3.1 Growth under different light conditions
In this study, microcystin producing PCC 7806 (WT) and its microcystin-deficient mutant (MT) were compared with respect to light intensity-dependent growth and pigment contents under different light conditions. We did not observe significant differences between the light and growth curves (Fig. 1) and the maximum specific growth rate (Table 1) of MT and WT. Thus, the lack of microcystins in MT had no effect on the growth under the tested light conditions. However, the low-maximum specific growth rates measured for both variants differentiate them from other strains of M. aeruginosa[18,19]. Similarly, light saturation intensities (Isat: light intensity at μ=0.95 μm) of approximately 32 μE m−2 s−1 for the WT, and 38 μE m−2 s−1 for the MT were low compared to other Microcystis strains that require higher intensities (single cell strains 50 μE m−2 s−1, colony-forming strains 80 μE m−2s−1, ) to reach light saturation conditions.
Figure 1. Light intensity-dependent growth of PCC 7806 WT and MT, means (n=25) and confidence limits; Values of filled symbols were tested with Student's t-test (P=0.05) for significant differences at comparable light supply. Grey filled symbols – tested values without significant differences; black filled symbols – tested values with significant difference; open symbols – not tested; Light–growth curves were estimated by non-linear regression to a modified Mitscherlich model  (μ=μm(1−exp(−ln2(I−i0)/(KI−i0)).
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Table 1. Model parameters of estimated light–growth curves (Fig. 2)
|μm (day−1)||0.211 (0.205–0.217)a||0.208 (0.202–0.214)|
|i0 (μE m−2 s−1)||1.85 (0.73–2.97)||0.61 (−0.43–1.70)|
|KI (μE m−2 s−1)||8.79 (8.16–9.42)||9.44 (8.60–10.28)|
|Isat (μE m−2 s−1)||31.8||37.7|
3.2 Biovolume vs absorption
We could not detect significant differences in the size of cells between WT and MT. Surprisingly, WT and MT variants exhibited significant differences in the biovolume vs light extinction curves (Fig. 2). At the same biovolume a 30% higher optical signal was measured from the WT culture compared to the MT.
Light reflection, refraction and absorption are the components that constitute light attenuation of culture solutions during photometric biovolume measurements. While light absorption by pigments (chl a at 436 nm) represents only a minor proportion of this measurement and light reflection is mainly influenced by cellular surface structure, refraction is determined by the internal organisation of membrane systems, the nature and size of inclusions and the occurrence of gas vesicles within the cell. The double-frame technique for measuring photometric signals, used in this study, is based on light attenuation as a consequence of light refraction (scattering). Therefore, the significantly lower photometric absorbance signals observed in MT cultures in comparison to the WT, are an indication of internal structural differences in MT cells.
3.3 Pigment composition
Structural changes in the MT are also suggested by the significantly higher specific absorbance values (for individual pigments), but lower chl a and carotenoid contents (Fig. 3, Table 2). To investigate possible adaptive responses to different light intensities, the pigment contents of WT and MT cells were analysed by HPLC. Clear separation of peaks was seen for chl a, the carotenoids β-carotene (β-car) and zeaxanthin (zea), the ketocarotenoid echinenone (ech), and the carotenoid-glycoside myxoxanthophyll (myx). The detected crocetindial (cro) represents an artificial degradation product of β-car or zea due to oxidative splitting by the carotene oxygenase enzyme complex . Due to the highly variable occurrence of cro in the samples, the sum of β-car, zea and cro was used to compare WT and MT.
Figure 3. Light intensity-dependent pigment contents (A, B, C and D) and pigment/chl a-ratios (E, F and G) of PCC 7806 WT and MT, means (n=6) and standard deviations; Values of filled symbols were tested with Student's t-test (P=0.05) for significant differences at comparable light supply. Grey filled symbols – tested values without significant differences; black filled symbols – tested values with significant difference; open symbols – not tested;
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Table 2. Cytophotometrically determined values of specific absorbances of light limited and nearly saturated WT and MT cells
|Specific absorbance (μm−1)||WT (5.3 μE m−2−1)||MT (4.0 μE m−2 s−1)|
|Total specific absorbance (420–700 nm)||7.1a||10.1a|
| ||WT (34.6 μE m−2 s−1)||MT (31.0 μE m−2 s−1)|
|total specific absorbance (420–700 nm)||5.9||5.8|
The observed cellular pigment concentrations reflect adaptation of the pigment apparatus to light intensities between 4 and 110 μE m−2s−1. Both the WT and the MT adapted to light by altering their intracellular concentrations of chl a and carotenoids. Chl a, β-car+zea+cro and ech decreased with increasing light intensity, especially in the low light range, while high irradiance resulted in higher myx concentrations. When normalised to chl a, all pigment ratios increased with increasing light supply, largely reflecting a much greater reduction in chl a relative to accessory carotenoids. The carotenoid/chl a ratios were identical for both variants.
While the adaptation to different light intensities was shown to be the same in MT and WT, the pigment concentrations revealed differences between the two variants independent of light intensities (Fig. 3). The cellular concentrations (means) of all pigments were approximately 20% lower in the MT compared to the WT. Significant differences were found at light limited conditions for chl a and ech, and at light saturation for myx.
The cytophotometrically determined absorbance data are displayed in Table 2. Under nearly light saturated conditions, there were no significant differences in the specific absorbances, the ratio of E625/E675 and total specific absorbances between 420 and 700 nm. However, the cytophotometric in vivo absorbance spectra revealed a significantly higher PC content (E625/E675) relative to chl a in the MT cells under light limitation. PC is the main component of the phycobilisomes in M. aeruginosa, which act as light-harvesting systems and transfer collected light energy to the photosynthetic reaction centres, primarily to PS II .
The lower chl a content, compared to the WT, and the simultaneously increasing ratio of PC to chl a, exhibited under light limited conditions in MT cells, point toward a changed PS I/PS II ratio. In cyanobacteria light adaptation mechanisms are guided by variations in the PS I/PS II ratio . Therefore, it can be speculated that microcystins could play a role in light adaptation processes. The light-regulated transcription of the microcystin synthetase gene cluster  and the subcellular localisation of microcystins on thylakoid membranes  support such assumptions. In this case, presumably, compounds with similar chemical structure (i.e. cyanopeptolines, microginins ) serve similar functions in non-microcystin-containing WT strains.
On the other hand our observations could be explained by structural changes in the cellular membrane system caused by the lack of membrane bound microcystin. Then the assumed change in the PS I/PS II ratio could be a consequence of the lack of space for the photosynthetic reaction centres and the expanded antennae assembly under light-deficient conditions.
It is likely that more information will be gained, when the second cyclic peptide present in PCC 7806 cyanopeptoline is absent also.