Algal production of extra and intra-cellular polysaccharides as an adaptive response to the toxin crude extract of Microcystis aeruginosa

This is an investigation concerned with studying the possible adaptive response of four different unicellular algae, Anabaena PCC 7120, Oscillatoria angustissima, Scendesmus obliquus and Chlorella vulgaris, to the toxin of Microcystis aeruginosa (Kützing). The effects of four different concentrations, 25, 50, 100 and 200 μg mL-1 of microcystins crude extract of M. aeruginosa, on both intra and extra-cellular polysaccharide levels, in log phase, of the four tested algae were studied. The obtained results showed differential increase in the production levels for both intra and extra-cellular polysaccharides by the tested algae, compared with the control. S. obliquus and C. vulgaris showed a resistance to crude toxin higher than Anabaena PCC 7120 and O. angustissima. The highly production of polysaccharides by green algal species under this toxic stress indicated the involvement of these polysaccharides in protecting the algal cells against toxic species and, reflect the biological behavior of particular algal species to the environmental stresses.


Introduction
Microcystis aeruginosa is a common hepatotoxic cyanobacterium living in eutrophic freshwaters [1]. The inhibition of competitors by the release of compounds, a process known as allelopathy, may be important in plank tonic systems [2]. Allelopathy has been hypothesized to play a role in species succession [3], the formation of harmful algal blooms in water resources [4], and the establishment of invasive species [5]. Allelopathy is defined as any process involving secondary metabolites produced by plants and microorganisms that influence the growth and development of biological systems, including positive and negative effects [6]. These secondary metabolites are called allelochemicals and play a major role on growth and development in both natural and agro-ecosystems [7]. Most allelopathic are compounds biodegradable and at the same time natural toxins [8].
The production of biologically active substances which promotes the growth of algae and other plant organisms has been reported [9].
The algal cells can release extra-cellular polysaccharides (EPS) into the environment; these EPS are ecologically important through their influence on carbon cycle and microbial diversity [10]. They may enhance the bacterial growth and activity leading to the release of inorganic substances useful for microalgae in the same environment [11]. In addition, these polysaccharides can also make complexes with inorganic ions and thus reducing their toxicity to aquatic organisms [12].
Some green algae can develop a defense system by the production of polysaccharides to cope with oxidative stress induced by microcystin. The results of in vitro assay of antioxidant activity revealed that these polysaccharides had different activities, depending on their sulfate contents [13]. On the other hand, toxic blooms of Nodularia spumigena can provide a potential food source for the heterotrophic food chain [14]. Furthermore, bacteria can efficiently degrade microcystins in natural waters with previous cyanobacterial history, and heterotrophic nanoflagellates respond quickly to the bacterial growth [15]. However, some species of green algae e.g. Scenedesmus sp. coexist and even flourish in the presence of either toxic cyanobacteria or their toxins [16]. Mass-occurrences of cyanobacteria have been reported to increase in frequency, as well as in intensity, due to eutrophication [17]. It is therefore important to investigate how cyanobacteria influence on the structure and functioning of other surrounding organisms in the aquatic ecosystem since River water quality zoning could provide essential information for developing river water quality management policies [18].
Therefore, the aim of this study was to investigate the response of several unicellular algae to the stress of crude toxin of Microcystis aeruginosa through the production of intra and extra-cellular polysaccharides.

Test organisms
Two unicellular blue green algae (Anabaena PCC. 7120 and Oscillatoria angustissima) and two green algae (Scendesmus obliquus and Chlorella vulgaris) were kindly provided from Phycology Laboratory, Faculty of Science, Tanta University.

Culture and crude extract of cyanobacterium Microcystis aeruginosa
Microcystis aeruginosa was isolated from fresh water samples from Nile river channel near Tanta city, Egypt, and spread with an inoculating needle on the surface of sterilized Petridishescontaining solidified, sterilemedia (Allen's, [19]). The cultures of M. aeruginosa purified and prepared in axenic unialgal cultures by Venkataraman [20].
M. aeruginosa was cultured in 1 liter conical flasks containing 400 mL medium (Allen's, [19]) and kept in controlled conditions of continuous light (45 μmol/ms) and temperature (25±2°C). Algal cells were harvested at the end of log growth phase, lyophilized and kept in deep freeze (−20°C).
The crude extracts were prepared by suspending 100 mg lyophilized cells in 10 mL of 75% methanol according to [21] with ultrasonication (ultrasonic probe with characteristics of~60 W and~20 KHZ) for 2 min followed by intermittent shaking for 1 h. Debris was removed by centrifugation for 10 min at 4000 rpm. The pellet was reextracted and the combined supernatants were evaporated to dryness at 30°C using a rotary evaporator (Perfit, India).

Treatments
All experiments were carried out in 250 mL conical flasks, containing 100 mL Allen's and Stanier medium which was adjusted to contain 0 (control), 25, 50, 100 and 200 μg/mL of lyophilized cell extract of previously mentioned cyanobacteria species. Culture conditions of the tested algae were the same as previously mentioned. The experimental cultures were harvested at 3, 6, 9, 12 and 15 days for the determination of intra and extracellular polysaccharides. Cultures were grown in triplicate for statistical analysis.

Measurement of intra-cellular polysaccharides (IPS)
During the growth, 100 mL of the tested algae cultures were pipette out and centrifuged at 3000 rpm for 10 min. The filtrate was used to estimate extra-cellular polysaccharides and the pellets were dried and then used to estimate intra-cellular polysaccharide as described by [22]. Intra-cellular polysaccharide (IPS) was extracted by homogenizing the derided pellet in distilled water (50 mL). The homogenates were then heated in water

Measurement of extra-cellular polysaccharide (EPS)
The extra-cellular polysaccharides were estimated according to [23]. To precipitate proteins from the algal culture, trichloroacetic acid (TCA) was added in a final concentration of 4% and the algal filtrate was stirred for 2 h. Precipitated proteins were removed by centrifugation. The clear supernatant was collected which contains EPS. Extra-cellular polysaccharides was precipitated by ethanol and determined as described in IPS.

Statistical analysis
Results are presented as mean ±SD (standard deviation) for three replicates. Data obtained were analyzed statistically to determine the degree of significance between treatments using one, two and three way analysis of variance (ANOVA) at P ≤ 0.001. The statistical analyses were carried out using SAS program [24] version 6.12.

Results
Data in Table 1 show the effect of various concentrations of microcystins crude extract of M. aeruginosa (25, 50, 100 and 200 μg/mL), in log phase, on both extracellular polysaccharides (EPS) and intra-cellular polysaccharides (IPS) of Anabaena PCC.7120. Results showed a significant increase of both EPS and IPS as compared with control. Thus, the crude microcystins extract could induce the production of polysaccharides and their      (Table 5). On the other hand, increasing in EPS and IPS of C. vulgaris amounted by (56%, 133%, 188% and 308.6%) and (25%, 60%, 309% and 478%), respectively over than control ( Table 6). Analysis of variance revealed high significant increase in extra and intra-cellular polysaccharides of Sc. obliquus and Chlorella vulgaris at (P ≤ 0. 001) except at 25 μg/ml the increases in IPS of Sc. obliquus and EPS of Chlorella vulgaris (Tables 5 and 6, respectively) were insignificant at the days 6, 9 and 12, and the IPS of C.vulgaris was insignificant at days 6 and 9 as EPS of Sc. obliquus.
From the suggested statistical model (concentrations, days and interception between them), it could be observed that, the deviation for EPS and IPS content of Sc. obliquus was 98.89% and 99.68%, respectively (Table 7) and for C. vulgaris was 99.32% and 99.48%, respectively (Table 8).

Discussion
Microcystis aeruginosa blooms are hazardous to freshwater flora and fauna due to the production of toxins [25]. M. aeruginosa produces a wide range of toxic metabolites. The physiological and ecological role of these compounds remains largely unknown. Some studies have suggested that these compounds may have allelochemical roles, such as compounds that may inhibit competing sympatric macrophytes, algae and microbes [26,27]. The adverse effects of crude microcystins (MCs) could be due to the synergistic interactions among MCs variants or to the presence of unidentified toxic other than MCs in the crude extracts [28]. Leflaive and Ten-Hage [29] reported that cellular extracts containing toxins are often more active than purified toxin, they suggests that cellular extracts contain a mix of active toxin that may act synergistically.
The possible adaptive response of four unicellular algae (Anabaena PCC. 7120 and O. angustissima as blue green algae and S. obliquus; C. vulgaris as green algae) to M. aeruginosa producing toxin were studied. The results indicated that the crude microcystins extract induces the production of extra and intra-cellular polysaccharides of the tested algae and their amounts increased with increasing the microcystins crude extract concentration. The production of polysaccharide by green algal species under toxic stress indicated the involvement of this polysaccharide in protecting the algal cells against toxic species [13].
Inhibition in the growth of Anabaena PCC 7120; Oscillatoria angustissima; Scendesmus obliquus and Chlorella vulgaris at different microcystin crude extract concentrations was previously represented by [27]. Similar results were recorded by Abdel-Rahman [30] found that growth and the physiological activities, except amino acids biosynthesis, of both Chlorococcum humicola and Chlorella vulgaris were inhibited by crude extracts of the two cyanobacteria species Microcystis and Nodularia. Singh et al. [31] reported that the crude extract of M. aeruginosa provides toxicity to green algae (Chlorella sp. and Scenedesmus sp.) and cyanobacteria (Anabaena BT1 and Nostoc muscorum). As represented by [13] pure and crude microcystins significantly decreased the growth of Scenedesmus quadricauda and Chlorella vulgaris, and these results correlated with polysaccharide contents of toxin-treated cultures. This results was in accordance with our results which indicated that S. obliquus and C. vulgaris having a resistant system to crude toxin by the production of polysaccharide more than Anabaena PCC. 7120 and O. angustissima.
The increasing in polysaccharides contents in MCs treated cultures in all experimented algae indicated that these polysaccharides may be involved in certain defense mechanisms in response to toxin stress. Some studies have reported the protective role of polysaccharides  against oxidative stress and their ability in scavenging ROS in plant cells [32]. The antioxidant mechanisms may be due to the supply of hydrogen by these polysaccharides which combines with radicals and forms more stable products to terminate radical chain reaction [33].
Microcystins have been shown to induce formation of reactive oxygen species (ROS) that might cause serious cellular damage such as peroxidation of lipid membranes, genotoxicity, or modulation of apoptosis [13]. During the present study the EPS and IPS were detected in the algal medium increased with increase of MCs concentration. It has been suggested that polysaccharides are produced inside the cells during oxidative stress to scavenge the free radicals and remove them from the cells to the medium [32]. However, a variety of other mechanisms such as extra-cellular detoxification, reduced uptake, efflux, sequestration by polysaccharides have been proposed to explain algal tolerance to oxidative stress [34].
Extrusion of EPS can serve as a boundary between cells and the surrounding environment; they could fulfill a protective role against desiccation, antibacterial agents or predation by protozoan [35]. Hence, the results of present study showed that some algae can develop a defense system by the production of polysaccharides to cope with oxidative stress induced by MCs, and this may explain why some plank tonic algae present in close proximity with toxic cyanobacteria or their toxins are not affected by these toxins at environmentally relevant concentrations (1-10 μL) [36].

Conclusion
Here, we showed a physiological model of algal life sustainability under the stress of natural cyanobacterial toxins. The presented results showed the production of polysaccharides by tested algae as a response to the cyanobacterial toxins, microcystins. on the other hand, the release of these polysaccharides into the culture medium and most likely in the natural environment, is of ecologically importance because they may increase the polysaccharide contents of the water column and the growth of heterotrophic bacteria, and complex with heavy metals to reduce their toxicity to aquatic organisms. Therefore, in future studies involving in vitro cultures more attention should be paid to the role of algal exudates, in order to improve the significance of the results with the aim of using them as models of the real environment.