Impact of aging of primary and secondary polystyrene nanoplastics on the transmission of antibiotic resistance genes in anaerobic digestion.

Sewage sludge is a significant reservoir of nano/microplastics (NPs/MPs) and antibiotic resistance genes (ARGs). Research has revealed that NPs/MPs may exert an inhibitory effect on anaerobic digestion (AD) of sludge. Moreover, NPs/MPs can influence microbial community diversity and composition, potentially increasing ARGs dissemination. The morphological changes to NPs/MPs surface due to aging contribute to modifying hydrophobic properties. To date, there is limited comprehension regarding how various surface properties of NPs influence ARGs dissemination during AD. This study investigated the impact of primary aged/non-aged and secondary aged/non-aged polystyrene nanoplastics (PSNPs) on ARGs and mobile genetic elements (MGEs) propagation during AD. The findings indicated that the UV-aging process resulted in surface oxidation and distinct morphological characteristics in both primary and secondary PSNPs, while the surface oxidation effect was more pronounced in the secondary aged PSNPs. High concentrations (150 µg/L) of primary and secondary PSNPs inhibited methane production, with secondary PSNPs causing greater inhibition by 16 to 20 % compared to control. In contrast, low concentration (25 µg/L) had negligible or slightly positive effects on methane production. PSNPs at 150 µg/L reduced total VFA concentration, indicating an inhibitory effect on the fermentation step in the AD process. Primary and secondary PSNPs exhibited changes in EPS characteristics. ARGs abundance was enriched in reactors amended with PSNPs, with the highest abundance of 8.54 × 105 copies/g sludge observed in the secondary aged PSNPs (150 µg/L) reactor. Reactors exposed to aged PSNPs exhibited a relatively higher abundance of ARGs compared to reactors exposed to non-aged PSNPs. Exposure to PSNPs increased the microbial community diversity within the digesters and triggered the enrichment of Comamonadaceae and Syntrophaceae, belonging to Proteobacteria phylum. On the other hand, archaeal communities tended to shift towards hydrogenotrophic methanogens in PSNPs reactors. The correlation analysis showed that Comamonadaceae were positively correlated with the majority of ARGs and intl1. A positive correlation was observed between MGEs and most ARGs, suggesting that the increased proliferation of ARGs under PSNPs exposure may be linked to the abundance of MGEs, which in turn promotes the growth of hosts carrying ARGs. These findings suggest that aged and non-aged NPs could substantially impact the spread of ARGs and MGEs, which also led to notable alterations in the composition of the microbial community. Overall, this study provides valuable insights into the multifaceted impacts of PSNPs with various characteristics on AD processes, microbial communities, and ARGs proliferation, highlighting the urgent need for comprehensive assessments of NPs pollutants in the environment.

Pilot-scale investigation of conductive carbon cloth amendment for enhancing high-solids anaerobic digestion and mitigating antibiotic resistance.

This study examined the effectiveness of introducing conductive carbon cloth into a pilot-scale high-solids anaerobic digestion (HSAD) system. Adding carbon cloth increased methane production by 22 % and improved the maximum methane production rate by 39 %. Microbial community characterization indicated a possible direct interspecies electron transfer-based syntrophic association among microbes. Using carbon cloth also enhanced microbial richness, diversity, and evenness. Carbon cloth effectively reduced the total abundance of antibiotic resistance genes (ARGs) by 44.6 %, mainly by inhibiting horizontal gene transfer, as shown by the significant decrease in the relative abundance of integron genes (particularly intl1). The multivariate analysis further demonstrated strong correlations of intl1 with most of the targeted ARGs. These findings suggest that carbon cloth amendment can promote efficient methane production and attenuate the spread of ARGs in HSAD systems.

Antibiotic resistance genes proliferation under anaerobic degradation of polylactic acid and polyhydroxy butyrate bioplastics.

As the global concern over plastic pollution grows, efforts are underway to find environmentally friendly alternatives to traditional plastics. Bioplastics are being extensively researched and developed as a possible solution. This study compared the impact of two bioplastics, polylactic acid (PLA) and polyhydroxy butyrate (PHB), on the proliferation of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) during anaerobic digestion (AD). Both bioplastics (250-500 particles) could be degraded to a certain extent over 79 days, as indicated by higher methane production than the control without bioplastic particles. The PHB 500 reactor showed the highest methane yield along with the highest biodegradation efficiency (91 %) than other reactors amended with PHB and PLA particles. The highest ARG and MGE abundances were also observed in PLA 500, and the lowest ARG abundance was in PLA 250. Conversely, PHB reactors showed a relatively lower ARG abundance than the control. The correlation analysis suggested that most ARGs were positively correlated with PLA and negatively correlated with PHB (except for tetA, tetB, and tetX). Moreover, a correlation between MGEs and ARGs in PLA and PHB reactors was revealed by correlation analysis. These results show that AD responds differently to the different types/levels of bioplastics, which can ultimately influence the behavior of ARG proliferation. Thus, bioplastics may also pose a potential risk for spreading antibiotic resistance. These findings can be used as a basis for setting environmental standards for bioplastics and creating monitoring and control measures to prevent potential negative impacts on public health.

Granular activated carbon remediates antibiotic resistance propagation and methanogenic inhibition induced by polystyrene nanoplastics in sludge anaerobic digestion.

Nano/microplastics (NPs/MPs) in sewage sludge can induce oxidative stress to the anaerobic digestion (AD) and also proliferate antibiotic resistance genes (ARGs). Recently, granular activated carbon (GAC) has been used as an additive to enhance methane production in AD via direct interspecies electron transfer (DIET); however, its impact on AD exposed to NPs/MPs is yet to be studied. This study examined the effect of GAC (5 and 15 g/L) on sludge AD exposed to 150 µg/L of polystyrene nanoplastics (PsNPs). PsNPs decreased methane yield by 32.3% due to elevated levels of reactive oxygen species. However, GAC addition counteracted this adverse effect and improved methane production, attributed to the potential enrichment of DIET-active microbes and the adsorption of PsNPs by GAC. Moreover, GAC reduced the total abundance of ARGs, which was increased by PsNPs exposure. Thus, GAC can provide dual benefits in mitigating methanogenic inhibition caused by PsNPs and ARG spread.

Fate of intracellular, extracellular polymeric substances-associated, and cell-free antibiotic resistance genes in anaerobic digestion of thermally hydrolyzed sludge.

Thermal hydrolysis of sludge is a promising approach to mitigate antibiotic resistance genes (ARGs) propagation in anaerobic digestion (AD). Although ARGs in sludge may be fractioned into intracellular, extracellular polymeric substance (EPS)-associated, and cell-free ARGs, the fate of these different fractions in AD has never been investigated. This study presents a detailed characterization of intracellular and extracellular ARGs in AD of sludge thermally hydrolyzed at 90 °C and 140 °C. EPS-associated ARGs represented the major fraction of the total extracellular ARGs in all samples, while its lowest abundance was observed for thermal hydrolysis at 140 °C along with the lowest EPS levels. The results suggested a positive correlation between EPS-associated ARGs with intracellular and cell-free ARGs. Furthermore, various EPS components, such as proteins and e-DNA, were positively correlated with ß-lactam resistance genes. sul1 dominated all samples as an EPS-associated resistance gene. These results provide new insights into the significance of different ARGs fractions in their overall dissemination in AD integrated with thermal hydrolysis.

Exploration of machine learning algorithms for predicting the changes in abundance of antibiotic resistance genes in anaerobic digestion.

The land application of digestate from anaerobic digestion (AD) is considered a significant route for transmitting antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) to ecosystems. To date, efforts towards understanding complex non-linear interactions between AD operating parameters with ARG/MGE abundances rely on experimental investigations due to a lack of mechanistic models. Herein, three different machine learning (ML) algorithms, Random Forest (RF), eXtreme Gradient Boosting (XGBoost), and Artificial Neural Network (ANN), were compared for their predictive capacities in simulating ARG/MGE abundance changes during AD. The models were trained and cross-validated using experimental data collected from 33 published literature. The comparison of model performance using coefficients of determination (R2) and root mean squared errors (RMSE) indicated that ANN was more reliable than RF and XGBoost. The mode of operation (batch/semi-continuous), co-digestion of food waste and sewage sludge, and residence time were identified as the three most critical features in predicting ARG/MGE abundance changes. Moreover, the trained ANN model could simulate non-linear interactions between operational parameters and ARG/MGE abundance changes that could be interpreted intuitively based on existing knowledge. Overall, this study demonstrates that machine learning can enable a reliable predictive model that can provide a holistic optimization tool for mitigating the ARG/MGE transmission potential of AD.

A critical review of process parameters influencing the fate of antibiotic resistance genes in the anaerobic digestion of organic waste.

The overuse and inappropriate disposal of antibiotics raised severe public health risks worldwide. Specifically, the incomplete antibiotics metabolism in human and animal bodies contributes to the significant release of antibiotics into the natural ecosystems and the proliferation of antibiotic-resistant bacteria carrying antibiotic-resistant genes. Moreover, the organic feedstocks used for anaerobic digestion are often highly-rich in residual antibiotics and antibiotic-resistant genes. Hence, understanding their fate during anaerobic digestion has become a significant research focus recently. Previous studies demonstrated that various process parameters could considerably influence the propagation of the antibiotic-resistant genes during anaerobic digestion and their transmission via land application of digestate. This review article scrutinizes the influences of process parameters on antibiotic-resistant genes propagation in anaerobic digestion and the inherent fundamentals behind their effects. Based on the literature review, critical research gaps and challenges are summarized to guide the prospects for future studies.

Propagation of antibiotic resistance genes during anaerobic digestion of thermally hydrolyzed sludge and their correlation with extracellular polymeric substances.

The positive impact of the thermal hydrolysis process (THP) of sewage sludge on antibiotic resistance genes (ARGs) removal during anaerobic digestion (AD) has been reported in the literature. However, little information is available on how changes in different extracellular polymeric substances (EPS) due to THP can influence ARG propagation during AD. This study focused on systematically correlating EPS components and ARG abundance in AD of sewage sludge pretreated with THP (80 °C, 110 °C, 140 °C, 170 °C). THP under different conditions improved sludge solubilization followed by improved methane yields in the biochemical methane potential (BMP) test. The highest methane yield of 275 ± 11.5 ml CH4/g COD was observed for THP-140 °C, which was 40.5 ± 2.5% higher than the control. Increasing THP operating temperatures showed a non-linear response of ARG propagation in AD due to the rebound effect. The highest ARGs removal in AD was achieved with THP at 140 °C. The multivariate analysis showed that EPS polysaccharides positively correlated with most ARGs and integrons, except for macrolides resistance genes. In contrast, EPS protein was only strongly correlated with ß-lactam resistance genes. These results suggest that manipulating THP operating conditions targeting specific EPS components will be critical to effectively mitigating the dissemination of particular ARG types in AD.