Enhancing the Efficiency of Anaerobic Digestion Treatment of Tetracycline-Containing Wastewater

Abstract

To improve the efficiency of AD for tetracycline-containing wastewater, this study systematically investigated the effects of ZVI and PAC on pollutant removal, VFA metabolism, biogas production, and sludge physicochemical characteristics. RSM was employed to optimize the co-dosing conditions of ZVI and PAC. The results suggested that ZVI and PAC could significantly facilitate COD degradation, and the optimal dosages for COD removal were 1000 mg/L ZVI and 2000 mg/L PAC with a removal efficiency of 61.03% and 56.9%, respectively. Both additives effectively accelerated the catabolism of typical VFAs, thereby mitigating the accumulation of intermediate metabolites that may cause AD system instability. In terms of biogas production, 1000 mg/L ZVI and 2000 mg/L PAC enhanced methane yield by 55.9% and 35.0% compared to the control group, with ZVI exhibiting a more prominent enhancement effect. Mechanistic analysis suggested that ZVI and PAC reinforced the AD process through multiple pathways: enhancing the electrical conductivity of the AD system, possibly facilitating the electron transfer pathways, and stimulating the secretion of EPSs by anaerobic microbes. RSM optimization yielded the optimal co-dosing parameters: 1000 mg/L ZVI and 1200 mg/L PAC. Under these conditions, the methane yield was increased by 71.18% relative to the control group, and the model validation accuracy reached 97.94%. This study provides a viable technical strategy and theoretical basis for enhancing the efficiency of anaerobic treatment of tetracycline-containing wastewater.

1. Introduction

Antibiotics, as pivotal therapeutic agents for inhibiting bacterial growth or eradicating bacterial pathogens, are extensively applied in the fields of clinical medicine, agriculture, and aquaculture [1]. The global total production capacity of antibiotic active pharmaceutical ingredients reached approximately 286,000 tons in 2023. Globally, the consumption of antibiotics has exhibited a continuous upward trend, with a cumulative increase of 16.3% during the period from 2016 to 2023 [2]. Notably, parts of antibiotics are discharged into the environment in their biologically active forms, thereby giving rise to a series of ecological risks. The ecological and environmental impacts of antibiotic residues are substantial: they not only alter the composition and structure of microbial communities in aquatic and terrestrial ecosystems but also interfere with critical biogeochemical processes such as the nitrogen cycle [3]. More importantly, antibiotic residues can induce the horizontal transfer of ARGs, which facilitates the dissemination of ARB and poses a potential threat of a “silent epidemic”. An antibiotic pollution distribution map, constructed based on the usage and discharge data of various antibiotics across 58 river basins in China, reveals that economically developed regions, including Guangdong, Jiangsu, Zhejiang, and Hebei provinces, are confronted with severe antibiotic pollution [4]. Long-term exposure to environmental antibiotic residues can accelerate the proliferation and dissemination of ARB and ARGs in the environment, induce multi-drug resistance in pathogenic bacteria, and consequently pose a significant threat to both human health and ecological security [5].

Antibiotic wastewater poses significant challenges in treatment processes, primarily attributed to its complex composition, high toxicity, and poor biodegradability, as well as the presence of antibiotic residues and ARGs [6]. Existing treatment technologies are predominantly categorized into three types, physical, chemical and biological processes, along with their combined systems [3]. Among these, anaerobic treatment technology exhibits distinct and prominent advantages in the treatment of such wastewater. It is well-suited to the characteristics of antibiotic wastewater, such as high COD and high organic matter concentration. Additionally, it enables energy recovery, features low operational costs, and demonstrates strong environmental adaptability [7].

Anaerobic digestion (AD) is a sequential microbial degradation process consisting of hydrolysis, acidogenesis, acetogenesis, and methanogenesis, which enables resourceful utilization of organic wastewater. Each stage exhibits distinct sensitivity to antibiotic inhibition. Tetracycline exerts severe inhibitory effects on AD systems by disrupting microbial community structure and metabolic pathways. It inhibits microbial protein synthesis, reduces the abundance of functional microorganisms such as acetogenic bacteria and methanogens, and impairs VFA catabolism [8]. Excessive accumulation of intermediate metabolites like propionate further suppresses subsequent methanation, significantly reducing methane yield and pollutant removal efficiency. Direct interspecies electron transfer (DIET) has been proven as an efficient pathway to mitigate antibiotic-induced inhibition. Different from traditional hydrogen-mediated electron transfer, DIET relies on conductive materials, microbial c-type cytochromes and conductive pili to realize direct electron exchange between syntrophic bacteria and methanogens [9]. It effectively improves electron transfer efficiency, enhances microbial extracellular polymeric substance secretion and system conductivity, and repairs inhibited metabolic functions. Facilitated DIET can accelerate VFA degradation, relieve metabolite accumulation, and remarkably strengthen the stability and methane production performance of tetracycline-containing wastewater anaerobic digestion systems [10].

Conductive mediators can significantly enhance the treatment efficiency of anaerobic systems. Currently, iron-based and carbon-based materials are the most extensively studied conductive mediators for improving the operational efficiency and microbial activity of anaerobic systems [11]. Iron-based materials employed for enhancing anaerobic systems include ZVI, magnetite, iron oxides, iron powder, and iron filings [12]. The particle size of the utilized iron-based materials is generally less than 200 nm, with the dosage typically ranging from several mg/L to several hundred mg/L [13]. Substrates for anaerobic fermentation encompass wastewater, excess sludge, municipal solid waste, and cow dung, among others. The enhancement effect on methane production varies from 4% to 105%, which is primarily associated with mediator type, dosage, substrate properties, and anaerobic digestion conditions [14]. As a reducing agent, ZVI can facilitate the establishment of an anaerobic microenvironment, improve the operational performance of anaerobic processes, and alter the dominant microbial community structure of the anaerobic system. Carbon-based materials mainly utilized for enhancing DIET-driven methane production in anaerobic systems include GAC, PAC, carbon cloth, carbon nanotubes, biochar, graphite, and graphene [15]. These materials all possess high electrical conductivity, favorable biocompatibility, and excellent chemical stability [16]. The particle size of the adopted carbon-based materials generally ranges from several μm to several tens of mm, and the dosage varies from several mg/L to 10 g/L. The enhancement effect on methane production is in the range of 11.7% to 70.6%, which is related to the dosage, type, and surface morphology of carbon-based mediators [17]. Carbon-based materials facilitate methane generation chiefly by elevating DIET efficiency among microbial populations. The removal rate of tetracycline in the anaerobic digestion system ranges from 14.8% to 90%, while iron-based and carbon-based materials can further enhance the removal efficiency by means of adsorption and promoting interspecies electron transfer [8]. Though related studies have made great progress, many key problems still await solutions. Iron and carbon materials tend to aggregate and fit poorly with anaerobic systems. The optimal dosage of iron–carbon composites differs greatly with substrate characteristics [18]. The internal mechanisms driving antibiotic degradation and methane improvement remain unclear. In addition, combined effects and relevant principles of various conductive mediators in antibiotic wastewater treatment also need further systematic research. This study innovatively explored the enhancement effect of ZVI and PAC on anaerobic digestion of tetracycline wastewater, and suggested its mechanism for enhancing pollutant removal and methane production.

The objectives of the present study were to (1) compare the enhanced efficacy of PAC and ZVI in the anaerobic digestion process of tetracycline-containing wastewater; (2) explore the intrinsic mechanisms underlying the promotion of tetracycline anaerobic degradation and methane production by PAC and ZVI; (3) clarify the combined effect of PAC-ZVI coupling on enhancing the treatment performance of tetracycline-containing wastewater. The research results are expected to provide a theoretical basis and practical operational guidance for the anaerobic treatment of antibiotic-containing wastewater.

2. Results and Discussion

2.1. Pollutant Removal and Transformation

2.1.1. COD and Tetracycline Removal

As illustrated in Figure 1A,B, the system COD concentration continuously decreased and stabilized at approximately 80 h. All groups showed slow and similar COD degradation rates within the initial 6 h, as anaerobic microorganisms underwent environmental adaptation under tetracycline stress, with limited organic matter metabolism prioritized for microbial physiological recovery rather than substrate degradation. From 6 h to 36 h, ZVI and PAC supplementation significantly promoted COD degradation, with distinct efficiency differences between the control and experimental groups due to enhanced microbial metabolic activity and mediator-mediated electron transfer. The COD degradation slowed down from 36 h to 64 h and subsequently plateaued, which was attributed to the low inoculated sludge concentration that caused a kinetic bottleneck and left refractory organic matters in the system. ZVI exhibited a dose-dependent promotion effect on COD removal with an optimal dosage of 1000 mg/L, achieving a removal rate of 61.03% (11.23% higher than the control). Excessive ZVI might weaken the promotion effect because nanoparticle aggregation hindered microbial substrate contact and induced microbial toxicity. In contrast, PAC’s promotional effect continuously improved with increasing dosage, reaching a 56.9% removal rate at 2000 mg/L. With superior biocompatibility and no toxic effects, PAC sustained efficient inter-microbial electron transfer. Both mediators served as microbial carriers; additionally, ZVI-released Fe²⁺ further boosted microbial metabolic activity [19].

As shown in Figure 1C,D, tetracycline degraded much faster than COD and stabilized at 48 h. Rapid initial tetracycline removal within 12 h was mainly driven by sludge EPSs and mediator adsorption, followed by slow biodegradation until stabilization, with a final removal efficiency over 94%. All groups maintained high tetracycline removal rates with insignificant dosage-dependent differences. The rapid tetracycline elimination might be due to selective microbial inhibition: tetracycline primarily suppresses acetogens and methanogens rather than hydrolytic bacteria that dominate tetracycline degradation [8,20]. Overall, sludge intrinsic adsorption dominated tetracycline removal, and ZVI/PAC exerted limited auxiliary effects.

2.1.2. VFA Production

VFAs are core intermediate metabolites that determine the stability and efficiency of anaerobic digestion. This study investigated the temporal evolution of acetic and butyric acids under ZVI and PAC regulation as shown in Figure 2A–D. The two typical VFAs followed a consistent four-stage variation trend of rapid accumulation, rapid degradation, slow degradation and final stabilization, with the peak acetic acid concentration ranging from 1700 to 1800 mg/L across treatments. Under optimal dosages (1000 mg/L ZVI and 2000 mg/L PAC), both mediators effectively mitigated VFA accumulation. Specifically, ZVI and PAC reduced acetic acid content by 24.2% and 26.1%, and decreased butyric acid concentration by 15.4% and 26.8%, respectively. Consistent with previous research, ZVI improved VFA degradation and methanogenesis by enhancing microbial interspecific electron transfer [21]. The digestion system exhibited an acetic acid-dominated VFA metabolic pattern, with negligible propionic and valeric acid accumulation, which was favorable for methanation. Notably, the optimal dosages and promoting effects of ZVI and PAC on VFA metabolism were highly consistent with their COD removal performance. This demonstrates that exogenous mediators achieve systematic enhancement of anaerobic digestion by simultaneously promoting organic matter degradation and intermediate VFA metabolism, validating the reliability of the experimental findings.

2.1.3. Degradation Byproducts of Tetracycline

Figure 3A compares the liquid chromatograms of raw tetracycline wastewater and anaerobic effluent. The weakened tetracycline characteristic peak in effluent suggested effective antibiotic removal, while two new chromatographic peaks at retention times of 66 s and 80 s were detected and might be putative transformation products (Substance A and Substance B). Due to the failure of baseline separation under current detection conditions, the peak height was adopted for semi-quantitative analysis of the two intermediates.

Figure 3B,C illustrate their dynamic variations throughout the reaction. Substance A in all groups consistently increased to the peak at 12 h and then gradually degraded. The ZVI group possessed the lowest peak abundance (1694) and final residue (74), followed by the PAC group, demonstrating that both mediators effectively promoted Substance A transformation, with ZVI achieving a better improvement effect. Substance B remained stable in the control group, while PAC and ZVI supplementation continuously reduced Substance B content from the early reaction stage to the end. Overall, ZVI and PAC exhibited stronger promotion efficiency for Substance B degradation than for Substance A, and ZVI performed better than PAC at identical dosages. Mechanistically, both intermediates possess low ORP characteristics. PAC optimized the anaerobic environment by reducing system ORP to facilitate biodegradation, while ZVI further strengthened ORP regulation and provided surface catalytic effects, thereby achieving superior degradation enhancement performance [22].

2.2. Biogas Production and Composition

The modified Gompertz equation was used for nonlinear fitting and kinetic analysis of gas production data in this study. The cumulative gas production curves of control, ZVI and PAC groups presented consistent staged variations in Figure 4A,B. All groups showed low gas production within the initial 6 h due to microbial environmental acclimation with weak organic degradation capacity, which matched the trend of COD degradation. The gas production rate peaked significantly from 6 h to 24 h with distinct group differences, as exogenous mediators effectively promoted anaerobic digestion of tetracycline wastewater. The gas production gradually slowed down from 24 h to 60 h, while cumulative gas volume still increased slightly, and the gap between experimental and control groups gradually narrowed. The gas production finally stabilized after 60 h with negligible incremental output. Consistent with COD degradation results, ZVI exhibited a concentration-dependent promotion effect with an optimal dosage of 1000 mg/L, improving interspecific electron syntrophy to accelerate VFA conversion and gas production, achieving a cumulative gas output of 136 mL (14.3% higher than the control). In contrast, PAC’s promotional effect increased continuously with dosage, reaching the maximum gas production of 145 mL at 2000 mg/L, which was 21.8% higher than the control. Overall, 2000 mg/L PAC and 1000 mg/L ZVI were the optimal dosages for enhancing the gas production performance of tetracycline wastewater anaerobic digestion.

The modified Gompertz equation was applied to fit methane production data for kinetic parameter analysis in Figure 4C,D. Methane generation followed four sequential stages: lag phase (0–6 h), rapid production phase (6–36 h), slow production phase (36–60 h), and stationary phase (60–84 h). Unlike total gas production, methane generation presented an obvious delay, since early-produced gas was dominated by H₂ and CO₂, which were further utilized as substrates for subsequent methanogenesis. Both PAC and ZVI effectively promoted cumulative methane production in a dose-dependent manner. At optimal dosages, PAC (2000 mg/L) and ZVI (1000 mg/L) achieved cumulative methane yields of 49 mL and 56.6 mL, corresponding to 172.2 and 185.5 mL CH₄/g COD removed, increasing by 35.0% and 55.9% relative to the control, respectively. ZVI exhibited a superior enhancement performance. Compared with total gas generation, the mediators exerted a more prominent promoting effect on methane accumulation, indicating that exogenous additives altered methanogenic metabolic pathways and facilitated the hydrogen-driven reduction of CO₂ into CH₄ [23]. The prominent promotional effect can be attributed to the long-term inhibition of anaerobic sludge by tetracycline (TC) and its degradation intermediates in wastewater, which inherently weakened the methanogenic activity of microbial consortia. The introduction of PAC and ZVI alleviated the toxic stress of TC and its metabolites on methanogens mainly via adsorption.

The modified Gompertz model was employed to kinetically fit total gas and methane production data (

Table 1. Fitting results of gas production process with Gompertz equation.

System	Total Gas					Methane
	Pmax (mL/gVSS)	Rmax mL/(gVSS.h)	λ/h	R2	RMSE (mL/gVSS)	Pmax (mL/gVSS)	Rmax (mL/gVSS.h)	λ/h	R2	RMSE (mL/gVSS)
Control	287.5	15.6	4.36	0.99	2.1	91.5	3.05	7.36	0.99	0.6
2000 mg/L PAC	346.1 (+20.4%)	19.3 (+23.7%)	3.23	0.98	3.3	122.8 (+34.2%)	4.02 (+24.1%)	6.30	0.99	1.2
1000 mg/L ZVI	331.2 (+15.2%)	25.2 (+61.5%)	3.12	0.99	5.4	138.8 (+51.7%)	4.70 (+54.1%)	5.03	0.98	1.7

). The fitting results exhibited high reliability, with the coefficient of determination (R²) above 0.97, root-mean-square error (RMSE) lower than 10 mL/gVSS, 95% parameter confidence intervals, and significant p-values (p < 0.05), demonstrating that the established model well matched the experimental data. For the control group, the maximum total gas and methane yields were 287.5 mL/gVSS and 91.5 mL/gVSS, with corresponding maximum production rates of 15.6 mL/(gVSS·h) and 3.05 mL/(gVSS·h), respectively. The supplementation of 2000 mg/L PAC effectively improved digestion performance, increasing the maximum total gas and methane yields to 346.1 mL/gVSS and 122.8 mL/gVSS (20.4% and 34.2% higher than the control), while the maximum production rates were elevated to 19.3 mL/(gVSS·h) and 4.02 mL/(gVSS·h), corresponding to 23.7% and 24.1% increments. Comparatively, 1000 mg/L ZVI addition achieved superior enhancement effects. The maximum total gas and methane yields reached 331.2 mL/gVSS and 138.8 mL/gVSS, which were 15.2% and 51.7% higher than those of the control group. Meanwhile, ZVI supplementation increased the maximum total gas and methane production rates to 25.2 mL/(gVSS·h) and 4.7 mL/(gVSS·h), with prominent increments of 61.5% and 54.1%, respectively. These quantitative results confirm that ZVI possesses a more robust capacity to facilitate gas production and methane generation than PAC. The methane promotion efficiencies of ZVI and PAC in this study fell within the reported ranges of 4–105% and 11.7–70.6%, consistent with previous findings and verifying the reliability of the results [14,17]. Tetracycline toxicity inherently inhibits the methanogenic potential of inoculated sludge. By means of adsorption and enhancement of microbial interspecies electron transfer, ZVI and PAC can mitigate such inhibitory effects and consequently facilitate methane generation. Additionally, both mediators effectively shortened the lag phases of gas and methane generation. PAC (2000 mg/L) reduced the lag periods from 4.36 h and 7.36 h to 3.23 h and 4.02 h, whereas ZVI (1000 mg/L) decreased them to 3.12 h and 5.03 h. As efficient substrate carriers, PAC and ZVI facilitate continuous contact and interaction between microorganisms and substrates, effectively accelerating the anaerobic digestion response [24].

2.3. Sludge Characteristics and Strengthening Mechanism

As illustrated in Figure 5A, the microbial community of the control group was dominated by bacilli exhibiting a bamboo-like morphological structure, which might be Methanobacterium on the basis of its characteristic morphology. This archaeal genus is known to mediate methanogenesis through the acetoclastic pathway, with acetic acid serving as the primary substrate. Notably, only a minor fraction of cocci was detected within this community. Concomitantly, distinct intercellular gaps were observed among the microbial cells. Under such a spatial configuration, IET is presumably accomplished via either H₂/acetic acid as metabolic electron carriers or extracellularly secreted electron shuttle substances. Owing to these indirect electron transfer mechanisms, the efficiency of intercellular electron exchange in the control group remained relatively low.

PAC is an amorphous carbonaceous material with heterogeneous morphologies including blocky, sheet-like, and spherical structures, exhibiting a wide particle size distribution ranging from nanoscale to several micrometers (Figure 5B). In PAC-amended systems, nanoscale PAC particles readily adhere to or even encapsulate bacterial cells and fill intercellular voids. Such structural characteristics enable PAC to serve as a conductive bridge for physically connecting adjacent microbial cells. Small-sized PAC fractions are speculated to possess superior capacity for facilitating interspecies electron transfer relative to larger particles. Furthermore, the macroporous structure of PAC can link spatially isolated microbial aggregates, integrating dispersed anaerobic sludge into a unified metabolic system. This structural optimization strengthens metabolic interactions among functional microorganisms, thereby improving the biodegradation efficiency of target pollutants.

For the ZVI-supplemented group (Figure 5C,D), microscopic observations suggested that the overwhelming majority of bacterial cells were coated and encapsulated by spherical ZVI particles, with only a small proportion of cells remaining exposed. The intimate contact between ZVI and microbial cells drastically reduces the intercellular distance, thereby favoring the possible enhancement of electron transfer pathways [13]. Furthermore, a morphological transformation of partial ZVI particles was observed, with the original spherical structure transitioning to a lanceolate shape. A plausible mechanism underlying this morphological change involves the oxidation of ZVI to Fe²⁺, which subsequently reacts with dissolved CO₂ in the aqueous matrix to form siderite (FeCO₃) precipitates.

As shown in Figure 6A, the FTIR spectra of anaerobic sludge from the control and experimental groups presented identical major characteristic peak positions, indicating that mediator addition did not change the basic functional group composition of sludge. Both mediator-amended groups exhibited higher absorbance intensity at 3300 cm⁻¹ than the control. Mediators might facilitate microbial interspecific electron transfer, enhance the metabolic activity of functional microbes, and promote the secretion of protein and fulvic acid-dominated extracellular polymeric substances. The ZVI group achieved the highest absorbance at 3300 cm⁻¹ (142% of the control), consistent with its optimal anaerobic digestion performance for tetracycline wastewater. By contrast, mediator addition might reduce the absorbance at 1450 cm⁻¹ by stimulating hydrogenotrophic methanogens to consume carbonate substances for methanogenesis. For iron-based groups, the absorbance reduction was not positively correlated with methane yield, because slowly released Fe²⁺ reacted with dissolved CO₂ to form siderite precipitates, which offset the absorbance decline. The PAC group had the lowest 1450 cm⁻¹ absorbance (61.8% of the control), which agreed with previous analytical results.

Figure 6B presents the electrical conductivity of the exogenous mediator–anaerobic sludge composites. In comparison with the control group, all experimental groups exhibited significantly higher conductivity values, indicating that the addition of exogenous mediators possibly enhances electron transfer pathways, thereby improving the efficiency of tetracycline wastewater anaerobic digestion. The control group had a conductivity of 3.77 μS/cm, whereas the PAC- and ZVI-supplemented groups displayed conductivities of 30.02 μS/cm and 684.93 μS/cm, respectively—representing 7-fold and 180-fold increases relative to the control. This substantial conductivity enhancement can be ascribed to the excellent intrinsic electrical conductivity of PAC and ZVI, which directly elevates the conductive properties of the mediator–sludge composite system. GAC/NZVI exposure to anaerobic sludge significantly enhanced its conductivity to 498.8 mS/cm, and enhanced electron transfer activity by more than 55%, effectively accelerating the process of organic matter degradation and methane production, similarly to this study [25].

Polysaccharides (PSs) and proteins (PNs) are pivotal components of EPSs, accounting for 70–80% of the total EPS content. Thus, variations in the concentrations of these two components can be employed as key indicators to characterize the dynamics of EPS secretion. The temporal changes in EPS content across different experimental systems are illustrated in Figure 6C,D. The control group had LB-EPS and TB-EPS concentrations of 74 mg/g MLVSS and 227 mg/g MLVSS, respectively. Among all experimental groups, the ZVI-supplemented system exhibited the most pronounced increase in EPS secretion, with LB-EPS and TB-EPS concentrations reaching 165 mg/g MLVSS and 595 mg/g MLVSS—corresponding to 123.3% and 161.6% increases relative to the control. This finding indicates that ZVI addition significantly stimulates EPS secretion by anaerobic microorganisms, thereby enhancing the capacity for IET among microbiota and ultimately improving the anaerobic digestion efficiency of tetracycline wastewater. ZVI and PAC could stimulate the microbial cell membranes moderately, promoting the synthesis of PN and PS, increasing the total amount of EPSs and the ratio of PN/PS, enhancing the flocculation property and structural stability of the sludge. Under an appropriate dosage, the EPS concentration of the treatment group is 30–60% higher than that of the control group, and the degree of sludge granulation is also higher [26]. In the PAC-supplemented group, LB-EPS and TB-EPS concentrations increased to 131 mg/g MLVSS and 410 mg/g MLVSS, representing 75.8% and 80.5% increases relative to the control. The more potent effect of ZVI than PAC in promoting EPS secretion aligns with the superior tetracycline wastewater digestion performance of iron-based mediator systems, and is also consistent with the elevated relative absorbance intensity at 3300 cm⁻¹ in the FTIR spectra of the corresponding composites. These results collectively demonstrate that the addition of an optimal dose of mediators can effectively stimulate microbial EPS secretion.

The CV curves of supernatants from different experimental systems are presented in Figure 6E,F. In comparison with the control group, the mediator-supplemented systems exhibited more distinct oxidation–reduction peaks, indicating enhanced redox activity. The oxidation peak potentials of the control, PAC-supplemented, and ZVI-supplemented groups were 0.18 V, 0.22 V and 0.25 V, respectively, while their reduction peak potentials were 0.48 V, 0.57 V and 0.49 V. Correspondingly, the oxidation peak currents were −0.42 μA, −0.57 μA and −0.67 μA, and the reduction peak currents were 0.75 μA, 0.97 μA and 1.34 μA. The higher peak currents observed in the experimental groups relative to the control indicate that the mediator–sludge composite systems possess stronger redox activity, which is attributed to the presence of a greater abundance of redox-active substances in the aqueous phase. This phenomenon may be explained by the fact that exogenous mediators induce the secretion of soluble redox mediators (i.e., electron shuttles) by microorganisms, thereby accelerating extracellular indirect electron transfer and enhancing the anaerobic digestion efficacy of tetracycline-containing wastewater [27].

2.4. Optimization of Composite Conditions via Response Surface Methodology

As the dosages of ZVI and PAC increase, the cumulative CH₄ production also increases. However, the promoting effect of ZVI dosage on the cumulative CH₄ production is more significant than that of PAC dosage. Using Design Expert 8.0.5 to perform multiple regression fitting on the data in

Table 2. Response surface optimization of methane production experimental scheme and results.

Experimental Number	Exogenous Vector Content (mg/L)		Cumulative Methane Production (mL)
	PAC	ZVI
1	1000	1000	62.55
2	2000	500	61.73
3	1000	500	57.73
4	2000	500	58.95
5	1000	0	49.54
6	0	1000	56.57
7	1000	1000	69.41
8	0	500	51.73
9	0	0	36.3
10	0	500	45
11	1000	500	58.33
12	2000	0	49
13	1000	500	55.85
14	1000	0	45
15	1000	500	57.96
16	2000	1000	55.73

, the multiple regression equation for cumulative CH₄ production (Y) with respect to PAC (A) and ZVI (B) was obtained: Y = 34.470 + 0.017 × A + 0.035 × B − 6.772 × 10⁻⁶ × AB − 4.560 × 10⁻⁶ × A² − 1.192 × 10⁻⁵ × B².

The regression model is highly significant while the test for the non-fitting term is not significant, indicating that the fitted model is appropriate. The model has been successfully fitted and no further higher-order terms need to be introduced. The p-values of the corresponding terms in the regression model can reflect the significance of their influence on the experimental response values (

Table 3. Regression model variance analysis.

Sources of Variation	Sum of Squares	Degree of Freedom	Mean Square	F Value	p-Value	Significance
Model	869.58	5	173.92	13.27	0.0004	Significant
A-PAC	186.12	1	186.12	14.2	0.0037	Significant
B-ZVI	518.89	1	518.89	39.59	<0.0001	Significant
A × B	45.86	1	45.86	3.5	0.0909	Not significant
A2	83.18	1	83.18	6.35	0.0304	Significant
B2	35.53	1	35.53	2.71	0.1307	Not significant
Surplus	131.05	10	13.11
Lack of Fit	69.63	3	23.21	2.65	0.1305	Not significant
Pure Error	61.42	7	8.77

When p < 0.05, it indicates a significant difference; when p < 0.01, it indicates an extremely significant difference.

). Thus, it can be known that in this experiment, the first-order terms ZVI and PAC are significant, the second-order term PAC² is significant, ZVI² is not significant, and the interaction term ZVI × PAC is also not significant. The absolute value of the regression coefficient of the first-order term in the regression model, ZVI, is greater than that of PAC, indicating that the influence of ZVI concentration on methane production is greater than that of PAC concentration on methane production.

On the basis of the derived regression equation, a three-dimensional response surface contour plot (Figure 7) was constructed, which visually delineates the functional relationship between cumulative CH₄ production and the concentrations of ZVI and PAC. This plot explicitly reflects the interactive effects exerted by ZVI and PAC concentrations on the target response variable. Notably, cumulative CH₄ production exhibited a positive correlation with the increasing dosages of both ZVI and PAC, whereas the enhancing effect of ZVI dosage was found to be more pronounced than that of PAC.

Design-Expert software (V8.0.6.1) was utilized for statistical analysis and numerical computation, which yielded the optimal mediator dosage combination for maximizing CH₄ production as follows: PAC = 1157.67 mg/L and ZVI = 1000 mg/L. Nevertheless, to streamline operational protocols in practical anaerobic reactor applications while satisfying the statistical prerequisites of ANOVA, the final optimized mediator dosage combination was adjusted to PAC = 1200 mg/L and ZVI = 1000 mg/L. Under these modified optimal conditions, the regression model predicted a cumulative CH₄ production of 63.45 mL. To further validate the predictive accuracy of the regression model, triplicate validation experiments were executed using the optimized ZVI and PAC dosages. The results suggested a satisfactory consistency between the predicted and experimentally determined cumulative CH₄ yields, with a fitting accuracy of 97.94%. Specifically, the average cumulative CH₄ yield across the three replicate experiments reached 62.14 mL, which corroborates the reliability and robustness of the constructed regression model. In comparison with the control group, the cumulative CH₄ production in the system supplemented with the optimized ZVI-PAC combination was significantly elevated by 71.18%. This finding confirms that the application of ZVI and PAC at the optimized dosages constitutes an efficient and rational strategy for enhancing methanogenic activity in anaerobic digestion systems.

3. Materials and Methods

3.1. Experimental Materials and Water Quality

The PAC and ZVI employed in this experiment were of analytical grade, procured from Tianjin Standard Chemical Reagent Company (Tianjin, China) and Dekedaojin Co., Ltd. (Beijing, China)., respectively. The inoculated sludge consisted entirely of anaerobic granular sludge derived from an efficiently operating EGSB reactor, which was dedicated to the treatment of tetracycline-containing wastewater and had an MLVSS of 22.38 g/L and MLVSS/MLSS of 0.63. In accordance with the principal composition of real-world tetracycline wastewater, synthetic wastewater was utilized for laboratory-scale experiments. Saccharose was used as a carbon source with the concentration of 4.46 g/L with an initial COD concentration of approximately 5000 mg/L [26]. In addition, 1.98 g NH₄Cl and 0.5 g K₂HPO₄ were added (per liter) to supply necessary nutrients for microbial growth with a carbon-to-nitrogen-to-phosphorus ratio of 200:5:1. A total of 0.105 g of tetracycline was introduced to the wastewater to serve as the antibiotic source at a concentration of 105 mg/L. Additionally, 1 mL of trace element solution [5 mL/L 37% HCl; 0.5 g/L of H₃BO₃, CoCl₂.6H₂O, AlCl₃.6H₂O, NiCl.6H₂O, ZnCl₂, (NH₄)₆Mo₇O₂₄.4H₂O and CuSO₄.5H₂O; 1 g/L NaSeO₃.5H₂O; 1.5 g/L FeCl₃.6H₂O; 5 g/L MnCl₂.4H₂O] was added per liter of wastewater to provide essential nutrients for microbial growth [27]. The pH of synthetic wastewater was adjusted to 8.0 ± 0.3 using Na₂CO₃ and HCl solution.

3.2. Experimental Setup and Procedures

The batch mode experimental system comprised three components: the reaction unit, the gas collection unit, and a constant-temperature shaker [28]. The reaction unit consisted of 150 mL screw-capped anaerobic bottles with a 50 mL headspace. A rubber stopper fitted to each anaerobic bottle was penetrated by two needles (a long needle and a short needle). The long needle was submerged below the liquid surface, serving dual purposes of connecting to a nitrogen cylinder for nitrogen sparging and facilitating sample collection. In contrast, the short needle was used to expel residual air from the anaerobic bottle and function as the gas collection pipeline. For each 150 mL anaerobic bottle, 100 mL of tetracycline wastewater was added, followed by the addition of 10 mL of anaerobic granular sludge acclimated to tetracycline wastewater. The concentrations of the exogenous mediators (PAC and ZVI) were maintained at 100 mg/L, 200 mg/L, 500 mg/L, 1000 mg/L and 2000 mg/L, with a sludge concentration of 4 g MLVSS/L. Each experimental group had three parallel runs. A control group without the addition of exogenous mediators was also established. After sealing the bottle cap, the anaerobic bottle was sparged with nitrogen at a flow rate of 2.0 L/min for 5 min to establish an anaerobic environment. Subsequently, the nitrogen cylinder was turned off gradually to avoid effluent overflow from the long needle due to elevated internal pressure. Immediately thereafter, the short needle was connected to a gas sampling bag. Finally, the anaerobic bottle, with the gas collection unit attached, was placed in the constant-temperature shaker. The temperature inside the shaker was controlled at 35 ± 1 °C, and the rotation speed was maintained at 130 r/min. In total, 5 mL gas and 2 mL liquid samples were collected at 6 h, 12 h, 24 h, 30 h, 36 h, 48 h, 60 h and 84 h for subsequent analyses, then 2 mL of tetracycline wastewater was added to keep the total volume constant. Based on the changing trends of COD, TC and gas production volume in the pre-experiment, this study selected an experimental duration of 84 h. At this point, the influence of the intermediate agent on the anaerobic digestion system could be observed, while the system still contained high levels of VFAs, and a longer period of time would be needed to consume them in a future study. For gas samples, the gas production volume was recorded, and the gas composition was analyzed. For liquid samples, the COD, VFA content and TC of both influents and at different reaction times were determined. Once the water quality in the anaerobic bottle stabilized, the sludge–water mixture was centrifuged, and the supernatant was filtered to determine the cyclic voltammetry curve. The anaerobic granular sludge was then harvested from the mixture to characterize its surface morphology, internal structure, electrical conductivity, and EPS content.

3.3. Analytical Methods

In accordance with the predefined sampling schedule, aqueous and gaseous samples were systematically collected for each experimental group. Prior to the quantification of COD, VFAs and TC, the aqueous samples were subjected to centrifugation and filtration for solid–liquid separation, followed by gradient dilution at an appropriate ratio to meet the detection requirements. Specifically, for VFA analysis, 100 μL of formic acid was added to the pretreated samples to adjust the pH to the optimal range for detection. The COD concentration was determined strictly following the standard methods recommended by the American Public Health Association [29]. The VFA composition and contents in the aqueous samples were analyzed using a gas chromatograph (Agilent GC 7890A, Santa Clara, CA, USA) equipped with an FID and a HP-INNOWAX capillary column, with the analytical procedure referencing the method described by Ambuchi [30]. Meanwhile, the tetracycline concentration was quantified via high-performance liquid chromatography (Shimadzu LC-2010, Kyoto, Japan) coupled with a variable-wavelength UV–visible detector, using a C18 reversed-phase column (4.6 × 150 mm) as the stationary phase, and the operational parameters were set according to the protocol in our previous study [25]. The mobile phase was a mixture of 60% acetonitrile and 40% deionized water (containing 0.1% formic acid) at a flow rate of 1.0 mL/min, and the detection wavelength was 273 nm. The injection volume was 10.0 μL, and the column temperature was set at 30 °C. The calibration range was 0.2–20 mg/L, and the limit of detection and limit of quantification were 0.02 and 0.05 mg/L. The recovery rate was 92–98% at a concentration of 0.5 mg/L.

The gaseous parameters were characterized by two key indices, namely biogas yield and gas composition. The daily biogas production was measured volumetrically using a gas-tight syringe, whereas the contents of target gaseous components (H₂, CH₄ and CO₂) were determined by gas chromatography (Agilent 6890A, Santa Clara, CA, USA), with the analytical method adopted from the study of Sheng and Yu [31]. The surface morphology and structure of granule sludge at the final stage were analyzed by SEM with EDS following the procedure outlined by our previous study [32].

The electrochemical properties of the supernatant were determined using the AUTOLAB electrochemical workstation. The experimental system was a conventional three-electrode electrolytic cell system. A glassy carbon electrode was used as the working electrode, a platinum sheet electrode as the counter electrode, and a common SCE as the reference electrode. Before the experiment, the anaerobic sludge/external mediator composite system was centrifuged at 5000 r/min for 15 min. Then, the supernatant was filtered and the filtrate was transferred to the electrolytic cell. Before the experiment, the system was purged with nitrogen for 30 min to eliminate the influence of oxygen on the electrode reaction. A negative scan from 1 to −1 V was performed, with a scan speed of 10 mV/s, and the scan was repeated 4 times.

The electrical conductivity of sludge was measured using a four-probe conductivity meter. The sludge samples were collected by centrifugation at 8000 rpm for 5 min and washed three times with 0.1 M NaCl. The pellets were placed on the circle mold and freeze-dried for 24 h. The functional groups contained in the anaerobic granular sludge were determined by Fourier infrared spectroscopy. It was firstly placed in a drying dish for drying, then the sample and potassium bromide were pressed into a tablet under a pressure of 15–20 kPa, and finally the sample was placed in the infrared spectrometer for measurement. The scanning was conducted in the range of 4000~400 cm⁻¹, with a resolution of 4 cm⁻¹. The EPSs of anaerobic sludge could be categorized into two distinct fractions, namely LB-EPSs and TB-EPSs. In the present study, a two-step thermal extraction protocol was employed to sequentially isolate LB-EPSs and TB-EPSs from the anaerobic sludge matrix. Proteins and polysaccharides in EPSs were determined by the modified Lowry method and the phenol–sulfuric acid method respectively.

3.4. Calculation and Fitting Methods

The modified Gompertz equation was employed to simulate and analyze the gas production process under different medium conditions, and the equation is presented as follows:

$$P=P_{max}exp\left\{-exp\left[{\displaystyle \frac{R_{max}e(\lambda -t)}{P_{max}}}+1\right]\right\}$$

(1)

In the formula, P is the cumulative gas production, mL/gMLVSS; P_max is the maximum cumulative gas production during the reaction, mL/gMLVSS; R_max is the maximum gas production rate during the reaction, mL/(gMLVSS. h); λ is the reaction lag time, h; t is the reaction time, h; e is 2.71828. Notably, the kinetic parameters P_max, R_max, and λ could be derived via nonlinear regression fitting of experimental data obtained from the batch anaerobic digestion of tetracycline-containing wastewater.

An RSM experimental design was employed with Design-Expert software (V8.0.6.1), where the dosages of PAC and ZVI were designated as independent variables, and cumulative methane (CH₄) production was defined as the response variable. Experimental datasets were subjected to multiple regression fitting, followed by analysis of variance (ANOVA) to assess the significance and fitness of the established regression model. By interrogating the topological characteristics of the response surfaces generated by the model, the optimal experimental conditions corresponding to the maximum response value were identified and subsequently verified.

4. Conclusions

This study systematically explored the enhancement effects and mechanisms of ZVI and PAC on the AD of tetracycline-containing wastewater and optimized their dosage conditions via RSM. Single-mediator tests identified optimal dosages of 1000 mg/L ZVI and 2000 mg/L PAC for organic pollutant removal. Both mediators accelerated acetic and butyric acid degradation to alleviate acidification risks and improved methane production, with ZVI achieving a more prominent promotion effect. Mechanistically, ZVI and PAC enhanced system conductivity, might have facilitated electron transfer-related processes, and stimulated EPS secretion. RSM optimization determined the optimal combined dosage of 1000 mg/L ZVI and 1200 mg/L PAC, which improved methane yield by 71.18% with a model validation accuracy of 97.94%. This study clarifies the enhancement mechanism of mediator-modified AD, providing theoretical and technical support for the treatment of antibiotic-containing wastewater. However, this study only focuses on the short-term enhancement effect of ZVI and PAC on the anaerobic digestion process. The long-term promoting effect needs to be verified through long-term continuous-flow experiments.