Effect of Microplastics on Anaerobic Digestion Process with Rapidly Degradable Organic Matter

Abstract

The increasing presence of microplastics (MPs) in wastewater sludge raises concerns about their potential interference with anaerobic digestion (AD), a key process for energy recovery and sludge stabilization. This study investigated the impact of three common MPs, polystyrene (PS), polyethylene terephthalate (PET), and high-density polyethylene (HDPE), on the anaerobic degradation of a synthetic, rapidly biodegradable substrate under controlled batch conditions with the biomass from an anaerobic digester as inoculum. Biogas production, intermediate metabolic parameters, and microbial community dynamics were comprehensively assessed. The results showed a moderate inhibition of methane yield in the presence of MPs, with HDPE causing the most significant reduction (up to 24%) in biogas generation. PS exhibited the lowest impact, independent of the concentration added (0.5 and 1.0 g·L⁻¹). The microbial community structure demonstrated robustness, with Firmicutes and Bacteroidota maintaining dominance and methanogenic populations largely unaffected, except in the presence of HDPE. Raman spectroscopy indicated that none of the MPs underwent substantial structural degradation, but the subtle spectral shifts—particularly in PET—suggested the initial stages of physicochemical alteration. These findings offer new insights into the short-term resilience and adaptability of anaerobic microbiomes in the presence of MPs while revealing potential signals of process disruption.

1. Introduction

Microplastics (MPs), defined as plastic particles smaller than 5 mm, have emerged as persistent environmental contaminants due to their widespread presence in various ecosystems [1]. These particles originate from the degradation of larger plastic debris or are manufactured intentionally for use in products such as cosmetics and cleaning agents [2]. The persistence of MPs in the environment, coupled with their potential to adsorb and transport hazardous chemicals, presents important concerns regarding their impact on ecological and human health [3]. Wastewater treatment plants (WWTPs) are critical in the management of MPs [4].

While conventional treatment processes can remove a substantial fraction of the MPs from influent wastewater, a significant proportion—often exceeding 75%—is retained in the generated sewage sludge [5]. This accumulation highlights the importance of focusing on the sludge line within WWTPs when addressing MP contamination.

Recent studies have emphasized the prevalence of MPs in sewage sludge samples. For instance, Ormaniec & Baczyński (2025) [6] reported that the average MP content in sewage sludge can range from 13,748 to 116,307 MPs·kg⁻¹ dw. Another study reported that pre-digested sewage sludge and digestate exhibited 7600 ± 6800 and 7200 ± 1100 MPs·g⁻¹ dw, respectively, indicating that anaerobic digestion processes do not significantly reduce MP concentrations [7].

The presence of MPs in sewage sludge presents challenges for its management and disposal. Its complex matrix, rich in organic matter and characterized by a heterogeneous composition, facilitates the entrapment and potential interactions of MPs with organic and inorganic constituents [8]. Such interactions may influence the physicochemical properties of the sludge, affecting its dewaterability, stability, and suitability for applications such as land spreading [9].

Anaerobic digestion is a widely adopted strategy for the biological stabilization of WWTP sludge and the recovery of energy in the form of biogas [10]. In order to enhance biogas production, anaerobic digesters are also fed with rapidly biodegradable substrates coming from industries. However, the presence of MPs in the AD process introduces uncertainties regarding their impact on microbial communities and process efficiency. The literature indicates that MPs can adversely affect AD performance, leading to reductions in methane yield and alterations in microbial community structures. For instance, the presence of polyethylene (PE) MPs has been associated with a decrease in methane production by up to 13.8%, attributed to the disruption of microbial activity [11].

Multiple mechanisms underly the inhibitory effects of MPs on AD processes [12]. Potential factors include the physical obstruction of microbial access to substrates, the leaching of toxic additives from MPs, and the adsorption of essential nutrients or enzymes onto MP surfaces [13]. These interactions can compromise the metabolic activities of anaerobic microorganisms, disrupting digestion performance [14]

As the addition of highly biodegradable substrates is becoming an increasingly common practice, the specific effects of different MP types on AD processes, especially on the microbial population of the digesters, have to be studied. In fact, as far as the authors are aware, these effects have not been examined in previous studies. To address this, the present study focuses on the anaerobic digestion of a highly biodegradable substrate, serving as a simplified model to elucidate the interactions between MPs and microbial communities. By employing batch experiments with three distinct polymer types (high-density polyethylene (HDPE), polyethylene terephthalate (PET), and polystyrene PS-)), this research aims to assess the immediate impacts of MPs on anaerobic digesters receiving highly biodegradable substrates. For this study, metagenomic sequencing was used to examine how MPs influence the structure of microbial communities during anaerobic digestion.

The outcomes of this investigation are anticipated to enhance our understanding of MPs’ behavior in anaerobic environments and of their adverse effects on sludge treatment processes. Such knowledge is crucial for optimizing the energy recovery potential of WWTPs and for ensuring sustainable sludge management in the context of increasing plastic pollution.

2. Materials and Methods

2.1. Sample Collection and Initial Characterization

Digested sludge (DS), used as the inoculum, was collected from a municipal WWTP located near Valencia (Spain). Immediately after collection, the DS samples were purged with nitrogen gas to remove oxygen and stored in airtight bottles to maintain anaerobic conditions. Prior to their use in anaerobic digestion experiments, the inoculum was pre-acclimatized for 10 days at 37 °C to eliminate any residual biodegradable organic matter capable of producing biogas.

The initial characterization of the inoculum included the determination of the total solid (TS) and total volatile solid (TVS) concentrations. These parameters were measured in accordance with standard methods [15]. Additionally, soluble chemical oxygen demand (SCOD), sulfate (SO₄²⁻) concentration, and pH were determined. SCOD and SO₄²⁻ were measured using commercial Merck kits (Merck, Darmstadt, Germany), while pH was measured using a GLP 21+ pH meter (CRISON instruments, Barcelona, Spain).

2.2. Synthetic Biodegradable Substrate

A synthetic, highly biodegradable substrate was used as the feedstock for anaerobic digestion. It was composed of a mixture of peptone, meat extract, glucose, and K₂HPO₄ (ITW Reagents Panreac, Barcelona, Spain). To maintain a stable pH around 7.5 during digestion, a sodium bicarbonate buffer solution (NaHCO₃, ITW Reagents Panreac, Barcelona, Spain) was added. The synthetic substrate solution was characterized by measuring the total chemical oxygen demand (TCOD), SCOD, total nitrogen (NTotal), total phosphor (PTotal), and pH. For these nutrients’ characterization, samples were prefiltered through 0.45 µm using BRANCHIA SFMC-245-100 syringe filters (Labbox, Barcelona, Spain).

2.3. Anaerobic Digestion Setup, Operation and Monitoring

Batch anaerobic digestion tests were conducted in triplicate in 250 mL glass bottles with a working volume of 200 mL. The food-to-microorganism (F·M⁻¹) ratio was set at 0.5 g TCOD substrate per g TVS of inoculum. Two experimental series were conducted to assess the impact of MPs on anaerobic digestion performance. In all cases, the MPs consisted of fragments prepared in the laboratory. The plastics were cleaned, cut with stainless-steel scissors, and subsequently ground using an ultracentrifugal mill (Retsch ZM 1000, Haan, Germany) with liquid nitrogen to ensure brittle fracture. A rotor speed of 800 rpm was applied to obtain particles with sizes between 200 and 500 µm, following the methodology described by Mompó-Curell et al. (2024) [16]. Visual sorting of the MPs was carried out by a stereomicroscope MZ APO (LEICA, Wetzlar, Germany) to compare the MPs’ state before and after milling. Additionally, a subsample of 175 microparticles was measured in order to estimate their real size. The measured sizes were the following: 230.19 ± 131.48 µm for HDPE MPs, 432.29 ± 302.39 µm for PET MPs, and 386.97 ± 192.74 µm for PS MPs. In the first series, the influence of PS concentration was evaluated by testing two concentrations: 0.5 and 1.0 g·L⁻¹. In the second series, three different polymer types—PS, PET and HDPE—were assessed at a fixed concentration of 1.0 g·L⁻¹. These conditions were selected to examine both the concentration-dependent effects and the polymer-specific impacts of MPs on the digestion process. They are summarized in

Table 1. MPs present in anaerobic digesters in series of experiments.

First Series			Second Series
Digester Nomenclature	Polymer Type	Polymer Concentration	Digester Nomenclature	Polymer Type	Polymer Concentration
Control-FirstS	-	-	Control-SecondS	-	-
PS-0.5-FirstS	PS	0.5 g·L−1	PS-1-SecondS	PS	1 g·L−1
PS-0.5-FirstS	PS	1 g·L−1	PET-1.0-SecondS	PET	1 g·L−1
			HDPE-1.0-SecondS	HDPE	1 g·L−1

.

Prior to the start of the experiments, the headspace of each digester was purged with nitrogen gas for 5 min using a septum to ensure anaerobic conditions and to seal the system against further oxygen intrusion. The digesters were then maintained at 37 ± 1 °C during the entire experimental period. All anaerobic digestion assays were conducted in triplicate to ensure reproducibility, and blank digesters were included under identical conditions but without the addition of MPs.

The anaerobic digestion process was monitored by measuring several key operational parameters as biogas production and composition. Biogas production was quantified by recording the headspace overpressure using a handheld GMH 5100 series pressure meter (GREISINGER, Linz, Austria); after each measurement, the pressure was released to reset the system to zero. Biogas composition was analyzed using the following approaches:

• CO₂ and H₂S concentrations were determined with a gas sampling pump set GV-100S (GASTEC, Berlin, Germany) in combination with GASTEC detector tubes 2HH (CO₂) and 4L (H₂S).
• Volatile Fatty acids (VFAs) were analyzed using LCK365 Organic Fatty Acids cuvette tests (HACH LANGE, Hospitalet de Llobregat, Spain). For FA determination, 3 mL of digestate was withdrawn from each digester and filtered through 0.45 µm membrane filters prior to analysis.

At the end of the experiment, biogas composition, specifically CH₄, H₂ and CO₂, was measured by gas chromatography (GC) using a Bruker 450GC Rapid Refinery Gas Analyzer (RRGA) at the Instituto de Tecnología Química (ITQ, Valencia, Spain). In addition, pH, TS and TVS were determined for each digester.

To assess whether the MPs underwent structural changes during the anaerobic digestion process, their final characteristics were analyzed. The method is described elsewhere [17]. Specifically, MPs were subjected to an oxidation step with H₂O₂ at 60 °C for 2 h, followed by filtration using glass watch filters and drying in an oven at 60 °C prior to Raman characterization. Analyses were carried out using a Witec ALPHA300 M+ Raman microscope (Witec, Ulm, Germany) at three laser excitation wavelengths: 488, 532, and 633 nm. For each measurement, 20 accumulations were recorded with an integration time of 0.5 s to improve signal quality and ensure the accurate detection of possible changes in polymer structure.

2.4. Microbial Community Analysis of the Anaerobic Digestion Process

Triplicate samples of mixed liquor from anaerobic digesters, specifically from the first series, particularly Control-FirstS, PS-0.5-FirstS, and PS-1.0-FirstS; and from the second series, specifically Control-SecondS, PS-1.0-SecondS, HDPE-1.0-SecondS, and PET-1.0-SecondS, were prepared for deoxyribonucleic acid (DNA) extraction. A volume of 1 mL from each sample was centrifuged at 8000 rpm for 2 min. The resulting pellets were then resuspended in 978 µL of sodium phosphate buffer. DNA extraction from the aerobic digester samples was conducted following the methodology described by Bretas Alvim et al. (2021) [18]. Briefly, homogenization was performed using a FastPrep-24^® instrument (MP Biomedicals, Irvine, CA, USA) set at 6.5 m·s⁻¹ for 60 s, utilizing Lysing Matrix E tubes. The total genomic DNA was subsequently isolated with a FastDNA^® SPIN kit for soil (MP Biomedicals, USA), adhering to the manufacturer’s protocol. The purified DNA was eluted in 100 µL of elution buffer and further purified using a OneStep™ Polymerase Chain Reaction (PCR) Inhibitor Removal Kit (Zymo Research, Irvine, CA, USA). The resulting DNA samples were sent to the FISABIO sequencing service (Valencia, Spain) for analysis using via the Illumina MiSeq platform. Amplicon libraries targeting the V4 region of the 16S rRNA gene were prepared using primers 515F and 806R [19]. Sequence data analysis was performed using QIIME version 1.9.1 [20], supplemented with scripts from MicrobiomeHelper virtual box v0.4 [21]. The taxonomic classification of operational taxonomic units (OTUs) was carried out using the MiDAS v5.3 database [22], which is specifically designed for sewage treatment microbiomes, with a similarity threshold of 97%.

2.5. Statistical Analysis

A statistical analysis of relative abundance across various taxonomic levels was performed to assess the effect of PS, PE and PET particles on the microbial communities within anaerobic digesters. One-way analysis of variance (ANOVA) was conducted using Statgraphics Plus 4.0 software. The analysis yielded F-ratio values and p-values, with significance set at a 95% confidence level (p < 0.05).

3. Results and Discussion

3.1. Inoculum and Substrate Characterization

The physicochemical characteristics of the inoculum and the synthetic substrate are summarized in

Table 2. Characteristics of inoculum in first and second series.

	First Series	Second Series
TS (mg·L−1)	16,108 ± 119	15,940 ± 85
TVS (mg·L−1)	10,976 ± 187	10,320 ± 10
SCOD (mg·L−1)	470 ± 14	519 ± 16
SO42− (mg·L−1)	201 ± 6	169 ± 4
pH	8.15 ± 0.03	7.98 ± 0.6

and

Table 3. Characteristics of substrates in first and second series.

	First Series	Second Series
SCOD (mg·L−1)	5620 ± 31	5920 ± 46
Ntotal (mg·L−1)	710 ± 12	630 ± 22
Ptotal (mg·L−1)	62 ± 7	49 ± 9
pH	7.47 ± 0.07	7.39 ± 0.02

. The inoculum exhibited consistent total and volatile solids, SCOD, pH and sulfate concentration values across both experimental series, indicating stable and reproducible starting conditions. These parameters confirmed the effectiveness of the acclimation step in reducing background biodegradability and minimizing biogas production prior to carrying out the batch anaerobic digestion tests.

The substrate formulation, composed of simple proteins and carbohydrates, provided a readily biodegradable carbon source. Its nutrient content and buffering capacity were sufficient to maintain optimal conditions for microbial growth and activity. As shown in Table 3, the nitrogen and phosphorus concentrations were appropriate for supporting balanced anaerobic digestion.

This physicochemical uniformity between series was essential for isolating the effects of MP concentration and polymer type. The baseline values of the substrate are consistent with those reported in previous studies using controlled batch assays with sewage sludge as feed [7], validating the experimental approach adopted in this work.

3.2. Biogas Production and Intermediate Digester Development

The cumulative biogas production (at 37 °C) per g of COD in the substrate throughout the digestion process is presented in Figure 1 for both experimental series. It should be noted that the feed was a rapidly biodegradable substrate. Thus, the degradation time was consequently short. A slight general decrease in gas production was observed in the presence of MPs compared to the control, suggesting a negative impact on anaerobic digestion performance. However, within the first series, no significant differences were detected between the two concentrations of PS (0.5 and 1.0 g·L⁻¹), indicating that the observed inhibition was not strongly concentration-dependent within the tested range for this polymer. This points to a possible threshold effect or saturation of the inhibitory mechanisms at relatively low MP concentrations, as previously suggested in batch anaerobic assays [23].

The second experimental series enabled a comparative assessment of the effects exerted by different polymer types at equal concentrations. As shown in Figure 1b, different tendencies were observed in biogas production, suggesting that the impact of MPs on the anaerobic process was polymer-specific. Among the three tested materials, HDPE induced the most pronounced reduction in gas production relative to the control, followed by PET. In contrast, PS exhibited the least inhibitory effect under the same conditions. These results highlight the relevance of polymer properties—such as chemical composition—in modulating microbial accessibility and potential toxicity. Similar polymer-specific trends have been described in recent studies, where HDPE has been associated with greater microbial inhibition due to its higher hydrophobicity and persistence [24,25,26].

The intermediate gas composition during the digestion period after 75 h is summarized in

Table 4. Biogas composition and FA analysis in the experiments.

	First Series			Second Series
	Control-FirstS	PS-0.5-FirstS	PS-1.0-FirstS	Control-SecondS	PS-1.0-SecondS	PET-1.0-SecondS	HDPE-1.0-SecondS
CO2 (% vol)	21.20 ± 2.73	25.23 ± 3.96	25.50 ± 0.95	22.78 ± 2.47	23.69 ± 1.11	23.18 ± 1.93	21.99 ± 2.88
SH2 (ppm)	15	20	20	15	18	22	22
FA (mg CH3-COOH·L−1)	135.33 ± 20.60	75.50 ± 3.54	53.13 ± 6.07	122.67 ± 7.37	63.67 ± 16.56	57.33 ± 14.57	78.00 ± 8.54

. The volumetric fraction of CO₂ was practically the same in all the experiments. Only a slight increase in CO₂ concentration in the biogas in the presence of PS was observed, suggesting either a shift toward acidogenesis or a partial arrest of methanogenesis [27,28]. The concentration of H₂S in the biogas remained similar across all digesters, with no clear trend attributable to the presence of MPs. High H₂S concentrations have been associated with toxicity effects that impair the methanogenic consortia and enzymatic systems [29], and their rise should be closely monitored as a potential secondary indicator of MP inhibition. However, the measured H₂S values were too low to jeopardize the efficiency of the anaerobic process.

The lower concentrations of FAs, measured as acetic acid equivalents and observed in the MP-doped digesters, than in the control (Table 4) may be attributed to the following mechanism: The presence of MPs may not only inhibit methanogenic activity but also interfere with upstream acidogenic processes, thereby reducing the formation of FAs from proteins and other long-chain organic molecules [30]. This would result in a lower overall accumulation of FAs throughout the digestion process. The reason for this behavior could be the lower availability of the hydrolytic exoenzymes, like protease, that are responsible for the hydrolysis step in anaerobic digestion, which could be attributed to their adsorption onto the MPs of the reactors. Wei et al. (2019) [23] observed a reduction in protease activity in anaerobic reactors with PVC MPs, although these authors did not detail the mechanism responsible for this inhibition. However, this trend contrasts that in other previous studies, which have generally reported an increase in FA accumulation in the presence of MPs, attributed to the inhibition of methanogenic activity and the resulting disruption of FA consumption pathways [31].

The lower FA accumulation with MPs contrasts the findings of some researchers, who observed acidification in the presence of MPs as a result of inhibited VFA consumption during the methanogenic phase [32]. Therefore, monitoring FAs over time would help to better understand how MPs affect each stage of the digestion process. This discrepancy may also be related to the nature of the substrate used: unlike sewage sludge, the synthetic substrate applied in this study is rapidly biodegradable, allowing faster progression through the digestion stages, which could mask the accumulation of intermediate compounds such as VFAs.

3.3. Post-Digestion Characterization

At the end of the digestion period, key physicochemical parameters were analyses in the digested sludge to assess the extent of organic matter degradation and the effects of MPs on process efficiency.

As shown in

Table 5. Characteristics of anaerobic digesters at the end of the tests.

	First Series			Second Series
	Control-FirstS	PS-0.5-FirstS	PS-1.0-FirstS	Control-SecondS	PS-1.0-SecondS	PET-1.0-SecondS	HDPE-1.0-SecondS
pH	7.64 ± 0.05	7.61 ± 0.02	7.61 ± 0.03	7.65 ± 0.02	7.61 ± 0.02	7.63 ± 0.02	7.64 ± 0.02
TS (mg·L−1)	605.9 ± 500	7680 ± 600	7480 ± 532	6267 ± 106	6800 ± 116	6720 ± 164	7307 ± 712
TVS (mg·L−1)	2383 ± 217	3100 ± 763	3160 ± 503	2360 ± 80	2653 ± 41	2653 ± 60	2820 ± 298

, the pH remained within the optimal range for methanogenesis (6.6–7.8) [33] in all cases, indicating that the systems maintained acid–base balance and sufficient buffering capacity, even under the influence of MPs.

Moreover, the TS and TVS contents were consistently higher in the digesters containing MPs. This increase was due to two factors: on the one hand, the reduced conversion of organic matter to biogas, which was confirmed by the lower biogas production in the digesters with MPs (Figure 1); on the other hand, the MPs added contributed to the TS and the TVS since they were organic matter as well. A perfect mass balance is not possible since some of the MPs remain attached to the tank walls, as reported by Miloloža et al. (2025) and Rasmussen et al. (2021) [34,35].

Table 6. Biogas chromatography at the end of each test.

	First Series			Second Series
	Control-FirstS	PS-0.5-FirstS	PS-1.0-FirstS	Control-SecondS	PS-1.0-SecondS	PET-1.0-SecondS	HDPE-1.0-SecondS
CH4 (% vol)	77.41 ± 0.37	76.94 ± 2.23	77.23 ± 1.75	72.89 ± 4.86	74.14 ± 0.99	72.69 ± 0.15	75.71 ± 3.13
CO2 (% vol)	21.86 ± 0.22	22.99 ± 2.2	22.44 ± 2.06	23.90 ± 0.65	22.69 ± 1.52	22.60 ± 2.14	20.29 ± 2.57
H2 (% vol)	0.73 ± 0.58	0.07 ± 0.04	0.25 ± 0.20	3.15 ± 0.28	3.17 ± 0.53	3.56 ± 0.35	4.01 ± 0.56
Ratio CH4/CO2	3.54	3.35	3.44	3.05	3.27	3.22	3.73

shows the final biogas composition measured by gas chromatography. The methane content ranged between 72% and 78. Interestingly, HDPE not only maintained but slightly improved the CH₄/CO₂ ratio compared to the second series control (3.73 vs. 3.05, respectively), which indicated either enhanced carbon conversion or preferential microbial adaptation. Conversely, the lowest CH₄/CO₂ ratio was observed in the PET digesters (3.22), suggesting a partial inhibition of methanogenic pathways [30].

The H₂ concentrations in the biogas were low in all treatments, remaining below 4% and 4.1%. Although slight variations were observed—particularly a modest increase in the H₂ in PET and HDPE digesters in the second series—these values fell within the range of normal fluctuations for batch systems and were not indicative of major metabolic disruption.

3.4. Microplastic Characterization After Anaerobic Digestion

The post-digestion characterization of MPs was performed by Raman spectroscopy to assess potential structural changes after exposure to anaerobic conditions. The spectra obtained for each polymer type are shown in Figure 2, and representative peaks were analyzed to detect signs of chemical degradation or molecular rearrangement.

No substantial spectral alterations were observed in the HDPE samples, suggesting high structural stability and resistance to degradation under mesophilic anaerobic conditions. This result aligns with the existing literature describing HDPE as one of the most inert plastic types in anaerobic environments [36].

For PET, minor spectral variations were identified, including the appearance of or shift in characteristic peaks at 1544 cm⁻¹, 1776 cm⁻¹, and around 3076 cm⁻¹. The latter corresponds to the C–H stretching of aromatic rings, and its presence indicated subtle changes in surface chemistry or weak interactions with surrounding organic matter [37]. However, the limited magnitude of these shifts prevented any conclusion on significant degradation.

In the case of PS, different weak signals were detected at 910 cm⁻¹ and 1501 cm⁻¹. These bands may be associated with out-of-plane C–H deformations and C=C stretching vibrations, respectively [38]. Although their appearance could reflect superficial modifications or microbial interaction with the polymer surface, the evidence was insufficient to confirm chemical alteration.

Overall, the Raman spectra indicate that none of the tested MPs experienced extensive structural degradation during the digestion process. However, the detection of subtle spectral shifts, particularly in PET and PS, suggests that, even under relatively short experiment times and mesophilic conditions, minor modifications can occur. These may be associated with superficial oxidation or early-stage microbial interaction with the polymer surface [14].

These findings are relevant because they show that changes in MPs can begin to occur even under moderate conditions and short digestion times. Therefore, chemical characterization via spectroscopy could be useful in detecting early signs of polymer alteration [39,40]. Further studies using longer retention times or under more demanding conditions could help clarify how MPs behave and transform during anaerobic treatment.

3.5. Microbial Community Analysis

Illumina sequencing was performed of the AD of the first and second experimental series to assess the impacts of PS, HDPE, and PET particles on the microbial community during anaerobic digestion. The MIDAS 5.3 database was used to analyze the microbiome in 9 and 12 anaerobic digester samples from the first and second experimental series, respectively. The 9 and 12 Illumina libraries of bacterial and archaea 16S rRNA genes from the first and second experimental series yielded 3,348,804 and 5,030,720 reads after quality filtering and removal of chimeric sequences. All samples were rarefied at 291,780 reads in the first series and 291,780 reads in the second series. The relative abundance (%) was calculated as taxonomic classification counts divided by total reads. The relative abundances at different taxonomic categories from the first experimental series (Table S1) and second experimental series (Table S2) can be found in the Supplementary Material.

The 16S rRNA gene sequencing showed that the predominant phyla in both experimental series were Firmicutes, Chloroflexi, Bacteroidota, Proteobacteria, and Actinobacteriota (Figure 3a,b). In the conducted experiments, members of the phylum Firmicutes demonstrated predominant dominance within the microbial communities. Specifically, in the first series, Firmicutes accounted for 23.14% (Control-FirstS), 26.71% (PS-0.5-FirstS), and 27.22% (PS-1.0-FirstS) of the total bacterial and archaeal populations. In the second series, their relative abundance was 22.2% (Control- SecondS), 19.82% (PS-1.0-SecondS), 22.71% (HDPE-1.0-SecondS), and 20.91% (PET-1.0-SecondS). The phylum Chloroflexi emerged as the second most prevalent group (first series: 17.74–18.25% and second series: 16.10–18.18%), followed by Bacteroidota (first series: 17.74–18.25% and second series: 10.96–12.77%). Other significant phyla included Proteobacteria, Actinobacteriota, Synergistota, and Desulfobacterota, each comprising approximately 6% of the core community. The seven most dominant phyla constituted 81.97% and 78.84% of the core bacterial communities in first series and second series, respectively. Among these, Firmicutes, Bacteroidota, Proteobacteria, Actinobacteriota, and Synergistota have been consistently observed in various anaerobic digestion systems, indicating their potential functional relevance in such processes [41,42]. Firmicutes were the predominant bacterial phylum under conditions of stable process performance, whereas Bacteroidota became more dominant under conditions of organic overloading [43].

The ratio of Firmicutes to Bacteroidota (F/B ratio) has been proposed as an indicator of anaerobic digestion performance based on observed correlations between this microbial abundance ratio and key operational parameters [43,44]. The Firmicutes-to-Bacteroidetes (F/B) ratios in the first series were 1.47 (Control-FirstS), 2.08 (PS-0.5-FirstS), and 2.14 (PS-1.0-FirstS), while in the second series, the ratios were 2.02 (Control- SecondS), 1.55 (PS-1.0-SecondS), 1.81 (HDPE-1.0-SecondS), and 1.79 (PET-1.0-SecondS). High F/B ratios are associated with higher specific biogas yields [45]. The values of the F/B ratio indicate that the presence of MPs, compared to the control, did not significantly affect the anaerobic digestion process.

The anaerobic digestion of organic compounds comprises four primary stages: hydrolysis, acidogenesis, acetogenesis, and methanogenesis [46]. Among the various microbial processes involved in anaerobic digestion, archaeal communities uniquely carry out methanogenesis. In contrast, bacterial communities are responsible for the remaining stages, including hydrolysis, acidogenesis, and acetogenesis. Notably, specific bacterial groups often play a critical role in determining the overall efficiency and stability of the anaerobic digestion process [43].

The relative abundance of the representative genera for the acidogenesis, acetogenesis, and methanogenesis steps, as reported in the recent literature [47,48], from the first and second series are shown in Figure 4a and Figure 4b, respectively. Multiple bacterial genera associated with hydrolysis, acidogenesis, acetogenesis, and syntrophic interactions were identified, each exhibiting a relative abundance exceeding 1% in at least one sample. Among these, Romboutsia was the most dominant genus, with relative abundances ranging from 11.15% to 13.32% in the first series and from 9.98% to 12.5% in the second series. This genus is characterized by extensive metabolic versatility, including the utilization of carbohydrates, the fermentation of individual amino acids, anaerobic respiration, and the production of diverse metabolic end products [49]. The second most abundant genus was DMER64, belonging to the family Rikenellaceae, with relative abundances of 8.41–10.01% in the first series and 4.20–5.09% in the second series. DMER64 has been suggested to participate in syntrophic interactions within anaerobic microbial communities [50]. Other notable genera included midas_g_944 from the class Anaerolineae (6.54–6.86% in the first series and 6.40–7.23% in the second series) and Paraclostridium from the family Peptostreptococcaceae (4.13–5.41% in the first series and 10.96–12.77% in the second series). Previous studies have demonstrated that bioaugmentation with Paraclostridium species, in combination with multi-enzyme treatments, can enhance methane production from kitchen waste [51]. Members of the family Anaerolineaceae have also been identified as key participants in the AD process, particularly in the degradation of carbohydrates and proteins, often in association with methanogenic archaea [52]. Among archaeal methanogens, Methanothrix (family Methanosaetaceae) was the most abundant methanogenic archaea, with relative abundances between 2.46 and 2.67% in the first series and 2.34 and 2.92% in the second series. Methanothrix is recognized as a major methane producer in various anoxic environments, including soils, sediments, and anaerobic digesters, primarily due to its high affinity for acetate and its capacity for acetoclastic methanogenesis [53].

In this study, the relative abundance of methanogens did not differ significantly (p > 0.05) among the Control-FirstS, PS-0.5-FirstS, and PS-1.0-FirstS AD samples in the first series (Figure 5a). This result was expected since a highly biodegradable substrate should enhance microbial activity and overall digestion efficiency, helping to overcome the negative impacts of the MPs present in the feed (HDS) due to its nutrient composition and biodegradability [54]. However, in the second series of experiments, the abundances of methanogens in HDPE-1.0-SecondS AD were significantly lower than in PS-1.0-SecondS, PET-1.0-SecondS and Control-SecondS. (Figure 5b). In fact, this situation is similar to the one reported by Wang et al. (2022) [55], who observed that PE MPs (10 mg·L⁻¹) inhibited methane production by 30.71%. The impact of MPs on methane production is highly variable and depends on the type of MP [56]. In our work, only HPDE produced a significant decrease in methanogens.

The F ratio in the first experimental series was 0.27, and, in the second experimental series, it was 0.04. Since the p-value of the F test in experimental series two was less than 0.05, there was a statistically significant difference between the mean relative abundance from one sample level to another at the 95.0% confidence level. Fisher’s least significant difference (LSD) procedure showed that relative PE abundance was significantly lower than in the B control.

4. Conclusions

This study provides evidence that MPs, particularly PS, PET and HDPE, can influence AD performance even under conditions optimized for fast biodegradation. Among the tested polymers, HDPE caused the greatest decline in biogas production (up to 24%), which was corroborated by a significant decrease in the relative abundance of methanogens. PET also showed signs of microbial inhibition, while PS had a comparatively limited impact. Despite these performance shifts, the pH and methane content of the biogas remained within acceptable ranges, and no extensive polymer degradation was detected. The microbial community structure demonstrated robustness, with Firmicutes and Bacteroidota maintaining dominance and methanogenic populations largely unaffected, except in the presence of HDPE.

These findings underscore two key innovations: demonstrating that MPs have polymer-specific impacts on AD performance, even in co-digestion cases in which a rapidly biodegradable substrate is fed to the digesters to enhance biogas production, and identifying early microbial and spectroscopic indicators of process disturbance. The integration of Raman spectroscopy and advanced DNA sequencing provides a novel diagnostic approach for tracking MP-related shifts during sludge digestion. In the context of growing plastic pollution, this research suggests a method for assessing the risk posed to AD technologies, offering critical knowledge for wastewater treatment engineers aiming to improve energy recovery processes.