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Article

Effects of Different Microplastics on Methane Production and Microbial Community Structure in Anaerobic Digestion of Cattle Manure

by
Mengjiao Zhang
1,
Congxu Zhao
1,
Tian Yuan
1,
Qing Wang
1,
Qiuxian Zhang
1,
Shuangdui Yan
1,
Xiaohong Guo
2,
Yanzhuan Cao
1,* and
Hongyan Cheng
1,*
1
College of Resources and Environment, Shanxi Agricultural University, Taigu, Jinzhong 030800, China
2
College of Animal Science, Shanxi Agricultural University, Taigu, Jinzhong 030800, China
*
Authors to whom correspondence should be addressed.
Agronomy 2025, 15(1), 107; https://doi.org/10.3390/agronomy15010107
Submission received: 9 December 2024 / Revised: 31 December 2024 / Accepted: 1 January 2025 / Published: 3 January 2025
(This article belongs to the Section Agricultural Biosystem and Biological Engineering)
Browse Figures
Graphical abstract
"> Figure 1
<p>Cumulative methane production from cattle manure at different MPs.</p> "> Figure 2
<p>Effects of different MP exposures on the degradation of organic compounds (<b>a</b>) SCOD, (<b>b</b>) TOC, and (<b>c</b>) ammonia nitrogen.</p> "> Figure 3
<p>Effects of different MP exposures on total VFAs (including acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, and valeric acid).</p> "> Figure 4
<p>Effects of different MPs exposures on (<b>a</b>) ACK activity and (<b>b</b>) LDH release. Note: ACK, acetate kinase; LDH, lactate dehydrogenase.</p> "> Figure 5
<p>SEM images of MPs before and after AD.</p> "> Figure 6
<p>Relative abundances of (<b>a</b>) bacteria and (<b>b</b>) archaeal at phylum level; (<b>c</b>) Abundance of archaea at the Unweighted UniFrac metrics NMDS analysis; (<b>d</b>) archaeal community analysis genus level.</p> "> Figure 7
<p>(<b>a</b>) Spearman’s correlation between microorganisms and environmental factors. * 0.01 &lt; <span class="html-italic">p</span> ≤ 0.05, ** 0.001 &lt; <span class="html-italic">p</span> ≤ 0.01; (<b>b</b>) Heat map of relative abundance of acidification pathway and functional enzyme genes. (Relative abundance in ‰ (parts per thousand)).</p> ">
Versions Notes

Abstract

:
Microplastics (MPs) are widely distributed in the environment, and they inevitably enter animal bodies during livestock and poultry farming, leading to their presence in livestock and poultry manure. However, there is limited research on the effects of different types of MPs on the anaerobic digestion (AD) performance of livestock and poultry manure. Herein, we investigated the impact of four types of MPs (polyvinyl chloride (PVC), polypropylene (PP), polyethylene (PE), and polyhydroxyalkanoate (PHA)) on AD performance using cattle manure as a substrate. Results demonstrated that the cumulative methane production in the PE group reached 5568.05 mL, exhibiting an 11.97% increase compared to the control group. Conversely, the cumulative methane production was decreased by 5.52%, 9.69%, and 14.48% in the PP, PVC, and PHA groups, respectively. Physicochemical analyses showed that MPs promoted organic matter hydrolysis on day 4 of AD, leading to the accumulation of volatile fatty acids (VFAs) in the initial stage. Specifically, the acetic acid content of PE was 44.48–92.07 mL/L higher than that of the control during the first 8 days. PE MPs also enriched microorganisms associated with methane production. The abundance of Firmicutes was enhanced by 2.89–17.57%, Methanosaeta by 8.42–12.48%, and Methanospirillum by 10.91–16.89% in comparison to the control; whereas PHA MPs decreased the abundance of Methanosaeta by 8.14–31.40%. Moreover, PHA MPs inhibited methane production by suppressing acetate kinase activity while promoting lactate dehydrogenase release from microorganisms involved in the AD process. Based on changes observed in key enzyme functional gene abundances, PHA MPs reduced acetyl-CoA carboxylase functional gene abundance, negatively affecting the acetone cleavage methanogenesis pathway. Meanwhile, PE MPs significantly increased acetate-CoA ligase abundance, thereby promoting the acetic acid methanogenesis pathway. The results provide novel insights into the influence exerted by MPs on AD performance when applied to livestock manure.

Graphical Abstract">
Graphical Abstract

1. Introduction

Along with the massive use of plastic products, plastic pollution is increasingly serious and has become a global concern [1]. Through natural weathering, microbial degradation, and ultraviolet radiation [2,3], plastic blocks can be gradually converted into tiny particles of different sizes, which are then crushed into microplastics (MPs). MPs, a new pollutant owing to their ecotoxicity and environmental ubiquity, are tiny plastic particles ranging from 1 to 5 mm and were originally found in the oceans. After intensive research, the number of MPs detected in cattle manure was 74 per kilogram of wet matter [4], MPs were found in raw poultry feces in the concentration range of 74–88,333 n/kg [5,6] and even in human feces [7,8].
With the intensive and large-scale development of livestock and poultry farming, livestock and poultry manure production has also increased. According to the Ministry of Agriculture, the current annual production of livestock and poultry manure in China is approximately 3.8 billion tons, of which more than 40% is not effectively utilized, and the irrational use of livestock and poultry manure not only results in the waste of resources to aggravate environmental pressure but may even cause serious pollution problems [9,10]. In addition, livestock manure contains many trace elements, which can be rationalized as an important resource and reflect its utilization value in the development of agriculture. Resourceful recycling of livestock and poultry manure can be achieved by anaerobic digestion (AD), which is a renewable and sustainable energy technology. However, the microorganisms in AD systems exhibit mutual interactions, and the digestion process is unstable and susceptible to unfavorable factors [11]. As emerging contaminants, MPs may adversely affect functional microbial communities and pose new challenges to the operation of AD systems. For instance, MPs and their compounds can alter the composition of microbial communities and affect the activity of key enzymes, thereby influencing the transformation of organic matter and carbon cycling in different environments [12]. Therefore, the risks associated with MPs should not be underestimated.
Recently, some studies have shown that MP concentration has different effects on AD methanogenesis. Low MP doses have cell-protective and electron-transfer-promoting properties, increasing the secretion of extracellular polymeric substances [13,14], whereas high MP concentrations attenuate volatile fatty acid metabolism and inhibit microorganisms associated with methanogenesis. The impact of PS MPs on food waste AD was investigated [15]. The presence of low MP levels reduced the cumulative methane production from 1.46% to 18.11%. Furthermore, higher MP levels exhibited a significant decrease in cumulative methane production, with reductions ranging from 9.14% to 33.08%.
The particle size of MPs is another crucial factor influencing AD, with most findings indicating that small particle sizes have strong inhibitory effects [16]. With respect to anaerobic granular sludge, an increase in polyvinyl chloride (PVC) particle size led to a gradual enhancement of the inhibitory effect on cumulative methane production during AD, accompanied by disruption and reduction in microbial integrity as well as viability [17]. Different types of MPs exert varying effects on AD [18]. For instance, the impact of five different MPs (PVC, polyethylene terephthalate (PET), PE, Polystyrene (PS), and PP) on the AD process using anaerobic granular sludge was investigated [19] and it was found that these MPs promoted lactate dehydrogenase release while inhibiting related hydrolase activity. Moreover, distinct types of MPs may leach diverse additives or toxic chemicals [20]. These studies suggest that the effects of various MP types on AD might be attributed to their unique physicochemical properties. Notably, previous studies primarily focused on spent activated sludge and food waste samples; fewer investigations were conducted on livestock manure with high organic content.
The impact of MPs on the AD of livestock manure remains uncertain and requires further investigation. This study aimed to conduct a 30-day AD experiment using cattle manure to examine the effects of degradable MPs, such as PHA, and nondegradable MPs such as polyethylene (PE), polypropylene (PP), and PVC, on methane production. In addition, changes in the functional microorganisms and metabolic pathways following exposure to various MPs were analyzed using MiSeq high-throughput sequencing technology. This study provides new insights into the microbial responses during the digestion of livestock manure with MPs. It also establishes a foundation for subsequent efforts aimed at mitigating the adverse effects of MPs on AD.

2. Material and Methods

2.1. Experimental Materials

The cattle manure used in this study was obtained from the Animal Husbandry Station of Shanxi Agricultural University (Shanxi, China), thoroughly mixed, and then frozen at −24 °C before use. The inoculum was obtained from the operational fermentation tank in the laboratory. It was removed from the fermenter and allowed to sit for a few days until it no longer produced gas, after which it was used. Table 1 presents the main physical and chemical characteristics of the substrate and inoculum. Four MPs with a particle size of 120 μm—PVC, PP, PE, and polyhydroxyalkanoate (PHA)—were purchased from Goose Technology Co (Tianjin, China).

2.2. Biochemical Methane Potential Tests

The tests were performed in 1 L glass bottles with a working volume of 500 mL. Cattle manure (149.79 g) and inoculum (50 mL) were added to the reactor. Four experimental groups were added with 0.2 g of PVC, PP, PE, and PHA MPs in powder form, named PVC, PP, PE, and PHA. In addition, a blank group without added MPs was set up as CK, and three replicate samples were set up for each sample. The pH of the mixture was first adjusted to approximately 7.0 using a 2 mol/L solution of NaOH and HCl. All reactors were then purged with high-purity nitrogen for 2 min to ensure anaerobic conditions. The reactors were then tightly sealed with rubber dividers and plastic covers before being placed in a constant-temperature incubator (37 ± 2 °C) for AD. After 30 days of AD, the rate of increase in cumulative methane production in the reactor leveled off, indicating that most of the digestion of the cattle manure was complete. Samples were taken from the reactor on days 0, 4, 8, 12, 16, 20, 24, and 28 of fermentation for subsequent analysis.

2.3. Determination of Biogas Production

Total gas production from anaerobic fermentation was determined using a BMP-Test system (WAL-BMP-Test system 3150, WAL, Oldenburg, Germany), and biogas production was calculated from the absolute pressure difference from atmospheric pressure in a fermentation flask [15]. Methane content was determined using a Biogas 5000 gas analyzer (Geotech, Warwickshire, UK).
PV = nRT
V biogas = Δ P × V headspace × C R × T
where Vbiogas is the daily biogas volume (mL), ΔP is the absolute pressure difference (kPa), Vheadspace is the volume of the headspace (mL), C is the molar volume (22.41 L·mol−1 at 273.15 K, 101.325 kPa), R is the universal gas constant (8.314 L kPa·K−1·mol−1), and T is absolute temperature (K).

2.4. Kinetic Model Analysis

The kinetic parameters of methane production were analyzed using a modified Gompertz model, and the kinetic equation is as follows [21]:
P = P max [   1 −   exp   ( − R max ( t   −   λ ) P max ) ]
where P is the cumulative methane production (mL); Pmax and Rmax are the maximum methane production potential (mL) and maximum methane production rate (mL·d−1); λ and t are the lag time (d) and AD time (d), respectively; e is a constant of 2.71828; and λ, Pmax, and Rmax were obtained by fitting the experimental data.

2.5. Analytical Methods

The fermentation broth was centrifuged at 12,000 rpm for 10 min, and the supernatant was stored at −24 °C for physicochemical indexes. Total solids (TS) was measured using a thermostatic drying oven (GZX-9246 MBE, Boxun Industries Co., Ltd., Shanghai, China) at 105 °C for 24 h. Volatile solids (VS) was burned at 600 °C for 2 h in a muffle furnace (LX0711, Labtery, Tianjin, China) and then weighed for determination. Soluble chemical organic oxygen demand (SCOD) was determined using a SCOD rapid detector (Lovibond E799718, Berlin, Germany) [22]. The total organic carbon (TOC) was measured using a TOC tester (ALLC 3100, Analytik Jena AG, Jena, Germany). Ammonia nitrogen concentration was measured using a segmented flow analyzer (AMS, Alliance, Paris, France). Acetate kinase (ACK) and lactate dehydrogenase (LDH) activity test kits were purchased from Suzhou Dream Rhinoceros Bio-medical Technology Co. Ltd. (Suzhou, China), and the concentration of volatile fatty acids (VFAs) was determined using a high-performance liquid chromatograph (TRACETM1300, Thermo Fisher Scientific, Shanghai, China) [23]. The surface morphology of MPs before and after digestion was observed via scanning electron microscopy (SEM, MDS-HDAF, Shanghai, China).

2.6. Microbial Community Analysis

Samples were collected in a reactor at different AD stages (days 0, 4, and 28). DNA was extracted using a DNA isolation kit, and the extracted genomic DNA was detected using 1% agarose gel electrophoresis. After DNA extraction, the bacterial 16S rRNA gene V3–V4 region was amplified using the primers 338F (5′-ACTCCTACGGGGAGGCAGCA-3′)/806R (5′-GGACTACHVGGGTWTCTAAT-3′) to amplify the V3–V4 region of the bacterial 16S rRNA gene, and 524F (5′-TGYCAGCCGCCGCGGGTAA-3′)/958R (5′-YCCGGCGTTGAVTCCAATT-3′) to amplify the V4–V5 region of the archaeal 16S rRNA gene. The PCR products were mixed and detected via 2% agarose gel electrophoresis, and the PCR products were recovered using the AxyPrep DNA Gel Recovery Kit (AXYGEN). The purified amplification products were sequenced using an Illumina MiSeq sequencing platform (Shanghai Personal Biotechnology Co., Ltd., Shanghai, China).

2.7. Statistical Analysis

The data were analyzed using Microsoft Excel 2010, and significance was checked using one-way ANOVA via SPSS 27.0 software, and p < 0.05 was considered significant. The kinetics were analyzed, and graphs were plotted in Origin 2021. The microbial analysis platform used was the Majorbio platform (https://www.majorbio.com/, accessed on 22 January 2024).

3. Results and Discussion

3.1. Effects of MPs on Methane Production

Figure 1 shows the cumulative methane production from the AD of cattle manure with the addition of four types of MPs for 30 days. Methane production in each reaction group increased rapidly in the first 15 days and more slowly in the latter 15 days. The cumulative methane production was 4972.59 ± 150.12 mL in CK for 30 days. With the presence of PE MPs, the cumulative methane production was 5568.05 ± 145.22 mL, which was significantly higher than CK by 11.97% (p < 0.05), and the results are similar to those of previous studies [24]. Meanwhile, PP, PVC, and PHA MPs exhibited inhibitory effects on methane production throughout the digestion process, resulting in cumulative methane productions of 4697.77 ± 150.12, 4540.23 ± 180.12, and 4252.80 ± 200.76 mL, respectively. Specifically, PP and PVC MPs demonstrated a reduction in cumulative methane production by 5.52% and 9.69%, respectively. These results are consistent with previous studies reporting that a microplastic digestion system with 8% PP MPs (6 mg/L) reduces methane recovery from wastewater by 23.65% [25], and that PVC MPs reduces cumulative methane production by 8.5% [26]. Both PP and PVC microplastics reduced archaeal abundance and thus methane production. PHA displayed the lowest cumulative methane production rate, which was significantly lower than CK (p < 0.05), exhibiting a decrease of approximately 14.48%.
In addition, the Gompertz model was employed with modifications to fit the cumulative methane production values of cattle manure AD with different MPs. The fitting results are presented in Table 2, providing insights into the maximum methane production potential value (Pm), maximum methane production rate (Rm), and lag time (λ). The R2 values for all five treatment groups exceeded 99%, indicating a good functional fit. Moreover, λ was less than 0, suggesting an efficient digestion system that effectively utilizes organic matter [27]. Under the influence of various MPs, Pm and Rm were reduced to varying extents in PE, PP, and PHA compared with the control group (CK). Specifically, compared with CK, Pm and Rm decreased by 7.54% and 7.01%, respectively, in PHA, indicating significant inhibition of the methanogenic process by PHA MPs (p < 0.05). Moreover, Pm and Rm increased by 6.39% and 8.20%, respectively, in PE, indicating significant promotion of the methanogenic process (p < 0.05).

3.2. Effect of MPs on the Performance of Anaerobic Fermentation System

3.2.1. Effects of MPs on Organic Metabolism

The degradation of macromolecular organic matter is a crucial indicator of the efficiency of AD [28,29]. During the 30 days of AD of cattle manure, SCOD and TOC in each reactor under different MPs are shown in Figure 2a, b. The SCOD concentration of each group gradually decreased within the first 12 days reaching its lowest value on day 12, indicating the rapid utilization of organic matter by microorganisms at the beginning. However, it began to rise after day 12, indicating that the rate of dissolution of organic matter exceeded its utilization rate at that time. The SCOD of PE increased by 11.22–61.51% compared with CK after the 16th day of AD (p < 0.05), and a similar trend was observed in PP. These results suggest that PE and PP MPs increase the utilization of organic matter by microorganisms and promote its conversion from solid to soluble fractions [16]. On days 24 and 28, PHA and PVC had lower values than CK; specifically, on day 28, PHA had the lowest SCOD value (1970 ± 33 mg/L) among all groups, indicating that PHA and PVC MPs inhibit hydrolysis toward organic matter in the later stages, while also prolonging digestion cycle time as confirmed by the Gompertz model fit results (Table 2), where λ lag time was greater for PHA and PVC than for CK.
The TOC can indicate the hydrolysis and utilization of organic matter. In AD, the TOC concentration generally follows a declining trend. During the early stage of the reaction, a significant reduction in TOC was observed in each experimental group owing to the microbial acid production process that consumed organic carbon. Moreover, throughout the digestion process, the TOC value of PHA consistently remained lower than that of CK, providing further evidence for PHA MPs inhibiting the hydrolysis of organic matter.
The ammonia nitrogen concentration of each treatment group showed an increasing trend throughout the AD period (Figure 2c). This can be attributed to the conversion of proteins or other nitrogenous substances in the substrate into amino acids via hydrolysis, which are further transformed into ammonia nitrogen [30]. The ammonia nitrogen concentration plays a crucial role in influencing the activity of methanogenic bacteria in the AD system [16,31]. Herein, although there was a continuous increase in the ammonia nitrogen concentration, it remained within the range of 296.10–538.13 mg/L, which is below the inhibition threshold (1500–3000 mg/L), indicating that MPs did not induce an ammonia inhibition phenomenon [32,33]. The average concentrations of ammonia nitrogen in PHA, PP, PVC, and PE were 384 ± 10.61, 383.13 ± 8.35, 393.68 ± 6.1, and 417.22 ± 7.09 mg/L, respectively, which indicated increases compared with CK (374.2 ± 12.31 mg/L). These findings suggest that MPs increased ammonia nitrogen production. By contrast, SCOD, TOC, and ammonia nitrogen concentration values were generally high in PE throughout the digestion period, suggesting a more rapid degradation of organic matter in PE, consistent with increased high total methane production.

3.2.2. System Stability Under MPs Exposure

During the AD process, complex organic matter macromolecules undergo hydrolysis and acidification to produce VFAs, which directly affect methane production [19,34]. The content and composition of VFAs were analyzed in each reactor with different MP additions, as shown in Figure 3. The total VFA concentration gradually decreased and then stabilized over time. On day 4 of digestion, the MPs group exhibited significantly higher VFA content than CK. This can be attributed to the increased release of SCOD, leading to greater availability of carbon sources for methanogenic bacteria. The hydrolytic acidification rate at this time was lower than that of methane production, corresponding to a faster rate of methane production in the early stage (Figure 1). The total VFA concentration of PE was 483.09 mg/L on day 8, which was significantly higher than that of CK by an increase of approximately 57.05%. This indicates that PE promoted organic matter hydrolysis and favored acid production.
The material composition of VFAs has an important influence on the subsequent anaerobic methane production, and VFAs such as acetic acid are the main raw materials for methane biosynthesis [35]. Figure 3 shows the changes in the proportion of VFAs, with an increased proportion of acetic acid followed by an overall increasing and then decreasing trend and a gradually decreasing proportion of propionic acid. The acetic acid concentrations of CK and PE peaked at 148.10 and 240.17 mg/L, respectively, on day 8, indicating that the presence of PE MPs significantly enhanced acetic acid accumulation by 62.17% compared to CK. Moreover, PP, PHA, and PVC exhibited their maximum concentrations of acetic acid earlier on day 4 at levels of 205.86, 190.34, and 209.31 mg/L, respectively, when introduced into the digestive system as MPs. This suggests that these three types of MPs accordingly promoted VFA accumulation on day 4. Furthermore, these findings imply that MPs may interfere with hydrolytic microorganisms responsible for degrading organic matter as well as acidogenic bacteria involved in VFA metabolism processes.
The activities of microbial enzymes are closely related to the performance of the digestive system. Among these enzymes, ACK plays a crucial role in catalyzing the conversion of acetyl coenzyme A into acetic acid, and its activity directly influences the amount of acetic acid produced [35]. During AD progression, ACK activity gradually decreases (Figure 4a), which is consistent with the observed changes in acetic acid concentration (Figure 3). At the initial stage of digestion (day 4), ACK activity in the MPs-treated group ranged from 46.42 ± 2.34 to 64.92 ± 1.7 nmol/min/mL, representing a decrease of 12.30–37.30% compared with that of CK (74.03 ± 3.64 nmol/min/mL). This finding does not entirely correlate with the amount of acetic acid on day 4, possibly because MPs promote extensive hydrolysis of organic matter resulting in more available substrates for fermentation reactions. Another reason may be that the acidification efficiency depends not only on the enzyme activity but also on the distribution of active sites on the enzyme surface [12]. At the end of digestion (day 28), PHA exhibited the lowest ACK activity at 6.43 ± 1.22 nmol·min−1·mL−1. These results collectively indicate that the addition of MPs inhibits ACK activity across all reactors; however, PP, PHA, and PVC MPs exhibit stronger inhibitory effects on ACK than PE MPs.
In addition, LDH is a stable cytoplasmic enzyme widely present in microorganisms. When the cell membrane is severely damaged, LDH present inside the cell is released outside the cell, and its leakage usually represents a change in the permeability of the cell membrane or cellular disruption; therefore, LDH is an important indicator to characterize the integrity of microbial cell membranes [36]. The presence of PHA, PP, and PVC MPs resulted in varying degrees of increased LDH release, which could be a key factor contributing to methanogenesis inhibition (Figure 4b). On day 4 and day 28, LDH released by the PHA reactor increased by 24.25% and 39.47% compared to CK, respectively, suggesting that the entry of PHA MPs into the AD system disrupts microbial cellular integrity, increasing the extracellular LDH concentration. By contrast, PE MPs did not promote LDH release, indicating their weaker impact on systemic microorganisms. The release of toxic additives such as bisphenol A, dibutyl phthalate, and polyfluoroalkyl substances, which are present in MPs [37,38], exert toxic effects on the microorganisms in the system [39]. Among the four types of MPs added, PE MPs leached acetyl tributyl citrate, which did not affect methane production. Unlike PE MPs, PVC MPs released toxic bisphenol A, which has toxic effects on the system. Moreover, PHA MPs are degradable MPs that may further degrade, producing more harmful substances, thereby inhibiting the activity of key enzymes or affecting the nutrient metabolism of microorganisms while reducing microbial community diversity and selectively enriching certain bacteria.

3.3. Properties of MPs Before and After AD

The changes in the surface microstructures of different MP samples before and after digestion were observed via SEM, as shown in Figure 5. The degradation levels of the four MPs have the following order: PHA > PVC > PE > PP MPs. Initially, the MP samples exhibited a smoother and more compact texture with intact particles (except for the PVC MPs standard samples that displayed some pits on the surface, and the PVC and PE MPs had some “peeling” phenomenon). However, after 30 days of AD, the surface of the MPs exhibited different degrees of change. The PHA MPs demonstrated significant deterioration with holes appearing on their surfaces because they are biodegradable MPs characterized by low crystallinity, resulting in porous surfaces that are susceptible to hydrolysis by diverse microorganisms [40,41]. The degradation of PHA during AD was investigated using O2, NO3−, and Fe3+ as electron acceptors [42], respectively, and it was found that the degradation efficiency of PHA MPs was approximately 50% after 43 days of AD. For the nondegradable MPs, breakage and small holes appeared in PP, wrinkles appeared on the surface of PE, the aging degree of PVC was more serious among the nondegradable MPs, the surface of PVC not only had increased pits but also had obvious irregular and deep cracks, and the surface had smaller raised structures and showed a tendency to peel off. This can be attributed to the fact that the organic acid enters the MP particles and enlarges the structural defects such as holes and cracks inside the particles. As a results, the surface material of the MPs peels off layer by layer, the internal structure is damaged and fragmented, and the entire structure becomes a pulverized.

3.4. Effects of Different MPs on the Microbial Community of AD System

3.4.1. Responses of Bacterial and Archaeal Phylum Changes to MPs

To further understand the mechanism by which MPs affect methane production, the microbial communities of mixed samples from anaerobically digested fermentation broths on days 0, 4, and 28 were analyzed via 16S rRNA high-throughput sequencing. Figure 6a shows the phylum level distribution of the top 10 abundances of bacterial communities in the reactor at different ADs. The dominant phyla in the initial samples were Firmicutes (51.99%), Bacteroidota (13.78%), Synergistota (1.82%), and Actinobacteriota (12.62%), which play predominant roles in the hydrolysis and acid production of AD [43,44,45]. The addition of MPs resulted in a significantly higher relative abundance of Firmicutes (51.97–56.09%) than that of CK (47.71%) during the early stage of digestion (day 4) (p < 0.05). At the terminal stage (day 28), the relative abundance of Firmicutes of the four treatments with MPs (36.62–41.09%) was higher than that of CK (35.59%) with the addition of MPs. Firmicutes secrete extracellular enzymes that facilitate the degradation of cellulose, leading to its conversion into lipids and proteins, which are subsequently transformed into VFAs [46,47]. This suggests that MPs may increase and delay the hydrolysis of organic matter (Figure 2a,b), leading to the accumulation of VFAs (Figure 3). Bacteroidota and Synergistota are common hydrolyzing bacteria in AD. Particularly, Bacteroidota is known for their efficient degradation of large molecular weight carbohydrates to produce VFAs, and hold pivotal roles in the meta-bolism of propionate, butyrate, and acetate [42,48,49]. As anaerobic digestion proceeded, the abundance of Bacteroidota in each treatment gradually increased. This was because the degradation of substances became increasingly difficult in the later stage of digestion, and Bacteroidota had a strong degradability for the materials. On the 4th day of digestion, MPs significantly reduced the relative abundance of Bacteroidota by 10.41–13.94% (p < 0.05), indicating that the presence of MPs had a certain inhibitory effect on Bacteroidota. It is worth noting that Synergistetes can also decompose organic acids into acetate, CO2, and H2 [46,50]. The decrease in Synergistetes is unfavorable for the conversion of other organic acids into acetic acid, which explains to some extent why the acetic acid content in PHA on the 4th day was lower than that in the other three MPs treatment groups (Figure 3). Additionally, the activity of acetate kinase in PHA diminished (Figure 4a), consequently leading to a relatively diminished methane output. At the late stage of digestion (day 28), the relative abundance of Chloroflexi in PE was 1.69% higher than that of the control, whereas its relative abundance decreased by 4.14–4.96% in the PHA, PP, and PVC samples. Previous studies have reported that Chloroflexi can decompose various organic matter such as polysaccharides and proteins [51]. The enrichment of Firmicutes at the start and Chloroflexi toward the end explains the high cumulative methane production observed with PE.
The final step of AD involves methane production through the activity of archaea. In this study, the predominant phyla of the archaea in all samples were Halobacterota and Euryarchaeota (Figure 6b), which have been frequently reported in previous research on AD and biogas production [52]. Together, Halobacterota and Euryarchaeota accounted for 87.53–99.49% of the total archaeal sequences across different reactors during both stages. On day 4, PE, PP, and PVC exhibited higher relative abundances of Halobacterota than Euryarchaeota, whereas PHA showed a decrease in Halobacterota abundance at both time points (days 4 and 28). Generally, most members of the phylum Euryarchaeota are hydrogenotrophic methanogens, whereas those within the phylum Halobacterota are acetotrophic methanogens [38]. These findings suggest that PHA may inhibit acetate-utilizing methanogenic archaea.

3.4.2. Function of Archaea Genus During AD

The nonmetric multidimensional scaling (NMDS) analysis, as shown in Figure 6c, is commonly used to assess the differences between communities, and its accuracy is typically evaluated based on the stress value. A stress value below 0.1 indicates high model reliability, and in this study, the stress value was 0.026. Moreover, a closer proximity of sample points suggests greater similarity in species composition between samples. Notably, microbial communities located outside the circles exhibited significant differences (p < 0.05) than those within the circles, indicating that MPs had a significant impact on the distribution of microbial communities at different time points. In addition, a complete shift in methanogen composition was observed during digestion compared with the initial period, with significant changes observed as the digestion time increased. Moreover, on day 28 of digestion, the PHA treatment resulted in a significantly distinct flora composition compared with the other treatments during the same period. On days 4 and 28, PE treatment indicated considerable dissimilarity from CK treatment with large species variations noted, thus suggesting that PE MPs exerted a more pronounced effect on archaea composition than the other three MPs tested here. Collectively, these findings indicate that PE and PHA MPs have a notable influence on archaea.
Figure 6d shows the community distribution of the archaeal genera. The predominant archaeal genera in CK during the initiation phase (day 0) were Methanosaeta (41%), Methanobacterium (27.91%), Methanobrevibacter (18.98%), and Methanosarcina (5.27%). However, at the end of digestion (day 28), the dominant archaeal shifted to Methanosaeta (27.01%), Methanobacterium (25.20%), and Methanocorpusculum (31.12%). Notably, there was a transition from Methanosaeta dominance to Methanocorpusculum dominance within the system over time. Acetic acid-based methanogens (Methanosaeta) produce methane by catabolizing acetic acid, whereas methylotrophic methanogens (Methanocorpusculum) utilize methyl analogs, methylamine analogs, and methylsulfur compounds to produce methane. Consequently, there was a shift in the dominant pathway of systemic methanogenesis from acetate to methylotrophic.
The relative abundance of Methanosaeta showed an overall increasing and decreasing trend, with PE MPs increasing the enrichment of Methanosaeta, while PHA MPs greatly reduced the abundance of the archaeal. Methanosaeta can only utilize acetic acid as a substrate for methanogenesis and are strictly acetic acid-type methanogens [53]. The decrease in the abundance of Methanosaeta in PHA corresponds to a decrease in Halobacterota (Figure 6b) and indicates that the acetic acid methanogenic pathway in PHA is inhibited. Although an augmentation in the relative abundance of VFAs-producing bacteria was observed in PP, PHA, and PVC during the predigestion period (Figure 6a), giving rise to substantial VFA production (Figure 3), the abundance of Methanosaeta declined. This attenuation inhibits the acetic acid methanogenic pathway and curtails methane production. Methanobrevibacter is a predominant hydrogenotrophic methanogenic archaeal found in animal gastrointestinal systems [54]; hence, its initial high abundance subsequently decreased over time. On day 28, the highest abundance of Methanospirillum in PE was 3.47%. Methanospirillum could effectively improve AD performance by reducing the involvement of CO2 in direct interspecies electron transfer during methane metabolism [55,56]. This suggests that the enrichment of Methanospirillum in PE enhanced the stimulatory effect on methane production.

3.4.3. Correlation Analysis of Environmental Factors with Microbia

Herein, the significant correlation between environmental factors and microorganisms was characterized using Spearman’s correlation coefficient (Figure 7a). The results indicated a positive and significant association between Firmicutes, Actinobacteriota, Patescibacteria (top 10 bacterial phyla), hydrolysis products (SCODs and VFAs), and ACK, suggesting their potential involvement in the synthesis of acetic and propionic acids. Bacteroidota showed a significant negative correlation with SCOD, TOC, and VFAs, indicating that the VFA production from organic matter hydrolysis depended on the hydrolysis activity of Firmicutes and Actinobacteriota, as discussed in Section 3.4.1. Methanosaeta, Methanobrevibacter, and Methanosphaera were significantly correlated with VFAs, SCOD, and ACK, indicating that these three archaea play important roles in the acetic acid methanogenesis pathway. On days 4 and 28, the three types of archaeal PE increased by 6.78% and 8.65% compared with CK, while those of PHA decreased by 4.20% and 28.05% (Figure 6d), respectively. These findings indicated that PE MPs enhanced the acetic acid methanogenesis pathway, while PHA MPs had an inhibitory effect that is consistent with methane production trends observed earlier. Five genera, such as Methanocorpusculum, Methanospirillum, and Methanomassiliicoccus, were found to be significantly correlated with NH4+–N content, implying their potential involvement in nitrogen transformation.

3.5. Shifts in Microbial Enzyme Function

Figure 7b shows changes in the key enzyme pathways related to VFA generation and methanogenesis in the presence of PHA and PE. Acetyl-CoA carboxylase (EC: 6.4.1.2) and ACK (EC: 2.7.2.1) are the key enzymes in the main metabolic pathway of acetic acid production. The addition of PHA MPs reduces their expression levels. Acetyl-CoA carboxylase (EC: 6.4.1.2) is the central node of the VFA generation pathway, such as the metabolism of carbohydrates and pyruvate to produce acetic acid and butyric acid [57]. As shown in Figure 3, after day 4, the total VFA content was lower in PHA than in CK, indicating that PHA MPs inhibited the conversion of intermediate products such as pyruvate to VFAs. Furthermore, the genes annotated in the CO2 generation pathways, such as pyruvate formate-lyase (EC: 2.3.1.54) and anaerobic carbon monoxide dehydrogenase (EC: 1.2.7.4), were upregulated in response to the presence of PE MPs, leading to enhanced CO2 generation.
It is widely recognized that the methanogenesis process comprises two pathways: acetate decarboxylation and CO2 reduction [58]. In the acetic acid decarboxylation pathway, PE and PHA MPs inhibited the gene abundances of ACK (EC: 2.7.2.1) and phosphate acetyltransferase (EC: 2.3.1.8). Conversely, PE MPs significantly increased the abundance of acetate-CoA ligase (EC: 6.2.1.1). With respect to the CO2 reduction pathway, the presence of PE MPs increased the gene abundances of enzymes related to CO2 reduction compared with CK. However, PHA MPs exhibited inhibitory effects instead. The enzymes related to CO2 reduction include methenyltetrahydromethanopterin cyclohydrolase (EC: 3.5.4.27), methylenetetrahydromethanopterin dehydrogenase (EC: 1.5.98.1), and 5,10-methylenetetrahydromethanopterin reductase (EC: 1.5.98.2). Notably, PE MPs increased the gene abundance of methyl coenzyme M reductase (EC: 2.8.4.1) involved in the conversion of 5-Methyl-THM(S)PT to methane, whereas PHA MPs decreased its abundance. The impact of MPs on key enzymes during the methanogenesis stage outweighs their influence on the VFA generation stage. Based on changes observed in gene abundances for key enzymes in the presence of PE MPs, the related pathways involved in methanogenesis via the acetic acid pathway, as well as CO2 reduction, were both strengthened, leading to increased methane production. On the contrary, in the presence of PHA MPs, both pathways for methanogenesis were suppressed, ultimately leading to decreased methane production.

4. Conclusions

This study investigated the effects of degradable MPs (PHA) and non-degradable MPs (PE, PP, and PVC) on methane production through a 30-day AD experiment using cattle manure. The findings indicated that distinct types of MPs exerted diverse influences on methane production within cattle manure during the AD process. The PE MPs accelerated methane production by shortening the lag phase, while the PP, PHA, and PVC MPs inhibited methane production. Among them, PHA MPs had the most detrimental impact on the reaction system. The degradation rate of the four types of MPs followed the order of PHA > PVC > PE > PP, with PHA MPs showing more severe shape destruction owing to their degradability. The presence of MPs in livestock and poultry manure promoted organic matter dissolution, hydrolysis, and VFA generation in the initial stage of AD. However, it also suppressed ACK activity and enhanced LDH release at later stages, negatively affecting the reaction system. Furthermore, PE MPs increased the abundances of Firmicutes, Methanosaeta, and Methanospirillum, thereby increasing methane production. Conversely, PHA MPs decreased the abundance of the Methanosaeta and weakened the acetic acid methanogenesis pathway. Therefore, this study provides valuable insights into the influence of MP exposure on AD performance in livestock and highlights the importance of plastic management in livestock farming.
However, this study solely considered the type of cattle manure as the digestion substrate and the added MPs. No additional research was carried out regarding other livestock and poultry manures, as well as the particle size and concentration of MPs. To a certain extent, these limitations have impacted the generalizability of the research findings. In the future, the scope of research on digestion substrates can be broadened, and the particle size and concentration of MPs can be diversified for a more comprehensive investigation, thus offering stronger support for the development of this field.

Author Contributions

M.Z.: Conceptualization, formal analysis, writing—original draft. C.Z.: Conceptualization, resources, writing—review & editing. T.Y.: Conceptualization, resources, writing—review & editing. Q.W.: Conceptualization, resources, writing—review & editing. Q.Z.: Conceptualization, resources, writing—review & editing. S.Y.: methodology, validation, investigation. Y.C.: Conceptualization, funding acquisition. X.G.: Investigation, writing—review & editing. H.C.: Methodology, validation, investigation. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Major Special Science and Technology Projects of Shanxi Province (202301140601015), the Shanxi Agricultural University “Special” and “Excellent” Agricultural High Quality Development Science and Technology Support Project (No.TYGC24-03), Key R&D Projects in Shanxi Province (No.201903D211013), and the Shanxi Province University Student Innovation and Entrepreneurship Training Program (20220166).

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Acknowledgments

We thank the anonymous reviewers for their constructive comments.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Cumulative methane production from cattle manure at different MPs.
Figure 1. Cumulative methane production from cattle manure at different MPs.
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Figure 2. Effects of different MP exposures on the degradation of organic compounds (a) SCOD, (b) TOC, and (c) ammonia nitrogen.
Figure 2. Effects of different MP exposures on the degradation of organic compounds (a) SCOD, (b) TOC, and (c) ammonia nitrogen.
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Figure 3. Effects of different MP exposures on total VFAs (including acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, and valeric acid).
Figure 3. Effects of different MP exposures on total VFAs (including acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, and valeric acid).
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Figure 4. Effects of different MPs exposures on (a) ACK activity and (b) LDH release. Note: ACK, acetate kinase; LDH, lactate dehydrogenase.
Figure 4. Effects of different MPs exposures on (a) ACK activity and (b) LDH release. Note: ACK, acetate kinase; LDH, lactate dehydrogenase.
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Figure 5. SEM images of MPs before and after AD.
Figure 5. SEM images of MPs before and after AD.
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Figure 6. Relative abundances of (a) bacteria and (b) archaeal at phylum level; (c) Abundance of archaea at the Unweighted UniFrac metrics NMDS analysis; (d) archaeal community analysis genus level.
Figure 6. Relative abundances of (a) bacteria and (b) archaeal at phylum level; (c) Abundance of archaea at the Unweighted UniFrac metrics NMDS analysis; (d) archaeal community analysis genus level.
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Figure 7. (a) Spearman’s correlation between microorganisms and environmental factors. * 0.01 < p ≤ 0.05, ** 0.001 < p ≤ 0.01; (b) Heat map of relative abundance of acidification pathway and functional enzyme genes. (Relative abundance in ‰ (parts per thousand)).
Figure 7. (a) Spearman’s correlation between microorganisms and environmental factors. * 0.01 < p ≤ 0.05, ** 0.001 < p ≤ 0.01; (b) Heat map of relative abundance of acidification pathway and functional enzyme genes. (Relative abundance in ‰ (parts per thousand)).
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Table 1. Characteristics of cattle manure and inoculum.
Table 1. Characteristics of cattle manure and inoculum.
Samples pH EC
(mS/cm) 		TS
(%) 		VS
(%) 		Organic Carbon
(g/Kg)
Cattle manure7.68 ± 0.023.11 ± 0.0116.69 ± 0.3378 ± 0.23382.39 ± 20
Inoculum--12.18 ± 0.3029.45 ± 0.20-
Note: pH is potential of hydrogen, EC is electrical conductivity, TS is total solids, VS is volatile solids.
Table 2. Fitting parameters and degree of fitting of the Gompertz model.
Table 2. Fitting parameters and degree of fitting of the Gompertz model.
Different
Treatments 		Pmax
(mL) 		Rmax
(mL/d) 		λ
(d) 		R2
(%)
CK9510 ± 62585 ± 12−0.87 ± 0.1799.71
PP9278 ± 59572 ± 12−0.87 ± 0.1899.72
PVC9063 ± 59565 ± 12−0.9 ± 0.1799.70
PHA8793 ± 60544 ± 12−0.92 ± 0.1899.68
PE10,118 ± 71633 ± 14−0.55 ± 0.1899.67
Note: Pmax is the maximum methane production potential (mL), Rmax is the maximum methane production rate (mL/d), and λ and t are the lag times (d).
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Zhang, M.; Zhao, C.; Yuan, T.; Wang, Q.; Zhang, Q.; Yan, S.; Guo, X.; Cao, Y.; Cheng, H. Effects of Different Microplastics on Methane Production and Microbial Community Structure in Anaerobic Digestion of Cattle Manure. Agronomy 2025, 15, 107. https://doi.org/10.3390/agronomy15010107

AMA Style

Zhang M, Zhao C, Yuan T, Wang Q, Zhang Q, Yan S, Guo X, Cao Y, Cheng H. Effects of Different Microplastics on Methane Production and Microbial Community Structure in Anaerobic Digestion of Cattle Manure. Agronomy. 2025; 15(1):107. https://doi.org/10.3390/agronomy15010107

Chicago/Turabian Style

Zhang, Mengjiao, Congxu Zhao, Tian Yuan, Qing Wang, Qiuxian Zhang, Shuangdui Yan, Xiaohong Guo, Yanzhuan Cao, and Hongyan Cheng. 2025. "Effects of Different Microplastics on Methane Production and Microbial Community Structure in Anaerobic Digestion of Cattle Manure" Agronomy 15, no. 1: 107. https://doi.org/10.3390/agronomy15010107

APA Style

Zhang, M., Zhao, C., Yuan, T., Wang, Q., Zhang, Q., Yan, S., Guo, X., Cao, Y., & Cheng, H. (2025). Effects of Different Microplastics on Methane Production and Microbial Community Structure in Anaerobic Digestion of Cattle Manure. Agronomy, 15(1), 107. https://doi.org/10.3390/agronomy15010107

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