Synergistic Ozone-Ultrasonication Pretreatment for Enhanced Algal Bioresource Recovery: Optimization and Detoxification

Abstract

Although algae possess a high capacity for carbon sequestration, the recalcitrant multilayered cell wall structure and residual microcystin toxicity associated with Microcystis aeruginosa significantly hinder the efficient recovery of algal biomass resources. This study developed a synergistic ozone-ultrasonication (O₃-US) pretreatment strategy, systematically comparing its cell-disruption efficacy with standalone O₃ or US, using harvested algal biomass from natural aquatic systems dominated by Microcystis aeruginosa. The synergistic effects revealed were: (1) O₃-mediated oxidation of extracellular polymeric substances and cell wall matrices, (2) the release of ultrasound-induced cavitation-enhancing intracellular components, and (3) an improvement in the O₃ mass transfer by hydrodynamic shear forces. Through response surface methodology optimization, the O₃-US process achieved maximal performance at 0.14 gO₃/gTSS, with a 4 W/mL ultrasonic intensity, and a 20 min duration. Remarkably, the released protein was 289.2 mg/gTSS, which was 4.3-fold and 1.9-fold, respectively, more than that released in O₃ pretreatment and US pretreatment, while the polysaccharide was 87.5 mg/gTSS, increased by 2.4-fold and 3.1-fold respectively, compared to O₃ alone and US alone. The released solubilized chemical oxygen demand (SCOD) was 1037.1 mg/gTSS, increased by 43.3% and 216.1%, respectively, relative to O₃ alone and US alone. DNA quantification further validated the synergistic cell disruption caused by O₃-US. Fluorescence excitation-emission matrix (EEM) spectroscopy identified biodegradable aromatic proteins (Regions I-II) and soluble microbial byproducts (Region IV) as dominant organic fractions, demonstrating enhanced bioavailability. The hybrid process reduced energy consumption by 33.3% in ultrasonic intensity and 60% in duration versus US alone, while achieving 94.5% microcystin-LR (MC-LR) degradation, which showed a 96.6% risk reduction compared to ultrasonic treatment. This work establishes an efficient, low-energy, and safe pretreatment technology for algal resource recovery, synergistically enhancing intracellular resource release while mitigating cyanotoxin hazards in algal biomass valorization.

1. Introduction

Algae have emerged as a pivotal bioresource for the sustainable production of high-value compounds, including biofuels, proteins, polyunsaturated fatty acids, and bioactive pigments, owing to their rapid growth rates and carbon-neutral lifecycle [1]. However, the recalcitrant multilayered cell walls of algae, composed of cellulose, hemicellulose, peptidoglycan (in the case of cyanobacteria), and glycoproteins, act as barriers to intracellular component extraction, necessitating efficient cell-disruption technology as the critical first step in downstream valorization processes [2].

Physical and chemical treatments, such as thermal treatments [3], ultrasonication [4], high-pressure homogenization treatments [5], acid/alkaline treatments [6,7], and chemical oxidation [8], are typically employed to release intracellular biomass. Unlike thermal treatments, which compromise product quality [9], ultrasonication achieves efficient cell disruption via cavitation effects without significantly raising the bulk temperature. In contrast to acid/alkaline treatments that rely on harsh chemicals [10], ultrasonic processing eliminates secondary pollution risks. Notably, ultrasonication has garnered substantial research interest due to its chemical-free operation and ability to generate localized high-pressure zones that effectively cause ruptures of Microcystis (Cyanobacteria) cell structures. Additionally, unlike high-pressure homogenization, ultrasonication produces in situ hydroxyl radicals (•OH), thereby improving subsequent ozone oxidation efficiency [11]. This technique establishes a unique reaction environment unattainable under conventional conditions [12]. Passos et al. [13] demonstrated that ultrasonic pretreatment of filamentous alga Hydrodictyon reticulatum, a Chlorophyta (eukaryotic alga), increased soluble chemical oxygen demand (COD) from 250 mg/L to 1000 mg/L at an energy input of 2500 J/mL. However, ultrasonic treatment has been demonstrated to be highly energy consuming and relatively ineffective for Chlorella pyrenoidosa (C.pyrenoidosa) and some other algae. An ultrasonic treatment at 35 kHz and 0.043 W/mL for 60 s only achieved a 7.9% cell disruption rate in C.pyrenoidosa [14]. To address these limitations, emerging hybrid strategies combine ultrasonication with chemical treatments to synergistically enhance disruption efficiency. This dual approach utilizes chemical agents for cell wall permeabilization while applying an ultrasound for precise mechanical disruption, optimizing both energy efficiency and product recovery. In lipid extraction, Hui et al. [15] demonstrated this synergy, achieving a 25.05 ± 0.92% crude lipid yield using ultrasound-assisted ethanol-2-MeTHF extraction. Similarly, our prior work [16] obtained a 360.42 mg/gTSS protein release from C. pyrenoidosa through ultrasonic-alkaline treatment. The hybrid strategy also shows promise in microbial control, as evidenced by Ren et al. [9], where ultrasound-enhanced slightly acidic electrolyzed water (SAEW) penetration significantly improved Listeria monocytogenes biofilm eradication through synergistic cavitation effects.

Ozone oxidation has gained prominence as an effective strategy for algal biomass valorization. Leveraging its well-established application in water treatment, ozone disrupts cellular integrity through three primary mechanisms: (1) enhancing membrane permeability, (2) promoting intracellular component release, and (3) degrading refractory organics [17]. Studies by Cardena et al. [18] and Pranowo et al. [19] corroborate its efficacy, demonstrating improved cell disruption, methane yield, and organic-matter mineralization. Moreover, ozone’s dual function—cyanotoxin degradation (e.g., microcystin-LR) and enhanced carbon assimilation—renders it a versatile solution for algal treatment [20,21,22]. However, its practical adoption faces challenges, including slow kinetics and limited intracellular penetration due to mass transfer constraints [23]. Overcoming these barriers, particularly in achieving simultaneous cell disruption and detoxification, remains a critical research priority [24].

The integration of ultrasonication with ozone (O₃-US) offers a transformative synergy. Ultrasonic cavitation generates localized microjets and shockwaves (>100 MPa) that physically disrupt cell membranes while enhancing ozone mass transfer via microbubble dispersion [25]. Concurrently, ozone pretreatment oxidizes EPS layers and selectively cleaves unsaturated bonds in cell wall polymers, exposing structural vulnerabilities for targeted ultrasonic attack [26]. This combination has demonstrated efficacy in sludge disruption: for example, a 54% reduction in excess sludge production in the SBR system [27]. When applied to microalgal (in particular, Cyanobacteria) systems, O₃-US not only enhances soluble organic release, but also neutralizes MC-LR through hydroxyl radical pathways, addressing bioavailability and biosafety challenges [28]. For instance, O₃-US achieved 100% inactivation of M. aeruginosa in 20 min [29]. The recovery rate of lipids and carbohydrates from mixed algal consortia reached 59% and 81% of the total lipids and carbohydrates, respectively [30]. Though these studies have shown promising results of O₃-US, reactivity and consequent optimal conditions toward species with different cell structures and physiological characteristics needs to be determined for in specific applications.

In freshwater ecosystems, excessive algal proliferation, particularly cyanobacterial blooms dominated by toxin-producing species like M. aeruginosa, not only poses severe environmental and public health risks, but also affects the reutilization process. For example, algal biomass harvested from Taihu Lake, China’s third-largest freshwater lake, is typically subjected to harvest followed by energy-intensive disposal methods (e.g., incineration, landfilling) or reutilization [31]. However, microcystin-LR (MC-LR) poses a potential risk for reutilization of such kinds of waste [32]. These practices incur high economic and carbon costs while failing to degrade intracellular MC-LR, which persist through conventional treatments and limit downstream utilization [33].

This study systematically investigates the feasibility of O₃-US treatment for enhancing resource recovery from Microcystis aeruginosa-dominated algal blooms. The integrated O₃-US pretreatment strategy synergistically combines chemical oxidation and physical disruption to overcome limitations of standalone technologies. This approach achieves: (1) enhanced microalgal cell wall disruption, (2) controlled release of intracellular organics, and (3) innovative toxin degradation—advancing beyond conventional pretreatment’s narrow focus on resource recovery to incorporate critical biosafety functions. Specifically addressing Microcystis aeruginosa recovery challenges, we developed an energy-efficient, safe, and economically viable protocol for natural bloom remediation, establishing a sustainable pathway for algal resource utilization that balances processing efficiency with environmental safety.

2. Materials and Methods

2.1. Materials and Reagents

The algal consortium used in this study, collected from Taihu Lake in Suzhou, Jiangsu Province, China, was authenticated by the Institute of Hydrobiology at the Chinese Academy of Sciences as a mixed community dominated by Microcystis aeruginosa (M. aeruginosa), comprising 91% of the total biomass. The algae were concentrated by 200—mesh sieve filtration and subsequently stored at 4 °C. The main physicochemical properties of the algae concentrates are shown in

Table 1. Main physicochemical properties of the algal concentrate.

Parameters	Average
pH	6.2
TS (Total Solid) (%)	1.7
TSS (Total Suspended Solids) (g/L)	17.4
VSS/TSS	0.9
TCOD (Total Chemical Oxygen Demand) (mg/gTSS)	1567.7
SCOD (Soluble Chemical Oxygen Demand) (mg/gTSS)	45.2
Soluble Protein (mg/gTSS)	3.1
Soluble Polysaccharides (mg/gTSS)	9.2

. Concentrated algae were diluted to 10.0 gTSS/L with ultrapure water before performing the experiments. This resulting mixture was referred to as the algae suspension.

Unless otherwise stated, all chemicals used were of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd., Shanghai, China, without additional purification. Solutions were prepared with ultrapure water.

2.2. Experiments

Using an algae suspension without any treatment as the control group, ultrasonication (US), ozonation (O₃), and ozonation-ultrasonication hybrid (O₃-US) treatments were conducted to evaluate the cell disruption performance. An ultrasonic processor with a frequency of 20 kHz (Scientz-IID, Ningbo Scientz Biotechnology Co., Ltd., Ningbo, China) and an ozone generator (COM-AD-02, Anseros, Anshan, China) were used to supply ultrasonic treatments and ozone, respectively. For the US treatments, the ultrasonic processor power levels were set at 200 W, 400 W, and 600 W, with programmable on/off cycles (at 2 s intervals). A 100 mL algae suspension in a beaker in a water bath, to keep temperature constant, underwent US at 4 min, 10 min, 20 min, 40 min, 60 min, and 100 min through the working cycle, generating effective ultrasonic times of 2 min, 5 min, 10 min, 20 min, 30 min, and 50 min, respectively.

Ozonation was performed with an ozone generator. Generated ozone was dispersed into a 100 mL reaction bottle containing an algae suspension with a sand core. During the reaction, the aeration flow rate was kept at 0.5 L/min. The ozone dose was obtained at the reaction durations of 2 min, 5 min, 7 min, 10 min, 15 min, 20 min, and 30 min. The ozone concentration in the algal suspension was monitored by the iodometric method [34]. Immediately following each treatment, samples were taken to test proteins, polysaccharides, microcystin-LR (MC-LR), soluble chemical oxygen demand (SCOD), Excitation-Emission Matrix Fluorescence Spectroscopy (EEMs), and DNA. Additionally, samples were prepared for subsequent drying procedures.

For O₃-US, the samples after ozonation with an ozone dose of 0.14 gO₃/gTSS were immediately subjected to ultrasonication to investigate the combined effect of ozone and ultrasound on algal cell disruption. The power density and reaction time used were the same with the US process.

In order to determine the effect of various operating parameters such as the ozone dose, ultrasound intensity, and ultrasound duration on the intracellular matters release, a Box-Behnken design with three factors and three levels was used to design a second-order polynomial model between the response variables and the process parameters and to optimize the optimum condition. Ultrasonic time (A = X₁, min), Sound intensity (B = X₂, W/mL), and Ozone dose (C = X₃, g/gTSS) were the controllable process parameters (independent variables) studied, while DD_SCOD was chosen as the response variables. The coded and actual levels of the process parameters used in the experiment are given in

Table 2. Response surface analysis factors and levels.

Variables	Coded	Max & Min Levels
		−1	0	1
Ultrasonic time (min)	A	10	20	30
Sound intensity (W/mL)	B	2	4	6
Ozone dose (g/gTSS)	C	0.07	0.14	0.21

.

The quadratic response surface model fitted Equation (1):

$$Y={\beta }_0+{\displaystyle \sum _{i=1}^k{\beta }_i{X}_i}+{\displaystyle \sum _{i=1}^k{\beta }_{ii}{X}_{ii}}+{\displaystyle \sum _{i=1}^k{\displaystyle \sum _{j=i+1}^k{\beta }_{ij}{X}_{ij}}}$$

(1)

where Y is the response; X₁, X₂, …, X_k are the process parameters; β₀ is the model constant coefficient; β_i is the linear coefficient; β_ii is the quadratic coefficient; β_ij is the interaction coefficient of variables i and j; and k is equal to the number of the tested factors.

Additionally, Analysis of Variance (ANOVA) was performed to evaluate the adjustment of the model with regard to the regression coefficients, the p-values of the regressions, and the lack of fit. Optimum conditions were established using RSM through three-dimensional graphs of the responses. Further, the optimal conditions were validated and confirmed.

2.3. Analytical Methods

2.3.1. Fluorescence EEMs Spectra

Analysis of dissolved organic matter components used Excitation-Emission Matrix Fluorescence Spectroscopy (EEMs) [35]. After cell disruption, the samples were first pretreated by centrifugation (11,000 rpm, 10 min) following a 0.22 μm membrane filtration to remove excess impurities (algal debris). Then, to avoid internal filtration effects during EEMs testing, all samples were diluted equally, to a sufficient degree (50 times dilution) to ensure that the absorbance at 254 nm of the sample to be tested was <0.3 [16]. The excitation and emission wavelengths were in ranges of 200 nm~400 nm and 250 nm~500 nm, respectively, with f 2- nm intervals.

2.3.2. Scanning Electron Microscopy

Changes in the structure of algal cells after cell disruption by different pretreatments were detected by scanning electron microscopy (SEM). The samples were centrifuged at 5000 rpm for 10 min, and the resulting precipitates were washed with 0.1 M phosphate buffered saline (pH 6.8) after pouring out the supernatant. They were then fixed in 2.5% glutaraldehyde at 4 °C overnight. The precipitate was again washed several times with a phosphate buffer and subsequently dehydrated with a gradient of 30%, 50%, 70%, 80%, 90%, 95% and 100% ethanol. The dehydrated samples were dried overnight in a vacuum-freeze dryer. The dried samples were gold-coated by cathodic spraying. Finally, the samples were observed and photographed under a scanning electron microscope.

2.3.3. Other Analysis Methods

Relevant water quality indicators in this experiment, total solids (TS), total suspended solids (TSS), and volatile suspended solids (VSS), were measured according to the gravimetric analysis specified in Standard Methods for the Examination of Water and Wastewater (SMWW) [36]. The pH value was measured using a portable pH meter (PHBJ-260 F, Shanghai Leici Co., Ltd., Shanghai, China). Two forms of COD, TCOD, and SCOD (after filtration through 0.45 μm) were determined by the semi-automated colorimetric method at 600 nm with a Cary 60 spectrophotometer (Agilent Technologies Inc., Santa Clara, CA, USA). The released protein and polysaccharides were determined by the Bradford method [37] and Anthron method [38]. Released DNA was analyzed by the diphenylamine method [39]. The degree of algal cell disruption was calculated by the following Equation (2):

$$D{D}_{SCOD}\left(\%\right)={\displaystyle \frac{SCO{D}_t-SCO{D}_0}{TCOD-SCO{D}_0}}\times 100\%$$

(2)

where SCOD_t is the SCOD concentration of algal suspension at time t (mg/L), SCOD₀ is the SCOD concentration of the initial algal suspension (mg/L), and TCOD is the total COD concentration of the initial algal suspension (mg/L).

Ozone concentration was determined by the iodometric method [34] and calculated by Equation (3). The determined ozone doses were 0.028 gO₃/gTSS, 0.07 gO₃/gTSS, 0.098 gO₃/gTSS, 0.14 gO₃/gTSS, 0.21 gO₃/gTSS, 0.28 gO₃/gTSS, and 0.42 gO₃/gTSS respectively.

$$d_{O_3}={\displaystyle \frac{Q_g\times t\times C_{O_3}}{1000\times V\times TSS}}$$

(3)

where d_O3 is the ozone dose (g/gTSS), Q_g is the ventilation flow (L/min), t is the reaction time (min), C_O3 is the ozone concentration in the algal suspension at time t (g/m³), V is the volume of algae suspension (L), and TSS is the total suspended solid in the algae suspension (g/L).

MC-LR concentration was determined by a time-resolved fluorescence immunoassay (TRFIA) using an enzyme immunoassay (ELISA) kit developed by the Institute of Hydrobiology at the Chinese Academy of Sciences in Wuhan, China [40]. This method is recommended by the National Standard of the People’s Republic of China GB/T 20466-2006.

In this study, all experiments were conducted in triplicate with data presented as mean values. Response Surface Methodology (RSM) analysis was performed using Design Expert 13 software (Stat-Ease, Inc., Minneapolis, MN, USA), with model adequacy verified through ANOVA including Fisher’s F-test and lack-of-fit. First-order kinetic models for protein and SCOD release were fitted using nonlinear regression analysis in OriginPro 2024 (OriginLab Corporation, Northampton, MA, USA). Error bars in graphical representations indicate standard deviation intervals based on triplicate measurements.

3. Result and Discussion

3.1. Study of Solubilization of Algal Polymers with Different Pretreatment

3.1.1. Release Properties of Proteins and Polysaccharides

Proteins and polysaccharides constitute two fundamental components of algal organic matter, predominantly distributed within algal extracellular polymers (EPS) and the interior of the cells; so, released proteins and polysaccharides can be used as an important indicator of algae cell disruption.

Ozonation pretreatment exhibited a bell-shaped dose-response relationship for protein and polysaccharide release, characterized by a critical optimal dose (0.14 gO₃/gTSS) that maximized intracellular substance release while minimizing subsequent oxidation losses (Figure 1a). At this optimal dose, ozonation released 66.6 mg/gTSS of protein (a 21.5-fold increase over the control group) and 87.5 mg/gTSS of polysaccharide (a 9.6-fold increase over the control group), reflecting an efficient disruption of the EPS and cell wall structures. The bell-shape release is attributed to the release of intracellular polymer substances (IPS) by attacking the cell itself and the subsequent oxidation of the released IPS. Besides, the activity of protein is higher than that of polysaccharides toward ozone, resulting in a significantly faster oxidation rate, which is also demonstrated in the literature [41].

Ultrasonication (US) pretreatment significantly increased the release of both proteins and polysaccharides from algal suspensions over time, gradually reaching a plateau at all settings of ultrasonic intensity (Figure 1b). With increased ultrasonic intensity from 2 to 6 W/mL, proteins elevated in concentration from 109.9 to 170.6 mg/gTSS in 20 min, representing a 35.5-fold to 55.0-fold increase over the untreated control (3.10 mg/gTSS), while polysaccharide concentrations increased from 53.2 mg/gTSS (5.9-fold) to 79.9 mg/gTSS (8.7-fold) in the first 20 min, gradually reaching a maximum in the range of 67.9 mg/gTSS to 87.8 mg/gTSS after the reaction. The dissolution of proteins is more pronounced due to the disruptive effect of ultrasonication, which not only damages the microbial cell membrane but also facilitates the release of proteins from microbial flocs, transforming them from a particulate state into a dissolved form [42].

The algae suspension using ozonation followed by ultrasonication with ozone residue (O₃-US) was studied, considering that ozone-induced cell disruption may be further enhanced by ultrasonication, while ozonation first minimizes the opportunity of intracellular matter mineralization. The results showed a O₃-US treatment significantly amplified the release of proteins and polysaccharides (Figure 1c). Initial exposure to O₃ (0.14 gO₃/gTSS) achieved 66.6 mg/gTSS and 87.5 mg/gTSS of protein and polysaccharide, respectively. Similar to the US process, O₃-US quickly released protein and polysaccharide in the first 20 min and then reached a plateau at all ultrasonic densities. Increasing the intensity to 6 W/mL induced only marginal gains (2.7% for proteins, 1.3% for polysaccharides), despite a 50% increase in energy input, indicating an energy-economic threshold. Compared with individual O₃ and US at an ultrasonic density of 4 W/mL, the protein release was 4.3 times and 1.9 times greater, respectively, while the released polysaccharides were 2.4 times and 3.1 times greater, respectively. Critically, the combined O₃-US yield exceeded the sum of individual treatments by 30% for proteins and 40% for polysaccharides, a statistically significant synergistic effect, confirming non-additive interactions [43]. These phenomena demonstrated that there was a synergistic effect on intracellular release caused by O₃-US treatment. The maximum release can be achieved in a short reaction time and at a lower ultrasonic density. The synergistic effect was induced by two factors. Firstly, initial ozonation makes the algal cell sensitive to the chemical and mechanical attack [44]. Secondly, ultrasonication enhances the mass transfer of ozone and facilitates continuous chemical attacks on the cell structures, thereby amplifying the overall cell disruption effect [25].

3.1.2. SCOD Release Characteristics

The soluble chemical oxygen demand (SCOD) and, consequently, the degree of algal cell disruption (DD_SCOD) in O₃, US, and O₃-US treatments are shown in Figure 2 and Figure 3. Figure 2a shows that the production rate of SCOD follows a rapid stage and subsequent slow stage. The SCOD increased linearly from 64.7 to 328.1 mg/g TSS at the dose in the range of 0.028 gO₃/gTSS to 0.14 gO₃/gTSS, while the SCOD in the solution slowly increased to 416.5 mg/g TSS. The variation of DD_SCOD followed a similar pattern, but the largest DD_SCOD value observed was only 24.4%. The value of specific SCOD production versus the initial ozone concentration (Figure 2b) shows that the ozone utilization was almost the same below the dose of 0.14 gO₃/gTSS, while continuously decreasing with a larger dose, indicating that there was a critical dose for both the algal cell disruption and the following oxidation of the produced SCOD. Specifically, the released intracellular polymer substances compete with ozone with for algal cells, resulting in the oxidation of organic matter [45].

Ultrasonication (US) pretreatment significantly enhanced SCOD release from algae, with sound intensity and treatment duration exhibiting dose-dependent effects (Figure 3a). SCOD concentrations increased rapidly during the initial 20 min of treatment across all tested intensities (2–6 W/mL), indicating immediate cell disruption and IPS release. At 20 min, SCOD levels reached 12.9, 16.0, and 20.1 times higher than controls for 2, 4, and 6 W/mL treatments, respectively, with DD_SCOD of 35.2%, 44.6%, and 56.6%.

Above 20 min, SCOD accumulation slowed markedly; extending treatment to 50 min yielded only 7.9–21.9% incremental gains. In the range of sound intensity from 2 W/mL to 6 W/mL, the degree of disrupting algal cells by cavitation effect and mechanical action was enhanced with the increase of sound intensity (Figure 3b). These results confirm ultrasonication’s efficacy in enhancing soluble organic matter extraction, though optimization of intensity and duration was critical to avoid energy over-consumption [46].

The O₃-US pretreatment markedly improved SCOD release and DD_SCOD, exhibiting clear kinetics and intensity-dependent responses (Figure 3c,d). Initial ozonation (0.14 gO₃/gTSS) achieved 328.1 mg/gTSS SCOD and 18.6% DD_SCOD, indicating preliminary cell wall permeabilization. Subsequent ultrasonication dramatically enhanced these effects, with SCOD increasing by 1.8-, 2.6-, and 2.9-fold (at 2, 4, and 6 W/mL, respectively) within 5 min compared to ozonation alone, corresponding to DD_SCOD values of 36.1%, 53.8%, and 60.9% (Figure 3c). Prolonging treatment to 20 min further elevated SCOD by 45.8% (2 W/mL), 20.0% (4 W/mL), and 12.4% (6 W/mL), with DD_SCOD reaching 55.3%, 65.2%, and 68.8%, respectively, demonstrating progressive cell disruption. Beyond 20 min, SCOD improvements diminished (3.7–9.8% increase at 50 min), with final DD_SCOD plateauing at 60.8%, 69.7%, and 71.5%, likely due to intracellular component retention and cellular debris aggregation [47]. Furthermore, when the ultrasonic density was between 2–4 W/mL within the treatment time of 20 min, the SCOD concentration of O₃-US was 2.5–24.2% higher than the sum of the individual O₃ and US treatments, reflecting the synergistic effect of ozone and ultrasonication, as well as the balance between cell disruption and degradation. This synergy arises from two possible mechanisms: (1) ultrasonication generates transient cavitation bubbles, whose collapse produces hydroxyl radicals (•OH) and extreme localized conditions, disrupting algal cell walls and amplifying oxidative reactions [48]; (2) mechanical shear forces fragment ozone into micro-bubbles, increasing gas-liquid interfacial area and mass transfer efficiency, thereby accelerating oxidative degradation [49].

3.1.3. Dynamic Analysis of Algal Cell Disruption Using a O₃-US Pretreatment

Cell disruption typically follows a first-order kinetic model proposed by Hetherington et al. [50] in their 1971 experiment on protein release from yeast cells. Therefore, a similar first-order kinetic model was used to describe the release behavior of proteins and SCOD from algal cells after disruption during ultrasound treatment, as detailed in the following Equations (4) and (5).

$$1-{\displaystyle \frac{R_m}{R_t}}=\exp \left(-K_{p}t\right)$$

(4)

$$1-{\displaystyle \frac{F_m}{F_t}}=\exp \left(-K_{c}t\right)$$

(5)

In this context, R_m denotes the maximum protein-release value (mg/g TSS), while R_t represents the protein-release value at time t (mg/g TSS). The protein-release rate constant is K_p (min⁻¹). Additionally, F_m signifies the maximum SCOD-release value (mg/g TSS), and F_t indicates the SCOD-release value at time t (mg/g TSS). The SCOD-release rate constant is K_c (min⁻¹).

Figure 4 shows the kinetic fitting curves of protein and SCOD fit well with the data obtained from the O₃-US cell-disruption experiments. Under different operating parameters, the protein-release rate constant K_p and the maximum release value R_m, as well as the SCOD-release rate constant K_c and the maximum release value F_m, are presented in

Table 3. Estimated constant K_p, R_m, and R² statistical values for protein release and estimated constant K_c, F_m, and R² statistical values for SCOD release in O₃-US experiments.

Parameters	Kp /(min−1)	Rm /(mg/g TSS)	R2 Statistical	Kc /(min−1)	Fc /(mg/g TSS)	R2 Statistical
0.14 g O3/g TSS + 2 W/mL	0.06193	211.1	0.993	0.11532	605.4	0.996
0.14 g O3/g TSS + 4 W/mL	0.1383	234.2	0.992	0.25452	741.4	0.995
0.14 g O3/g TSS + 6 W/mL	0.15835	239.2	0.992	0.35847	775.0	0.996

.

Increasing the ultrasonic density in O₃-US pretreatments enhanced both protein and SCOD-release rates, with a maximum achieved at 0.14 g O₃/g TSS combined with 6 W/mL (Table 3). The release rate of protein and SCOD more than doubled when the energy density was increased from 2 W/mL to 4 W/mL (0.06 to 0.13 min⁻¹ and 0.12 to 0.25 min⁻¹). The protein and SCOD maximum release value increased by 10.9% and 22.5%, respectively. Further escalation to 6 W/mL yielded marginal gains: the protein and SCOD maxima rose by only 2.2% and 4.5%, respectively, despite a 50% increase in energy input. This nonlinear efficiency gain underscored an energy-economic threshold near 4 W/mL, beyond which cell disruption efficiency plateaus due to cavitation saturation and microbubble shielding effects.

3.1.4. EEMs Spectrometry Analysis

Excitation-Emission Matrix Fluorescence Spectroscopy (EEMs) was employed to characterize released organics, categorizing them into five regions based on the fluorescence regional integration (FRI) method [51]: aromatic proteins (AP) (region I, Ex/Em: 220–250 nm/280–330 nm; region II, Ex/Em: 200–250 nm/330–380 nm), which are susceptible to microbial decomposition; fulvic acid-like (FA-like) substances (region III, Ex/Em: 200–250 nm/380–500 nm), which are non-biodegradable organics; the soluble microbial by-products (SMPs), including tyrosine-, tryptophan-, and protein-like substances (region IV, Ex/Em: 250–280 nm/200–380 nm) with good biodegradability; and finally the humic acid-like (HA-like) substances (region V, Ex/Em: 250–400 nm/380–500 nm), which are not readily biodegradable. Yu et al. [52] employed a similar EEM zoning pattern to characterize the composition of algal extracellular substances (AESs) and to analyze how ultrasound degradation of AESs can alleviate their impact on algal growth and metabolism.

In untreated algal biomass, the organics were primarily comprised of HA-like substances (region V) and minor FA-like compounds (region III), as shown in Figure 5a. Pretreatments markedly altered EEM profiles. Within an O₃ pretreatment, distinct peaks emerged in regions I, II, and IV in Figure 5b, which indicates a release of tyrosine-like proteins and soluble microbial metabolites into the solution, thereby enhancing the solution’s bioavailability. Concurrently, the amounts of HA-like substances and FA-like substances were degraded. After the US, maximized fluorescence was observed in regions I and II. After O₃-US pretreatment, the fluorescence intensity in regions I, II, and IV increased. Most of these substances were AP and SMPs, which are crucial organic components of EPS and microbial metabolites. These substances have already shown their effectiveness in promoting the biochemical hydrolysis process [53]. Additionally, SMPs, either in the free state or in the form of peptides, are highly biodegradable [54]. The results show that O₃-US treatment enhances the release of IPS and improves bioavailability, which is advantageous for subsequent utilization, such as promoting fermentation processes.

3.2. Optimization of Parameters for Combined O₃-US Treatment

To achieve the maximum release of IPS from the algae suspension, the key factors influencing the degree of disruption of algae cells during the O₃-US treatment were identified. Ultrasonic density, ozone dose, and ultrasonic time were optimized by response-surface analysis.

Based on these variables, a Box-Behnken response-surface optimization experiment with three factors and three levels was designed through Design Expert 13 software. The disruption degree, represented by DD_SCOD, was selected as the response index for parameter optimization. The detailed experimental results are presented in

Table 4. Response-surface analysis design and experimental results.

Process Group	A: Ultrasonic Time /(min)	B: Ultrasonic Density /(W/mL)	C: Ozone Dose /(g/g TSS)	DDSCOD /(%)
1	20	2	0.21	56.0
2	20	6	0.21	69.9
3	30	6	0.14	69.7
4	10	2	0.14	45.1
5	20	4	0.14	65.2
6	20	4	0.14	64.9
7	30	4	0.21	69.2
8	20	4	0.14	65.1
9	20	6	0.07	65.6
10	10	6	0.14	66.2
11	20	4	0.14	65.2
12	30	2	0.14	57.3
13	30	4	0.07	67.2
14	20	2	0.07	49.1
15	20	4	0.14	65.3
16	10	4	0.07	55.6
17	10	4	0.21	66.0

.

A regression was fitted to the experimental data in Table 4, and the following quadratic polynomial regression equation was obtained:

Y = 65.46 + 3.70A + 8.02B + 3.07C − 2.12AB − 1.85AC − 0.6375BC − 0.9017A² − 4.97B² − 0.3317C²

Analysis of Variance (ANOVA) was then employed to assess the validity of this regression equation, and the results are presented in

Table 5. Regression model analysis of variance.

Source	Sum of Squares	df	Mean Square	F-Value	p-Value
Model	844.90	9	93.88	197.35	<0.0001
A	109.82	1	109.82	230.86	<0.0001
B	514.56	1	514.56	1081.72	<0.0001
C	75.52	1	75.52	158.76	<0.0001
AB	18.02	1	18.02	37.88	0.0005
AC	13.73	1	13.73	28.86	0.0010
BC	1.63	1	1.63	3.42	0.1070
A2	3.42	1	3.42	7.20	0.0314
B2	104.08	1	104.08	218.79	<0.0001
C2	0.4634	1	0.4634	0.9742	0.3565
Residual	3.33	7	0.4757
Lack of fit	2.33	3	0.7768	3.11	0.1510
Pure Error	0.9995	4	0.2499
Cor Total	848.23	16

. The F-test, along with its corresponding p-value (significance level), is commonly utilized to evaluate whether the regression model and its factors have a significant impact on the response values [55]. The model F-values exceeded 197.35, and very low p-values (<0.0001) indicated that all models were significant. Additionally, the lack-of-fit term was not significant (F = 3.11, p = 0.1510 > 0.05), showing agreement between the experimental data and any predicted response value. This indicated that the model diagnostics were appropriate. The R² value for the regression model is 0.9945, and the adjusted R² (R_Adj²) is 0.9874, which indicates that the model was well-fitted; only 1.3% of the total variability could not be explained by the model.

Figure 6 shows the 3D response surface and contour plots of the model equations fitted to the data. It shows the relationship between the factors and helps determine the optimum level of each factor for maximum response. Figure 6a illustrates the interaction between ultrasonic times and ultrasonic density on DD_SCOD. The increase in ultrasonic time and ultrasonic density significantly enhanced the DD_SCOD. Under low ultrasonic-density conditions (≤4 W/mL), DD_SCOD exhibited rapid growth, but the rate of increase gradually plateaued as ultrasonic density exceeded 6 W/mL. Experimental results demonstrated that, at a fixed ultrasonic time of 30 min, elevating ultrasonic density from 4 W/mL to 6 W/mL yielded only a marginal 1.9% improvement in DD_SCOD. A similar diminishing return was observed with extended ultrasonic time. To optimize energy efficiency, it is critical to select a lower ultrasonic density (2–4 W/mL) and a shorter ultrasonic time (10–20 min).

The interaction between ultrasonic time and ozone dose exhibited significant synergistic effects on DD_SCOD (Figure 6b). DD_SCOD increased with both parameters, but the rate of increase gradually slowed. This nonlinear behavior likely stems from competitive oxidation mechanisms: excessive ozone reacted with proteins and polysaccharides released during initial disruption, leading to nonproductive degradation rather than further cell disruption.

The contour density (Figure 6c) confirms the ultrasonic density’s primacy over ozone dose, and the effect of cell disruption almost stopped increasing when ultrasonic density exceeded 4 W/mL. Moreover, keeping the ultrasonication conditions the same, ozone dose contributed little to the growth of DD_SCOD after it reached the threshold value of 0.14 gO₃/gTSS. Besides, excessive ultrasonic density could be detrimental to the lifetime of the ultrasonic probe. Therefore, blindly prolonging the ultrasonic time or increasing the ultrasonic density and ozone dose will not cause the algae disruption indefinitely, but rather increase the operation cost of the treatment process. The above results indicate that the effect of the interaction on DD_SCOD was, in descending order: ultrasonic time and ultrasonic density > ultrasonic time and ozone dose > ultrasonic density and ozone dose.

The regression equation was well fitted to the response surface experiment for DD_SCOD, considering the energy consumption of ultrasonication and the degradation of released organic matter by excessive ozone doses. The optimal cell disruption parameters obtained from the response-surface analysis were an ultrasonic time of 23.89 min, ultrasonic density of 3.78 W/mL, and ozone dose of 0.143 gO₃/gTSS, predictively achieving a DD_SCOD of 66.3%. Experimental validation under practical conditions (0.14 gO₃/gTSS, 4 W/mL, 20 min) achieved a 65.2% DD_SCOD, a slight deviation (1.1%) from predictions. Triplicate testing showed <1.2% variability, confirming model robustness for practical application.

3.3. Disruption of Algal Cell Structure by Different Pretreatments

3.3.1. Algae Morphology Before and After Pretreatment

SEM images of algae cells were taken after different pretreatment reactions. Untreated cells (control, Figure 7a) exhibited pristine structural integrity, with smooth surfaces and uniform spherical morphology. O₃ pretreatment (Figure 7b) showed significant shriveling, implying that ozone was able to oxidize algal cell wall structures. In contrast, the integrity of the cell was destroyed after US alone (Figure 7c), with lots of fragments. O₃-US pretreatment (Figure 7d) caused the most significant damage to algal cells, with more severe cell fragmentation, which resulted in a rough and loose structure.

3.3.2. DNA Release Characteristics

Extracellular DNA quantification served as a robust indicator of cellular integrity loss during pretreatment processes. Extracellular DNA in untreated samples measured 0.1 mg/gTSS, which may be due to cells aging and apoptosis, resulting in the release of small amounts of intracellular DNA into the supernatant. Figure 8a shows DNA release exhibited a bell shape, depending on the ozone dose in the O₃ pretreatment. A maximum DNA concentration of 2.2 mg/g TSS occurred at 0.21 gO₃/gTSS, whereas the DNA concentration began to decrease when the ozone dose continued to increase, which was due to the fact that the degradation of DNA was greater than the release at an ozone dose of 0.21 gO₃/ gTSS and above.

Figure 8b demonstrates the change in DNA concentration for the US pretreatment. The DNA concentration of algae treated with different ultrasonic intensities all increased with the ultrasonic time, and the DNA concentration increased to 2.6 mg/gTSS, 4.6 mg/gTSS, and 5.9 mg/gTSS at 5 min of treatment at ultrasonic intensities of 2 W/mL, 4 W/mL, and 6 W/mL, respectively, achieving the values of 5.6 mg/gTSS, 7.6 mg/gTSS, and 8.7 mg/gTSS after 20 min of treatment, respectively. Compared with the control group, the US pretreatment significantly increased the release of DNA, possibly attributed to prolonged shear stress and •OH radical attacks on cell walls [56].

The variation of DNA concentrations in the O₃-US pretreatment is shown in Figure 8c; they increased and then leveled off with the increase of ultrasonic time. Under an ultrasonic density of 2 W/mL, 4 W/mL, and 6 W/mL, the DNA concentrations after 5 min were detected to be 3.8 mg/g TSS, 7.1 mg/g TSS, and 9.0 mg/gTSS, respectively, which showed an improvement of 45.7%, 53.3%, and 51.7%, respectively, compared with US alone, and were 1.7 times, 3.3 times, and 4.1 times that of O₃ pretreatment alone, respectively. Continuing the treatment until 20 min, the DNA concentration increased to 10.6 mg/gTSS, 13.6 mg/gTSS, and 14.0 mg/gTSS, respectively. The results indicate that O₃-US pretreatment intensifies the disruption of cyanobacterial cell integrity. Under the synergistic effect of ultrasonication, ozone’s oxidative capacity is markedly enhanced, generating a greater abundance of highly reactive free radicals capable of penetrating cellular membranes. This process induces oxidative stress within the cells, leading to structural damage and the extensive release of intracellular DNA due to compromised cellular protection.

3.4. Other Risk Factors: Behavior of MC-LR in Different Pretreatment Processes

Microcystin-LR (MC-LR), a hepatotoxic cyclic peptide primarily localized within algal cells, requires careful monitoring during biomass processing. Figure 9 demonstrates that US alone induced structural disintegration of algal cells, triggering substantial release of intracellular constituents, including MC-LR, into the aqueous phase. Quantitative analysis revealed a 60.0% increase in extracellular MC-LR concentration compared to the untreated control, consistent with previous reports documenting ultrasonication-enhanced liberation of toxins [57]. Ozonation (0.14 g O₃/g TSS) achieved a 95.2% MC-LR removal relative to the control, attributed to ozone’s strong oxidative capacity toward unsaturated bonds in organic compounds [58]. The O₃-US pretreatment exhibited a synergistic efficacy, exhibiting a 94.5% MC-LR removal and 96.6% reduction relative to US-only treatments. These findings conclusively establish the dual advantage of O₃-US pretreatment, as simultaneous cell disruption and chemical detoxification.

4. Conclusions

This study demonstrates that the synergistic ozone-ultrasonication (O₃-US) pretreatment effectively overcomes the dual challenges of algal biomass valorization: recalcitrant cell wall barriers and residual cyanotoxin risks. Two key advancements were achieved:

• Synergistic disruption mechanism: The integration of ozone oxidation targeting EPS and cell wall polymers with ultrasonication treatment—inducing cavitation-driven mechanical disruption—significantly enhances the release of intracellular resources. Optimized conditions (0.14 gO₃/gTSS, 4 W/mL, 20 min) achieved a protein content of 289.2 mg/g TSS, representing 4.3-fold and 1.9-fold increases compared to standalone O₃ and US treatment, respectively. Additionally, polysaccharide content reached 87.5 mg/g TSS, corresponding to 2.4-fold and 3.1-fold enhancements over singular treatments, achieving 65.2% cell disruption efficiency. Fluorescence EEM spectroscopy further confirmed that biodegradable organic compounds predominate post-treatment, ensuring compatibility with subsequent downstream processes.
• Detoxification and energy efficiency: The synchronized degradation of MC-LR achieved a removal efficiency of 94.5%, representing a 96.6% reduction in risk compared to US alone, thereby addressing biosafety concerns. Additionally, this integrated approach reduced ultrasonic intensity by 33.3% and lowered treatment duration by 60% relative to systems employing US-only treatments.

The O₃-US pretreatment system emerges as an energy-efficient and effective approach for algal biomass processing, simultaneously achieving cell disruption and toxin degradation. Although laboratory-scale results are encouraging, scaling considerations must address: (1) ozone-production economics through dose optimization, and (2) equipment maintenance via improved system design and cleaning protocols. The method’s synergistic potential with conventional anaerobic digestion processes enhances both biogas production and operational safety, aligning with circular bioeconomy principles. Key research priorities include: pilot-scale verification, comprehensive techno-economic analysis, downstream process integration, and fundamental studies of ozone-ultrasound synergies. This integrated pretreatment strategy represents a sustainable platform for algal biorefining, combining operational efficiency with environmental safety.