Biochemical and proteomic response of the freshwater green alga Pseudochlorella pringsheimii to iron and salinity stressors.

BACKGROUND: Pseudochlorella pringsheimii (Ppr) is a green unicellular alga rich with chlorophyll, carotenoids, and antioxidants. As a widespread organism, Ppr must face, and adapt to, many environmental stresses and these are becoming more frequent and more extreme under the conditions of climate change. We therefore focused on salinity induced by NaCl and iron (Fe) variation stresses, which are commonly encountered by algae in their natural environment. RESULTS: The relatively low stress levels improved the biomass, growth rate, and biochemical components of Ppr. In addition, the radical-scavenging activity, reducing power, and chelating activity were stimulated by lower iron concentrations and all NaCl concentrations. We believe that the alga has adapted to the stressors by increasing certain biomolecules such as carotenoids, phenolics, proteins, and carbohydrates. These act as antioxidants and osmoregulators to protect cell membranes and other cellular components from the harmful effects of ions. We have used SDS-PAGE and 2D-PAGE in combination with tandem mass spectrometry to identify responsive proteins in the proteomes of stressed vs. non-stressed Ppr. The results of 2D-PAGE analysis showed a total of 67 differentially expressed proteins, and SDS-PAGE identified 559 peptides corresponding to 77 proteins. Of these, 15, 8, and 17 peptides were uniquely identified only under the control, iron, and salinity treatments, respectively. The peptides were classified into 12 functional categories: energy metabolism (the most notable proteins), carbohydrate metabolism, regulation, photosynthesis, protein synthesis, stress proteins, oxido-reductase proteins, transfer proteins, ribonucleic-associated proteins, hypothetical proteins, and unknown proteins. The number of identified peptides was higher under salinity stress compared to iron stress. CONCLUSIONS: A proposed mechanism for the adaptation of Ppr to stress is discussed based on the collected data. This data could serve as reference material for algal proteomics and the mechanisms involved in mediating stress tolerance.

Dietary Chlorella vulgaris effectively alleviates oxidative stress, immunosuppression, and enhances the resistance to Streptococcus agalactiae infection in cadmium-intoxicated Nile tilapia fingerlings.

Aquatic pollutants, including cadmium (Cd), cause oxidative stress on aquatic animals. The use of probiotics, including microalgae as a feed additive to alleviate the toxic impacts of heavy metals, is a much more interesting point. Hence, the current study investigated the oxidative stress and immunosuppression in Nile tilapia (Oreochromis niloticus) fingerlings caused by Cd toxicity as well as the preventive function of dietary Chlorella vulgaris against Cd toxicity. Accordingly, fish were fed on 0.0 (control), 5, and 15 g/kg diet of Chlorella up to satiation thrice a day, along with being exposed to 0.0 or 2.5 mg Cd/L for 60 days. Following the experimental procedure, fish from each group were intraperitoneally injected with Streptococcus agalactiae, and their survivability was observed for further ten days. Chlorella-supplemented diets meaningfully (P < 0.05) boosted the antioxidative capability of fish, which was evidenced by higher activities of hepatic superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione-S-transferase (GST) as well as higher levels of reduced glutathione (GSH) along with significant reductions in hepatic malondialdehyde levels. Moreover, the innate immunity indices [phagocytic activity (PA), respiratory burst activity (RBA), and alternative complement activity (ACH50)] were significantly higher in Chlorella-fed fish, particularly in the group of 15 g/kg diet. Additionally, serum of Chlorella-fed fish showed potent bactericidal activities against S. agalactiae, particularly at the treatment of a 15 g/kg diet. Feeding Chlorella diets to Nile tilapia fingerlings upregulated SOD, CAT, and GPx genes expression alongside the down-regulation of IL-1ß, IL-8, IL-10, TNF-α, and HSP70 genes expression. Conversely, Cd toxicity caused oxidative stress and suppressed the fish's innate immunity with upregulation of the expression of IL-1ß, IL-8, IL-10, TNF-α, and HSP70 genes. Feeding Cd-exposed fish on Chlorella-containing diets attenuated these adverse effects. The current research revealed that supplementing feeds with the treatment of 15 g/kg diet of C. vulgaris supports the antioxidant-immune responses and alleviates the Cd toxicity effects on Nile tilapia fingerlings.

Antioxidant enzymes of Pseudochlorella pringsheimii under two stressors: variation of SOD Isoforms activity.

Algae are always facing the challenge of exposure to different stress conditions, therefore raising challenges of adaptation for survival. In this context, the growth and the antioxidant enzymes of the green stress-tolerant alga Pseudochlorella pringsheimii were investigated under two environmental stresses viz. iron and salinity. The number of algal cells was moderately increased by iron treatment in the range of 0.025-0.09 mM of iron, yet, the number of cells decreased at high iron concentrations (0.18 to 0.7 mM Fe). Furthermore, the different NaCl concentrations (8.5-136.0 mM) had an inhibitory effect on the algal cell number, compared to the control.The superoxide dismutase (SOD) showed three isoforms namely; Mn, Fe, and Cu/Zn SOD. The in gel and in vitro (tube-test) activities of FeSOD were higher compared with the other SOD isoforms. The activity of total SOD and its isoforms increased significantly by the different concentrations of Fe and non-significantly by NaCl. The maximum SOD activity was recorded at 0.7 mM Fe (67.9% above control). The relative expression of FeSOD was high under iron and NaCl at 8.5 and 34 mM, respectively. However, FeSOD expression was reduced at the highest NaCl tested concentration (136 mM). In addition, the antioxidant enzyme activity of catalase (CAT) and peroxidase (POD) were accelerated by increasing iron and salinity stress which indicates the essential role of these enzymes under stress. The correlation between the investigated parameters was also investigated. A highly significant positive correlation between the activity of total SOD and its isoforms, and with the relative expression of FeSOD was observed.

Biosorption of cyanate by two strains of Chlamydomonas reinhardtii: evaluation of the removal efficiency and antioxidants activity.

Two strains of the chlorophyte Chlamydomonas reinhardtii, a wild type (WT) and a transgenic strain (C.CYN) contained an exogenous cyanase gene (CYN), were used to investigate the growth and cyanate biosorption capability through the analysis of the adsorption equilibrium isotherm. The potential antioxidants activity of the algal strains was also investigated under cyanate concentration. The antioxidants activity of both C.CYN and WT were enhanced by the application of cyanate.Two adsorption isotherm models and the sorption kinetics were used to check the efficiency of the cyanate removal process. The results showed the biosorbent efficiency of Chlamydomonas in the removal of KCNO from aqueous solution. The C.CYN strain has great efficiency to remove cyanate as compared to the WT. The maximum percentage of cyanate removal was 83.75% for the C.CYN and 50% for the WT as treated with 0.8 mg.ml-1 KCNO. The data were adapted to the nonlinear Langmuir model on the basis of the coefficient of determination. The calculated qmax was 0.54 and 0.42 µg.mg-1 for C.CYN and WT which correlated to the experimental one (0.67 and 0.4 µg.mg-1, respectively). Our data highlight the application of the transgenic algal strain toward the removal of highly toxic materials as cyanate.Novelty statement The main objective of this work is to find out an efficient genetically-modified Chlamydomonas strain to remove the highly toxic cyanate compound from contaminated area. Moreover, to evaluate the biosorption ability of this transgenic strain with its wild one via two adsorption isotherm (the Langmuir and Freundlich) models. Also, to estimate the antioxidants activity of these strains under the cyanate toxicity through four different assays.

Disparity of the carotenoids antioxidant properties of wild-type and D-PSY-transgenic Dunaliella parva strains under three environmental stresses.

Two strains of the halophilic alga Dunaliella parva, a wild type (WT) and a transgenic strain (D-PSY) containing an exogenous phytoene synthase gene (PSY), were used to investigate the growth, carotenoid accumulation, and carotenoid antioxidant properties under nitrogen starvation, cobalt and biochar treatments. D-PSY had higher carotenoid content (1.8 times) compared to the WT. The applied stressors stimulated the carotenoid content of both WT and D-PSY especially. The carotenoids were assayed for the potential antioxidant activities by five different assays. Generally, the antioxidant activities of D-PSY carotenoids were superior to that of WT. The biochar and nitrogen treatments generally enhanced the antioxidant activities of the carotenoids, whereas cobalt came third in this respect. The D-PSY transgenic algal strain has both high carotenoids content and antioxidant properties which enhanced under the relatively lower concentrations of the applied stressors. The results have shown to lead to an accurate application of the transgenic alga as a source of potent antioxidant compounds. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01077-0.

Molecular characterization and expression analysis of iron superoxide dismutase gene from Pseudochlorella pringsheimii (Trebouxiophyceae, Chlorophyta).

The FeSOD isoforms of Pseudochlorella pringsheimii were identified, a preliminary characterization of the enzyme was conducted, and the relationship among the FeSOD gene from P. pringsheimii and that of other organisms was examined. The FeSOD has an open reading frame of 612 bp that encodes 203 deduced amino acids with a molecular mass of 23 kDa. Expression of the recombinant FeSOD gene was done successfully in Escherichia coli. The purified FeSOD has a specific enzyme activity that reached 688 U mg-1 protein (in vitro assay). Alkaline conditions showed the highest activity for the recombinant FeSOD. Moreover, it showed a relative thermostability up to 50 °C, while at 50 and 70 °C, the activity was reduced by 32 and 68%, respectively, after 1 h as compared to the maximum. Phylogenetic analysis revealed three main clusters i.e., the prokaryotic Cyanophyta, bacteria, and the eukaryotic Chlorophyta intermingled with plant species and a dinoflagellate. P. pringsheimii was closely grouped with Chlorella pyrenoidosa, however, other species showed a relative disparity. Alignment of FeSOD gene sequences of the different species showed many conserved regions which could be used for FeSOD sequences among unexplored species and may be useful for the taxonomy of the revised coccoid Chlorella species.

Tolerance of Pseudochlorella pringsheimii to Cd and Pb stress: Role of antioxidants and biochemical contents in metal detoxification.

The tolerance and antioxidant response of the green alga P. pringsheimii to cadmium (Cd) and lead (Pb) was investigated. The algal biomass was constant at the relatively lower metal concentrations of Cd and Pb (5, 12 µM, and 2.5-200 µM, respectively), whereas higher concentrations severely inhibited the algal biomass yield. The pigment content of P. pringsheimii decreased due to the investigated metals, especially with Cd concentrations. However, the Pb concentrations of 2.5-200 µM enhanced the pigment content. The carotenoids content was highly repressed by the Cd concentrations. Nevertheless, Pb concentrations highly stimulated the carotenoids content, with the exception of 400 and 500 µM Pb. The biochemical contents of P. pringsheimii including phenolic, total soluble protein and carbohydrate contents responded variably to the investigated metals. The concentrations of Cd were found to be harmful to total soluble protein and carbohydrates, but not the phenolic contents. However, all biochemical contents were stimulated under relatively lower Pb concentrations. Markedly for Pb, the radical scavenging, reducing power, and chelating activities improved under the metals exposure excluding higher concentrations. The activities of the antioxidant enzymes (SOD, CAT, and POD) were highly stimulated with all treatments (except for CAT activities at the highest Cd and Pb concentrations, 300 and 500 µM, respectively). Remarkably, Cd treatments have higher antioxidant enzyme activities compared to that of Pb treatments. The antioxidants augmentation of P. pringsheimii under the metal stress may be exploited for future application in several fields.

Cooperative antioxidative defense of the blue-green alga Arthrospira (Spirulina) platensis under oxidative stress imposed by exogenous application of hydrogen peroxide.

Hydrogen peroxide (H2O2) is an environmentally-safe algaecide used to control harmful algal blooms and as a disinfectant in various domestic and industrial applications. It is produced naturally in sunny-water or as a by-product during growth, and metabolism of photosynthetic organisms. To assess the impact of H2O2 on Arthrospira platensis, several biochemical components, and antioxidant enzymes were analysed. The growth and biomass of A. platensis were decreased under the effect of H2O2. Whereas, the concentration up to 40 µM H2O2 non-significantly induced (at P < 0.05) the Chl a, C-phycocyanin (C-PC), total phycobiliprotein (PBP), and the radical scavenging activity of A. platensis. The half-maximal effective concentrations (EC50) for H2O2 were 57, 65, and 74 µM H2O2 with regards to the biomass yield, Chl a, and C-PC content, respectively. While, the total soluble protein, and soluble carbohydrates contents were significantly induced. However, the higher concentrations (60 and 80 µM) were lethal to these components, in parallel to the initiation of the lipid peroxidation process. Surprisingly, the carotenoids content was non-significantly increased by H2O2. Despite the relative consistency of catalase (CAT), the activities of superoxide dismutase (SOD) and peroxidase (POD) enzymes were boosted by all of the tested concentrations of H2O2. The relative transcript abundance of selected regulatory genes was also investigated. Except for the highest dose (80 µM), the tested concentrations had almost inhibitory effect on the relative transcripts of heat shock protein (HSP90), glutamate synthase (GOGAT), delta-9 desaturase (desC), iron-superoxide dismutase (FeSOD) and the Rubisco (the large subunit, rbcL) genes. The results demonstrated the importance of the non-enzymatic and enzymatic antioxidants for the cumulative tolerance of A. platensis.

Biosorption of uranium by immobilized Nostoc sp. and Scenedesmus sp.: kinetic and equilibrium modeling.

Different activities related to uranium mining and nuclear industry may have a negative impact on the environment. Bioremediation of nuclear pollutants using microorganisms is an effective, safe, and economic method. The present study compared the uranium biosorption efficiency of two immobilized algae: Nostoc sp. (cyanophyte) and Scenedesmus sp. (chlorophyte). Effects of metal concentration, contact time, pH, and biosorbent dosage were also studied. The maximum biosorption capacity (60%) by Nostoc sp. was obtained at 300 mg/l uranium solution, 60 min, pH 4.5, and 4.2 g/l algal dosage, whereas Scenedesmus sp. maximally absorbed uranium (65 %) at 150 mg/l uranium solution, 40 min, pH 4.5, and 5.6 g/l of algal dosage. The interaction of metal ions as Na2SO4, FeCl3, CuCl2, NiCl2, CoCl2, CdCl2, and AlCl3 did not support the uranium biosorption by algae. The obtained data was adapted to the linearized form of the Langmuir isotherm model. The experimental qmax values were 130 and 75 mg/g for Nostoc sp. and Scenedesmus sp., respectively. Moreover, the pseudo-second-order kinetic model was more applicable, as the calculated parameters were close to the experimental data. The biosorbents were also characterized by Fourier-transform infrared spectroscopy (ATR-FTIR), energy-dispersive X-ray spectroscopy (EDX), and scanning electron microscopy (SEM) analyses. The results suggest the applicability of algae, in their immobilized form, for recovery and biosorption of uranium from aqueous solution.

Biochemical composition and antioxidant activities of Arthrospira (Spirulina) platensis in response to gamma irradiation.

Arthrospira (Spirulina) platensis is a blue-green alga, rich with bioactive components and nutrients. To evaluate effect of gamma irradiation, A. platensis was exposed to different doses of 0.0, 0.5, 1.0, 1.5, 2.0 and 2.5kGy. The data showed that the phenolic and proline contents significantly increased with the increase of gamma irradiation doses up to 2.0kGy, above which a reduction was observed. The soluble proteins and malondialdehyde (MDA) contents were stimulated by all tested irradiation doses. Furthermore, the vitamins (A, K and B group) and mineral contents (N, P, Na, K, Ca, Mg and Fe) were stimulated by the irradiation doses compared with the control. The activities of some N-assimilating and antioxidant enzymes were significantly increased with the irradiation doses up to 2.0kGy. This study suggests the possible use of gamma irradiation as a stimulatory agent to raise the nutritive value and antioxidant activity of A. platensis.