Sludge Anaerobic Digestion Research Articles

Studies on the treatment of anaerobically digested sludge by white-rot fungi: evaluation of the effect of Phanerochaete chrysosporium and Trametes versicolor

BackgroundThe composition of anaerobically digested sludge is inherently complex, enriched with structurally complex organic compounds and nitrogenous constituents, which are refractory to biodegradation. These characteristics limit the subsequent rational utilization of resources from anaerobically digested sludge. White-rot fungi (WRF) have garnered significant research interest due to their exceptional capacity to degrade complex and recalcitrant organic pollutants. However, the exploration of WRF in the context of sludge treatment remains an under-investigated area within the scientific community. The present investigation explores the application of WRF in the treatment of anaerobically digested sludge, offering a novel approach for the valorization of sludge resources.ResultsIn this study, WRF enzymes, manganese peroxidase (MnP) and lignin peroxidase (LiP), exhibited sustained high activities of approximately 102 U/L and 26 U/L, respectively, within the anaerobically digested sludge under a controlled pH of 5.5 within the growth system. These conditions were found to significantly enhance the treatment efficacy of the anaerobic sludge. The removal of soluble chemical oxygen demand (COD) and Total COD by Trametes versicolor powder was better than that of Phanerochaete chrysosporium powder. The treatment of sludge samples with WRF, specifically Phanerochaete chrysosporium powder, resulted in a significant reduction of ultraviolet radiation (UV254). Fourier-transform infrared spectroscopy (FTIR) analysis revealed that the application of Trametes versicolor powder exerted a notably pronounced impact on the functional groups present in sludge samples. Specifically, there was a significant decrease in the peak intensities corresponding to the C-O bonds, indicative of saccharide degradation, alongside an observable increase in the intensities of amide peaks, which is suggestive of protein synthesis enhancement. Microbial community analysis demonstrated that Phanerochaete chrysosporium was the predominant fungal species, exerting a significant regulatory role within the sludge ecosystem.ConclusionIn conclusion, this research furnishes a robust scientific foundation for the utilization of WRF in the treatment of anaerobic digestion sludge. It elucidates the fungi’s capacity to ameliorate the physicochemical attributes and microbial community composition within the sludge. Furthermore, the study offers a certain reference for the subsequent use of WRF in the treatment of other types of sludge.

Metagenomic analysis reveals the effects of potassium ferrate and steel slag on fate of ARGs in anaerobic sludge digestion system.

Waste activated sludge (WAS) pose a potential risk for the spread of antibiotic resistance genes (ARGs). This study estimated the effect of sludge on antibiotic resistance genes (ARGs) in anaerobic sludge digestion process. Metagenomic analysis revealed anaerobic sludge with potassium ferrate (PF) and the modified PF loaded steel slag (MPF-SS) brought an increase of ARGs during digestion process. PF was found to effectively reduce most of the high-risk ARGs (i.e., acrB and mexW). Furthermore, network and correlation analysis among ARGs and genera verified that PF significantly increased the potential ARGs hosts. Mechanistic analysis revealed that PF induced oxidative stress behavior of anaerobic digestion microorganisms, and observably upregulated the relative genes about SOS response-related. These findings provide insights into the mechanism underlining PF for ARGs fate and its risk during anaerobic sludge digestion, which could offer practical guidance on the sustainable management of WAS.

Proximity-Ligation Metagenomic Sequence Analysis Reveals That the Antibiotic Resistome Makes Significant Transitions During Municipal Wastewater Treatment.

Shotgun and proximity-ligation metagenomic sequencing were used to generate thousands of metagenome assembled genomes (MAGs) from the untreated wastewater, activated sludge bioreactors, and anaerobic digesters from two full-scale municipal wastewater treatment facilities. Analysis of the antibiotic resistance genes (ARGs) in the pool of contigs from the shotgun metagenomic sequences revealed significantly different relative abundances and types of ARGs in the untreated wastewaster compared to the activated sludge bioreactors or the anaerobic digesters (p < 0.05). In contrast, these results were statistically similar when comparing the ARGs in the pool of MAGs, suggesting that proximity-ligation metagenomic sequencing is particularly useful for pairing ARGs with their hosts but less adept at discerning quantitative differences in ARG types and relative abundances. For example, numerous MAGs of the genera Acinetobacter, Enterococcus, Klebsiella and Pseudomonas were identified in the untreated wastewater, many of which harboured plasmid-borne and/or chromosomal-borne ARGs; none of these MAGs, however, were detected in the activated sludge bioreactors or anaerobic digesters. In conclusion, this research demonstrates that the antibiotic resistome undergoes significant transitions in both the relative abundance and the host organisms during the municipal wastewater treatment process.

Sustainable utilization of plastic-derived graphene for tetracycline wastewater treatment and its recycling for biogas and biochar production

While several studies have used plastic-derived graphene (PDG) for antibiotics adsorption from wastewater, there is a research gap in finding an environmental-friendly and technically feasible approach for the recycling of exhausted adsorbent. Hence, this study focuses on the utilization of PDG as an adsorbent for tetracycline removal from aqueous solutions, followed by the valorization of spent PDG for dual biogas and biochar production. In the first experiment, a tetracycline removal efficiency of 75.61 % was obtained under the optimum condition of PDG dosage = 0.03 mg/mL, initial concentration = 5.20 ppm, 77.0 min-adsorption time, and acidic pH. Supplementing the regenerated PDG to the anaerobic digestion of sludge and ethanol exhibited bio-CH4 yield and chemical oxygen demand (COD) removal efficiency of 200.60±9.40 mL/g COD and 57.11±3.01 %, respectively. The recycling of anaerobic digestate by pyrolysis showed a biochar yield of 0.58±0.09 g/g, with a high carbon content of 55.34 % w/w. The scalability of this adsorption/digestion/pyrolysis approach for managing 1 kg PDG could be economically feasible with a payback period of 5.02 years, NPV = 297.62 USD, and internal rate of return = 10 %. This project showed less detrimental impacts on the environment, regarding the life cycle assessment (LCA) impact categories related to terrestrial ecosystem and resource recovery.

Geographical Distribution and Risk of Antibiotic Resistance Genes in Sludge Anaerobic Digestion Process across China

Geographical Distribution and Risk of Antibiotic Resistance Genes in Sludge Anaerobic Digestion Process across China

Deciphering the potential mechanisms and influencing factors of the effects of micro(nano)plastics on microbe in sludge anaerobic digestion system

Deciphering the potential mechanisms and influencing factors of the effects of micro(nano)plastics on microbe in sludge anaerobic digestion system

INFLUENCE OF WASTEWATER TREATMENT TECHNOLOGY AND SLUDGE STABILIZATION ON THE SORPTION CAPACITY OF SEWAGE SLUDGE

This article investigates the influence of sewage sludge treatment processes and stabilizationmethods on metal-binding capacity and susceptibility to metal remobilization from sewagesludge. The materials (stabilized sewage sludge) were collected from two mechanical-biologicalsewage treatment plants located in an industrial area in southern Poland (Silesian Province):1) one with nutrient removal (nitrogen and phosphorus) and anaerobic sludge digestion (WWTPIII),and 2) a facility without nutrient removal and with aerobic sludge stabilization (WWTP-II).Concentrations of PTEs in sewage sludge samples were measured using the inductively coupledplasma optical emission spectrometry (ICP-OES). Sewage sludge from WWTP-III showeda significantly higher sorption capacity for Cu ions (20–50%) and Ni ions (50% to 2.5-fold) ascompared to sewage sludge from WWTP-II. However, sewage sludge from WWTP-II exhibiteda higher sorption capacity for Cd ions. The studied potentially toxic elements (PTEs), irrespectiveof anion type, formed unstable bonds with sewage sludge, which may lead to their desorption intothe environment. The high mobility of Cd, Cu, Ni and Zn ions in chloride and sulphate system, aswell as the low susceptibility to Cr ion release, should be considered in various applications of thetested sewage sludge.

Metagenomic Analysis Reveals the Effects of Microplastics on Antibiotic Resistance Genes in Sludge Anaerobic Digestion.

Sewage sludge is recognized as both a source and a reservoir for antibiotic resistance genes (ARGs). Within an anaerobic digestion (AD) system, the presence of microplastics (MPs) has been observed to potentially facilitate the proliferation of these ARGs. Understanding the influence of MPs on microbial behavior and horizontal gene transfer (HGT) within the AD system is crucial for effectively managing the dissemination of ARGs in the environment. This study utilized metagenomic approaches to analyze the dynamics of various types of ARGs and potential microbial mechanisms under exposure to MPs during the AD process. The findings indicated that MPs in the AD process can enhance the proliferation of ARGs, with the extent of this enhancement increasing with the dosage of MPs: polyethylene (PE), polyethylene terephthalate (PET), and polylactic acid (PLA) MPs increased the abundance of ARGs in the anaerobic digestion system by up to 29.90%, 18.64%, and 14.15%, respectively. Additionally, the presence of MPs increased the relative abundance of mobile genetic elements (MGEs) during the AD process. Network correlation analysis further revealed that plasmids represent the predominant category of MGEs involved in the HGT of ARGs. Propionibacterium and Alicycliphilus were identified as the primary potential hosts for these ARGs. The results of gene function annotation indicated that exposure to MPs led to an increased the relative abundance of genes related to the production of reactive oxygen species (ROS), alterations in membrane permeability, ATP synthesis, and the secretion of extracellular polymeric substances (EPS). These genes play crucial roles in influencing the HGT of ARGs.

A comparison of radio frequency and microwave heating methods for enhancing the removal and biotransformation of endocrine-disrupting triclocarban during advanced anaerobic digestion

Anaerobic digestion (AD) has very limited effectiveness against the removal of many emerging contaminants, including the pervasive antimicrobial triclocarban (TCC). This is the first study to compare two thermal pretreatment methods to evaluate the fate of persistent TCC and its transformation/degradation by-products during advanced AD. Two electromagnetic heating methods are employed: one uses an innovative radio frequency (RF) heating system at 13.56 MHz, while the other uses microwave (MW) heating at 2450 MHz. The biodegradation was assessed by biochemical methane potential (BMP) assays utilizing spiked secondary sludge. Overall, advanced digestion processes, combined with RF and MW pretreatments, were highly effective in increasing TCC elimination (up to 65 %) from digested sludge after 60 days of incubation, compared to conventional digestion (without pretreatment), which achieved only up to 20 % elimination. The contribution of RF and MW pretreatments to the total TCC removal efficiencies of the overall advanced digestion process was 26 ± 1 % and 22 ± 1 %, respectively. After pretreatment, during thermophilic anaerobic digestion, 34 ± 3 % and 30 ± 2 % additional TCC removals were achieved from the RF- and MW-pretreated samples, respectively. Significantly higher TCC removal and biotransformation occurred in advanced BMPs incubated at thermophilic temperatures compared to mesophilic temperatures. The chlorinated TCC was converted to less toxic carbanilide during digestion, reducing the environmental risk of TCC through the dechlorination mechanism. Considering the feasibility of RF and MW heating systems at full scale, the RF heating technology combined with anaerobic sludge digestion can be preferred to manage the municipal sludge contaminated with antimicrobials.

Trace concentrations of graphene oxide and magnetic graphene oxide rescue anaerobic municipal sludge digesters under stress

This study evaluated long-term performance of graphene oxide (GO) and magnetic GO (MGO) nanosheets in semi-continuous-flow anaerobic digestion (AD) of municipal sludge over 230 days. At organic loading rates (OLRs) of 2.6 and 3.4 g chemical oxygen demand (COD)fed/L/day, 20 and 200 mg/L of GO and MGO did not affect AD performance. However, at an OLR of 5.2 g CODfed/L/day, where control digesters failed, 20 and 200 mg/L of GO and MGO sustained biogas yields at 190 mL/g CODfed/day, similar to yields at lower OLRs (2.6 and 3.4 g CODfed/L/day). This performance persisted after thedaily nanosheet replenishment stopped. Improvements were due to enhanced conductivity and microbial syntropy. The results showed a strong correlation with previous biochemical methane potential (BMP) assays, positioning BMPs as a predictive tool for continuous-flow AD performance. Overall, this study demonstrated potential of GO/MGO nanosheets to improve the stability and efficiency of AD systems under stress.

Simultaneous devulcanization and denitrification: a novel approach for valorization of both ground tire rubber and nitrate-containing wastewater.

This study focused on a new approach for valorization of both ground tire rubber (GTR) and nitrate-containing wastewater via simultaneous devulcanization and denitrification. Initially, sulfur-based autotrophic denitrifiers were successfully enriched from three different seed sludge sources, biological nutrient removal (BNR) sludge, anaerobic digester sludge and BNR sludge of a leather organized industrial zone WWTP. Average nitrate removal efficiencies were 96-98%. Biological devulcanization (biodevulcanization) of GTR was later investigated with these enriched cultures. Results revealed that biodevulcanization was only achieved with the culture enriched from BNR sludge of the leather organized industrial zone WWTP, as 3.9% sulfur removal (desulfurization efficiency). Metal sulfate precipitation was speculated to cause an underestimation of the desulfurization ratio. Fourier-transform infrared spectroscopy (FTIR) results demonstrated a decrease in the intensity of the C-S bonds and an increase in intensity of S-O bonds in treated GTR samples. This was attributed to the oxidation of sulfidic crosslinks, i.e. verification of biodevulcanization. This study indicated that simultaneous biodevulcanization and denitrification could be a promising process for valorization of both GTR and nitrate-containing wastewater which in turn would support circular economy and sustainable development.

Technical–Economic Analyses of Electric Energy Generation by Biogas from Anaerobic Digestion of Sewage Sludge from an Aerobic Reactor with the Addition of Charcoal

This study aimed to obtain the energy recovery potential of the biogas produced from anaerobic digestion (AD) of the sludge from a wastewater treatment plant (WWTP), including the use of biochar as an additive for substrate co-digestion and catalyst for methane production. We carried out the following steps: chemical–physical laboratory analyses of sludge samples; the building, operation, and monitoring of an experimental prototype of a batch bioreactor of 2.5 L for the AD of the sludge (with and without the addition of charcoal); qualitative measurements of biogas; the study of charcoal morphology; and the projection of useful energy generation from the AD sludge after treatment. A study on the economic viability and avoided greenhouse gas (GHG) emissions was performed based on the experimental results. The substrate showed alterations in all the physicochemical parameters evaluated after AD, such as a reduction of 35% in the biochemical oxygen demand (BOD) analysis; the experiment carried out using biochar showed positive results regarding the speed of CH4 production and a greater potential for energy recovery. Enterprises from 2000 kW onwards would present an internal rate of return (IRR) equal to or higher than the minimum attractiveness rate (MAR) of 15%. The USD 95.28/MWh tariff presented economic feasibility for the studied scenarios. WWTPs that produce enough sludge to generate power of 2000 kW would need to process the waste of 117,200 inhabitants with charcoal addition and 136,000 without charcoal. It would be possible to avoid the emission of 2307.97 tCO2/year (2000 kW). According to the results obtained, this study revealed that using alternative energies based on anaerobic digestion and biochar can generate positive results regarding methane production, and its application as an energy source in a WWTP proved to be economically viable at a specific level of power production.

A review on the recovery of humic substances from anaerobic digestate of sludge as a potential fertilizer: Quantification, efficiency and interaction with pollutants.

This study reviewed the recovery of humic substances (HS) from anaerobic digestate of sludge as a potential fertilizer, focusing on the quantification of HS, the efficiency of HS recovery, and its interaction with pollutants. The potential pitfalls of current misunderstanding for HS quantification in sludge were pointed out. HS present in sludge showed potential to be used as a fertilizer, which solubilized insoluble phosphates for enhanced soil fertility. Pretreatment prior to anaerobic sludge digestion and composting of anaerobic digestate of sludge (ADS) improved the amount of HS in anaerobic digestate. But the amount of HS extracted from the retentate of ADS was much lower than the level required for the common HS fertilizer. Therefore, an additional concentration was required to process the retentate of ADS into HS liquid fertilizer. The quinone moieties in HS accepted electrons from anaerobic microbial respiration and their role in the degradation of organic pollutants were summarized. By binding with HS, the speciation of metals in sludge was changed from water-soluble and exchangeable to organic- and sulfide-bound fractions. Future research should focus on the novel application of machine learning for quantifying HS within sludge, offering a practical approach to interpret complex fluorescence data and enhance understanding of HS characteristics and distribution. Further studies should explore the application of hydrothermal humification to enhance HS content in ADS, offering an energy-efficient method for rapid fertilization in sustainable agriculture. This study offers a window into HS recovery from ADS.

Potentialities of semi-continuous anaerobic digestion for mitigating antibiotics in sludge.

The behavior and removal of roxithromycin (ROX), oxytetracycline (OTC), chlortetracycline (CTC), and enrofloxacin (ENR) were investigated during the steady state of sludge anaerobic digestion (AD) in semi-continuous mode (37°C). Sludge was spiked at realistic concentrations (50μg/L of each antibiotic) and then used to feed the bioreactor for 80days. Antibiotics were extracted from the substrate and digested sludge samples by accelerated solvent extraction (ASE). Accurate determination of antibiotics was obtained by the standard addition method (SAM) associated with the liquid chromatography-tandem mass spectrometry (LC-MS/MS). The presence of antibiotics at a concentration of 2.5μg/g TS had no inhibitory effects on methane (CH4) production, total and volatile solids (TS and VS) removal as well as chemical oxygen demand (COD) removal. During the steady-state, antibiotics were removed significantly by 50, 100, and 59% respectively for the ROX, OTC, and CTC. Furthermore, ENR removal was not statistically significant and was estimated at 36%. This study highlighted that AD process could partially remove parent compounds, but ROX, CTC, and ENR persisted in the digested sludge. Hence, AD could be considered as a sludge treatment for mitigating, but not suppressing, the release of antibiotics through sludge application.

Mechanistic insights into activation of peracetic acid by sludge biogas residue biochar for efficient sulfamethoxazole degradation in aqueous solution

The application of peracetic acid (PAA) in the advanced oxidation process has been demonstrated to be an effective approach for treating aqueous organic pollutants. In this study, it is the first time that biogas residue biochar (BRBC) derived from sludge anaerobic digestion plants was prepared and used as a PAA activator for sulfamethoxazole (SMX) degradation. The optimal SMX removal could achieve 92 % within 120 min under acidic conditions. The SMX degradation was slightly enhanced in the presence of Cl−, while it could be inhibited by HCO3−. Quenching experiment and EPR analysis demonstrated that both radical and non-radical processes contributed to SMX degradation. ECOSAR analysis showed a significant reduction in intermediate toxicity. Meanwhile, BRBC700 exhibited excellent reusability and stability even in real water matrices. The study presented an innovative approach for biogas residue application and provided a novel pretreatment for SMX-containing wastewater for further biological treatment method after simple acid-base regulation.

Total ammonia removal from anaerobic digestion effluents of municipal sewage sludge using Nordic microalgae

The treatment of organic waste using anaerobic digestion is a promising and well-matured organic waste management method. However, the effluent from anaerobic digestion has a significant discharge risk due to its high ammonium content. Microalgae could be a valuable solution to remove this nitrogen. This work aimed at evaluating the growth of three Nordic microalgae strains (Chlorella vulgaris, Chlorococcum sp. and Coelastrella sp.) in different concentrations of effluent from anaerobic digestion of municipal sewage sludge. None of the strains was able to grow in effluent diluted two times (X2) or three times (X3) due to the high ammonium content (600 and 400 mg L−1, respectively). While Chlorococcum sp. showed a lag phase of 7 and 11-days in 5 times (X5) and 7 times (X7) diluted effluent, respectively, this strain demonstrated 53 % and 86 % total ammonia nitrogen (TAN) removal efficiency after 15 days; in X10 its TAN removal was 100 %. Without any lag phase Coelastrella sp. showed the same TAN removal efficiencies in X5 and X7 as Chlorococcum sp. However, C. vulgaris had the highest TAN removal in X5 (90%) and X7 (90%). Furthermore, this strain showed the highest amount of biomass dry weight production in all media (1.1 g L−1 in X5). Therefore, C. vulgaris and Chlorococcum sp. are promising candidates for nitrogen removal and sustainable algae biomass production, resulting in mitigating the environmental issues of anaerobic digestion effluents in Nordic countries through the conversion of waste streams into resources.

Evaluating the Performance of Anaerobic Digestion with Upstream Thermal Hydrolysis—What Role Does the Activated Sludge Process Play?

The performance of anaerobic digestion of mixed sludge (MS) with upstream thermal hydrolysis of waste activated sludge (WAS) was evaluated and compared to conventional anaerobic digestion. In contrast to previous studies, this work focuses on the evaluation of the impact of the activated sludge process, which was assessed using a temperature-normalized solids retention time (SRTASP,T). For this purpose, data from a full-scale wastewater treatment plant related to SRTASP,T, primary sludge (PS) and WAS production were combined with experimental data from laboratory-scale anaerobic digestion of PS, WAS, thermally hydrolyzed WAS, and MS. The parameter SRTASP,T was used as a key link between the full-scale and experimental data. For WAS, SRTASP,T essentially influenced the efficacy of thermal hydrolysis on the performance of anaerobic digestion. The increase in methane yield was higher with increasing SRTASP,T. When considering MS, however, the increase was significantly lower and leveled out over the investigated range of SRTASP,T, mainly due to corresponding WAS/MS ratios. This study demonstrates that the knowledge of SRTASP,T, sludge production, and anaerobic degradability enables the assessment of the potential of thermal hydrolysis and its effect on anaerobic digestion.

Assessing the Amending and Fertilizing Properties of Digestates From Anaerobic Digestion of Faecal Sludge in Burkina Faso, West Africa

The aim of this work is to contribute to the agronomic utilization of digestate from the anaerobic treatment of faecal sludge at the Office National de l&amp;rsquo;Eau et de l&amp;rsquo;Assainissement (ONEA) Kossodo plant in Burkina Faso. Digestates were sampled and dried in the laboratory (25&amp;plusmn;2 &amp;deg;C). Physico-chemical and microbiological parameters were determined using standard methods. The degree of maturity of the digestate was determined using respirometry and humification tests. The effects of digestate on plants and on environment were studied using phytotoxicity, emergence, growth tests and microbiological analysis. The results revealed a pH of 8.54, electrical conductivity of 7300 &amp;micro;S/cm, 25.42% dry matter, 83.31% organic matter, and 489.8 g/kg total organic carbon. The C/N ratio was 9.7 and the NO3/NH4 ratio was 23.34, indicating mature compost despite other indicators suggesting that the digestate was only partially stabilized. Nutrient contents were 50.48 g/kg total nitrogen, 0.02 g/kg total phosphorus, and 4.72 g/kg total potassium. Water-soluble minerals included 1.97 g/kg ammonium, 45.99 g/kg nitrate, 0.02 g/kg nitrite, 0.35 g/kg phosphate, and 5.90 g/kg sulfate. Phytotoxicity tests showed that cabbage seeds had a germination rate of 89% with digestate filtrate compared to 100% in the control, while tomato seeds had a 100% germination rate with digestate filtrate. The germination indices were 137.58 for tomato and 82.10 for cabbage. Tests on tomato plant growth showed significant effects on biomass, height, leaf number, and branch number, with the 50% digestate proportion demonstrating the highest values. Microbiological analysis detected total aerobic flora at 4.75E+07 CFU/g, yeasts and molds at 7.77E+04 CFU/g, and coliforms at various levels, with potential pathogens such as S. aureus and Salmonella sp. reaching 6.14E+04 and 2.65E+06 CFU/g respectively. The presence of these pathogens suggests a need for further treatment of digestate to ensure safety before field application.

Non-antibiotic disinfectant synchronously interferes methane production and antibiotic resistance genes propagation during sludge anaerobic digestion: Activation of microbial adaptation and reconfiguration of bacteria-archaea synergies

Waste activated sludge (WAS) presents both resource recovery potential and pollution risks, making its efficient treatment challenging. Anaerobic digestion is broadly recognized as a green and sustainable approach to WAS treatment, whose efficiency is easily impacted by the exogeneous pollutants in WAS. However, the impact of polyhexamethylene guanidine (PHMG), as a widely-used non-antibiotic disinfectant, on WAS digestion under semi-continuous flow conditions remains unclear. In this study, CH4 production decreased from 16.1 mL/g volatile suspended solids (VSS) in the control to 13.2 mL/g VSS and 0.3 mL/g VSS under low and high PHMG exposure, respectively, while PHMG increased the number of antibiotic resistance gene (ARG) copies per bacterium by 4.6-12.7 %. Molecular docking analysis revealed that PHMG could spontaneously bind to and disintegrate WAS (binding energy:2.35 and -9.62 kcal/mol), increasing the likelihood of microbial exposure to PHMG. This led to an increase in bacterial abundance and a reduction in archaeal populations, resulting in bacterial dominance in ecological niches. The network topology index in PHMG-treated reactors was consistently lower than in the control, with a higher proportion of negatively correlated links, indicating a more antagonistic relationship between bacteria and archaea. Consequently, PHMG significantly interfered with key genes involved in CH4 biosynthesis (e.g., mch and mtd). Interestingly, methanogenic activity and archaeal chemotaxis (e.g., rfk and cheA) partially recovered under low PHMG exposure due to archaeal adaptation through quorum sensing and two-component systems. However, this adaptation process also contributed to the propagation of ARGs through horizontal gene transfer, facilitated by the enhancement of mobile genetic elements and ARGs hosts. These findings confirm the ecological risks of PHMG and highlight the need for effective WAS disposal strategies.

Assessment of trace organic chemicals in anaerobically digested sludge and their partitioning behaviour: Simultaneous Soxhlet chemical extraction and quantification via LC-MS/MS analysis

The increasing number of trace organic contaminants (TrOCs) detected in anaerobically digested sludge (ADS) is triggering increasing concern on its circular-economy reuse practices. A large scientific effort has been performed to define their concentration limits, partition behaviour, and innovative technologies for their removal, which require the definition of versatile and economically sustainable analytical methodologies. In this study, a Soxhlet extraction method coupled with LC-MS/MS analysis was developed to simultaneously determine 32 TrOCs in ADS, 11 of them being quantified in this matrix for the first time. The targeted TrOCs were selected based on the European Urban Wastewater Treatment Directive, and on their frequency of detection in municipal wastewater and/or sludge and chemical diversity. The use of methanol as solvent allowed good recovery efficiencies from ADS solid phase, with an extraction time of 3.5 h and without the need for subsequent clean-up procedures. The targeted LC-MS/MS method enabled high-sensitivity quantification of TrOCs in the liquid phase. At least 25 out of the 32 target compounds were detected in ADS samples from two wastewater treatment plants in Germany, providing their concentration data and highlighting the influence of TrOCs characteristics and sludge properties on contaminant partition coefficients (KD). The experimental outcomes highlight the versatility of the Soxhlet method, which is effective in extracting compounds characterized by diverse properties and structures, and opens new perspectives for the analysis of various substrates. This could support the European Sewage Sludge Directive, expanding its application to soils and cultivated foods and offering insights into TrOCs transfer among different substrates and their influence when used as fertilizer, aiding in the efficient definition of risk assessment methodologies and regulatory concentration limits.