Design and Validation of Primer Sets for the Detection and Quantification of Antibiotic Resistance Genes in Environmental Samples by Quantitative PCR.

The high prevalence of antibiotic resistant bacteria (ARB) in several environments is a great concern threatening human health. Particularly, wastewater treatment plants (WWTP) become important contributors to the dissemination of ARB to receiving water bodies, due to the inefficient management or treatment of highly antibiotic-concentrated wastewaters. Hence, it is vital to develop molecular tools that allow proper monitoring of the genes encoding resistances to these important therapeutic compounds (antibiotic resistant genes, ARGs). For an accurate quantification of ARGs, there is a need for sensitive and robust qPCR assays supported by a good design of primers and validated protocols. In this study, eleven relevant ARGs were selected as targets, including aadA and aadB (conferring resistance to aminoglycosides); ampC, blaTEM, blaSHV, and mecA (resistance to beta-lactams); dfrA1 (resistance to trimethoprim); ermB (resistance to macrolides); fosA (resistance to fosfomycin); qnrS (resistance to quinolones); and tetA(A) (resistance to tetracyclines). The in silico design of the new primer sets was performed based on the alignment of all the sequences of the target ARGs (orthology grade > 70%) deposited in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, allowing higher coverages of the ARGs' biodiversity than those of several primers described to date. The adequate design and performance of the new molecular tools were validated in six samples, retrieved from both natural and engineered environments related to wastewater treatment. The hallmarks of the optimized qPCR assays were high amplification efficiency (> 90%), good linearity of the standard curve (R2 > 0.980), repeatability and reproducibility across experiments, and a wide linear dynamic range. The new primer sets and methodology described here are valuable tools to upgrade the monitorization of the abundance and emergence of the targeted ARGs by qPCR in WWTPs and related environments.

Deciphering the Role of WWTPs in Cold Environments as Hotspots for the Dissemination of Antibiotic Resistance Genes.

Cold environments are the most widespread extreme habitats in the world. However, the role of wastewater treatment plants (WWTPs) in the cryosphere as hotspots in antibiotic resistance dissemination has not been well established. Hence, a snapshot of the resistomes of WWTPs in cold environments, below 5 °C, was provided to elucidate their role in disseminating antibiotic resistance genes (ARGs) to the receiving waterbodies. The resistomes of two natural environments from the cold biosphere were also determined. Quantitative PCR analysis of the aadA, aadB, ampC, blaSHV, blaTEM, dfrA1, ermB, fosA, mecA, qnrS, and tetA(A) genes indicated strong prevalences of these genetic determinants in the selected environments, except for the mecA gene, which was not found in any of the samples. Notably, high abundances of the aadA, ermB, and tetA(A) genes were found in the influents and activated sludge, highlighting that WWTPs of the cryosphere are critical hotspots for disseminating ARGs, potentially worsening the resistance of bacteria to some of the most commonly prescribed antibiotics. Besides, the samples from non-disturbed cold environments had large quantities of ARGs, although their ARG profiles were highly dissimilar. Hence, the high prevalences of ARGs lend support to the fact that antibiotic resistance is a common issue worldwide, including environmentally fragile cold ecosystems.

Effects of sulphur amino acids on the size and structure of microbial communities of aerobic granular sludge bioreactors.

Granular activated sludge has been described as a promising tool in treating wastewater. However, the effect of high concentrations of sulphur amino acids, cysteine and methionine, in the evolution, development and stability of AGS-SBRs (aerobic granular sludge in sequential batch reactors) and their microbial communities is not well-established. Therefore, this study aimed to evaluate microbial communities' size, structure and dynamics in two AGS-SBRs fed with two different concentrations of amino acids (50 and 100 mg L-1 of both amino acids). In addition, the impact of the higher level of amino acids was also determined under an acclimatization or shock strategy. While N removal efficiency decreased with amino acids, the removal of the organic matter was generally satisfactory. Moreover, the abrupt presence of both amino acids reduced even further the removal performance of N, whereas under progressive adaptation, the removal yield was higher. Besides, excellent removal rates of cysteine and methionine elimination were found, in all stages below 80% of the influent values. Generally considered, the addition of amino acids weakly impacts the microbial communities' total abundances. On the contrary, the presence of amino acids sharply modulated the dominant bacterial structures. Furthermore, the highest amino acid concentration under the shock strategy resulted in a severe change in the structure of the microbial community. Acidovorax, Flavobacterium, Methylophilus, Stenotrophomonas and Thauera stood out as the prominent bacteria to cope with the high presence of cysteine and methionine. Hence, the AGS-SBR technology is valuable for treating influents enriched in sulphur Aa inclusively when a shock strategy was used.

New concepts in anaerobic digestion processes: recent advances and biological aspects.

Waste treatment and the simultaneous production of energy have gained great interest in the world. In the last decades, scientific efforts have focused largely on improving and developing sustainable bioprocess solutions for energy recovery from challenging waste. Anaerobic digestion (AD) has been developed as a low-cost organic waste treatment technology with a simple setup and relatively limited investment and operating costs. Different technologies such as one-stage and two-stage AD have been developed. The viability and performance of these technologies have been extensively reported, showing the supremacy of two-stage AD in terms of overall energy recovery from biomass under different substrates, temperatures, and pH conditions. However, a comprehensive review of the advantages and disadvantages of these technologies is still lacking. Since microbial ecology is critical to developing successful AD, many studies have shown the structure and dynamics of archaeal and bacterial communities in this type of system. However, the role of Eukarya groups remains largely unknown to date. In this review, we provide a comprehensive review of the role, abundance, dynamics, and structure of archaeal, bacterial, and eukaryal communities during the AD process. The information provided could help researchers to select the adequate operational parameters to obtain the best performance and biogas production results.

Biomineralisation of carbonate and sulphate by the halophilic bacterium Halomonas maura at different manganese concentrations.

The ability of Halomonas maura to bioprecipitate carbonate and sulphate crystals in solid media at different manganese concentrations has been demonstrated in this study for the first time. The precipitated minerals were studied by X-ray diffraction, scanning and transmission electron microscopy, and energy-dispersive X-ray spectroscopy. The precipitated minerals were different based on the manganese concentration present in the medium and the incubation time. In the absence of manganese, H. maura formed pseudokutnahorite crystals; in the presence of manganese, the concentration in the culture medium determined the precipitation carbonates, such as rhodochrosite and dolomites. However, in the presence of low concentrations of manganese chloride (MnCl2) (5 g/l), kutnohorite crystals were also formed. Finally, when H. maura was grown in the presence of manganese, small amounts of sulphate crystals (such as bassanite and gypsum) were detected. Our study of the precipitated minerals showed an active role of H. maura in the biomineralisation process, but the geochemical conditions, and the manganese concentrations in particular, were clearly influential.

16S rRNA gene-based characterization of bacteria potentially associated with phosphate and carbonate precipitation from a granular autotrophic nitrogen removal bioreactor.

A bench-scale granular autotrophic nitrogen removal bioreactor (completely autotrophic nitrogen removal over nitrite (CANON) system) used for the treatment of synthetic wastewater was analyzed for the identification of microbiota with potential capacity for carbonate and phosphate biomineral formation. 16S ribosomal RNA (rRNA) gene-based studies revealed that different bacterial species found in the granular biomass could trigger the formation of phosphate and calcite minerals in the CANON bioreactor. iTag analysis of the microbial community in the granular biomass with potential ability to precipitate calcium carbonate and hydroxyapatite constituted around 0.79-1.32 % of total bacteria. Specifically, the possible hydroxyapatite-producing Candidatus Accumulibacter had a relative abundance of 0.36-0.38 % and was the highest phosphate-precipitating bacteria in the granular CANON system. With respect to calcite precipitation, the major potential producer was thought to be Stenotrophomonas with a 0.38-0.50 % relative abundance. In conclusion, our study showed evidences that the formation of hydroxyapatite and calcite crystals inside of the granular biomass of a CANON system for the treatment wastewater with high ammonium concentration was a biological process. Therefore, it could be suggested that microorganisms play an important role as a precipitation core and also modified the environment due to their metabolic activities.

Chemical Synthesis and Self-Assembly of a Ladderane Phospholipid.

Ladderane lipids produced by anammox bacteria constitute some of the most structurally fascinating yet poorly studied molecules among biological membrane lipids. Slow growth of the producing organism and the inherent difficulty of purifying complex lipid mixtures have prohibited isolation of useful amounts of natural ladderane lipids. We have devised a highly selective total synthesis of ladderane lipid tails and a full phosphatidylcholine to enable biophysical studies on chemically homogeneous samples of these molecules. Additionally, we report the first proof of absolute configuration of a natural ladderane.

New concepts of microbial treatment processes for the nitrogen removal: effect of protein and amino acids degradation.

High concentrations of proteins and amino acids can be found in wastewater and wastewater stream produced in anaerobic digesters, having shown that amino acids could persist over different managements for nitrogen removal affecting the nitrogen removal processes. Nitrogen removal is completely necessary because of their implications and the significant adverse environmental impact of ammonium such as eutrophication and toxicity to aquatic life on the receiving bodies. In the last decade, the treatment of effluents with high ammonium concentration through anammox-based bioprocesses has been enhanced because these biotechnologies are cheaper and more environmentally friendly than conventional technologies. However, it has been shown that the presence of important amounts of proteins and amino acids in the effluents seriously affects the microbial autotrophic consortia leading to important losses in terms of ammonium oxidation efficiency. Particularly the presence of sulfur amino acids such as methionine and cysteine has been reported to drastically decrease the autotrophic denitrification processes as well as affect the microbial community structure promoting the decline of ammonium oxidizing bacteria in favor of other phylotypes. In this context we discuss that new biotechnological processes that improve the degradation of protein and amino acids must be considered as a priority to increase the performance of the autotrophic denitrification biotechnologies.

Archaeal and bacterial community dynamics and bioprocess performance of a bench-scale two-stage anaerobic digester.

Two-stage technologies have been developed for anaerobic digestion of waste-activated sludge. In this study, the archaeal and bacterial community structure dynamics and bioprocess performance of a bench-scale two-stage anaerobic digester treating urban sewage sludge have been studied by the means of high-throughput sequencing techniques and physicochemical parameters such as pH, dried sludge, volatile dried sludge, acid concentration, alkalinity, and biogas generation. The coupled analyses of archaeal and bacterial communities and physicochemical parameters showed a direct relationship between archaeal and bacterial populations and bioprocess performance during start-up and working operation of a two-stage anaerobic digester. Moreover, results demonstrated that archaeal and bacterial community structure was affected by changes in the acid/alkalinity ratio in the bioprocess. Thus, a predominance of the acetoclastic methanogen Methanosaeta was observed in the methanogenic bioreactor at high-value acid/alkaline ratio, while a predominance of Methanomassilicoccaeceae archaea and Methanoculleus genus was observed in the methanogenic bioreactor at low-value acid/alkaline ratio. Biodiversity tag-iTag sequencing studies showed that methanogenic archaea can be also detected in the acidogenic bioreactor, although its biological activity was decreased after 4 months of operation as supported by physicochemical analyses. Also, studies of the VFA producers and VFA consumers microbial populations showed as these microbiota were directly affected by the physicochemical parameters generated in the bioreactors. We suggest that the results obtained in our study could be useful for future implementations of two-stage anaerobic digestion processes at both bench- and full-scale.

Microbial community analysis of a full-scale DEMON bioreactor.

Full-scale applications of autotrophic nitrogen removal technologies for the treatment of digested sludge liquor have proliferated during the last decade. Among these technologies, the aerobic/anoxic deammonification process (DEMON) is one of the major applied processes. This technology achieves nitrogen removal from wastewater through anammox metabolism inside a single bioreactor due to alternating cycles of aeration. To date, microbial community composition of full-scale DEMON bioreactors have never been reported. In this study, bacterial community structure of a full-scale DEMON bioreactor located at the Apeldoorn wastewater treatment plant was analyzed using pyrosequencing. This technique provided a higher-resolution study of the bacterial assemblage of the system compared to other techniques used in lab-scale DEMON bioreactors. Results showed that the DEMON bioreactor was a complex ecosystem where ammonium oxidizing bacteria, anammox bacteria and many other bacterial phylotypes coexist. The potential ecological role of all phylotypes found was discussed. Thus, metagenomic analysis through pyrosequencing offered new perspectives over the functioning of the DEMON bioreactor by exhaustive identification of microorganisms, which play a key role in the performance of bioreactors. In this way, pyrosequencing has been proven as a helpful tool for the in-depth investigation of the functioning of bioreactors at microbiological scale.

Promising bioprocesses for the efficient removal of antibiotics and antibiotic-resistance genes from urban and hospital wastewaters: Potentialities of aerobic granular systems.

The use, overuse, and improper use of antibiotics have resulted in higher levels of antibiotic-resistant bacteria (ARB) and antibiotic-resistance genes (ARGs), which have profoundly disturbed the equilibrium of the environment. Furthermore, once antibiotic agents are excreted in urine and feces, these substances often can reach wastewater treatment plants (WWTPs), in which improper treatments have been highlighted as the main reason for stronger dissemination of antibiotics, ARB, and ARGs to the receiving bodies. Hence, achieving better antibiotic removal capacities in WWTPs is proposed as an adequate approach to limit the spread of antibiotics, ARB, and ARGs into the environment. In this review, we highlight hospital wastewater (WW) as a critical hotspot for the dissemination of antibiotic resistance due to its high level of antibiotics and pathogens. Hence, monitoring the composition and structure of the bacterial communities related to hospital WW is a key factor in controlling the spread of ARGs. In addition, we discuss the advantages and drawbacks of the current biological WW treatments regarding the antibiotic-resistance phenomenon. Widely used conventional activated sludge technology has proved to be ineffective in mitigating the dissemination of ARB and ARGs to the environment. However, aerobic granular sludge (AGS) technology is a promising technology-with broad adaptability and excellent performance-that could successfully reduce antibiotics, ARB, and ARGs in the generated effluents. We also outline the main operational parameters involved in mitigating antibiotics, ARB, and ARGs in WWTPs. In this regard, WW operation under long hydraulic and solid retention times allows better removal of antibiotics, ARB, and ARGs independently of the WW technology employed. Finally, we address the current knowledge of the adsorption and degradation of antibiotics and their importance in removing ARB and ARGs. Notably, AGS can enhance the removal of antibiotics, ARB, and ARGs due to the complex microbial metabolism within the granular biomass.

Novel insights into the impact of anticancer drugs on the performance and microbial communities of a continuous-flow aerobic granular sludge system.

Anticancer drugs are frequently found in domestic wastewater, but knowledge of their impacts on wastewater treatment processes is limited. The effects of three levels of concentrations (low, medium, and high) of three anticancer drugs on physicochemical parameters and prokaryotic communities of a continuous-flow aerobic granular sludge (AGS) system were examined. Drugs at medium and high concentrations reduced the removal of total nitrogen and organic matter during the first 15 days of operation by approximately 15-20 % compared to a control, but these effects disappeared afterward. Removal efficiencies of drugs were in the range of 51.2-100 % depending on the concentration level. Drugs at medium and high concentrations reduced the abundance and diversity and altered the composition of prokaryotic communities. Specific taxa were linked to variations in performance parameters after the addition of the drugs. This study provides improved knowledge of the impacts of anticancer drugs in AGS systems operated in continuous-flow reactor.

A metabolomics perspective on the effect of environmental micro and nanoplastics on living organisms: A review.

The increasing trend regarding the use of plastics has arisen an exponential concern on the fate of their derived products to the environment. Among these derivatives, microplastics and nanoplastics (MNPs) have been featured for their associated environmental impact due to their low molecular size and high surface area, which has prompted their ubiquitous transference among all environmental interfaces. Due to the heterogenous chemical composition of MNPs, the study of these particles has focused a high number of studies, as a result of the myriad of associated physicochemical properties that contribute to the co-transference of a wide range of contaminants, thus becoming a major challenge for the scientific community. In this sense, both primary and secondary MNPs are well-known to be adscribed to industrial and urbanized areas, from which they are massively released to the environment through a multiscale level, involving the atmosphere, hydrosphere, and lithosphere. Consequently, much research has been conducted on the understanding of the interconnection between those interfaces, that motivate the spread of these contaminants to biological systems, being mostly represented by the biosphere, especially phytosphere and, finally, the anthroposphere. These findings have highlighted the potential hazardous risk for human health through different mechanisms from the environment, requiring a much deeper approach to define the real risk of MNPs exposure. As a result, there is a gap of knowledge regarding the environmental impact of MNPs from a high-throughput perspective. In this review, a metabolomics-based overview on the impact of MNPs to all environmental interfaces was proposed, considering this technology a highly valuable tool to decipher the real impact of MNPs on biological systems, thus opening a novel perspective on the study of these contaminants.

Anticancer drugs impact the performance and prokaryotic microbiome of an aerobic granular sludge system operated in a sequential batch reactor.

Increased concerns exist about the presence of anticancer drugs in wastewater. However, knowledge of the impacts of anticancer drugs on the performance of the system and microbial communities during wastewater treatment processes is limited. We examined the effect of three anticancer drugs commonly detected in influents of wastewater treatment plants applied at three different concentration levels on the performance, efficiency of anticancer drug removal, and prokaryotic microbiome in an aerobic granular sludge system (AGS) operated in a sequential batch reactor (SBR). We showed that an AGS can efficiently remove anticancer drugs, with removal rates in the range of 53-100% depending on the type of drug and concentration level. Anticancer drugs significantly decreased the abundance of total bacterial and archaeal communities, an effect that was linked to reduced nitrogen removal efficiency. Anticancer drugs also reduced the diversity, altered the prokaryotic community composition, reduced network complexity, and induced a decrease of a wide range of predicted bacterial functions. Specific bacterial taxa responsive to the addition of anticancer drugs with known roles in nitrification and denitrification were identified. This study shows anticancer drugs should be monitored in the future as they can induce changes in the performance and microbiome of wastewater treatment technologies.

Microbial Ecology of Granular Biofilm Technologies for Wastewater Treatment: A Review.

Nowadays, the discharge of wastewater is a global concern due to the damage caused to human and environmental health. Wastewater treatment has progressed to provide environmentally and economically sustainable technologies. The biological treatment of wastewater is one of the fundamental bases of this field, and the employment of new technologies based on granular biofilm systems is demonstrating success in tackling the environmental issues derived from the discharge of wastewater. The granular-conforming microorganisms must be evaluated as functional entities because their activities and functions for removing pollutants are interconnected with the surrounding microbiota. The deep knowledge of microbial communities allows for the improvement in system operation, as the proliferation of microorganisms in charge of metabolic roles could be modified by adjustments to operational conditions. This is why engineering must consider the intrinsic microbiological aspects of biological wastewater treatment systems to obtain the most effective performance. This review provides an extensive view of the microbial ecology of biological wastewater treatment technologies based on granular biofilms for mitigating water pollution.

Archaeal diversity in biofilm technologies applied to treat urban and industrial wastewater: recent advances and future prospects.

Biological wastewater treatment (WWT) frequently relies on biofilms for the removal of anthropogenic contaminants. The use of inert carrier materials to support biofilm development is often required, although under certain operating conditions microorganisms yield structures called granules, dense aggregates of self-immobilized cells with the characteristics of biofilms maintained in suspension. Molecular techniques have been successfully applied in recent years to identify the prokaryotic communities inhabiting biofilms in WWT plants. Although methanogenic Archaea are widely acknowledged as key players for the degradation of organic matter in anaerobic bioreactors, other biotechnological functions fulfilled by Archaea are less explored, and research on their significance and potential for WWT is largely needed. In addition, the occurrence of biofilms in WWT plants can sometimes be a source of operational problems. This is the case for membrane bioreactors (MBR), an advanced technology that combines conventional biological treatment with membrane filtration, which is strongly limited by biofouling, defined as the undesirable accumulation of microbial biofilms and other materials on membrane surfaces. The prevalence and spatial distribution of archaeal communities in biofilm-based WWT as well as their role in biofouling are reviewed here, in order to illustrate the significance of this prokaryotic cellular lineage in engineered environments devoted to WWT.

Anticancer drugs in wastewater and natural environments: A review on their occurrence, environmental persistence, treatment, and ecological risks.

The consumption of anticancer drugs (also known as chemotherapy drugs or antineoplastic drugs) has augmented over the last decades due to increased cancer incidence. Although there is an increasing concern about the presence of pharmaceutical compounds in natural environments and urban/domestic wastewater, anticancer drugs used in chemotherapy and anticancer medication have received less attention. In this review, the occurrence, environmental persistence, and known and potential ecological impacts of anticancer drugs is discussed. This review shows that these compounds are being increasingly detected in effluents of hospitals, influents and effluents of wastewater treatment plants, river surface water and sediments, groundwater, and even drinking water. Anticancer drugs can impact aquatic organisms such as algae, crustaceans, rotifers, and fish and may promote changes in soil and water microbial communities that may alter ecosystem functioning. Our knowledge of technologies for the removal of anticancer drugs is still limited, and these drugs can be dispersed in nature in a diffuse way in an uncontrolled manner. For this reason, an improved understanding of the presence, persistence, and ecological impacts of anticancer drugs in wastewater and natural environments is needed to help design management strategies, protect aquatic microorganisms, and mitigate potential ecological impacts.

Anticancer drugs drive changes in the performance, abundance, diversity, and composition of eukaryotic communities of an aerobic granular sludge system.

Anticancer drugs are emerging contaminants that are being increasingly detected in urban wastewater. However, there is limited knowledge on the use of biological wastewater treatments, such as granular sludge systems (AGSs), to remove these substances and on their impacts on the general performance of the system and the eukaryotic communities in the granules. We investigated the impacts of three anticancer drugs commonly found in wastewater treatment plants and applied at three different concentrations on the removal efficiency of anticancer drugs, physicochemical parameters, and the eukaryotic microbiome of an AGS operated in a sequential batch reactor (SBR). Anticancer drugs applied at medium and high concentrations significantly decreased the removal efficiency of total nitrogen, the granular biomass concentration, and the size and setting velocity of granules. However, these effects disappeared after not adding the drugs for about a month thus showing the plasticity of the system to return to original levels. Regardless of the concentration of anticancer drugs tested, the AGS technology was effective in removing these substances, with removal rates in the range of 68.5%-100%. The presence of anticancer drugs at medium and high concentrations significantly decreased the abundance of total fungi, an effect that was linked to changes in the physicochemical parameters. Anticancer drugs also induced decreases in the diversity of the eukaryotic community, altered the community composition, and reduced the network complexity when applied at medium and high concentrations. Taxa responsive to the presence of anticancer drugs were identified. The diversity and composition of the eukaryotic microbiome returned to original diversity levels after not adding the drugs for about a month. Overall, this study increases our understanding of the impacts of anticancer drugs on the performance and eukaryotic microbiome of an AGS and highlights the need for monitoring these substances.

The dangerous transporters: A study of microplastic-associated bacteria passing through municipal wastewater treatment.

Microplastics (MPs) provide a stable and protective habitat for diverse wastewater bacteria, including pathogenic and antibiotic-resistant species. Therefore, MPs may potentially transport these bacteria through wastewater treatment steps to the environment and far distances. This study investigated bacterial communities of MP-associated bacteria from different stages of municipal wastewater treatment processes to evaluate the potential negative effect of these biofilms on the environment. The results showed a high diversity of bacteria that were strongly attached to MPs. After all treatment steps, the core bacterial groups remained attached to MPs and escaped from the wastewater treatment plant with effluent water. Several pathogenic bacteria were identified in MP samples from all treatment steps, and most of them were found in effluent water. These data provide new insights into the possible impacts of wastewater-derived MPs on the environment. MP-associated biofilms were proved to be important sources of pathogens and antibiotic-resistant genes in natural waters.

Evaluating the nitrogen-contaminated groundwater treatment by a denitrifying granular sludge bioreactor: effect of organic matter loading.

A sequential bed granular bioreactor was adapted to treat nitrate-polluted synthetic groundwater under anaerobic conditions and agitation with denitrification gas, achieving very efficient performance in total nitrogen removal at influent organic carbon concentrations of 1 g L-1 (80-90%) and 0.5 g L-1 (70-80%) sodium acetate, but concentrations below 0.5 g L-1 caused accumulation of nitrite and nitrate and led to system failure (30-40% removal). Biomass size and settling velocity were higher above 0.5 g L-1 sodium acetate. Trichosporonaceae dominated the fungal populations at all times, while a dominance of terrestrial group Thaumarchaeota and Acidovorax at 1 and 0.5 g L-1 passed to a domination of Methanobrevibacter and an unclassified Comamonadaceae clone for NaAc lower than 0.5 g L-1. The results obtained pointed out that the denitrifying granular sludge technology is a feasible solution for the treatment of nitrogen-contaminated groundwater, and that influent organic matter plays an important role on the conformation of microbial communities within it and, therefore, on the overall efficiency of the system.