Effects of thermal alkaline hydrolysis and biochar addition on anaerobic digestion of sewage sludge and fate of antibiotic resistance genes.

This study investigated the effect of ultrasonic (US) pretreatment at three different contact times (30, 45, and 60 min) with a power of 240 W and frequency of 40 kHz on the fate of antibiotic resistance genes (ARGs), mobile genetic elements (MGEs), and enteric pathogens during anaerobic digestion (AD) of sludge. By using real time-qPCR, three MGEs (int1, int2, and tnpA) and seven ARGs (sul1, sul2, tetW, tetA, tetO, ermF, and aac(6')-lb) were quantified that have serious human health impacts and represent the most widely used antibiotics (tetracycline, sulfonamide, macrolide, and aminoglycoside). Results indicated that US pretreatment under different contact times improved the removal of ARGs and MGEs. Compared to 30 and 45 min of US pretreatment, 60 min of US pretreatment resulted in a higher reduction of ARGs with total ARG reduction of 41.70 ± 1.13%. Furthermore, the relative abundance of ARGs and MGEs after US pretreatment was reduced more effectively in anaerobic reactors than in a control AD without US pretreatment. The total ARGs and MGEs removal efficiency of control AD was 44.07 ± 0.72% and 63.69 ± 1.43%, and if US pretreatment at different times were applied, the total ARGs and MGEs removal efficiency of the whole pretreatment AD process improved to 59.71 ± 2.76-68.54 ± 1.58% and 69.82 ± 2.15-76.84 ± 0.22%. The highest removal of total ARGs (68.54 ± 1.58%) and MGEs (76.84 ± 0.22%) was achieved after AD with US pretreatment at 45 min. However, US pretreatment and AD with US pretreatment were not effective in inactivation of enteric pathogens (total coliforms and E. coli), suggesting that posttreatment is needed prior to land application of sludge to reduce the level of enteric pathogens. There was no detection of the studied ARGs and MGEs in the enteric pathogens after US pretreatment in subsequent AD. According to this study, long contact times of US pretreatment can mitigate ARGs and MGEs in AD processes, offering valuable insight into improving environmental safety and sustainable waste management. Additionally, the study highlights the need to investigate posttreatment techniques for reducing enteric pathogens in AD effluent, a crucial consideration for agricultural use and environmental protection.

Sewage sludge in the wastewater treatment plants contains considerable amount of antibiotic resistance genes (ARGs). A few studies have reported that anaerobic digestion (AD) could successfully remove some ARGs from sewage sludge, but information on the fate of ARGs in sludge pretreatment-AD process is still very limited. In this study, three sludge pretreatment methods, including alkaline, thermal hydrolysis and ultrasonic pretreatments, were compared to investigate the distribution and removal of ARGs in the sludge pretreatment-AD process. Results showed that the ARGs removal efficiency of AD itself was approximately 50.77%, and if these three sludge pretreatments were applied, the total ARGs removal efficiency of the whole pretreatment-AD process could be improved up to 52.50%–75.07%. The ultrasonic pretreatment was more efficient than alkaline and thermal hydrolysis pretreatments. Although thermal hydrolysis reduced ARGs obviously, the total ARGs rebounded considerably after inoculation and were only removed slightly in the subsequent AD process. Furthermore, it was found that the total ARGs concentration significantly correlated with the amount of 16S rRNA gene during the pretreatment and AD processes, and the bacteria carrying ARGs could be mainly affiliated with Proteobacteria.

The role of pretreatments in handling antibiotic resistance genes in anaerobic sludge digestion – A review

Enzymatic integrated in-situ advanced anaerobic digestion of sewage sludge for the removal of antibiotics and antibiotic resistance genes

Antibiotic resistance genes (ARGs) have emerged as a significant global health threat, contributing to fatalities worldwide. Wastewater treatment plants (WWTPs) and livestock farms serve as primary reservoirs for these genes due to the limited efficacy of existing treatment methods and microbial adaptation to environmental stressors. Anaerobic digestion (AD) stands as a prevalent biological treatment for managing sewage sludge and manure in these settings. Given the agricultural utility of AD digestate as biofertilizers, understanding ARGs’ fate within AD processes is essential to devise effective mitigation strategies. However, understanding the impact of various factors on ARGs occurrence, dissemination, and fate remains limited. This review article explores various AD treatment parameters and correlates to various resistance mechanisms and hotspots of ARGs in the environment. It further evaluates the dissemination and occurrence of ARGs in AD feedstocks and provides a comprehensive understanding of the fate of ARGs in AD systems. This review explores the influence of key AD parameters such as feedstock properties, pretreatments, additives, and operational strategies on ARGs. Results show that properties such as high solid content and optimum co-digestion ratios can enhance ARG removal, while the presence of heavy metals, microplastics, and antibiotics could elevate ARG abundance. Also, operational enhancements, such as employing two-stage digestion, have shown promise in improving ARG removal. However, certain pretreatment methods, like thermal hydrolysis, may exhibit a rebounding effect on ARG levels. Overall, this review systematically addresses current challenges and offers future perspectives associated with the fate of ARGs in AD systems.Graphical

Fate of antibiotic resistance genes during anaerobic digestion of sewage sludge: Role of solids retention times in different configurations

Antibiotic resistant bacteria (ARB) and their genes (ARGs) have become recognised as significant emerging environmental pollutants. ARB and ARGs in sewage sludge can be transmitted back to humans via the food chain when sludge is recycled to agricultural land, making sludge treatment key to control the release of ARB and ARGs to the environment. This study investigated the fate of antibiotic resistant Escherichia coli and a large set of antibiotic resistance genes (ARGs) during full scale anaerobic digestion (AD) of sewage sludge at two U.K. wastewater treatment plants and evaluated the impact of thermal hydrolysis (TH) pre-treatment on their abundance and diversity. Absolute abundance of 13 ARGs and the Class I integron gene intI1 was calculated using single gene quantitative (q) PCR. High through-put qPCR analysis was also used to determine the relative abundance of 370 ARGs and mobile genetic elements (MGEs). Results revealed that TH reduced the absolute abundance of all ARGs tested and intI1 by 10-12,000 fold. After subsequent AD, a rebound effect was seen in many ARGs. The fate of ARGs during AD without pre-treatment was variable. Relative abundance of most ARGs and MGEs decreased or fluctuated, with the exception of macrolide resistance genes, which were enriched at both plants, and tetracyline and glycopeptide resistance genes which were enriched in the plant employing TH. Diversity of ARGs and MGEs decreased in both plants during sludge treatment. Principal coordinates analysis revealed that ARGs are clearly distinguished according to treatment step, whereas MGEs in digested sludge cluster according to site. This study provides a comprehensive within-digestor analysis of the fate of ARGs, MGEs and antibiotic resistant E. coli and highlights the effectiveness of AD, particularly when TH is used as a pre-treatment, at reducing the abundance of most ARGs and MGEs in sludgeand preventing their release into the environment.

Antibiotic resistant bacteria (ARB) and their genes (ARGs) have become recognised as significant emerging environmental pollutants. ARB and ARGs in sewage sludge can be transmitted back to humans via the food chain when sludge is recycled to agricultural land, making sludge treatment key to control the release of ARB and ARGs to the environment. This study investigated the fate of antibiotic resistant Escherichia coli and a large set of antibiotic resistance genes (ARGs) during full scale anaerobic digestion (AD) of sewage sludge at two U.K. wastewater treatment plants and evaluated the impact of thermal hydrolysis (TH) pre-treatment on their abundance and diversity. Absolute abundance of 13 ARGs and the Class I integron gene intI1 was calculated using single gene quantitative (q) PCR. High through-put qPCR analysis was also used to determine the relative abundance of 370 ARGs and mobile genetic elements (MGEs). Results revealed that TH reduced the absolute abundance of all ARGs tested and intI1 by 10–12,000 fold. After subsequent AD, a rebound effect was seen in many ARGs. The fate of ARGs during AD without pre-treatment was variable. Relative abundance of most ARGs and MGEs decreased or fluctuated, with the exception of macrolide resistance genes, which were enriched at both plants, and tetracyline and glycopeptide resistance genes which were enriched in the plant employing TH. Diversity of ARGs and MGEs decreased in both plants during sludge treatment. Principal coordinates analysis revealed that ARGs are clearly distinguished according to treatment step, whereas MGEs in digested sludge cluster according to site. This study provides a comprehensive within-digestor analysis of the fate of ARGs, MGEs and antibiotic resistant E. coli and highlights the effectiveness of AD, particularly when TH is used as a pre-treatment, at reducing the abundance of most ARGs and MGEs in sludgeand preventing their release into the environment.

Impacts of norfloxacin on sewage sludge anaerobic digestion: Bioenergy generation and potential environmental risks

Fate of antibiotic resistance genes in mesophilic and thermophilic anaerobic digestion of chemically enhanced primary treatment (CEPT) sludge

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

Free ammonia pretreatment enhances the removal of antibiotic resistance genes in anaerobic sludge digestion

Microwave irradiation is a novel and very promising technology for sludge conditioning. As pretreatment, it has a verified beneficial effect on the microbial degradation and anaerobic digestion of sewage sludge, but in present work we dealt with the applicability of microwave pretreatments for food industrial sludge. However, studies cannot be found that specialize on the effects of the MW treatments with different intensities on the anaerobic digestion of sludge. In our work we focused on the examination of the effect of MW pretreatment for 0.5, 2.5, and 5 W/g on the carbonaceous biochemical oxygen demand (CBOD5), solubilization of organic matters (sCOD/tCOD), and the mesophilic anaerobic digestion of dairy sewage sludge. It can be concluded that the MW pretreatments were appropriate to enhance the efficiency of anaerobic digestion. With MW pretreatments the specific biogas product could be increased from 220 mL g−1 to more than 600 mL g−1 because of the increased solubility (from 9.7% to more than 40%), and the enhanced accessibility of organic compounds for decomposing bacteria. © 2010 American Institute of Chemical Engineers Environ Prog, 2011

Fate of antibiotic resistance genes during temperature-changed psychrophilic anaerobic digestion of municipal sludge

Sludge is a major by-product and the final reservoir of antibiotic resistance genes (ARGs) in wastewater treatment plants (WWTPs). Temperature-phased anaerobic digestion (TPAD), consisting of thermophilic anaerobic digestion (AD) (55 °C) and mesophilic AD processes (37 °C), has been implemented in WWTPs for sludge reduction while improving the biomethane production. However, the impact of TPAD on the ARGs' fate is still undiscovered in lab-scale experiments and full-scale WWTPs. This study, for the first time, investigated the fate of ARGs during the TPAD process across three seasons in a full-size WWTP. Ten typical ARGs and one integrase gene of class 1 integron (intI1) involving ARGs horizontal gene transfer were examined in sludge before and after each step of the TPAD process. TPAD reduced aac(6′)-Ib-cr, blaTEM, drfA1, sul1, sul2, ermb, mefA, tetA, tetB and tetX by 87.3–100.0 %. TPAD reduced the overall average absolute abundance of targeted ARGs and intI1 by 92.39 % and 92.50 %, respectively. The abundance of targeted ARGs in sludge was higher in winter than in summer and autumn before and after TPAD. During the TPAD processes, thermophilic AD played a major role in the removal of ARGs, contributing to >60 % removal of ARGs, while the subsequent mesophilic AD contributed to a further 31 % removal of ARGs. The microbial community analysis revealed that thermophilic AD reduced the absolute abundance of ARGs hosts, antibiotic resistant bacteria. In addition, thermophilic AD reduced the abundance of the intI1, while the intI1 did not reproduce during the mesophilic AD, also contributing to a decline in the absolute abundance of ARGs in TPAD. This study demonstrates that TPAD can effectively reduce the abundance of ARGs in sludge, which will suppress the transmission of ARGs from sludge into the natural environment and deliver environmental and health benefits to our society.