Effect of the Cellular Age of the Cyanobacterium Microcystis aeruginosa on the Efficacy of the UV/H₂O₂ Oxidative Process for Water Treatment

Abstract

Cyanobacteria, particularly Microcystis aeruginosa, can form dense blooms that impair water quality, and conventional treatment methods often fail to remove them effectively. This study evaluated the impact of cell age on the performance of the UV/H₂O₂ advanced oxidation process against M. aeruginosa. Cultures of M. aeruginosa were monitored over 64 days at an initial culture density of 1.20 × 10⁶ cells mL⁻¹. For the UV/H₂O₂ experiments, cells were adjusted to a density of 5.00 × 10⁵ cells mL⁻¹, and the growth and oxidative experiments were monitored using parameters such as hydrogen peroxide decay concentration, optical density at 730 nm (OD₇₃₀), cell density, and dissolved organic carbon (DOC). The hydrogen peroxide (H₂O₂) dosages used were 20 mg L⁻¹ and 50 mg L⁻¹, and the results showed that despite varying cell ages, H₂O₂ consumption remained stable at both dosages. While optical density and cell count indicate total cell removal, DOC levels increased due to cell lysis, resulting in contributions from both intracellular and extracellular fractions. A linear correlation was found between cell density and OD₇₃₀, and between total DOC and cell density. In conclusion, cell age did not influence the effectiveness of the UV/H₂O₂ process under the conditions tested. These findings indicate that UV/H₂O₂ can be an effective approach for managing cyanobacterial blooms in water treatment systems, with its performance being unaffected by cell age.

1. Introduction

Water availability is increasingly compromised by irresponsible consumption, rapid population growth, and the degradation of natural sources. Water pollution can occur due to factors such as the excessive use of agricultural fertilizers and pesticides [1], and the discharge of untreated industrial effluents and domestic sewage. These practices introduce excessive loads of organic matter, phosphorus, and nitrogen in water bodies. High concentrations of these compounds disrupt aquatic ecosystems, resulting in eutrophication [2].

The decomposition of excess organic matter leads to a progressive decrease in dissolved oxygen levels. Additionally, water pH values between 6 and 9, along with temperatures above 20 °C, create favorable conditions for the uncontrolled growth of algae, particularly cyanobacteria. This accelerated and excessive growth, also known as bloom, alters water quality and affects the population dynamics within aquatic ecosystems [3,4].

Cyanobacteria are photosynthetic microorganisms that exhibit high resistance to various physicochemical conditions in different environments. However, these microorganisms are more frequently found in freshwater [3,4]. Their proliferation in source waters presents significant challenges for Water Treatment Plants (WTPs). Beyond the unpleasant odor and taste characteristic of eutrophic waters, these microorganisms necessitate increased operational expenditure on coagulants and chemicals throughout the treatment stages [5,6]. Furthermore, cyanobacterial biomass frequently leads to filter clogging, compromising overall water treatment efficiency.

Conventional water treatment processes (chemical coagulation, flocculation, sedimentation, and filtration) may present limitations when treating water impacted by cyanobacterial contamination, which can compromise the quality of the treated water [7,8]. In this context, Advanced Oxidation Processes (AOPs) are widely studied for degradation processes in water treatment [9,10]. Among these, the UV/H₂O₂ process has shown efficient results [11] as it enables the degradation of even the most resistant pollutants and is also applicable to waters contaminated with cyanobacteria. This relies on the generation of hydroxyl radicals (•OH), which are non-selective and highly reactive with a high oxidation potential towards organic pollutants [12]. When applied to cyanobacteria, the combination of ultraviolet radiation and a strong oxidizing agent, depending on the dosage used, causes oxidative damage to the cells [13].

In this context, it is important to consider that during bloom events comprise cyanobacterial populations of varying cells ages are present and interspecific competition. Available studies on UV/H₂O₂ applied to cyanobacteria-contaminated waters have mainly focused on overall removal and by-product formation, while the influence of cyanobacterial cell age has received limited attention. Therefore, this study aimed to evaluate how the cellular age of Microcystis aeruginosa influences the performance of the UV/H₂O₂ oxidation process. The results obtained contribute to improving water treatment strategies for cyanobacteria-contaminated waters by considering the physiological variability of these microorganisms.

2. Materials and Methods

2.1. Chemical Reagents

The chemical reagents used in this study included monobasic potassium phosphate (KH₂PO₄, 99%, Synth, Diadema, Brazil), sodium hydroxide (NaOH, 98%, Synth, Diadema, Brazil), and phosphoric acid (H₃PO₄, 85%, Neon, Suzano, Brazil). Additionally, hydrogen peroxide (H₂O₂, 30%, AppliChem, Darmstadt, Germany), ammonium metavanadate solution (NH₄VO₃, 99%, Sigma-Aldrich, São Paulo, Brazil), and sodium sulfite (Na₂SO₃, 98%, Alphatec, São Bernardo do Campo, Brazil) were employed. All reagents were used as received, without further purification.

2.2. Cyanobacterial Cultivation

The Microcystis aeruginosa strain cultivated in ASM-1 medium and maintained in a climate-controlled chamber at 26 ± 1 °C with periodic manual aeration. The cultures were exposed to a 16/8-h light/dark photoperiod to simulate natural diurnal cycles.

2.3. Study Water

Synthetic study water was prepared using a phosphate-buffered solution. To reach a final volume of 2000 mL, 500 mL of monobasic potassium phosphate solution was mixed with 391 mL of 0.2 M sodium hydroxide solution in a volumetric flask and then diluted with deionized water.

Before preparing the study water, the culture was centrifuged at 4000 rpm for 10 min using a Daiki DT 4500 centrifuge (Daiki, Hunan, China). The supernatant fraction was discarded, and the resulting pellet was resuspended in an aliquot of the study water and homogenized using a vortex mixer. The study water pH was maintained at 7.4, with adjustment made as necessary, using 0.2 M H₃PO₄ or NaOH. For the essays, the phosphate-buffered solution was inoculated with M. aeruginosa cells (obtained after centrifugation) to achieve a cell density of 5.00 × 10⁵ cells mL⁻¹.

The M. aeruginosa cell density was determined by using an optical microscope and a Neubauer chamber to determine the exact volume of inoculum (resuspended cells) required for the 2 L assays.

2.4. Experimental Procedure

The study was conducted in two distinct stages: a growth phase characterization of M. aeruginosa and subsequent photo-oxidative assays. Growth curve monitoring was performed in duplicate and included cell density determination, optical density, pH, and dissolved organic carbon (DOC), evaluated as extracellular and total fractions. For the photo-oxidative assays, two H₂O₂ dosages were applied (20 mg L⁻¹ and 50 mg L⁻¹). All experiments were conducted in duplicate using study water prepared with a standardized initial cell density of 5.00 × 10⁵ cells mL⁻¹. The effect of culture age was assessed by comparative analysis of the temporal trends of hydrogen peroxide decay, optical density, cell density, and extracellular DOC under identical operational conditions.

2.4.1. Evaluation of the Cell Growth of Microcystis aeruginosa

Cell growth was monitored in duplicate over a period of 64 days, starting at day 0 and with sampling at 7-day intervals on days 8, 15, 22, 29, 36, 43, 50, 57, and 64. The initial cell density used for culture evaluation was 1.20 × 10⁶ cells mL⁻¹. Samples were analyzed for cell density to assess the increase in cell numbers during this period, as well as for optical density at 730 nm (OD₇₃₀), which also provides information on the species’ growth. Additionally, pH measurements were performed to verify whether there was an increase in pH in the medium over the cultivation period, since elevated pH values can reduce the effectiveness of water treatment processes in drinking water treatment plants [14].

To quantify extracellular DOC, approximately 30 mL of samples were collected from the culture medium and filtered through a 0.45 μm cellulose nitrate membrane and stored in carbon-free vials. The samples were subsequently acidified to pH < 2.0 using phosphoric acid (10% H₃PO₄) to eliminate any inorganic carbon present. All samples were kept refrigerated at 4 °C until analysis.

Total DOC includes both the extracellular and intracellular organic fractions. For this analysis, raw culture samples were subjected to three freeze–thaw cycles to ensure cell rupture and release of intracellular content. Subsequently, the samples were filtered and stored as previously described for extracellular DOC analysis.

2.4.2. Photo-Oxidative Assays

The photo-oxidative experiments were conducted in a 0.75 L bench-scale reactor, equipped with a submerged high-pressure mercury vapor lamp (254 nm, 125 W) protected by a double quartz tube. To ensure thermal stability and sample homogeneity, the solution was continuously stirred and maintained at 25 ± 1 °C using a recirculating thermostatic bath. For safety reasons, the system was operated inside a protective cabinet to prevent user exposure to UV radiation. All photo-oxidative experiments were conducted in duplicate.

The study water (previously inoculated with M. aeruginosa cells at a density of 5.00 × 10⁵ cells mL⁻¹) was exposed to the UV/H₂O₂ oxidative process for 60 min, with hydrogen peroxide added at dosages of 20 mg L⁻¹ and 50 mg L⁻¹. To evaluate the decomposition of the oxidizing agent during the 60-min UV exposure, aliquots of 4 mL were taken at 2.5, 5, 10, 20, 30, 45, and 60 min were transferred into test tubes containing distilled water and ammonium metavanadate solution to quench the reaction and quantify residual H₂O₂ [15].

For cell density analysis, 8 mL aliquots were taken at 0, 5, 10, 20, and 60 min. For optical density analysis, samples were collected at 0, 5, 10, 20, 30, 45, and 60 min. These analyses aimed to assess the integrity of the cells when subjected to the oxidative process.

For extracellular DOC evaluation, 30 mL aliquots were collected at 0, 5, 10, 20, and 60 min into carbon-free vials. To quench residual H₂O₂, a 1 g L⁻¹ sodium sulfite solution was added at a stoichiometric ration of [3.7 mg L⁻¹] Na₂SO₃/g H₂O₂. Finally, samples were processed according to the filtration and storage procedure described in Section 2.4.1.

2.5. Analytical Methods

2.5.1. Cell Counting

M. aeruginosa cell density was determined using a Neubauer counting chamber (Kasvi, São Paulo, Brazil). A 45 mL culture aliquot was centrifuged at 4000 rpm for 10 min using a centrifuge (Daiki DT4500). The supernatant was discarded, and the pellet was resuspended in the study water. The sample was then homogenized using a vortex tube shaker, and a 1:10 dilution was prepared. A 10 µL volume of the diluted sample was then loaded into the chamber for microscopic counting. The cell density (cells mL⁻¹) was calculated according to Equation (1):

$$\mathrm{Number} \mathrm{of} \mathrm{cells} \left({\mathrm{mL}}^{-1}\right)=C \times D \times {10}^4$$

(1)

were C represents the average cell count per chamber quadrant, D is the dilution factor used, and 10⁴ is the chamber correction factor.

2.5.2. Determination of H₂O₂ Concentration

A stock solution of hydrogen peroxide (50 mg L⁻¹) was prepared, and subsequent dilutions were made to construct the calibration curve (Figure S1), relating absorbance readings to H₂O₂ concentration. For the analysis, 4 mL of the sample was mixed with 1.6 mL of ammonium metavanadate solution and 4.4 mL of distilled water. After homogenization, the H₂O₂ concentration was determined by measuring the absorbance at 450 nm using a UV-Vis spectrophotometer (UV-5100 Global Trade Technology, Serial number: 20150706, China) [15].

2.5.3. Determination of Optical Density

For this analysis, 4 mL aliquots were collected at regular intervals throughout both the growth curve and the oxidative process. The optical density (OD₇₃₀) was determined by measuring sample at 730 nm [16] using a UV-Vis spectrophotometer (UV-5100 Global Trade Technology).

2.5.4. Determination of Extracellular and Total Dissolved Organic Carbon

To ensure carbon-free conditions, all glassware used for dissolved organic carbon (DOC) determination was pre-treated by heating in a muffle furnace at 550 °C for four hours. For each assay, approximately 30 mL of sample was collected at the predetermined intervals for the different H₂O₂ dosages, including the control sample without oxidation. Sodium sulfite aliquots were added to the duplicate samples to stop the reaction with hydrogen peroxide. Following filtration, the samples were transferred to glass vials, acidified, and refrigerated for a maximum of one week prior to analysis.

Both extracellular and total DOC samples were quantified via the high-temperature combustion method (680 °C) with CO₂ detection using a Thermo HiperTOC analyzer (Thermo Scientific, Cambridge, UK) [17].

2.5.5. Determination of the Extracellular DOC Fraction in Total DOC

To determine extracellular DOC, a fraction of each sample collected during the UV/H₂O₂ experiments was filtered through a 0.45 μm cellulose nitrate membrane and stored in carbon-free vials. Fo the total DOC, the remaining unfiltered sample was underwent three freeze–thaw cycles to ensure cell lysis and the release of all intracellular content; these lysed samples were then filtered. The samples were subsequently acidified to pH < 2.0 using 10% phosphoric acid (H₃PO₄) to eliminate any inorganic carbon. All samples were stored at 4 °C until analysis, following the protocol described in Section 2.5.4.

3. Results and Discussion

3.1. Growth Curve of Microcystis aeruginosa

Figure 1 shows the correlation between cell density and optical density at 730 nm, yielding a coefficient of determination (R²) of 0.9798. This result confirms a linear relationship between the methodologies evaluated. Thus, measuring optical density at 730 nm proved to be a viable and more practical alternative for monitoring the M. aeruginosa growth when compared to direct cell counting under a microscope. A previous study [18] also established a relationship between optical density and cell density in cyanobacteria, demonstrating the reliability of OD measurements for estimating cell density.

The growth kinetics and pH profile of Microcystis aeruginosa, are depicted in Figure 2. Starting from an initial cell density of 1.20 × 10⁶ cells mL⁻¹, the culture remained in the exponential growth phase throughout the monitoring period. The absence of stationary or decline phases suggests that the growth parameters (temperature, light exposure time, and agitation) were adequate to maintain the cultivation of M. aeruginosa for extended periods. In contrast, previous study [19] using the same ASM-1 and a similar initial cell density of 1.50 × 10⁶ cells mL⁻¹ observed a full growth cycle (lag, exponential, stationary, and decline) within 30 days. This difference in results may be related to the operational conditions employed or to the strain of M. aeruginosa used.

Figure 3 shows the relationship between optical density and the concentrations of extracellular and total dissolved organic carbon. At a peak of OD₇₃₀ of 0.644 cm⁻¹, the maximum values recorded were 10.7 mg L⁻¹ for extracellular DOC and 42.7 mg L⁻¹ for total DOC. Approximately 30% of the total DOC originated from extracellular DOC, while the remaining portion came from intracellular content released by cell lysis and from the cellular structure itself.

A linear correlation (R² = 0.9804) was observed between the total DOC concentration and M. aeruginosa cell density (Figure 4). This correlation allowed for the estimation of the amount of DOC per cell by relating the total DOC concentration to cell density (Equation (2)), with the results summarized in

Table 1. Cultivation time and total DOC/cell ratio.

Growth Time (Days)	Total DOC (mg L−1)	Cell Density (Cell mL−1)	The DOC/Cell Ratio
8	±10.4	1.29 × 106	2.33 × 10−6
15	±10.0	4.48 × 106	2.23 × 10−6
22	±15.0	7.15 × 106	2.10 × 10−6
29	±19.9	7.85 × 106	2.53 × 10−6
36	±20.1	10.9 × 106	1.84 × 10−6
43	±26.9	12.5 × 106	2.15 × 10−6
50	±32.1	14.1 × 106	2.27 × 10−6
57	±35.7	17.1 × 106	2.09 × 10−6
64	±35.8	17.4 × 106	2.22 × 10−6

. For this calculation, time zero (initial cultivation time) was excluded, as it showed atypical DOC per cell values, possibly due to the high concentration of nutrients and organic matter in the culture medium at that point. Thus, the average DOC per cell value obtained was 2.20 × 10⁻⁶ mg cell⁻¹, indicating that each cultivated M. aeruginosa cell contains, on average, 2.20 × 10⁻⁶ mg of total dissolved organic carbon, distributed between intracellular and extracellular content. Through this analysis, it was possible to estimate the potential formation of DOC resulting from cyanobacterial cell lysis during oxidative processes, thereby supporting the selection of appropriate parameters for advanced oxidation processes.

$${\displaystyle \frac{\mathit{Total} \mathit{DOC}}{\mathit{Cell}} = \frac{\mathit{Total} \mathit{DOC}}{\mathit{Cell} \mathit{density}} \frac{(\mathrm{mg} {\mathrm{L}}^{-1})}{(\mathrm{cell} {\mathrm{L}}^{-1})}}$$

(2)

3.2. Photo-Oxidative Experiments

3.2.1. Residual H₂O₂

Figure 5 illustrates the residual H₂O₂ concentrations for the initial dosages of 20 and 50 mg L⁻¹. For both hydrogen peroxide concentrations used, complete degradation of residual peroxide was achieved within 45 min, with residual levels falling below 0.1 mg L⁻¹. Greater data dispersion was observed at 20 mg L⁻¹ dosage compared to 50 mg L⁻¹. Although temperature was controlled at 25 ± 1 °C, minor fluctuations in the refrigeration system cannot be ruled out and may have contributed to the observed variability. This interpretation is therefore presented as a qualitative hypothesis rather than a confirmed causal factor. According to Mattos et al. [20], hydrogen peroxide solutions with concentrations between 35% and 52% (w/v) are stable under controlled temperatures but become unstable when exposed to increased temperatures. While low H₂O₂ dosages are recommended for the elimination of cyanobacteria [21], excessive concentrations may trigger H₂O₂ acting as a scavenger of hydroxyl radicals, converting them into hydroperoxyl radicals (HO₂•). This effect reduces the efficiency of the oxidation process, as the HO₂• radical is less reactive than the OH• radical [22]. Another study [23] evaluating the effectiveness of peroxide in controlling cyanobacteria demonstrated that both the peroxide dosage and the initial biomass significantly influence process efficiency. Additionally, for both hydrogen peroxide concentrations used, the cell age of the M. aeruginosa culture did not affect hydrogen peroxide decay.

3.2.2. Dissolved Organic Carbon

Figure 6 illustrates the DOC profiles for 20 and 50 mg L⁻¹ hydrogen peroxide dosages. An increase in DOC was observed during the first 10 min at the 20 mg L⁻¹ dosage, which may be related to the release of intracellular organic matter following initial cell damage. Similar results have been reported previously [24], which observed increases in DOC within the first 5 min of treatment using hydrogen peroxide dosages of 5 and 20 mg L⁻¹. By the end of 60 min of treatment, DOC values ranged from 2.7 to 7.3 mg L⁻¹, with a mean concentration of 5.1 mg L⁻¹. These results are consistent with a previous study [25] that evaluated the suppression of cyanobacteria using H₂O₂ under different light intensities and reported that H₂O₂ caused an increase in extracellular microcystin as well as a reduction in cell size.

Figure 6b presents the results obtained with a higher oxidant dosage, showing generally lower final DOC values at 50 mg L⁻¹ compared to 20 mg L⁻¹, although overlapping ranges between the two dosages were observed. Similar results were reported by Siquerolo et al. [26], who attributed this behavior to a superior mineralization capacity. The higher concentration of H₂O₂ likely facilitated a grater generation of hydroxyl radicals (OH•) during the oxidation process, promoting the complete oxidation of dissolved organic matter into inorganic constituents. Following the 60 min of treatment, DOC values ranged from 3.8 to 5.6 mg L⁻¹.

For all dosages tested, an increase in DOC was observed compared to time zero, which can be attributed to cell lysis and the subsequent release of intracellular organic matter. These elevated DOC values after 60 min of treatment suggests that the applied H₂O₂ dosages were insufficient to achieve complete oxidation of the organic material present, including the additional content released from lysed cells. The release of DOC during UV/H₂O₂ treatment underscores the importance of evaluating potential risks associated with toxin release and disinfection by-product formation in drinking water treatment systems.

3.2.3. Cell Density

Figure 7 shows the decay in M. aeruginosa cell density at H₂O₂ dosages of 20 and 50 mg L⁻¹, with initial values ranging from 5.02 × 10⁵ cells mL⁻¹ to 5.95 × 10⁵ cells mL⁻¹. Cell age did not influence the decay of H₂O₂ concentration, as all ages evaluated showed the same trend of greater reduction within the first 20 min of the photo-oxidative assay. Furthermore, after 60 min of treatment, cell density was reduced to zero, indicating the complete disruption of cellular structure regardless of culture age. The decrease in cell density and the absence of cells at 60 min suggest that cell lysis occurred during the process, releasing intracellular contents into the medium and consequently affecting extracellular DOC values (as shown in Figure 4). The 20 mg L⁻¹ dosage showed a greater effect on the M. aeruginosa cells, as evidenced by the lower cell densities observed at all sampling times when compared to the 50 mg L⁻¹ H₂O₂ dosage. This behavior is consistent with the action of H₂O₂ as a hydroxyl radical scavenger at higher dosages, as discussed previously.

3.2.4. Optical Density

Figure 8 illustrate the optical density decay curve for the 20 mg L⁻¹ and 50 mg L⁻¹ dosages. The greatest decrease in OD₇₃₀ occurred within the first 20 min, with an average decrease of 66% compared to time zero. After around 45 min, the observed values averaged 0.001 cm⁻¹, indicating a near absence of cells in the sample. A previous study [27] investigated the combined application of UV-C radiation and hydrogen peroxide on M. aeruginosa and found that this treatment affects both the cell membrane and photosystems. This may have contributed to the effective removal of the cells observed in this study. Additionally, OD at 264 nm was used in a previous study [28] to evaluate the inactivation of M. aeruginosa during the photocatalytic process. An increase in OD₂₆₄ was observed up to 150 min of treatment, followed by a decrease at 180 min. According to the authors, this behavior was attributed to the release of extracellular exudates resulting from membrane damage (150 min), followed by cell death (180 min) and the degradation of these compounds by reactive oxygen species. These findings reinforce that membrane disruption not only facilitates cell removal but also promotes the release of intracellular material into the medium.

4. Conclusions

These findings indicate that cell age does not affect the effectiveness of the UV/H₂O₂ oxidation process, regardless of the hydrogen peroxide dosage or the analytical parameter used. This supports the viability of the process for treating water, even during blooms containing cyanobacteria of varying ages. Through the linear correlation between cell density and total DOC, it was possible to determine the DOC/cell ratio for M. aeruginosa, allowing for an estimation of dissolved organic matter formation during the oxidation processes studied. It was observed that the 50 mg L⁻¹ dosage showed a tendency toward greater DOC reduction compared to 20 mg L⁻¹. Moreover, the reductions observed in cell density and optical density indicate that the DOC at the end of the UV/H₂O₂ process consists of both extracellular and intracellular fractions released during treatment. This finding is supported by the absence of intact cells in the medium at the end of the process. It was also found that the greatest decrease in these parameters occurred between 0 and 20 min of UV/H₂O₂ treatment, suggesting that cyanobacterial cell lysis mainly took place during this time interval. This observation underscores the importance of considering the implications of cyanobacterial cell lysis in full-scale water treatment. This study contributes to the understanding of the effects of the UV/H₂O₂ process on M. aeruginosa, providing important insights for the practical application of advanced oxidation processes in managing cyanobacterial blooms.