Tatiana Chiriac

ZnO and TiO₂ nanoparticles are increasingly biofunctionalized to improve stability, biological recognition, targeting, and compatibility. However, whether biofunctionalization inherently makes these nanoparticles safer remains unresolved. Rather than cataloguing biofunctionalization strategies, this critical review evaluates whether toxicological changes can be attributed specifically to deliberate biomolecular surface modification and whether any resulting safety advantage persists during exposure-relevant transformations. A structured literature search identified 60 publications for critical synthesis, including 10 comparative studies providing direct evidence on toxicological changes attributable to deliberate biofunctionalization. The available evidence does not support a uniform toxicity-reducing effect. Depending on the nanoparticle-biointerface combination and exposure context, biofunctionalization can reduce toxicity, produce coating-dependent changes, increase biological accessibility, or redirect reactivity toward selected biological targets. These outcomes arise from changes in surface charge, aggregation, colloidal stability, cellular interactions, and biointerface evolution and are material-specific: ZnO additionally requires consideration of dissolution and Zn²⁺ release, whereas TiO₂ behavior is more strongly influenced by particulate persistence and surface-mediated reactivity. We therefore conceptualize toxicological identity as an evolving, exposure-dependent profile rather than an intrinsic material property. Within a safe-by-design framework, biofunctionalization is advantageous only when it reduces non-target toxicity, preserves or improves the intended function, and maintains this advantage after exposure-relevant transformations.

Zinc oxide nanostructures can induce highly variable biological responses depending on their physicochemical properties, concentration, and exposure conditions. This study investigated the effects of multipod ZnO nanostructures (ZnO-MPs) on Limnospira platensis, considering both concentration and application timing. ZnO-MPs (0.01–30 mg/L) were applied at 0, 72, or 120 h after inoculation, and the final biomass and the contents of proteins, carbohydrates, lipids, phycobiliproteins, phycocyanin, chlorophyll, and carotenoids were determined. Final biomass showed relatively limited and non-monotonic variation, whereas biochemical composition was substantially altered. Low concentrations (0.1–1 mg/L), particularly when applied at inoculation, stimulated total phycobiliprotein and phycocyanin accumulation. Higher concentrations strongly reduced protein and phycobiliprotein contents, while promoting carbohydrate and/or lipid accumulation under specific application conditions. Chlorophyll and carotenoids exhibited comparatively moderate and non-monotonic responses. Overall, the results demonstrate that the effects of ZnO-MPs on Limnospira platensis are strongly modulated by concentration and application timing, with biochemical responses being more pronounced than changes in final biomass. These findings emphasize the importance of considering both nanomaterial concentration and application timing when evaluating nanomaterial–cyanobacterium interactions.

The chemical form of copper is a key determinant of metal bioavailability and may profoundly influence cyanobacterial metabolism and the biological activity of culture-derived products. In this study, Nostoc linckia was cultivated in the presence of metallic copper nanoparticles (CuNPs), copper oxide nanoparticles (CuONPs), or CuSO₄·5H₂O (15 mg Cu L⁻¹). Biomass productivity, biochemical composition, and antioxidant capacity, together with the physicochemical and biochemical characteristics of the culture liquid, were determined. The culture liquid was subsequently evaluated through the seed priming of four maize (Zea mays L.) hybrids. The different copper forms induced distinct metabolic responses in Nostoc linckia. CuONPs promoted biomass accumulation while maintaining protein and photosynthetic pigment contents and inducing an adaptive oxidative response, whereas CuNPs, and particularly CuSO₄, impaired biomass production and primary metabolism. These metabolic changes were reflected in the culture liquid properties and were associated with genotype-dependent differences in maize germination dynamics, seedling vigor, and biochemical composition. Integrated correlation, hierarchical clustering, and principal component analyses revealed coordinated relationships linking cyanobacterial physiology, culture liquid profiles, and plant responses. Collectively, these findings demonstrate that the chemical form of copper drives metabolic remodeling in Nostoc linckia, thereby shaping the biochemical composition and biostimulant properties of its culture liquid.

Background: Extracts from microalgae and cyanobacteria represent important sources of bioactive compounds with antioxidant potential. Their efficacy depends on their chemical nature and behavior in different environments. For assessing activity in lipid systems, the lowdensity lipoprotein (LDL) oxidation inhibition assay is an appropriate functional model for comparing the capacity to prevent lipid peroxidation. Aim: To comparatively evaluate the inhibitory effect on LDL oxidation of selected bioactive preparations of cyanobacterial and microalgal origin. Materials and methods: Phycobiliproteins from Porphyridium cruentum and Arthrospira platensis (aqueous extracts), as well as astaxanthin from Haematococcus pluvialis (ethanolic extract from aplanospores), were analyzed. The inhibitory effect on LDL oxidation was assessed using a spectrophotometric assay, with results expressed as percentage inhibition at defined concentrations of the tested preparations. Results: All three preparations exhibited inhibitory activity against LDL oxidation, with variations depending on the chemical nature of the bioactive compounds and their concentration. Astaxanthin from Haematococcus pluvialis showed the strongest inhibitory effect, reaching 67.3% at 5 μg/mL, indicating high antioxidant potency at very low concentrations. This effect may be attributed to the specific chemical structure of astaxanthin, which enables activity in lipid systems by localizing within hydrophobic membrane regions and at the lipid-water interface, the primary site of LDL lipid peroxidation initiation. Phycobiliproteins from Arthrospira platensis showed an inhibitory effect of 27.9% at 3.99 μg/mL, suggesting high specific activity at low concentrations. Phycobiliproteins from Porphyridium cruentum induced 43.4% inhibition at 136.6 μg/mL. These differences in potency may be related to the distinct compositions of phycobilin chromophores (phycocyanobilin vs. phycoerythrobilin), which influence radical-scavenging efficiency and electron-transfer processes. Phycocyanin exhibits high activity at low concentrations through chain-breaking antioxidant mechanisms in the early stages of oxidation. In contrast, phycoerythrin may exhibit higher activity at elevated concentrations due to structural differences that affect interactions at the lipid-water interface of LDL. Conclusions: The study demonstrates that algal and cyanobacterial preparations inhibit LDL oxidation to varying degrees, depending on compound type and concentration. Astaxanthin from Haematococcus pluvialis was the most potent inhibitor at μg/mL levels, while phycobiliproteins from Arthrospira platensis and Porphyridium cruentum also exhibited relevant antioxidant activity. The LDL oxidation assay proved to be a suitable model for evaluating antioxidant potential in lipid systems.

Background: Interactions between engineered nanoparticles (NPs) and photosynthetic microorganisms represent a rapidly developing field at the interface of nanotechnology and microbial biotechnology. Metal and metal oxide nanoparticles can act as both stressors and modulators of cellular metabolism, inducing oxidative stress responses that may lead to either growth inhibition or adaptive metabolic redirection. Aim of the study: The study aims to elucidate the physiological and biochemical responses of selected photosynthetic microorganisms (cyanobacteria and microalgae) to exposure to engineered nanoparticles, with a focus on oxidative stress modulation and its impact on biomass productivity and metabolite accumulation. Materials and methods: Model strains of cyanobacteria and microalgae (e.g., Arthrospira platensis, Nostoc linckia, Haematococcus lacustris) were cultivated under controlled laboratory conditions and exposed to metal and metal oxide nanoparticles of defined sizes and concentrations. Growth parameters, pigment content (chlorophylls, carotenoids), and oxidative stress markers were monitored. Spectrophotometric and biochemical assays were used to assess cellular responses, and comparative analyses between treatments and controls were performed. Results: Exposure to engineered NPs induced dose- and size-dependent responses across all tested strains. At higher concentrations, NPs inhibited growth, increased oxidative stress, degraded pigments, and impaired photosynthetic activity. In contrast, sublethal concentrations triggered hormetic effects, reflected in moderate stimulation of growth and metabolism. Photosynthetic microorganisms activated antioxidant defensemechanisms, leading to increased levels of protective pigments and improved redox balance. In Haematococcus lacustris, controlled nanoparticle exposure promoted a shift toward carotenoid biosynthesis, resulting in enhanced astaxanthin accumulation under moderate stress conditions. Cyanobacteria such as Arthrospira platensis and Nostoc linckia exhibited adaptive responses mediated by extracellular polymeric substances (EPS), which reduced nanoparticle bioavailability and toxicity. Nanoparticle size also influenced biological effects, with smaller particles inducing stronger responses. Overall, the results highlight the dual role of nanoparticles as both stressors and modulators of metabolism, with controlled oxidative stress enhancing the production of valuable metabolites. Conclusions: The results demonstrate that controlled nanoparticle-induced stress can be exploited as a biotechnological tool to modulate metabolic pathways in photosynthetic microorganisms. Understanding the balance between toxicity and hormesis is essential for developing sustainable biotechnological applications, including high-value metabolite production.

Background: Porphyridium purpureum is a red microalga with significant biotechnological potential, whose biomass composition can be modulated under controlled culture conditions. Increased nitrogen availability and optimized light intensity stimulate protein accumulation, reflecting adaptive metabolic responses to environmental variations. This metabolic plasticity enables the redirection of biosynthetic pathways toward target compounds. Moreover, the resulting biomass can serve as a biological matrix for generating reducing biomolecules applicable in the functionalization of silver nanoparticles and the development of stable, biocompatible nanobiomaterials. Aim of the study: To identify optimal nutritional and illumination conditions for maximizing protein accumulation in Porphyridium purpureum and to validate the obtained biomass as a source of targeted proteins for subsequent use in the biofunctionalization of silver nanoparticles. Materials and methods: Porphyridium purpureum was cultivated in three nutrient media under controlled temperature, pH, and continuous illumination to determine the optimal growth conditions. Nitrogen source optimization was performed using a single-factor experimental design followed by the Box-Wilson method. The illumination regime was adjusted stepwise according to the growth phases (adaptation and exponential). Biomass productivity and protein accumulation were assessed by protein quantification, MDA content evaluation, and antioxidant activity assays (DPPH and FRAP) using specific spectrophotometric methods. Results: Optimization of cultivation conditions - by increasing nitrogen from 5.5 to 6.5 g/L and applying phased illumination (56 μmol photons·m⁻²·s⁻¹ for 6 days, then 72 μmol photons·m⁻²·s⁻¹ during the exponential phase) - significantly enhanced protein content compared to standard conditions, while maintaining low oxidative stress (reduced MDA). The optimized biomass reached ~45% protein versus ~33% under conventional conditions. Proteins were extracted via alkaline solubilization, followed by centrifugation and pH adjustment, yielding a soluble fraction of ~16 g/100 g extract. The extract showed antioxidant and reducing activity (DPPH inhibition ~20%; FRAP ~27 mg ascorbic acid equivalents/g extract), confirming its suitability as a biological matrix. Conclusions: Optimization of nutritional and illumination conditions significantly enhances protein accumulation in Porphyridium purpureum without inducing substantial oxidative stress. The resulting protein extract exhibits antioxidant and reducing capacity, supporting its applicability as a functional biological matrix for the biofunctionalization of silver nanoparticles.

Background: Sulfated polysaccharides (SPS) are biopolymers located on cell surfaces and in the extracellular matrix of microalgae and cyanobacteria. They are known for antioxidant, antiviral, and immunomodulatory properties. Due to their high sulphation and polyanionic nature, SPS interact with plant cells as natural elicitors and biostimulants. Their application can enhance seed germination, stimulate root and shoot growth, improve nutrient uptake, strengthen defense responses, and increase tolerance to abiotic stress. These effects are relevant for tomato seedlings in the Czech Republic, which are vulnerable to low spring temperatures and to summer heat and drought stress. Aim of the study: The main objective of this study is to establish baseline methodologies and assess seed treatments with SPS biostimulants derived from Arthrospira platensis (spirulina), developed at IMB UTM, for enhancing tomato resilience to temperature extremes and water deficit, in relation to cumulative Growing Degree Days (GDD). Materials and methods: For this purpose, SPS were extracted from spirulina biomass using distilled water at a ratio of 1:45 (w/v) at 90 °C for 2 h, followed by centrifugation to remove residual biomass. The resulting supernatant was subsequently treated with cetyltrimethylammonium bromide to precipitate the polysaccharide fraction, purified, and redissolved in distilled water. Tomato seeds were treated with obtained SPS before sowing. Results: Under controlled greenhouse conditions, treated seeds exhibited faster, more synchronized emergence, with a reduction in mean germination time of approximately 2-3 days and a 25-35% increase in seedling vigor index compared to the untreated control. These responses were associated with enhanced root elongation and shoot growth. The most pronounced effects of the biostimulant application were observed at the seedling stage, where treated plants showed more vigorous and structurally balanced development, including a betterdeveloped root system and improved physiological readiness for transplanting. Although the risk of late-spring frosts constrained transplanting dates, seedlings were successfully transplanted into open-field conditions in the Elbe Lowland region of the Czech Republic. Following transplanting, treated plants demonstrated faster field establishment, characterized by more rapid root anchorage and an earlier resumption of growth. Moreover, biostimulanttreated seedlings exhibited increased tolerance to early-season environmental variability, particularly spring temperature fluctuations, resulting in reduced transplanting stress and improved early field performance. Conclusions: These findings highlight the relevance of SPS biostimulants for improving tomato development and adaptation to extreme temperature and water deficit.

Background: Zinc oxide-based nanomaterials exhibit distinct physicochemical properties that influence their interactions with biological systems. In microalgae and cyanobacteria, these interactions exert effects that are strongly dependent on morphology, concentration, and the timing of application relative to the culture's physiological state. Aim of the study: To investigate interaction models of ZnO multipod nanoparticles with cyanobacterium Arthrospira platensis and red microalga Porphyridium purpureum, focusing on metabolic response and oxidative stress. Material and methods: Arthrospira platensis and Porphyridium purpureum cultures were grown under controlled conditions and exposed to ZnO multipod nanoparticles at concentrations ranging from 0.1 to 30 mg/L. Nanoparticles were applied at different growth stages (lag, exponential, and pre-stationary/tardo-exponential phases). Biomass, biochemical composition (proteins, carbohydrates, lipids, photosynthetic pigments, and phycobiliproteins), and oxidative stress marker (MDA) were assessed. Statistical analysis was performed using Student's t-test (p ≤ 0.05). Results: The interaction of ZnO multipod nanoparticles with microalgal systems follows a general dose-dependent model, differentiated according to the cultures' physiological state. In Arthrospira platensis, the response is phase-dependent. It follows a sequence of patterns: an initial adaptation model characterized by minor changes at low doses, followed by a compensatory model in the exponential phase, defined by the accumulation of carbohydrates and lipids and a reduction in phycobiliproteins, and finally an advanced oxidative stress model marked by decreased protein content and increased lipid peroxidation markers. In Porphyridium purpureum, the interaction is predominantly dose-dependent and less influenced by growth phase, characterized by an early and persistent oxidative stress model, with lipid reduction, pigment variations, and a continuous increase in malondialdehyde levels. At low doses, a moderate metabolic adjustment model is observed, whereas at high concentrations, phycobiliproteins undergo progressive degradation. Conclusion. The data obtained indicate that the interaction between ZnO multipod nanoparticles and microalgae and cyanobacteria follows a sequence of functional patterns: initial adaptation → compensatory response → advanced/persistent oxidative stress, characterized by correlated changes in biomolecules and oxidative stress markers. These results support the controlled use of nanomaterials to modulate microalgal metabolism, provided that the applied concentrations are strictly optimized.

The release of nano-sized metal particles into the environment, along with the associated risk of their migration through the food web, is linked to the widespread application of nanomaterials in industry and agriculture. In this study Mentha spicata L. plants were exposed to silver nanoparticles (1–2 nm) at concentrations of 1, 5, 10, 50, and 100 mg/L over a 28-day period, employing two distinct application methods: root application and foliar spraying. Silver content in plant tissues, soil samples, and herbal infusions prepared from exposed spearmint leaves was determined via atomic absorption spectrometry. In root-applied treatments, silver predominantly accumulated in the soil; however, translocation to the leaves was also detected, reaching a maximum concentration of 16.9 mg/kg in leaves at the highest exposure level (100 mg/L Ag nanoparticles). Silver nanoparticles significantly reduced the soil basal respiration rate compared to the control on the 7 and 30 days of incubation. When silver nanoparticles were applied to the foliage, accumulation in the leaves (5.04–140.4 mg/kg) was observed, alongside excretion through the root system into the rhizosphere (up to 2.41 mg/kg). Interestingly, silver nanoparticles enhanced photosynthetic efficiency in the plants by increasing β-carotene and chlorophyll content, and they also stimulated higher antioxidant activity compared to untreated plants. During brewing, silver extraction into the infusion was below 1% for soil-applied treatments but ranged from 22 to 98% for foliar applications. Risk assessment indicated that prolonged consumption of mint infusion containing silver concentrations exceeding 0.348 mg/L could pose adverse health effects.