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Soil Aquifer Treatment (SAT) is a robust technology to increase groundwater recharge and to improve reclaimed water quality. SAT reduces dissolved organic carbon, contaminants of emerging concern, nutrients, and colloidal matter, including pathogen indicators, but little is known about its ability to reduce loads of antibiotic resistance genes (ARGs) from reclaimed waters. Here we test six pilot SAT systems to eliminate various biological hazards from the secondary effluents of a wastewater treatment plant (WWTP), equipped with reactive barriers (RBs) including different sorptive materials. Using flow cytometry, qPCR and 16S rRNA gene amplicon sequencing methods, we determined that all six SAT systems reduced total loads of bacteria by 80 to 95 % and of clinically relevant ARGs by 85 to 99.9 %. These efficiencies are similar to those reported for UV/oxidation or membrane-based tertiary treatments, which require much more energy and resources. The presence and composition of reactive barriers, the season of sampling (June 2020, October 2020, and September 2021), or the flow regime (continuous versus pulsating) did not affect ARG removal efficiency, although they did alter the microbial community composition. This suggests that an adequate design of the SAT reactive barriers may significantly increase their performance. Under a mechanistic point of view, we observed an ecological succession of bacterial groups, linked to the changing physical-chemical conditions along the SAT, and likely correlated to the removal of ARGs. We concluded that SAT is as cost-efficient technology able to dramatically reduce ARG loads and other biological hazards from WWTP secondary effluents., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The plastisphere, a unique microbial biofilm community colonizing plastic debris and microplastics (MPs) in aquatic environments, has attracted increasing attention owing to its ecological and public health implications. This review consolidates current state of knowledge on freshwater plastisphere, focussing on its biodiversity, community assembly, and interactions with environmental factors. Current biomolecular approaches revealed a variety of prokaryotic and eukaryotic taxa associated with plastic surfaces. Despite their ecological importance, the presence of potentially pathogenic bacteria and mobile genetic elements (i.e., antibiotic resistance genes) raises concerns for ecosystem and human health. However, the extent of these risks and their implications remain unclear. Advanced sequencing technologies are promising for elucidating the functions of plastisphere, particularly in plastic biodegradation processes. Overall, this review emphasizes the need for comprehensive studies to understand plastisphere dynamics in freshwater and to support effective management strategies to mitigate the impact of plastic pollution on freshwater resources., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Bocci, Galafassi, Levantesi, Crognale, Amalfitano, Congestri, Matturro, Rossetti and Di Pippo.)

Introduction: Once dispersed in water, plastic materials become promptly colonized by biofilm-forming microorganisms, commonly known as plastisphere., Methods: By combining DNA sequencing and Confocal Laser Scanning Microscopy (CLSM), we investigated the plastisphere colonization patterns following exposure to natural lake waters (up to 77 days) of either petrochemical or biodegradable plastic materials (low density polyethylene - LDPE, polyethylene terephthalate - PET, polylactic acid - PLA, and the starch-based MaterBi® - Mb) in comparison to planktonic community composition. Chemical composition, water wettability, and morphology of plastic surfaces were evaluated, through Transform Infrared Spectroscopy (ATR-FTIR), Scanning Electron Microscopy (SEM), and static contact angle analysis, to assess the possible effects of microbial colonization and biodegradation activity., Results and Discussion: The phylogenetic composition of plastisphere and planktonic communities was notably different. Pioneering microbial colonisers, likely selected from lake waters, were found associated with all plastic materials, along with a core of more than 30 abundant bacterial families associated with all polymers. The different plastic materials, either derived from petrochemical hydrocarbons (i.e., LDPE and PET) or biodegradable (PLA and Mb), were used by opportunistic aquatic microorganisms as adhesion surfaces rather than carbon sources. The Mb-associated microorganisms (i.e. mostly members of the family Burkholderiaceae) were likely able to degrade the starch residues on the polymer surfaces, although the Mb matrix maintained its original chemical structure and morphology. Overall, our findings provide insights into the complex interactions between aquatic microorganisms and plastic materials found in lake waters, highlighting the importance of understanding the plastisphere dynamics to better manage the fate of plastic debris in the environment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Di Pippo, Bocci, Amalfitano, Crognale, Levantesi, Pietrelli, Di Lisio, Martinelli and Rossetti.)

Microbial chain elongation has emerged as a valuable bioprocess for obtaining marketable products, such as medium chain fatty acids usable in several industrial applications, from organic waste. The understanding of the microbiology and microbial ecology in these systems is crucial to apply these microbiomes in reliable production processes controlling microbial pathways to promote favourable metabolic processes, which will in turn increase product specificity and yields. In this research, the dynamics, cooperation/competition and potentialities of bacterial communities involved in the long-term lactate-based chain elongation process from food waste extract were evaluated under different operating conditions by DNA/RNA amplicon sequencing and functional profile prediction. The feeding strategies and the applied organic loading rates strongly affected the microbial community composition. The use of food waste extract promoted the selection of primary fermenters (i.e., Olsenella , Lactobacillus ) responsible for the in situ production of electron donors (i.e., lactate). The discontinuous feeding and the organic loading rate 15 gCOD L -1  d -1 selected the best performing microbiome in which microbes coexist and cooperate to complete the chain elongation process. Both at DNA and RNA level, this microbiome was composed by the lactate producer Olsenella , the short chain fatty acids producers Anaerostipes, Clostridium sensu stricto 7, C. sensu stricto 12, Corynebacterium, Erysipelotrichaceae UCG-004, F0332, Leuconostoc , and the chain elongator Caproiciproducens . This microbiome also showed the highest predicted abundance of short-chain acyl-CoA dehydrogenase, the functional enzyme responsible for the chain elongation process. The combined approach herein used allowed to study the microbial ecology of chain elongation process from food waste by identifying the main functional groups, establishing the presence of potential biotic interactions within the microbiomes, and predicting metabolic potentialities. This study provided pivotal indications for the selection of high-performance microbiome involved in caproate production from food waste that can serve as a basis for further improving system performance and engineering the process scale-up., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Crognale, Massimi, Sbicego, Braguglia, Gallipoli, Gazzola, Gianico, Tonanzi, Di Pippo and Rossetti.)

Once dispersed in water, microplastic (MP) particles are rapidly colonised by aquatic microbes, which can adhere and grow onto solid surfaces in the form of biofilms. This study provides new insights on microbial diversity and biofilm structure of plastisphere in lake waters. By combining Fourier Confocal Laser Scanning Microscopy (CLSM), Transform Infrared Spectroscopy (FT-IR) and high-throughput DNA sequencing, we investigated the microbial colonization patterns on floating MPs and, for the first time, the occurrence of eukaryotic core members and their possible relations with biofilm-forming bacterial taxa within the plastisphere of four different lakes. Through PCR-based methods (qPCR, LAMP-PCR), we also evaluated the role of lake plastisphere as long-term dispersal vectors of potentially harmful organisms (including pathogens) and antibiotic resistance genes (ARGs) in freshwater ecosystems. Consistent variation patterns of the microbial community composition occurred between water and among the plastisphere samples of the different lakes. The eukaryotic core microbiome was mainly composed by typical freshwater biofilm colonizers, such as diatoms (Pennales, Bacillariophyceaea) and green algae (Chlorophyceae), which interact with eukaryotic and prokaryotic microbes of different trophic levels. Results also showed that MPs are suitable vectors of biofilm-forming opportunistic pathogens and a hotspot for horizontal gene transfer, likely facilitating antibiotic resistance spread in the environments., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

In this study, marine biofilms were cultured in a flow-lane, semi-continuous photobioreactor at different irradiances and flows to evaluate their combined effect on biofilms' phototrophic composition and photosynthetic activity. Taxon richness, evaluated by different microscopy techniques, including transmission and scanning electron microscopy, resulted to be heavily reduced from source communities to mature cultures. The strongest decrease was observed for diatoms, which were overcome by cyanobacteria and green algae over time. Photosynthetic performance was investigated by pulse amplitude modulated fluorescence. Irradiance was the main driver of data distribution of the photosynthetic parameters rel.ETR max and I k, while flow rate affected α and ΔF/F m '. The combination of irradiance and flow rate affected ΔF/F m ' reflecting the photosynthetic performance of the most relatively abundant taxa. Higher ΔF/F m ' was attained when cyanobacteria and green algae were dominating, whilst lower ΔF/F m ' when diatoms occurred in the initial phase of biofilm development., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Fluorescence in situ hybridization (FISH) enables the detection and enumeration of microorganisms in a diversity of samples. Short-length oligonucleotide DNA probes complementary to 16S or 23S rRNA sequences are generally used to target different phylogenetic levels. The protocol for the application of FISH to aggregated or suspended cells in mixed microbial communities is described in this chapter, with a special emphasis on environmental samples.

In this study, 16S rRNA gene high throughput sequencing and Fluorescence In Situ Hybridization (FISH) combined with confocal laser scanning microscopy (CLSM) were used to assess for the first time biodiversity and structure of microplastic-associated biofilms (plastisphere) collected from Italian lentic ecosystems. The analysis revealed clear differences in microbial community composition among biofilms and corresponding planktonic populations indicating a selective adhesion on microplastics (MP). Although geographical variations in taxa composition were observed, a plastisphere core microbiome, composed by known biofilm formers found in freshwater ecosystems (e.g. Sphingorhabdus, Sphingomonas, Rhodobacter, Aquabacterium and Acidovorax genera) was found. Species composition of plastisphere did not substantially differ between the diverse polymers, while a clear link with the MP exposure time was found by Fourier Transform Infrared spectroscopy (FT-IR) and Scanning Electron Microscopy (SEM) analysis. Generalist planktonic taxa (e.g. members of the families Sphingomonadaceae and Rhodobacteraceae) were found on MPs with the lowest degradation level whereas the biodiversity increased with the increase of MP degradation. FISH-CLSM analysis confirmed the Burkolderiaceae dominance in most of the analyzed plastisphere samples and revealed a patchy microbial colonization and a complex biofilm architecture with bacterial micro-colonies and cyanobacterial aggregates occurring together with microalgae assemblages., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier Ltd.)

Photosynthetic performance of algal-bacterial biofilms from an Italian wastewater treatment plant was studied in a flow-lane photobioreactor at different irradiances, temperatures, and flow regime to evaluate the effects of these environmental parameters on biofilms' functioning, in view of application of these communities in wastewater biological treatment. Pulse amplitude modulated fluorescence was used to estimate the effective quantum yield of PSII (ΔF/F m ') of the light-acclimated biofilms and to perform rapid light curves (RLCs) for the determination of the photosynthetic parameters (rel.ETR max , α, I k ). Chl a , ash free dry weight (AFDW), and dry weight (DW) were measured to assess phototrophic and whole biofilm biomass development over time. From the analysis of photosynthetic parameter variation with light intensity, temperature and flow rate, it was possible to identify the set of experimental values favoring biofilm photosynthetic activity. Biomass increased over time, especially at the highest irradiances, where substrata were fastly colonized and mature biofilms developed at all temperatures and flow conditions tested.

Biological nutrient removal performances and kinetics of autochthonous marine biomass in forms of activated sludge and aerobic granular sludge were investigated under different salinity and sludge retention time (SRT). Both the biomasses, cultivated from a fish-canning wastewater, were subjected to stepwise increases in salinity (+2 gNaCl L -1 ), from 30 gNaCl L -1 up to 50 gNaCl L -1 with the aim to evaluate the maximum potential in withstanding salinity by the autochthonous marine biomass. Microbial marine species belonging to the genus of Cryomorphaceae and of Rhodobacteraceae were found dominant in both the systems at the maximum salinity tested (50 gNaCl L -1 ). The organic carbon was removed with a yield of approximately 98%, irrespective of the salinity. Similarly, nitrogen removal occurred via nitritation-denitritation and was not affected by salinity. The ammonium utilization rate and the nitrite utilization rate were approximately of 3.60 mgNH 4 -N gVSS -1 h -1 and 10.0 mgNO 2 -N gVSS -1 h -1 , respectively, indicating a high activity of nitrifying and denitrifying bacteria. The granulation process did not provide significant improvements in the nutrients removal process likely due to the stepwise salinity increase strategy. Biomass activity and performances resulted affected by long SRT (27 days) due to salt accumulation within the activated sludge flocs and granules. In contrast, a lower SRT (14 days) favoured the discharge of the granules and flocs with higher inert content, thereby enhancing the biomass renewing. The obtained results demonstrated that the use of autochthonous-halophilic bacteria represents a valuable solution for the treatment of high-strength carbon and nitrogen saline wastewater in a wide range of salinity. Besides, the stepwise increase in salinity and the operation at low SRT enabled high metabolic activity and to avoid excessive accumulation of salt within the biomass aggregates, limiting their physical destructuration due to the increase in loosely-bound exopolymers., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

A microcosm designed for culturing aquatic phototrophic biofilms on artificial substrata was used to perform experiments with microphytobenthos sampled during summer toxic outbreaks of Ostreopsis cf. ovata along the Middle Tyrrhenian coast. This dynamic approach aimed at exploring the unique and complex nature of O. cf. ovata bloom development in the benthic system. Epibenthic assemblages were used as inocula for co-cultures of bloom organisms on polycarbonate slides at controlled environmental conditions. Biofilm surface adhesion, growth, and spatial structure were evaluated along with shifts in composition and matrix production in a low disturbance regime, simulating source habitat. Initial adhesion and substratum colonisation appeared as stochastic processes, then community structure and physiognomy markedly changed with time. Dominance of filamentous cyanobacteria and diatoms, and dense clusters of Amphidinium cf. carterae at the mature biofilm phases, were recorded by light and confocal microscopy, whilst O. cf. ovata growth was visibly limited in the late culture phases. Life-form strategies, competitiveness for resources, and possibly allelopathic interactions shaped biofilm structure during culture growth. HPLC (High Performance Liquid Chromatography) analysis of exopolysaccharidic matrix revealed variations in sugar total amounts and composition. No toxic compounds were detected in the final communities tested by LC-MS (Liquid Chromatography- Mass Spectrometry) and MALDI-TOF MS (Matrix Assisted Laser Desorption Ionization Time OF Flight Mass Spectroscopy) techniques., Competing Interests: The authors declare no conflict of interest.

In this study, monthly variations in biomass of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) were analysed over a 1-year period by fluorescence in situ hybridization (FISH) at the full-scale Fusina WWTP. The nitrification capacity of the plant was also monitored using periodic respirometric batch tests and by an automated on-line titrimetric instrument (TITrimetric Automated ANalyser). The percentage of nitrifying bacteria in the plant was the highest in summer and was in the range of 10-15 % of the active biomass. The maximum nitrosation rate varied in the range 2.0-4.0 mg NH4 g(-1) VSS h(-1) (0.048-0.096 kg TKN kg(-1) VSS day(-1)): values obtained by laboratory measurements and the on-line instrument were similar and significantly correlated. The activity measurements provided a valuable tool for estimating the maximum total Kjeldahl nitrogen (TKN) loading possible at the plant and provided an early warning of whether the TKN was approaching its limiting value. The FISH analysis permitted determination of the nitrifying biomass present. The main operational parameter affecting both the population dynamics and the maximum nitrosation activity was mixed liquor volatile suspended solids (MLVSS) concentration and was negatively correlated with ammonia-oxidizing bacteria (AOB) (p = 0.029) and (NOB) (p = 0.01) abundances and positively correlated with maximum nitrosation rates (p = 0.035). Increases in concentrations led to decreases in nitrifying bacteria abundance, but their nitrosation activity was higher. These results demonstrate the importance of MLVSS concentration as key factor in the development and activity of nitrifying communities in wastewater treatment plants (WWTPs). Operational data on VSS and sludge volume index (SVI) values are also presented on 11-year basis observations.

Aims: This work reports on one of the first attempts to use biofilm-forming cyanobacteria for biomass and lipid production., Methods and Results: Three isolates of filamentous cyanobacteria were obtained from biofilms at different Italian sites and characterized by a polyphasic approach, involving microscopic observations, ecology and genetic diversity (studying the 16S rRNA gene). The isolates were grown in batch systems and in a semi-continuous flow incubator, specifically designed for biofilms development. Culture system affected biomass and lipid production, but did not influence the fatty acid profile. The composition of fatty acids was mainly palmitic acid (>50%) and less amounts of other saturated and monounsaturated fatty acids. Only two isolates contained two polyunsaturated fatty acids., Conclusions: Data obtained from the flow-lane incubator system would support a more economical and sustainable use of the benthic micro-organisms for biomass production. The produced lipids contained fatty acids suitable for a high-quality biodiesel production, showing high proportions of saturated and monounsaturated fatty acids., Significance and Impact of the Study: Data seem promising when taking into account the savings in cost and time derived from easy procedures for biomass harvesting, especially when being able to obtain the co-production of other valuable by-products., (© 2012 The Authors Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.)

The responses of cultured phototrophic biofilms to diverse phosphorus (P) regimes were assessed using a semi-continuous flow incubator. Three biofilms were grown over 18 days under three different P regimes: replete inorganic P, organic P-only and limited inorganic P. Assessing the response of the biofilms took into account the rate of phosphomonoesterase and phosphodiesterase activities, biofilm nutrient contents and biomass accrual across the growth period. Phosphorus limitation was indicated by slower biomass accumulation and higher phosphatase activities of the organic P-only and P-limited biofilms compared to the P-replete biofilms. The cyanobacterium Phormidium sp. dominated the later stages in all the treatments forming a dense layer at the biofilm-medium interface. This layer possibly led to a reduction of light and nutrient diffusion to sub-surface cells and may account for the production of phosphatases under P replete conditions. In addition, the Phormidium-layer possibly produced a top-heavy P (and N) distribution and could explain the large reductions in areal nutrient concentrations. End-product repression and de-repression of phosphatase activity was suggested to be a main controlling factor of phosphatase activity. Consequently, it is proposed that for efficient nutrient removal from wastewaters that biofilms should be regularly removed to continually maintain biofilms at the initial stages (3-7 days)., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The present work reports on the first attempt to study water mobility in phototrophic biofilms, applying the (1)H-NMR relaxometry technique to closely monitored microbial communities grown in a microcosm under controlled ambient conditions. Longitudinal water proton relaxation times exhibited a bi-exponential behavior in all biofilm samples, indicating two types of water molecules with diverging dynamic properties, confined to different compartments of the biofilm. The fast-relaxing component can be attributed to water molecules tightly bound to the intracellular matrix, while the slow-relaxing component could reflect the behavior of water embedded in the biopolymer matrix, confined into matrix pores and channels. The results are discussed with respect to a possible key role of exopolysaccharides and uronic acids in water binding in phototrophic biofilms.