Overview

Cyanobacteria, also known as blue-green algae, are one of the first photosynthetic organisms to have evolved on Earth. Cyanobacteria are regarded as photoautotrophic prokaryotes, having internal membranes with thylakoids, where photosynthesis is performed. Besides possessing these photosynthetic structures, cyanobacteria differ from other bacteria in many ways: size (cell sizes are comparatively larger), distribution (they are only present in moist areas), locomotion (they lack flagella and any other locomotory organ) and presence of heterocysts (Fig.1).

1. Reception of bacterial samples

A world-wide increase in the incidence of toxin-producing, harmful cyanobacterial blooms (cyanoHABs) over the last two decades has prompted a great deal of research into the triggers of their excessive growth. Massive surface blooms are known to decrease light penetration through the water, cause depletion of dissolved oxygen following bacterial mineralization of blooms, and cause mortality of aquatic life following ingestion of prey with high concentrations of toxins.
Additionally, humans coming in contact with the water may develop digestive and skin diseases, and it may affect the drinking water (Berg et al.,2015).

The five cyanobacteria species were supplied from Pasteur Institute, The Pasteur Culture Collection of Cyanobacteria. The species were the following:

• PCC 7121-Nostoc (Nostoc muscorum)
• PCC 7512-Gloeocapsa- CB1
• PCC 7425-Cyanothece (Synechococcus sp.)- CB2
• PCC 6602-Oscillatoria
• PCC 7942-Synechococcus- CB3

These species were chosen as they are all widespread types of cyanobacteria in freshwaters, which contribute to the nitrogen fixation process in the aquatic environment, forming organic compounds as a result of this mechanism. Moreover, they are considered to be safe, as they do not release the toxic compounds described above.

Each species were placed in a 5 mL tube. PCC 7512, PCC 7425, PCC 7942 were suspended in the growth medium (BG11), PCC 7121 was placed in agar gel and PCC 6602 was in a compacted, circular shape (Fig.2)

*Note: The annotations CB1, CB2, CB3 were made for this study for an easier identification of the cyanobacteria species used in each experiment

All species were grown in BG-11 medium (firma, tara), which contains the nutrients: H3BO3 (0.00287%), MnCl2•4H2O (0.00181%), ZnSO4•7H2O (0.00022%), Na2MoO4 (0.00039%), CuSO4•5H2O (0.00008%), NaNO3, (0.15000%), CaCl2•2H2O (0.00270%), C6H5 4yFexNyO7 (0.00120%), EDTA (0.00010%), K2HPO4 (0.00390%), MgSO4•7H2O(0.00750%), Na2CO3•1 H2O(0.00200%), at a ratio of 1:10 initial cyanobacteria to growth media.

2. Transfer of received specimens into growth medium

The strains were transferred immediately after arrival to create specific culture conditions so that they would not have released their pigments. Each 5 mL tube of cyanobacteria was split into 4 flasks (each containing 1,25 mL cyanobacteria and 12,5 mL medium). (Fig.3).

Fig.2.-Cyanobacteria specimens (Pasteur Institute, France) (from left to right): PCC 7121, PCC 6602, PCC 7942, PCC 7425, PCC 7512

Fig.3-Each cyanobacteria was split into 4 tubes

3. Cultivation

Before measuring any properties, cultures were grown for two weeks to observe their growing cycle. For this initial time period, samples were kept at room temperature (≈ 23°C), under indirect sunlight, according to the day/night cycle. The day after transfer, it could be seen that bubbles of oxygen were formed inside the flasks, indicating oxygen production, which continued to be seen in this form over the 2 weeks (Fig.4).

In addition to this, another visible change was that the bacteria did not remain homogenized with the culture media, instead it settled on the bottom of the flask. After one week of growth, there was a slight colour change (pale yellow) for BG-11 medium, which indicated a consumption of nutrients. The medium was changed by extracting 6,25 mL of the used solution and replacing it with the same quantity of BG-11 fresh growth media.

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Fig.4-First day after transfer: oxygen formation as a result of photosynthesis for PCC 7512

4. Studying cyanobacteria growth dynamic

Principle: measuring the optical density of different concentrations of cells from each species of cyanobacteria Before measuring other properties, it is needed to know the growth dynamic of each species in order to correlate it with any changes associated with it. Light absorption, as described by Beer–Lambert law states that it is directly proportional to the optical path length and concentration of the solution. In the case of this experiment, optical density, which takes into account the absorption of the light of a specific wavelength, is proportional to the number of cyanobacteria in each sample.

Methods: Beginning the experiment, cultures were grown within a controlled environment in a fume hood (Esco Frontier® Acela Fume Hood) at 22° C, following a light regime of 12 h light/ 12 h dark, with the light intensity of 886 lux, with the light being provided by the LED lamp of the fume hood. To maintain these conditions, flasks were placed on top of an electric heating mat (LERWAY 24, UK) with controllable temperature and a programmable power outlet (Well, 220 - 240V, 16A) was used for maintaining light conditions. (Fig.5) The 12h:12h photoperiod was chosen as studies conclude that most biomolecules accumulate after exposure of over 8h of light, with lipids, being accumulated after 12h (Sánchez-Bayo et al.,2020). This period also provides the closest representation to the natural light/dark cycle

Fig.5-The growth conditions for cyanobacteria

Initially, the cyanobacteria concentration of each analysed species was determined using an Improved Neubauer cytometer (Hirschmann, 0,100 mm depth, 0,0025 mm2). A suitable concentration for counting was achieved with a 1:10 dilution (10 µL bacteria suspension to 90 µL H2O), which was placed in an Eppendorf tube. A 10 µL volume of the diluted suspension containing the cyanobacteria was placed under the coverslip, and the sample was analysed using a microscope (Levenhuk, Inc., USA).

The number of cells was determined using the following equation (1), where f represents the concentration-dilution factor. All measurements were performed in duplicate and results shown in tables are based on the average corrected with standard deviation.

Results are described in the table below:

Table 1-Number of cells in 10µL of each cyanobacteria sample number of cells/quadrant

A 24 well VWR Standard Multiwell Cell Culture Plate 0.38 - 0.57 mL (Avantor, United States) was used to perform the optical density analysis. Three constant cell numbers of each bacteria sample (50.000, 150.000, 300.000 cells/well) were chosen to be plated, using the equation (2)

The remaining well volume was filled with BG-11 medium.

Table 2- Volumes of cyanobacteria suspension and growth medium to be plated in each well calculated using equation (2)

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Fig.6- Culture plate placed according to values in Fig.6, where N1, N2, N3 represent the three constant numbers for each cyanobacteria

4.1 Spectral analysis of cyanobacteria samples

The optical density of the species was measured using a SpectraMax iD3 Multi-Mode Microplate Reader (Molecular Devices, United States) A spectrum read was done to detect peak signals.

The optical density of the species was measured using a SpectraMax iD3 Multi-Mode Microplate Reader (Molecular Devices, United States) A spectrum read was done to detect peak signals.

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Fig.7- Signals emitted by BG-11 and cyanobacteria (a) and signals emitted by BG-11 in comparison with BG-11 and cyanobacteria (b)

Based on this graphic representation, wavelengths of possible interest can be determined for future studies. Detection sensitivity is higher where peaks appear: The growth medium containing bacteria also has specific absorbance peaks (440-450 nm, 620-640 nm, 670-690 nm, 750-760 nm, 800 nm, 870-880 nm, 950-980 nm). These peaks appear as a result of specific cyanobacteria signals, whereas for the BG-11 culture medium peaks are given only by the medium specific absorptions (670-690 nm, 750-760 nm, 870-88 nm, 950-980 nm).

Six different wavelengths were chosen for this study: 440, 620, 680, 750, 870, 970 nm.

Spectral reading was done twice a day as described in the scheme below during the light cycle following a repeated 8h/16h interval.

Fig.8- Spectral reading in 24h following the light/dark cycle of the bacteria

Six different wavelengths were chosen for this study: 440, 620, 680, 750, 870, 970 nm.

Results and discussions

Fig.9- Growth curve for PCC 7512 at different wavelengths and different concentrations

4.2 Determining optimal cell concentration for absorbance detection

For PCC 7512, it can be seen that for the smallest cell concentration, a clear growth pattern cannot be observed as trendline slopes vary between linear, exponential and polynomial and the scatter plot does not accurately match the line. For 150.000 cells/well as well as 300.000 cells/well the growth curve is linear. The overall highest growth curve was seen for 440 nm, in the blue part of the spectrum due to presence of chlorophyll a similar with other studies (Luimstra et al.,2018), which has an absorbance peak for this wavelength.

After performing spectral analysis for PCC 7512 and identifying 150.000 and 300.000 cells/well as the constant number with the most predictable growth pattern, similar conditions were considered also for PCC 7425 and PCC 7942. The following experiments used these two constant numbers of cyanobacteria to be plated and analysed. The tables below represent all 3 cyanobacteria species (CB1 – PCC7512, CB2- PCC7525, CB3 – PCC7942) compared at a constant wavelength. The values under the detection limit are not represented in the figures (PCC 7942 having the most values under the detection limit - for 750 nm, 870 nm, 970 nm values were not detectable for the first 64h).

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Fig.10- Growth curve for PCC 7512 (a), PCC 7425 (b), PCC 7942 (c) at 440 nm with optimized cell concentrations

4.3.1 Spectral analysis at 440 nm

Similar growth patterns are observed for PCC 7425 and PCC 7942, with PCC 7425 reaching higher values for both cell concentrations. For the lower cell concentration, all species multiplied exponentially and for the higher concentration, the same pattern was observed, except for PCC 7512 which had a linear growth (for 300,000 cells/well).

4.3.2 Spectral analysis at 620 nm

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Fig.11- Growth curves for PCC 7512 (a), PCC 7425 (b), PCC 7942 (c) at 620 nm with optimized cell concentrations

At 620 nm, the species reaching the highest absorbance values was the same as for the previous wavelength, PCC 7425. PCC 7512 has a linear growth for both concentrations of cells, while PCC 7425 and PCC 7942 have different growth patterns between cell concentrations (exponential for PCC 7425 and polynomial for PCC 7942 - lower cell concentration).

4.3.3 Spectral analysis at 680 nm

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Fig.12- Growth curve for PCC 7512 (a), PCC 7425 (b), PCC 7942 (c) at 680 nm with optimized cell concentrations

Similar to the previous wavelength, PCC 7512 has a linear growth for both cell concentrations. For the higher cell concentration, strains PCC 7425 and PCC 7942 have an exponential growth. For the lower cell concentration, PCC 7425 has an exponential growth, whereas PCC 7942 has a polynomial growth.

4.3.4 Spectral analysis at 750 nm

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Fig.13- Growth curve for PCC 7512 (a), PCC 7425 (b), PCC 7942 (c) at 750 nm with optimized cell concentrations

For the higher cell concentration, PCC 7425 and PCC 7942 have a polynomial growth, while for the lower cell concentration, strains have a different growth pattern, but unlike for other wavelengths, PCC 7425 has a polynomial growth and PCC 7942 has an exponential value, although R2 has a low value, indicating a lack of consistency. As for PCC 7512 at this wavelength, neither cell concentrations expressed a growth pattern and absorbance values were low, as the peak for this wavelength was not clearly seen in the spectral analysis.

4.3.5 Spectral analysis at 870 nm

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Fig.14- Growth curve for PCC 7512 (a), PCC 7425 (b), PCC 7942 (c) at 870 nm with optimized cell concentrations

For PCC 7512, the cells have a linear growth for both concentrations. For the higher concentration, both PCC 7425 and PCC 7942 have an exponential growth, while for the lower concentration both have a similar pattern as for the previous wavelength.

4.3.6 Spectral analysis at 970 nm

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Fig.15- Growth curve for PCC 7512 (a), PCC 7425 (b), PCC 7942 (c) at 970 nm with optimized cell concentrations

For PCC 7512, both cell concentrations have a linear growth. For the higher concentration, PCC 7425 and PCC 7942 have an exponential growth, whereas for the lower concentration, PCC 7425 has a polynomial growth and PCC 7942 an exponential growth.

Tables below represent the absorbance measured for PCC 7512 for a period of one week for all 6 wavelengths measured. The absorbance values below detection limit are not represented, the last omission value being for 24h at 870 nm.

Considering R², the wavelength with the highest overall value is 440 nm, which indicates that for this wavelength the growth pattern is described the most accurately as above, with all cyanobacteria strains following a predictable growing pattern. Both 620 nm and 680 nm also have good R2, similar with to each other. At 750 nm, the data is difficult to analyse, therefore for the red portion of the spectrum, the most suitable wavelengths are 620 nm and 680 nm. Another parameter which indicates an optimal growth is the absorbance dependency on the cell concentration.

The cyanobacteria with the highest final absorbance value for all wavelengths are PCC 7425, suggesting a fast growth and increased cell concentration during the experiment.

5. Influence of different environmental factors on the growth rate

Five principal drivers emerged as important determinants of cyanobacterial blooms in a review of the global literature on factors influencing cyanobacteria blooms and toxin production (Kaushik et al., 1994).

These include:

• Water temperature

• Water column irradiance and water clarity

• Stratified water column coupled with long residence times

• Availability of N and P - in non-limiting amounts; scientific consensus is lacking on the importance of N: P ratios as a driver for cyanoHABs

• Salinity regime

5.1 Thermal treatment-the influence of temperature on the growth rate

Principle: Another parameter that is taken into consideration for the growth rate analysis of cyanobacteria is temperature. Elevated temperature may also affect membrane fluidity and could determine protein denaturation, which can also lead to a decline in photosynthetic efficiency (Erik et al.,2019) .

Methods: After one week of studying the growth rate under the recommended temperature, the same 24-well plate was subjected to a temperature increase regulated by the heating mat, reaching a temperature of 29° C. This temperature was achieved gradually over a period of 6 hours. The spectral reading was done for the same wavelengths as described before to compare results before and after heat treatment. The time frame was the following: 0h, 15h, 24h, 40h (0h represents the first spectral analysis after samples reached 29° C). Results were analysed for constant wavelength at different bacteria.

Fig. a - representation of temperature increase

Results: All values for 0h are compared with the last value at the recommended temperature.

5.1.1 Spectral analysis at 440 nm

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Fig.16- Growth curve for PCC 7512 (a), PCC 7425 (b), PCC 7942 (c) at 440 nm after temperature increased

For PCC 7512 for the lowest cell concentration the absorbance value decreased from 0,04 to 0,02 in 8h and, considering that before the heating the growth curve was exponential, the growth no longer continued. This was most likely caused by the partial evaporation of the already small working volume, changes of the cell properties. For the other two concentrations, the absorbance values continued to increase with significant changes after the first 24h (for 150.000 cells/well a 10% increase and for 300.000 cells/well a 20% increase, versus time 0). For PCC 7425 the absorbance value decreased for both cell concentrations and there were not any significant changes over the time period observed. For PCC 7942 there was little to no difference between the last absorbance value before and after heat increase. Both concentrations had a similar evolution, which is comparable with the one for PCC 7512 for the medium cell concentration.

5.1.2 Spectral analysis at 620 nm

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Fig.17- Growth curve for PCC 7512 (a), PCC 7425 (b), PCC 7942 (c) at 620 nm after temperature increase

Results for 620 nm are the same as the ones above, considering the absorbance value modification, the only difference being for PCC 7942 where for the higher cell concentration the absorbance initially decreased from 0,1123 to 0,0899, but over the next 24h a clear linear increase could be observed as suggested by the R2 value (>0,99), as absorbance increased by 51%, indicating a doubling of the cell number.

5.1.3 Spectral analysis at 680 nm

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Fig.18- Growth curve for PCC 7512 (a0, PCC 7425 (b), PCC 7942 (c) at 680 nm after temperature increase)

For PCC 7512 differences to previous wavelengths were observed as only the highest cell concentrations had an increased absorbance after the heat treatment and for 150.000 cells the value remained similar (0,06 with optimal temperature and 0,062 after heating treatment). For PCC 7425 the absorbance decreased and remained linear after that. For PCC 7942 results were similar to the ones for 620 nm. The optica density for the samples containing the higher cell concentration increased by 54% over the course of 40h.

5.1.4 Spectral analysis at 750 nm

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Fig.19- Growth curve for PCC 7512 (a), PCC 7425 (b), PCC 7942 (c) at 750 nm after temperature increase

At 750 nm significant changes could be observed for PCC 7512 where for the highest cell concentration the value after temperature increase changed by 75%, with a decrease in the absorbance value. After that, the cells remaining multiplied starting from 15h with a value of 0,064 and reaching a final value of 0,1008. For PCC 7942 absorbance value did not change as much as for the other wavelengths after 40h.

5.1.5 Spectral analysis at 870 nm

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Fig.20- Growth curve for PCC 7512 (a), PCC 7425 (b), PCC 7942 (c) at 870 nm after temperature increase

For PCC 7512 the concentration with biggest absorbance value modification was 300.000 cells/well. The starting value for this concentration and for 150.000 cells/well was similar, however after 40h, the absorbance value for the higher concentration was proportional to the cell number , with a linear growth (R2>0,99). For the other two bacteria the growth dynamic was similar with the one for 750 nm.

5.1.6 Spectral analysis at 970 nm

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Fig.21- Growth curve for PCC 7512, PCC 7425, PCC 7942 at 970 nm after temperature increase

For PCC 7512, the absorbance for 50.000 cell/well decreased after the temperature change and increase for the other two concentrations, which had a similar growth pattern during the first 24h. This changed as the value for 150.000 cells/well remained relatively constant, with an increase of only 6%, whereas for 300.000 cells/well, the concentration changed more, with the absorbance increase being 35%. Results for the other two species were similar with the ones for 750 nm and 870 nm.

Discussion:

Photosynthetic organisms, such as algae and plants, have the ability to carry out photosynthesis efficiently within a range of temperatures. This adaptive capacity allows them to cope with variations in environmental conditions. Some acclimation strategies include balancing the electron flow through the electron transport chain (ETC), which a critical component of the photosynthetic process (Luimstra et al.,2018).

Overall, the bacteria with the most noticeable changes was PCC 7512 (150.000 and 300.000 cells/well), which continued to have an increased absorbance after the temperature change that still maintained that pattern, for most wavelengths. The highest increase could be seen after 24h. Higher temperatures promote metabolic acceleration (Rahman et al, 2023), which is a direct response to increased temperature. PCC 7425 remained static over the time period analysed for all wavelengths. As for PCC 7942 although the values decreased after the temperature change the final values increased, following a constant growth pattern. It should be noted that a change in temperature impacts the cell membrane composition, structure and permeability, with higher temperatures promoting increased fluidity. This results in an initial protein denaturation, which affects their function. After that, different heat shock proteins, which are induced as a temperature response, act as proteases repairing the protein structure (Mackey et al.,2013). After being exposed to heat shock, bacteria cells acquire thermotolerance, which is a feature proportional to the rate of expression of heat shock proteins.

For PCC 7942, several heat shock protein families (Hsp100 and Hsp90) have been studied before, with two main genes being responsible for: acquired thermotolerance (clpBI) and acquired and innate thermotolerance, as well as protection of the photosynthetic apparatus (htpG) (Rajaram et al., 2014). The initial absorbance decrease is a result of the heat shock, which initially damaged the cell membrane, before the proteins were activated.

5.2. The influence of pH on the growth rate

Principle: In culture media, the optimal pH for the growth of cyanobacteria ranges from 7.5 – 10, with a lower limit of 6.5 – 7.0. Among soil properties, pH is a very important factor in the growth, establishment and diversity of cyanobacteria, which have generally been reported to prefer neutral to slightly alkaline pH for optimum growth (Roger et al.,1979). The growth of cyanobacteria cultured under different pH conditions was analysed with a spectrophotometric method.

Methods: Since the previous experiment showed that PCC 7425 has a stagnant growth rate after increasing the temperature, this species was chosen to see the extent its growth is influenced by other factors such as the pH. Four pH adjustments were created for the BG-11 culture medium (pH3, pH5, pH8, and pH10). The volume of each prepared solution was 100mL. Each sample was treated with 0.1 M hydrochloric acid (HCl) or 0.1 M sodium hydroxide (NaOH) solution to obtain the desired pH while stirring the sample at 250rpm and the pH value was measured with a pH meter pH 50 VioLab (ROTH,Germany).A 10mL bacteria suspension was added to a 72 mL of each culture media. The pipetted volume in each well was 300 µL, with triplicates for each condition. The spectral reading was done for 440nm and 620 nm, for a time period of 48 hours, using 96 well VWR Standard Multiwell Cell Culture Plate.

Results and discussion:After 24 hours the cell number ( proportional to the absorbance value) remained relatively constant as the cyanobacteria was still adjusting to the new characteristics of the culture medium (lag phase). However, after 48 hours there is a clear increase of cell growth for pH 10 can be seen, suggesting that bacteria are positively influenced by an alkaline medium. Although, the growth pattern remains constant in BG-11, for the experimental condition at pH 10 the absorbance value rapidly increases. Samples grown in pH 8 increase had a similar increase in absorbance values over the time.

Fig.22- Growth curve for PCC 7425 at 440 nm in different pH conditions

Fig.23- Growth curve for PCC 7425 at 620 nm in different pH conditions

Results for 620 nm are similar as for 440 nm, with pH 10 resulting in the highest absorbance values. Cells grown in BG-11 culture medium have an exponential growth pattern, with a high coefficient of determination (R2=0,9977). It is interesting to observe however that pH 3 and pH 8 values almost overlap after 48 hours, indicating that the bacteria can grow both in acidic and basic medium. Several studies have analysed the optimal pH for cyanobacteria development, indicating that low pH values are not stable over time and it is not yet clear if cyanobacteria cells can proliferate in acidic mediums. It has been hypothesised that cyanobacteria can also exist in acidic medium due to their highly efficient CO2-concentrating mechanism (CCM) that allows them to take up CO2 and bicarbonate as an inorganic carbon source. This mechanism helps them maintain an elevated intracellular CO2 level around the enzyme RuBisCO, responsible for carbon fixation during photosynthesis (Xing et al.,2017).

In soil-culture experiments, soils having slightly alkaline reaction were more favourable, while in natural environments cyanobacteria prefer neutral to alkaline pH (Roger et al.,1979; De et al.,1939; Nayak et al.,2007). The development of soil acidity is generally believed to be associated with the base unsaturation caused by leaching out of bases and genesis from base-poor acidic rocks. The dissolved or free acidic substances, such as sulphuric acid and ferric and aluminium sulphate, accentuate acidity in acid sulphate soils (Dominic et al.,1999; Nayak et al.,2007).

Acidic soils are therefore one of the stressed environments for these organisms and they are normally absent at pH values below 4 or 5; eukaryotic algae, however, flourish under these conditions. Soil pH is also known to have a selective effect on the indigenous algal flora, especially cyanobacteria and their succession and abundance in soil (Nayak et al.,2007).

The effect of pH on algal flora is generally difficult to evaluate as it is often correlated with other factors, for e.g. arid soils are almost universally alkaline and many continuously wet soils acidic. Among correlations between the relative abundance of the individual groups of heterocystous cyanobacteria and soil physico-chemical properties, only the correlation between pH and the relative abundance of Nostoc was found to be statistically significant, but a degree of bias was introduced when dry and wet samples were tested separately (Roger et al.,1987; Nayak et al.,2007). Contradictory reports regarding the occurrence in acid and very acid environments are available.

However, one of the most acid lakes (pH 2.9) was observed to be inhabited by Oscillatoria/Limnothrix and Spirulina (Steinberg et al.,1997; Nayak et al.,2007). Despite the reference for neutral-highly alkaline environments, acidic soils do exhibit low diversity and abundance of cyanobacteria (Kulasooriya et al.1998; Nayak et al.,2007).

6. Description of the process of photosynthesis in cyanobacteria - analysis of the optimal method of extracting photosynthetic pigments using qualitative methods

Principle: Cyanobacteria differ from other bacteria as they are capable of photosynthesis because they contain a variety of photosynthetic pigments. Chlorophyll is a green pigment, which absorbs the red and blue spectrum of visible light and transmits green light. Due to the reflection of green light, all the chlorophyll-containing tissues or organelles appear green-coloured. The green colour of the leaves and stems is also due to this chlorophyll pigment. ChlorOphyll a and b have similar structures, with some differences that explain their different absorbance in the visible spectrum:

• Chlorophyll-a consists of a methyl group (-CH3) in the side chain and absorbs more red light of the visible spectrum.
• Chlorophyll-b consists of the aldehyde group (-CHO) in the side chain and absorbs more violet-blue light of the visible spectrum.

Fig.24 -Molecular structure of chlorophyll (https://biologyreader.com/chlorophyll-in-plants.html)

Chl-a is a primary pigment that absorbs the light energy (photons) from the sun (carries a bundle of photons) and passes it to the other pigment molecules till it outreaches a reaction centre. In a photosystem, a reaction centre functions as an electron donor that transfers the photons to the electron acceptor molecule for further cellular activities. An accessory pigment (chlorophyll-b) expands the light-absorbing capacity of the light-absorbing particles (https://biologyreader.com/chlorophyll-in-plants.html).

Light harvesting is performed by chlorophyll a (Chl a) pigment molecules that are associated with two photosystems (PSI and PSII) that comprise the centers of the photosynthetic process which starts with the liberation of an electron from the splitting of water and ends with the production of ATP (Berg et al., 2015). What makes the cyanobacteria unique is that they have a second light-harvesting antenna complex peripheral to the thylakoid membrane that is water soluble (e.g. not membrane bound). This pigment complex, comprised of pigmented proteins arranged in rods fanning out from a core attached to the thylakoid membrane, called the phycobilisome (PBS), is what gives cyanobacteria their name (Grossman et al.,1993; Grossman 2003). Similar to the carotenoid pigments, the PBS chromophores absorb light in between the Chl a absorption peaks of 440 nm and 670 nm (Grossman et al.,1993). Interestingly, the PBS proteins are not exclusive to cyanobacteria; they also occur in photosynthetic eukaryotes.

The extraction of photosynthetic pigments from cyanobacteria can reveal valuable information regarding the organism's growth rate, as viable cells can be assessed depending on pigment quantities. There are several extraction methods, most of which require polar solvents that can take up water. One method which allows visualising the components of the extract is TLC(thin layer chromatography), involving two phases: a stationary phase also known as the adsorbent bound to the TLC plate (represented in this case by silica) and a mobile phase which is the solvent carrying the sample. The components of the extract are separated based on polarity.

Methods: Chlorophyll was extracted with four different solvents in order to determine which one helps achieving the highest amount of chlorophyll:

• methanol-CH₃OH
• acetone- C3H6O
• 80% acetone
• ethanol- C₂H₆O

Results and discussion

Fig.25- TLC Silica gel 60 plate with extraction using four solvents(from left to right): 80% acetone (1), acetone (2), ethanol (3) and methanol (4) in visible light(a) and under UV light(b)

Fig.26- TLC Silica gel 60 RP18F256F plate with extraction using four solvents(from left to right): 80% acetone (1), acetone (2), ethanol (3) and methanol (4) in visible light(a) and under UV light(b)

The pigment separation begun approximately five minutes after the plate was introduced into the eluent. As extraction was done from a small volume of cyanobacteria and the extract was not concentrated, pigments appear as very thin lines. However, the lines can be clearly seen, the green ones for chlorophyll (most likely chlorophyll a as this type is the most abundant) and the light orange for carotenoids. Carotenoids appear higher than chlorophyll as they are more soluble and have smaller molecules, which means they are less absorbed by the TLC plate, making them migrate faster.

Under UV light, the pigments from the extraction made with pure methanol appear to be the most intense (chlorophyll fluorescence in red light), suggesting that this solvent was the most efficient. Ethanol also worked well, as the line is similar to the one for the methanol extraction. This result is also in direct correlation with another study which concludes that among different solvents (diethyl ether, 5% ethanol, pure acetone, 20% acetone, pure methanol and 10% methanol), pure methanol was the best for extraction of chlorophyll a (Ahmadi et al.,2019).

7. Analysis of the concentration of photosynthetic pigments using optimized extraction method

Principle: Pigments from cyanobacteria are determined by spectral analysis, the results being normalized according to the biomass. The photosynthesis process in cyanobacteria is controlled by various photosynthetic pigments such as chlorophyll a, chlorophyll b and carotenoids found in the cell’s cytoplasm.

Methods: The pigments were extracted using the following method. A 3 mL sample from each of three species analysed (PCC 7942, PCC 7425, PCC 7512) was centrifuged at 4000 rpm for 6 min, the supernatant was discarded and after that each pellet was resuspended in 3 mL of 100% methanol. Samples were placed in an ultrasonic bath (PCE Instruments, UK) and sonicated for 5 min and centrifuged again for 6 min at 4000 rpm.

Fig.27- Pigments extracted in supernatant after the second centrifugation for PCC 7942 (a), PCC 7425 (b), PCC 7512 (c)

The supernatant was separated from the white pellet and placed in a 96 well VWR Standard Multiwell Cell Culture Plate (Avantor, United States, 0.075 - 0.20 mL-250 µL/well) to be subjected to a spectrophotometric analysis (in duplicates) at different wavelengths 665 nm (A665), 652 nm (A652), 468 nm (A468) and turbidity corrections (A750) The quantity of pigments was determined using equations 3-6: (Adochite et al.,2021). Values were calculated for each duplicated sample and the average was expressed between the two values.

Chlorophyll-a ^μg⁄_mL=[ 16,72(A665-A750)-9,16(A652-A750)] (3)
Chlorophyll-b ^μg⁄_mL=[ 34,09(A652-A750)-15,28(A665-A750)] (4)
Total chlorophyll ^μg⁄_mL=[ 1,44(A665-A750)+24,93(A652-A750)] (5)
Carotenoids ^μg⁄_mL=[ 1000·(A468-A750)-1.63·Chl a-104.96·Chl b]/221 (6)

Pigments were assessed according to biomass, which was extracted from 3mL bacteria suspension using a vacuum pump (Labbox Labware, Spain). The solution was placed on a 150 mm qualitative filter paper, which was dried for 24 hours and weighed to determine the mass of extracted cells.

Fig.28- Photosynthetic pigments quantification(µg/mL): chlorophyll a, chlorophyll b, total chlorophyll and carotenoids for PCC 7942, PCC 7425, PCC 7512

Results and discussion Photosynthetic pigments chlorophyll a, chlorophyll b, total chlorophyll and carotenoids were quantified for PCC 7942, PCC 7425, PCC 7512 cyanobacteria and the results are indicated bellow (Fig. 28).

Chlorophyll a is responsible for photosynthesis, as the electrons surrounding a stable molecule move freely, therefore the molecule is able to capture electrons, transporting the sunlight energy. Chlorophyll b is also involved in photosynthesis by expanding the absorbance spectrum of light, as these pigments only react to certain wavelengths. According to the UC Museum of Palaeontology, carotenoids function in conjunction with chlorophyll as they pass the stored energy to be further used in the photosynthetic process. In figure 28, it can be seen that PCC 7512 has the highest concentration of photosynthetic pigments, while PCC 7942 and PCC 7425 have similar quantities. For each species chlorophyll a accounts for the highest value of pigments, while chlorophyll b has the lowest value, representing 22,5% for PCC 7942, 18% for PCC 7425 and only 7,3% for PCC 7512 out of the total chlorophyll value.

According to a study which analyses pigment accumulation of cyanobacteria under different light colours (Kim et al.,2014), chlorophyll b absorbs mostly blue light, with values for white light (used in the described experiment as well), being the lowest out of all LED colours analysed. PCC 7512 had the lowest initial cell number, however the cells for these bacteria were clearly defined and had a larger diameter than for the other two species, which appeared punctiform. This structural cell difference could represent a cause for the significant pigment variation between species.

8. Exposure of cyanobacteria species to different growth media variations

Analysis of the growth of cyanobacteria in different growth media

Principle : Different nutrients found in lake water (e.g. phosphorus and nitrogen) favour the growth of cellular mass for cyanobacteria. Phosphorus (P) is an important nutrient central to storing and the exchange of energy and information in the cell (Solovchenko et al.,2020), while nitrogen fixation is a result of the photosynthetic process of cyanobacteria, which uptake the nitrogen from sources such as nitrate and ammonium. Spectrophotometric analysis was performed to determine development of bacteria, which were grown in different culture media, containing lake water and BG-11 medium.

Methods: Four different medium solutions were created for each species of cyanobacteria, mixing in different ratio the volumes of the recommended media (BG-11) and a waterlake source (v:v): BG-11+cyanobacteria, WL (lake water) + cyanobacteria, 1:1 BG-11: WL + cyanobacteria, and 2:1 BG-11: WL + cyanobacteria. The lake water, which was collected from a lake in Brasov city (Lake Noua) was filtered before being pipetted. The spectral reading was done for the 6 different wavelengths described in the previous experiments, for a time period of 72 hours, using 96 well VWR Standard Multiwell Cell Culture Plate. Each well had a 300 µL volume of samples, which was pipetted from the recipients that contained a total volume of 3 mL of the aforementioned growth media (prepared before the study). A constant number of 300.000 cells was chosen as a reference as the growth curve could be best observed at this concentration according to the previous experiments.

The ratio of cyanobacteria to growth medium to be plated was calculated in accordance with the chosen constant cell number and initial cell concentration (from cell counting). Therefore each well contained 40 µl cyanobacteria and 260 µl different media. The proportional values of 300 µl and 3 mL were chosen to have direct proportionality between optical density values and pigments, which were later extracted from the 3 mL solutions.

The ratio of cyanobacteria to growth medium to be plated was calculated in accordance with the chosen constant cell number and initial cell concentration (from cell counting). Therefore each well contained 40 µl cyanobacteria and 260 µl different media. The proportional values of 300 µl and 3 mL were chosen to have direct proportionality between optical density values and pigments, which were later extracted from the 3 mL solutions.

The experiment was done in triplicates as presented in the scheme below:

Fig.29- Plating of cyanobacteria species and different growth media for spectral analysis

Results: The plate was read immediately after placing the corresponding quantities of medium and cyanobacteria into the wells, and analysed after 16 hours, 24 hours, 48 hours, and 72 hours. The following graphics depict the results of the measurements. Values that were under the detection limit were eliminated from the representations.

8.1. Spectral analysis at 440 nm

For PCC 7512 it can be seen that the 2:1 and the BG-11 cells have grown the most, which is expected, as they have the highest quantities of BG-11 medium, which is the most benefic for the bacteria. The rest of the results indicate an exponential growth, with the final values ranging between 0.10 and 0.16.

For PCC 7425 it can be seen that the BG-11 average indicates a clear exponential growth, while the rest of the media also have this type of growth. This could be due to the interaction between a larger amount of water and a smaller amount of medium. The final values are in a similar range to the PCC 7512 graph (0.11-0.16).

For PCC 7942 shows that the WL, 1:1, and 2:1 have a very similar growth path, while the BG-11 results indicate the highest multiplication rates, as expected. BG-11 and WL have clear exponential growth, while 2:1 and 1:1 have a lower but still good coefficient of determination. The other final values are in a similar range as for the previous two graphs (0.11-0.17).

Fig.30- Growth curve for PCC 7512, PCC 7425, PCC 7942 at 440 nm in different media

Overall, the most significant changes for the media containing different lake water concentrations can be seen after 48 hours, this being possibly explained by the uptake of nutrients promoting growth, which the bacteria did not interact with initially, as a result of sudden media change.

8.2. Spectral analysis at 620 nm

For PCC 7512 the growth is exponential once more, with BG-11 having the highest values for each time interval measured. The final values, range from 0.06 to 0.11. After 48 hours, the bacteria grown in lake water reached similar values to the one grown in the 2:1 ratio, which doubled its absorbance values between 24 and 48 hours.

The 620nm graph for PCC 7425 indicates that all media had an exponential growth, with BG-11 having the highest R2 value. However, its last value is considerably higher compared to other graphs. The values range from 0.05 to 0.11. Considering the last values, this bacteria species preferred lake water as growth medium, the most intense cell development being between 48 and 72 hours.

For PCC 7942 indicates an exponential growth once again, however, this time, the last values are distant from each other, ranging from 0.06 to 0.12, indicating a clear difference between the media. BG-11 has a high perfect coefficient of determination.

Fig.31- Growth curve for PCC 7512, PCC 7425, PCC 7942 at 620 nm in different media

8.3. Spectral analysis at 680 nm

For PCC 7512 indicates once again that BG-11 facilitates the best growth rate, as expected. The rest of the values also have an exponential growth with good coefficients of determination. The final values range between 0.05 and 0.10.

BG-11 has had lower final values for PCC 7425 for the analysed wavelengths, which could be due to the fact that this cyanobacteria strain has colonies of smaller sizes, that might not benefit as much from the medium as the others do. However, as it is illustrated in this graph, it still has a clear exponential growth. The other media also have an exponential growth similar to BG-11 until the last values, which are higher. Overall, final values range from 0.06 to 0.11.

For PCC 7942 the table shows an interesting growth path of 1:1. The first 4 values follow a linear growth (this can be observed in the other graphs for PCC 7942 for 1:1 too, but it is far more noticeable here), while the last value is triple in comparison with the one measured before, therefore classifying the trendline as exponential. This could be due to the interaction between an equal quantity of BG-11 and lake water, which could need a few days to stabilize and reach an optimal growth rate. The other media also have an exponential growth rate, with BG-11 showing once again the highest multiplication rate.

Fig.32- Growth curve for PCC 7512, PCC 7425, PCC 7942 at 680 nm in different media

8.4. Spectral analysis at 750 nm

The 750nm graph for PCC 7512 illustrates an exponential growth once again for all mediums with BG-11 having the highest final value. Similar to other graphs, after 48 hours cyanobacteria grown in lake water reached similar values with those grown in BG-11. Moreover, for each measurement, the difference between the 1:1 ratio and the 2:1 ratio is no more than 13%, indicating that the lake water is a viable growth medium as well. The final values range between 0.03 and 0.08.

For PCC 7425 indicates that the lake water was the most beneficial for this cyanobacteria strain, having by far the highest final value. The most significant increase for the absorbance value for the lake water can be seen between 48 and 72 hours. The other media had an exponential growth which had a polynomial growth path.

The 750nm graph for PCC 7942 indicates an exponential growth for all media with BG-11 having the highest final value and with lake water and 1:1 having very similar growth paths, with the 1:1 ratio reaching however a higher final absorbance value.

Fig.33- Growth curve for PCC 7512, PCC 7425, PCC 7942 at 750 nm in different media

8.5. Spectral analysis at 870 nm

For PCC 7512 the graph indicates once again an exponential growth path, with BG-11 having the highest values for each time interval measured. The final values, range between 0.02 and 0.08.

The 870nm graph for PCC 7425 is very similar to the graphs for other wavelengths for this bacteria, with lake water and 2:1 having the highest values, and with all media having an exponential growth,which has a polynomial growth, but still has significant values, having a final value almost as high as BG-11. The final values range between 0.05 and 0.09.

For PCC 7942 it can be noticed once again that BG-11 is the most beneficial medium for this cyanobacteria strain, having by far the highest final value. All media have an exponential growth path. The values range between 0.025 and 0.075. In this case, all media reach media reach similar values only after 16 hours and start to differentiate afterwards.

Fig.34- Growth curve for PCC 7512, PCC 7425, PCC 7942 at 870 nm in different media

8.6. Spectral analysis at 970 nm

The 970nm graph for PCC 7512 significantly different from other wavelengths, as 2:1 has a clear polynomial growth, being the first time a medium has this type of growth, which could be due to the increased wavelength. A clear difference between the final values is seen again, with BG-11 having by far the highest one. The ratio 1:1 did not have the same increasing values as for the other wavelengths, instead they remained almost constant.

For PCC 7425 an exponential growth for all the media can be seen once again. The final values range from 0.04 and 0.09.

For PCC 7942 it is shown once again that BG-11 is the optimal medium for this cyanobacteria strain, having by far the highest latest value. The lake water condition and 1:1 had similar growth paths, while 2:1 had a polynomial growth. For this wavelength the lake water and the 1:1 ratio had a similar final absorbance. As for the rest of the final values, the range is between 0.025 and 0.08.

Fig.35- Growth curve for PCC 7512, PCC 7425, PCC 7942 at 970 nm in different media

As a general conclusion, the strain that best adapted the medium conditions change is PCC 7425, which showed the best cell development rate(considering optical density) for the lake water as a growth medium, closely followed by the 2:1 ratio.

Multiple studies analysed the metabolic process for Cyanothece as they display the ability to fix nitrogen. According to Bandyopadhyay A, PCC 7425 only fixes nitrogen in anaerobic conditions, as compared to other species of Cyanothece (Bandyopadhyay et al.,2011). This could explain the optimal development in lake water in the case of this experiment as the plate with different culture conditions was maintained lidded for the entire duration of the experiment.

Another thing to observe is that generally the absorbance value modified the most after 48 hours, which indicates that this is an approximate time period in which cells adapt to the growth medium change.

9. Correlation of growth medium variations with changes in multiplication rate and photosynthetic pigments quantity

Principle: Chlorophyll production is influenced by a variety of parameters, including different growth medium. One of the factors influencing different photosynthetic pigment production is the pH level of the growth culture medium, which directly affects the uptake of phosphorous, one of the major nutrients for cyanobacteria. Moreover, phosphorous levels influence in turn other nutrients consumption [10], resulting in a circular process which is directly depending on exposure of cyanobacteria to varying culture conditions. As a result from the previous experiment, PCC 7425 was chosen to be subjected to the photosynthetic pigments production analysis in relation to different growth media due to the fact that this species proved to be highly adaptable. Additionally, after performing a supplementary cell counting, PCC 7425 had the highest cell number compared to the other two species (540⋅105 cells/mL).

9.1. Growth rate of cyanobacteria in different media

Methods: Five different culture conditions were created for the PCC 7425:

• BG-11+cyanobacteria
• WL (lake water) + cyanobacteria
• 1:2 BG-11: WL + cyanobacteria (33% BG-11)
• 1:1 BG-11: WL + cyanobacteria (50% BG-11)
• 2:1 BG-11: WL + cyanobacteria (66% BG-11)

Fig.36-Lake Noua, Brasov-top view

The water lake was filtered before being pipetted.

Fig. 37- PCC 7942 in 9 different culture conditions- each flask contains 36mL of culture, with 2 flasks for each culture condition

Samples were analysed with the spectrophotometric method used before, described in section 7. The spectral reading was done for the 6 different wavelengths described in the previous experiments, for a time period of 96 hours, using 96 well VWR Standard Multiwell Cell Culture Plate. The volume of each prepared solution was 100mL. A 10mL bacteria suspension was added to a 72 mL of each culture media. A volume of 300 µL was pipetted into each well. The experiment was done in triplicates and corrected values were calculated with negative control.

Results and discussion: The graphics below depict results for the initial five growth media measured for 0, 24, 48, 72 and 96 hours. However, in order to better identify significant value changes between values from different conditions, a Kruskal Wallis test was performed between data sets. To determine values comparatively, all conditions were analysed between each other using Dunn's multiple comparisons test (using GraphPad Prism v.10). In the right section of the graphic, there is an additional representation of the test done for each interval (until statistical significance is observed-p< 0.05). Graphics are represented up to the point where statistical significance is observed (for 970 nm statistical significance appears starting from 0h and therefore no other graphics are represented). As a general explanation, values with p>0.05 have no statistical significance, therefore this means that the cyanobacteria still adapts to the culture medium change and is not yet influenced by it. Values below detection limit were omitted in the representation.

9.1.1. Spectral analysis at 440 nm

Fig.38- Growth curve for PCC 7425 at 440 nm in different media. BG-11+cyanobacteria; WL (lake water) + cyanobacteria; 1:2 BG-1: WL + cyanobacteria (33% BG-11); 1:1 BG-11: WL + cyanobacteria (50% BG-11); 2:1 BG-11: WL + cyanobacteria (66% BG-11)

For 440 nm it can be seen that for BG-11 the cells multiply exponentially, with the R² coefficient being similar to the one found in 7.1., whereas the water lake does not influence the absorbance value, meaning that cells stagnate. What is interesting to observe is that although bacteria prefers the BG-11 culture medium, bacteria grown in the solution with a lower BG-11 concentration have the highest absorbance value, increasing by 90% over the course of 48 hours, compared to the initial absorbance value. This pattern can be also for the 1:1 and 2:1 concentrations as after 48 hours, absorbance values increase.

10. Fluorescence analysis of cyanobacteria growth and pigments

10.1. Fluorescence analysis of cyanobacteria growth

Principle: Fluorescence spectroscopy is an efficient, precise investigation method that analyses energy levels of samples, using a specific excitation wavelength that causes molecules to emit light at another wavelength. Cyanobacteria has absorption peaks in the blue and red part of the spectrum (440 and 680 nm) due to chlorophyll a (Chl a), and in the orange part (620 nm) due to phycocyanin (Luimstra et al.,2018). However, different media have different absorption wavelengths. The purpose of this experiment is to analyse the specific emission wavelengths of different culture media (mentioned in the experiments above), which was chosen as a reference for the previous experiments in comparison with wavelengths emitted by the different growth culture media containing cyanobacteria.

Methods: The samples used were the same as for experiment 9.1. For the fluorescent spectral analysis, a 96 well, F- bottom black plate was used (Greiner, Austria). A volume of 300 µL was pipetted into each well. The plate was analysed spectrally with SpectraMax iD3 Multi-Mode Microplate Reader (Molecular Devices, United States) to detect peak signals. The excitation wavelength chosen was 460 nm.

Fig.46- Spectrum read for excitation wavelength 460nm and emission range 500 nm-850 nm

Results and discussion:

(a)

(b)

(c)

(d)

(e)

Fig.47-Fluorescence spectral analysis for different media and for PCC 7425 grown in different media: BG-11(a), WL(b), BG-11:WL= 1:2 (c), BG-11:WL =1:1(d), BG-11:WL= 2:1 (e)

Except for the BG-11 culture media, all other conditions containing PCC 7425 have an emission peak at 690 nm. Cyanobacteria emit mean autofluorescence light at 645-665 nm while algae emit mean autofluorescence light at 680-690 nm (Zucker 2017 et al.,2017). As this was seen for all conditions with lake water, it can be hypothesised that the absorbance for lake water is given by various microorganisms found in the sample. Moreover, lake water specific absorbance can be influenced by dissolved organic matter (DOM), which has distinct fluorescent properties. DOM has inhomogeneous structures with a complex mixture, due to bonding with various functional groups such as amide, carboxyl, hydroxyl, and ketone (Hur et al.,2011).

Another peak was observed in the 710-720 nm range, which represent the wavelengths for Photosystem I(PS I), indicating this way the presence of photosynthetic pigments(Wei & Jongbloets et al.,2016).

It can be seen that differences between the reference culture media and culture media containing cyanobacteria can be seen mostly in the 600-800 nm wavelength range, therefore the cyanobacteria emission spectra ranges between these values.

Fig.48-Fluorescence spectral analysis for different media and for PCC 7425 grown in different media: BG-11, WL, BG-11:WL= 1:2, BG-11:WL= 1:1, BG-11:WL 2:1

As seen in the figure above, cyanobacteria emit similar signals regardless of the growth culture medium, which is influenced by absorbance peaks of these organisms.

11. Future perspectives and applications using cyanobacteria

Cyanobacteria can efficiently harvest CO2 as a carbon source, powering their metabolic processes by absorption of sunlight, the most abundant form of renewable energy.

Cyanobacteria are promising microorganisms for sustainable biotechnologies, yet unlocking their potential requires radical re-engineering and application of cutting-edge synthetic biology techniques (Santos Merion, Singh & Ducat, 2019).

The available tools and methodologies for characterising and modifying cyanobacteria have been increasing recently.

The successful utilization of cyanobacterial species for industrial production depends on the development and availability of accurate large-scale cultivation facilities. In addition to improving product yields, it is important to develop efficient and cost-effective photosynthetic bioreactors (Lau et al., 2015), as well as technologies to harvest the end products (Knoot et al., 2018) to minimize operation costs. Technological improvements in the manipulation of cyanobacteria have been developed using the advantages of metabolic engineering and synthetic biology, potentially setting the stage for cyanobacteria to significantly contribute as crop species.

Synthetic biology principles promote a bottom-up approach to designing biological systems, recombining defined parts or modules to restructure existing systems or build new pathways de novo (Sengupta et al., 2018)

Host for biomolecule production

Cyanobacteria could be sourced for different compounds as fiber, being considered as food (Santos Merion, Singh & Ducat, 2019). Many cyanobacterial strains also produce a wide spectrum of secondary metabolites with high-value commercial properties, such as pigments, vitamins, amino acids, fatty acids, lipopeptides, and amides (Lau et al., 2015).

Like many genera of eubacteria, cyanobacteria can synthesize polyhydroxyalkanoates, a thermoplastic class of biodegradable polyesters that includes polyhydroxybutyrate (Quintana et al., 2011).

Reporter proteins

Because of strong chlorophyll autofluorescence, the use of red fluorophores is not recommended, but many other reporters including GFPmut3B (a mutant of green fluorescent protein) and EYFP (enhanced yellow fluorescent protein) have been routinely used (Huang et al., 2010; Yang et al., 2010; Huang and Lindblad, 2013; Landry et al., 2013; Cohen et al., 2014).

Improving carbon fixation rates

Cyanobacteria are characterized by highly efficient carbon concentration mechanisms (CCM), compared to algae and plants (Price et al., 2011). The cyanobacterial CCM is efficient in part because it uses bicarbonate transporters to actively transport bicarbonate into the cell, which effectively overcomes the slower (104-fold) diffusion rates of CO2 in water compared to air (Price et al., 2011).

Bioindicators

Cyanobacteria, as primary producers with a key role in the N and C cycles, are useful bioindicators, given that any detrimental effect on this phototrophic community may have a negative effect on nutrient availability to organisms at higher trophic levels (Mateo et al, 2015).

Some countries use the full or a wide range of periphyton taxa, including cyanobacteria, for routine monitoring programs, such as several countries in central Europe, including Austria, Germany, Czech Republic and Poland (Kelly 2013; Whitton 2013). Studies of water quality in rivers of Iran confirmed the use of cyanobacterial species as bioindicators for monitoring eutrophication (Soltani et al. 2012).

Bioreporters

The specific bioreporters are able to detect a specific (or group of similar) pollutant as they have a fusion between a pollutant-responsive gene(s) regulatory element and the promoterless reporter system; they are usually turn-on strains but in some cases, such as some promoters responsive to nutrient deficiency used to develop bioreporters of nutrient bioavailability (see below), turn-off strains are constructed (Mateo et al, 2015).

The environmental importance of cyanobacteria as primary producers both in freshwater and marine environments makes cyanobacterial bioreporters a useful tool to assess nutrient bioavailability in water bodies. It was observed that an excess of P and/or N may lead to the eutrophication of water bodies and development of algal blooms which may lead to toxin production by cyanobacteria (Dodds ,2006).

One cyanobacterial bioreporter able to detect heavy metals was constructed by Erbe et al. (1996). It was based on the smt locus of the unicellular Synechococcus sp. PCC 7942, this locus consists of the cyanobacterial metallothionein gene smtA and smtB, a divergently transcribed gene encoding the transcriptional repressor of smtA; binding of metal ions by smtB induces conformational changes in it that promote RNA polymerase accessibility to smtA (Huckle et al. 1993; Morby et al. 1993). smtA transcription is induced in the presence of several metals: Zn (as the preferred metal), Cd, Cu, Hg, Co and Ni (Huckle et al. 1993; Osman and Cavet 2010).

Regarding cyanobacteria as bioreporters, they offer a low-cost, low maintenance alternative to heterotrophic bacterial bioreporters. Cyanobacterial bioreporters could serve as hosts for sensing elements from other bacteria, new reporter systems should be evaluated (most of them are bioluminescent, with fluorescence/other reporter elements seldom used) and also, given the fact that over 100 cyanobacterial genomes have been sequenced, novel genetic elements responsive to pollutants could be identified and used to construct new and useful bioreporters (Mateo et al, 2015).