Extraction of Proteins from Municipal Wastewater and Activated Sludge.

Wastewater treatment plants (WWTPs) are the main barrier to cope with the increased pressure of municipal and industrial wastewater on natural water resources in terms of both polluting load and produced volumes. For this reason, WWTP's efficiency should be the highest; thus, their monitoring becomes critical. In conventional WWTPs, biodegradation of pollutants mainly occurs in the biological reactors, and an increasing interest in a deeper characterization of the biomasses involved in these processes (made of biofilms, granules, and suspended activated sludge) rose up in recent years. In this sense, the meta-omics approaches were recently developed to investigate the entire set of biomolecules of a given class in a microbial community with the same general objective: the identification of the biomolecules through the sequence similarity of high degree in the already available databases. Particularly, metaproteomics concerns the identification of all proteins in a microbial community in a given moment or condition. In this chapter, a protocol for the extraction and separation of proteins from activate sludge sampled at WWTPs is proposed.

Uptake and translocation of pharmaceutically active compounds by olive tree (Olea europaea L.) irrigated with treated municipal wastewater.

Introduction: The use of treated municipal wastewater (TWW) represents a relevant opportunity for irrigation of agricultural crops in semi-arid regions to counter the increasing water scarcity. Pharmaceutically active compounds (PhACs) are often detected in treated wastewater, posing a risk to humans and the environment. PhACs can accumulate in soils and translocate into different plant tissues, reaching, in some cases, edible organs and entering the food chain. Methods: This study evaluated the uptake and translocation processes of 10 PhACs by olive trees irrigated with TWW, investigating their accumulation in different plant organs. The experiment was conducted in southern Italy, in 2-year-old plants irrigated with three different types of water: freshwater (FW), TWW spiked with 10 PhACs at a concentration of 200 µg L-1 (1× TWW), and at a triple dose (3× TWW), from July to October 2021. The concentration of PhACs in soil and plant organs was assessed, collecting samples of root, stem, shoot, leaf, fruit, and kernel at 0 (T0), 50 (T1), and 107 (T2) days of irrigation. PhACs extraction from soil and plant organs was carried out using the QuEChERS method, and their concentrations were determined by high-resolution mass spectrometry coupled with liquid chromatography. Results: Results of uptake factors (UF) showed a different behavior between compounds according to their physicochemical properties, highlighting PhACs accumulation and translocation in different plant organs (also edible part) in 1× TWW and 3× TWW compared to FW. Two PhACs, carbamazepine and fluconazole, showed interactions with the soil-plant system, translocating also in the aerial part of the plant, with a translocation factor (TF) greater than 1, which indicates high root-to-leaf translocation. Discussion: Findings highlight that only few PhACs among the selected compounds can be uptaken by woody plants and accumulated in edible parts at low concentration. No effects of PhACs exposure on plant growth have been detected. Despite the attention to be paid to the few compounds that translocate into edible organs, these results are promising for adapting wastewater irrigation in crops. Increasing knowledge about PhACs behavior in woody plants can be important for developing optimized wastewater irrigation and soil management strategies to reduce PhACs accumulation and translocation in plants.

Uptake and accumulation of emerging contaminants in processing tomato irrigated with tertiary treated wastewater effluent: a pilot-scale study.

The reuse of treated wastewater for crop irrigation is vital in water-scarce semi-arid regions. However, concerns arise regarding emerging contaminants (ECs) that persist in treated wastewater and may accumulate in irrigated crops, potentially entering the food chain and the environment. This pilot-scale study conducted in southern Italy focused on tomato plants (Solanum lycopersicum L. cv Taylor F1) irrigated with treated wastewater to investigate EC uptake, accumulation, and translocation processes. The experiment spanned from June to September 2021 and involved three irrigation strategies: conventional water (FW), treated wastewater spiked with 10 target contaminants at the European average dose (TWWx1), and tertiary WWTP effluent spiked with the target contaminants at a triple dose (TWWx3). The results showed distinct behavior and distribution of ECs between the TWWx1 and TWWx3 strategies. In the TWWx3 strategy, clarithromycin, carbamazepine, metoprolol, fluconazole, and climbazole exhibited interactions with the soil-plant system, with varying degradation rates, soil accumulation rates, and plant accumulation rates. In contrast, naproxen, ketoprofen, diclofenac, sulfamethoxazole, and trimethoprim showed degradation. These findings imply that some ECs may be actively taken up by plants, potentially introducing them into the food chain and raising concerns for humans and the environment.

Role of Mesh Pore Size in Dynamic Membrane Bioreactors.

Two identical bench-scale Self-Forming Dynamic Membrane BioReactors (SFD MBR) were set-up and operated for the treatment of real urban wastewater. The two bioreactors were equipped with meshes of different mesh pore size. Meshes having pore size values of 20 and 50 µm were tested under solid retention time (SRT) of 15 d, whereas meshes with 50 and 100 µm pore sizes were compared under SRT of 50 d. The results of long-term experiments showed very good overall performances by all systems at the steady state. High flux (in the range 61-71 L m-2 h-1) and very good effluent quality were obtained, with average suspended solids and chemical oxygen demanding values below 10 mg L-1 and 35 mg L-1, respectively. The mesh pore size did not have a major influence on the average cleaning frequency. However, the pore size affected the effluent quality in correspondence of two particular conditions: (i) immediately after mesh cleaning; and (ii) during operation under high suction pressures (mesh clogging not promptly removed through cleaning). Moreover, the mesh cleaning frequency was observed to be dependent on the SRT. In tests with 50 d SRT, the cleaning requirements were very low (one every five days), and this limited the influence of the mesh pore size on the effluent quality. In conclusion, in SFD MBR, the role of the mesh pore size on the effluent quality may be more or less relevant depending on the operating conditions that directly influence the Dynamic Membrane formation.

A global multinational survey of cefotaxime-resistant coliforms in urban wastewater treatment plants.

The World Health Organization Global Action Plan recommends integrated surveillance programs as crucial strategies for monitoring antibiotic resistance. Although several national surveillance programs are in place for clinical and veterinary settings, no such schemes exist for monitoring antibiotic-resistant bacteria in the environment. In this transnational study, we developed, validated, and tested a low-cost surveillance and easy to implement approach to evaluate antibiotic resistance in wastewater treatment plants (WWTPs) by targeting cefotaxime-resistant (CTX-R) coliforms as indicators. The rationale for this approach was: i) coliform quantification methods are internationally accepted as indicators of fecal contamination in recreational waters and are therefore routinely applied in analytical labs; ii) CTX-R coliforms are clinically relevant, associated with extended-spectrum ß-lactamases (ESBLs), and are rare in pristine environments. We analyzed 57 WWTPs in 22 countries across Europe, Asia, Africa, Australia, and North America. CTX-R coliforms were ubiquitous in raw sewage and their relative abundance varied significantly (<0.1% to 38.3%), being positively correlated (p < 0.001) with regional atmospheric temperatures. Although most WWTPs removed large proportions of CTX-R coliforms, loads over 103 colony-forming units per mL were occasionally observed in final effluents. We demonstrate that CTX-R coliform monitoring is a feasible and affordable approach to assess wastewater antibiotic resistance status.

Reuse of ultrafiltered effluents for crop irrigation: On-site flow cytometry unveiled microbial removal patterns across a full-scale tertiary treatment.

Reuse of treated wastewater for crop irrigation has been widely adopted to mitigate the effects of water scarcity on agricultural yields and to help preserving the integrity of aquatic ecosystems. This paper presents the outcomes of one-year monitoring of a full-scale agro-industrial wastewater treatment plant designed for water reuse, with a multistage tertiary treatment based on sand filtration, membrane ultrafiltration, storage and on-demand UV disinfection. We aimed to test flow cytometry as a monitoring tool to provide on-site indications on tertiary treatment performances and on the quality of treated wastewater along the treatment scheme. Membrane ultrafiltration retained prokaryotic cells and E. coli (>3 log). During storage of treated effluents, a significant decay of E. coli was observed together with the growth of prokaryotic and eukaryotic cells, and the UV disinfection was effective only against fecal indicators. The microbial quality of the treated effluent was comparable to the control groundwater locally used for irrigation. On-site rapid assessments by flow cytometry allowed unveiling crucial aspects affecting the microbiological quality of ultrafiltration permeate and treated effluent immediately after sampling, including plant operating performances and microbial removal patterns across the treatment train.

The Self-Forming Dynamic Membrane BioReactor (SFD MBR) as a suitable technology for agro-industrial wastewater treatment.

Two bench-scale Self-Forming Dynamic Membrane BioReactors (SFD MBR), equipped with 50 µm nylon meshes were set up and operated under aerobic conditions in order to treat canning and winery wastewaters. The results showed different behaviors of the two systems, confirming the strong dependence of SFD MBR performance on the type of biomass and, in turn, on the type of stream being treated. Both plants achieved good results in terms of effluent quality, demonstrating the suitability of the proposed technology. Median values of effluent turbidity were 2.7 and 15.4 NTU (Nephelometric Turbidity Units) in the reactors fed with canning wastewater and winery wastewater, respectively. The removal of organic matter (as COD, Chemical Oxygen Demand) was consistently above 90 %, although the retention of suspended solids was variable and somewhat dependent on operating conditions and feed composition. The activated sludge characteristics were observed to affect filtration performance and in particular the capillary suction time (CST) was a possible indicator of efficiency, with a threshold value of 11 s above which filtration performance decreased. This parameter is proposed as an early warning tool for changes in the filtration performance of an SFD MBR, both for effluent quality and cleaning requirements.

Functional Response of MBR Microbial Consortia to Substrate Stress as Revealed by Metaproteomics.

Bacterial consortia have a primary role in the biological degradations occurring in activated sludge for wastewater treatment, for their capacities to metabolize the polluting matter. Therefore, the knowledge of the main metabolic pathways for the degradation of pollutants becomes critical for a correct design and operation of wastewater treatment plants. The metabolic activity of the different bacterial groups in activated sludge is commonly investigated through respirometry. Furthermore, in the last years, the development of "omic" approaches has offered more opportunities to integrate or substitute the conventional microbiological assays and to deeply understand the taxonomy and dynamics of complex microbial consortia. In the present work, an experimental membrane bioreactor (MBR) was set up and operated for the treatment of municipal wastewater, and the effects of a sudden decrease of the organic supply on the activated sludge were investigated. Both respirometric and metaproteomic approaches revealed a resistance of autotrophic bacteria to the substrate stress, and particularly of nitrifying bacteria. Furthermore, metaproteomics allowed the identification of the taxonomy of the microbial consortium based on its protein expression, unveiling the prevalence of Sorangium and Nitrosomonas genera both before and after the organic load decrease. Moreover, it confirmed the results obtained through respirometry and revealed a general expression of proteins involved in metabolism and transport of nitrogen, or belonging to nitrifying species like Nitrosomonas europeae, Nitrosomonas sp. AL212, or Nitrospira defluvii.

Sludge cake and biofilm formation as valuable tools in wastewater treatment by coupling Integrated Fixed-film Activated Sludge (IFAS) with Self Forming Dynamic Membrane BioReactors (SFD-MBR).

Two lab-scale Self Forming Dynamic Membrane BioReactors (SFD-MBR), equipped with 50 µm nylon meshes were set up and operated for the treatment of real municipal wastewater. Plastic carriers were added in one of the two bioreactors to generate a combination of the Integrated Fixed-film Activated Sludge (IFAS) and the SFD-MBR technologies. Overall, the two systems performed very well, achieving excellent effluent quality under steady state conditions and showing good resilience to extreme organic loading conditions. Continuous air scouring and periodical mesh cleaning by jet rinsing with tap water were effective in maintaining stable and high productivity (membrane flux around 67 L m2 h-1) over a period of 140 days. The application of the IFAS process resulted in lower production of excess sludge and improved denitrification. On the other hand, under the tested conditions the combined IFAS-SFD-MBR showed a higher tendency to mesh clogging with respect to the SFD-MBR.

Influence of air scouring on the performance of a Self Forming Dynamic Membrane BioReactor (SFD MBR) for municipal wastewater treatment.

The Membrane BioReactor (MBR) is a well-established filtration-based technology for wastewater treatment. Despite the high quality of the effluent produced, one of the main drawbacks of the MBR is membrane fouling. In this context, a possible evolution towards systems having potentially lower installation and operating costs is the Self Forming Dynamic Membrane BioReactor (SFD MBR). Key of this technology is the self-formation of a biological filtering layer on a support of inert material. In this work, a lab-scale aerobic SFD MBR equipped with a nylon mesh was operated at approximately 95Lm-2h-1. Two mesh pore sizes (20 and 50µm) and three air scouring flow rates (150, 250, and 500mLairmin-1) were tested at steady state. Under all the tested conditions, the SFD MBR effectively treated real municipal wastewater. The quality of the produced effluent increased for lower mesh size and lower air scouring intensity.

Metaproteomics Applied to Activated Sludge for Industrial Wastewater Treatment Revealed a Dominant Methylotrophic Metabolism of Hyphomicrobium zavarzinii.

In biological wastewater treatments, microbial populations of the so-called activated sludge work together in the abatement of pollutants. In this work, the metabolic behavior of the biomass of a lab-scale plant treating industrial pharmaceutical wastewater was investigated through a metaproteomic approach. The complete treatment process included a membrane biological reactor (MBR) coupled with an advanced oxidation process (AOP) for partial breakdown of non-biodegradable molecules. Proteins from biomass samples collected pre- and post-AOP application were investigated by two-dimensional gel electrophoresis (2DE), mass spectrometry (MS), and finally identified by database search. Results showed that most proteins remained constant between pre- and post-AOP. Methanol dehydrogenase (MDH) belonging to Hyphomicrobium zavarzinii appeared as the most constantly expressed protein in the studied consortium. Other identified proteins belonging to Hyphomicrobium spp. revealed a predominant methylotrophic metabolism, and H. zavarzinii appeared as a key actor in the studied microbial community.