosynthetic apparatus designed for the in-situ detection of cyanobacteria and presented a commercially available diveable instru-

We elaborate a strict procedure for recording and processing single-cell fluorescence emission spectra, which eliminates the most of mentioned above difficulties and has a quite high classification accuracy. As well as according to our technique the fluorescence spectroscopic information is obtained via CLSM, the initial data has less variations and can be accurately sorted. Any objectionable and unpredictable impact can be eliminated at the first step of obtaining fluorescence spectra. Since noninvasive and nondestructive method is used the information about vital cell operation (e.g., light harvesting) can be additionally taken into account. All

this allows one to obtain the desirable result directly following the procedure.

tures of light harvesting complex in specified cyanobacterial strain.

not impede the species discrimination [98, 101].

ment for on-line monitoring of phytoplankton structure [21].

It can be concluded with confidence that the results obtained via the CLSM technique are correct, and the reduction of the photosynthetic activity and dumping of single-cell physiological state occur as a reply on the external ultrasonic action. The results presented here demonstrate the experiments conducted with the strain Synechocystis CALU 1336 aquatilis; however, similar results were already obtained for another unicellular cyanobacterial species (*Microcystis CALU 398*). Thus, the treatment presented here may refer to a rather diverse group of unicellular cyanobacteria.

It should be noted that ultrasonic treatment is widely used not only for inhibition of cyanobacteria growth diring harmful blooms, but also for enhancing of protein content and the whole biomass in the industrially cultured strains [91, 92], depending on power-frequency characteristics. Thus, it is very important to obtain on-line correct information about the influence of the ultrasound with given power and frequency on the specified cyanobacterial strain. The noninvasive fluorescent technique presented here gives the opportunity to detect any weak variations in the physiological state of single cyanobacterial cells in real time during sonication.
