Continuous fungal treatment of non-sterile veterinary hospital effluent: pharmaceuticals removal and microbial community assessment.

Source point treatment of effluents with a high load of pharmaceutical active compounds (PhACs), such as hospital wastewater, is a matter of discussion among the scientific community. Fungal treatments have been reported to be successful in degrading this type of pollutants and, therefore, the white-rot fungus Trametes versicolor was applied for the removal of PhACs from veterinary hospital wastewater. Sixty-six percent removal was achieved in a non-sterile batch bioreactor inoculated with T. versicolor pellets. On the other hand, the study of microbial communities by means of DGGE and phylogenetic analyses led us to identify some microbial interactions and helped us moving to a continuous process. PhAC removal efficiency achieved in the fungal treatment operated in non-sterile continuous mode was 44 % after adjusting the C/N ratio with respect to the previously calculated one for sterile treatments. Fungal and bacterial communities in the continuous bioreactors were monitored as well.

Degradation of UV filters in sewage sludge and 4-MBC in liquid medium by the ligninolytic fungus Trametes versicolor.

Ultraviolet (UV) filters are xenobiotic compounds that can enter the environment through the liquid effluent of wastewater treatment plants (WWTPs) and through adsorption in the sludge by-product because of their high hydrophobicity, as the sludge is subsequently applied as a fertiliser. A solid-state treatment of WWTP sludge with the white-rot fungus Trametes versicolor is reported in the present work as a feasible method for UV filter degradation, with reductions ranging from 87% in the case of 3-(4'-methylbenzylidene) camphor (4-MBC) to 100% for benzophenone-3 (BP3) and its metabolite 4,4'-dihydroxybenzophenone (4DHB). This study represents a first step in the development of a future fungal treatment for UV filters; thus, it is essential to prove that elimination is due only to the action of the fungus and not that of other microorganisms. To this end, the sludge was sterilised prior to fungal treatment. Biological assays indicate that T. versicolor readily eliminates oestrogenic activity, although it may be inefficient at eliminating other compounds, including some with dioxin-like activity. Degradation studies of 4-MBC in liquid media were also performed, and complete removal was achieved in less than 24 h. The main metabolites were identified, and the first steps of the transformation pathway were elucidated: a mono- or di-hydroxylation by cytochrome P450 and a subsequent conjugation with a pentose. None of 4-MBC transformation products was found to be responsible for increased dioxin-like activity in the sludge.

Decreasing environmental impact of landfill leachate treatment by MBR, RO and EDR hybrid treatment.

A prototype pilot plant testing for a novel complete treatment strategy for landfill leachate aimed to decrease its environmental impact was studied. Pre-treatment of leachate was performed by means of a membrane biore-actor (MBR) decreasing inorganic carbon concentration by 92 ± 8% and achieving N removals of 85%. Suspended solids removal in the MBR >99.9% conditioned leachate for the next membrane step. Spiral-would reverse osmosis (RO) regenerated membranes were used to treat the MBR effluent. This RO unit achieved a global recovery of 84% along with operation and rejections of >95% for most of the analyzed compounds. Since RO permeate did not meet discharge standards, promising results were obtained after a second RO pass was applied. The RO brine produced was further concentrated by an electrodialysis reversal (EDR) unit, achieving an averaged recovery of 67% throughout the operation. The average recovery of the whole pilot plant system was >90%. The reduction of global brine volume together with the use of regenerated membranes are key to the environmental impact of the process and contribute to closing the loop of the circular economy. Life Cycle Assessment (LCA), performed according to ILCD Handbook guidelines, demonstrated that proposed new treatment had lower environmental impact than conventional treatments currently used in landfill facilities. Concretely, for the nine impact categories evaluated, the proposed treatment presented an average impact reduction of 93% compared to an advanced oxidation system and an average reduction of 26% when compared to a conventional RO treatment.

Hydrolysis and Methanogenesis in UASB-AnMBR Treating Municipal Wastewater Under Psychrophilic Conditions: Importance of Reactor Configuration and Inoculum.

Three upflow anaerobic sludge blanket (UASB) pilot scale reactors with different configurations and inocula: flocculent biomass (F-UASB), flocculent biomass and membrane solids separation (F-AnMBR) and granular biomass and membrane solids separation (G-AnMBR) were operated to compare start-up, solids hydrolysis and effluent quality. The parallel operation of UASBs with these different configurations at low temperatures (9.7 ± 2.4°C) and the low COD content (sCOD 54.1 ± 10.3 mg/L and pCOD 84.1 ± 48.5 mg/L), was novel and not previously reported. A quick start-up was observed for the three reactors and could be attributed to the previous acclimation of the seed sludge to the settled wastewater and to low temperatures. The results obtained for the first 45 days of operation showed that solids management was critical to reach a high effluent quality. Overall, the F-AnMBR showed higher rates of hydrolysis per solid removed (38%) among the three different UASB configurations tested. Flocculent biomass promoted slightly higher hydrolysis than granular biomass. The effluent quality obtained in the F-AnMBR was 38.0 ± 5.9 mg pCOD/L, 0.4 ± 0.9 mg sCOD/L, 9.9 ± 1.3 mg BOD5/L and <1 mg TSS/L. The microbial diversity of the biomass was also assessed. Bacteroidales and Clostridiales were the major bacterial fermenter orders detected and a relative high abundance of syntrophic bacteria was also detected. Additionally, an elevated abundance of sulfate reducing bacteria (SRB) was also identified and was attributed to the low COD/SO4 2- ratio of the wastewater (0.5). Also, the coexistence of acetoclastic and hydrogenotrophic methanogenesis was suggested. Overall this study demonstrates the suitability of UASB reactors coupled with membrane can achieve a high effluent quality when treating municipal wastewater under psychrophilic temperatures with F-AnMBR promoting slightly higher hydrolysis rates.

Coagulation-flocculation and moving bed biofilm reactor as pre-treatment for water recycling in the petrochemical industry.

Water recycling and reuse is of important value in water-using sectors like petrochemical industry. The aim of this research was to optimise the pre-treatment of petrochemical wastewater to undergo a further membrane treatment, with the final objective of water recycling within the same industry. Laboratory coagulation-flocculation tests prior to biological treatment were performed using Actiflo® Veolia commercial technology and an optimal coagulant dose of 30 mg/L ferric chloride was obtained. A bench-scale Moving Bed Biofilm Reactor (MBBR) system with two sequential reactors with working volumes of 5 L was filled with Z-carriers at 35% of their working volume. Organic loading rate (OLR) was varied from 0.2 to 3.25 kg/(m3 d) and the hydraulic retention time (HRT) ranged from 23.4 h to 4.5 h. High soluble chemical oxygen demand (sCOD) removals were obtained in stationary states (80-90%) and the calculated maximum sCOD that the system could degrade was 4.96 ±â€¯0.01 kg/(m3 d) at 23 ±â€¯2 °C. Changes in feed composition did not decrease sCOD removals showing that MBBR is a robust technology and the coagulation-flocculation step could be by-passed. Further removal of total suspended solids (TSS) and turbidity from the MBBR effluent would be required before a reverse osmosis (RO) step could be performed. A biofilm-forming genus, Haliscomenobacter spp., and an oil degrading genus Flavobacterium spp. were found in all the attached biomass samples. Acinetobacter spp. was the major bacterial genera found in suspended biomass. Proteobacteria and Bacteroidetes were the major phyla detected in the carrier samples while Proteobacteria the main one detected in the suspended biomass. The lack of fungal annotated sequences in databases led to a major proportion of fungal sequences being categorized as unclassified Fungi. The results obtained indicate that MBBR is an appropriate technology for hydrocarbon-degrading microorganism growth and, thus, for petrochemical wastewater pre-treatment for water regeneration.

Glutamate as sole carbon source for enhanced biological phosphorus removal.

Enhanced Biological Phosphorus Removal (EBPR) is based on the enrichment of sludge in polyphosphate accumulating organisms (PAO). Candidatus Accumulibacter is the bacterial community member most commonly identified as PAO in EBPR systems when volatile fatty acids (VFA) are the carbon source. However, it is necessary to understand the role of non-Accumulibacter PAO in the case of wastewater with low VFA content. This work shows the first successful long-term operation of an EBPR system with glutamate as sole carbon and nitrogen source, resulting in the enrichment of sludge in the genus Thiothrix (37%), the family Comamonadaceae (15.6%) and Accumulibacter (7.7%). The enrichment was performed in an anaerobic/anoxic/oxic (A2/O) continuous pilot plant, obtaining stable biological N and P removal. This microbial community performed anaerobic P-release with only 18-29% of the observed PHA storage in Accumulibacter-enriched sludge and with slight glycogen storage instead of consumption, indicating the involvement of other carbon storage routes not related to PHA and glycogen. Thiothrix could be clearly involved in P-removal because it is able of accumulating Poly-P, probably without PHA synthesis, but with glutamate involvement. On the other hand, Comamonadaceae could participate in degradation of glutamate and denitrification, but its involvement in P-uptake cannot be reliably concluded.

Fungal treatment for the removal of endocrine disrupting compounds from reverse osmosis concentrate: Identification and monitoring of transformation products of benzotriazoles.

The removal of 27 endocrine-disrupting compounds and related compounds (suspect effect) from a reverse osmosis concentrate using an alternative decontamination method based on a fungal treatment involving Trametes versicolor was assessed. In addition to chemical analysis, the toxicity of the treated water during the treatment was monitored using a bioluminescence inhibition test and estrogenic and anti-estrogenic tests. The compounds 1H-benzotriazole (BTZ) and two tolyltriazoles (TTZs), 4-methyl-1H-benzotriazole (4-MBTZ) and 5-methyl-1H-benzotriazole (5-MBTZ), were present in the reverse osmosis concentrate at the highest concentrations (7.4 and 12.8 µg L-1, respectively) and were partially removed by the fungal treatment under sterile conditions (58% for BTZ and 92% for TTZs) and non-sterile conditions, although to lesser extents (32% for BTZ and 50% for TTZs). Individual biotransformation studies of BTZ and the TTZs by T. versicolor in a synthetic medium and further analysis via on-line turbulent flow chromatography coupled to an HRMS-Orbitrap allowed the tentative identification of the transformation products (TPs). Six TPs were postulated for BTZ, two TPs were postulated for 4-MBTZ, and four TPs were postulated for 5-MBTZ. Most of these TPs are suggested to have been generated by conjugation with some sugars and via the methylation of the triazole group. Only TP 148 A, postulated to be derived from the biotransformation of BTZ, was observed in the effluent of the bioreactor treating the reverse osmosis concentrate.

Study of the effect of the bacterial and fungal communities present in real wastewater effluents on the performance of fungal treatments.

The use of the ligninolytic fungi Trametes versicolor for the degradation of micropollutants has been widely studied. However, few studies have addressed the treatment of real wastewater containing pharmaceutically active compounds (PhAC) under non-sterile conditions. The main drawback of performing such treatments is the difficulty for the inoculated fungus to successfully compete with the other microorganisms growing in the bioreactor. In the present study, several fungal treatments were performed under non-sterile conditions in continuous operational mode with two types of real wastewater effluent, namely, a reverse osmosis concentrate (ROC) from a wastewater treatment plant and a veterinary hospital wastewater (VHW). In all cases, the setup consisted of two parallel reactors: one inoculated with T. versicolor and one non-inoculated, which was used as the control. The main objective of this work was to correlate the operational conditions and traditional monitoring parameters, such as laccase activity, with PhAC removal and the composition of the microbial communities developed inside the bioreactors. For that purpose a variety of biochemical and molecular biology analyses were performed: phospholipid fatty acids analysis (PLFA), quantitative PCR (qPCR) and denaturing gradient gel electrophoresis (DGGE) followed by sequencing. The results show that many indigenous fungi (and not only bacteria, which were the focus of the majority of previously published research) can successfully compete with the inoculated fungi (i.e., Trichoderma asperellum overtook T. versicolor in the ROC treatment). We also showed that the wastewater origin and the operational conditions had a stronger impact on the diversity of microbial communities developed in the bioreactors than the inoculation or not with T. versicolor.

Suspect screening of emerging pollutants and their major transformation products in wastewaters treated with fungi by liquid chromatography coupled to a high resolution mass spectrometry.

A new approach for the screening of 33 pharmaceuticals and 113 of their known transformation products in wastewaters was developed. The methodology is based on the analysis of samples by liquid chromatography coupled to high resolution mass spectrometry (HRMS) followed by data processing using specific software and manual confirmation. A home-made library was built with the transformation products reported in literature for the target pharmaceuticals after treatment with various fungi. The method was applied to the search of these contaminants in 67 samples generated along treatment of wastewaters with white-rot fungus Trametes versicolor. The screening methodology allowed the detection of different transformation products (TPs) generated from degradation of parent compounds after fungal treatment. This approach can be a useful tool for the rapid screening and tentative detection of emerging contaminants during water treatment in both full and batch-scale studies when pure standards are not available.

Identification of some factors affecting pharmaceutical active compounds (PhACs) removal in real wastewater. Case study of fungal treatment of reverse osmosis concentrate.

Many technologies are being developed for the efficient removal of micropollutants from wastewater and, among them, fungal degradation is one of the possible alternative biological treatments. In this article, some factors that might affect pharmaceutically active compounds (PhACs) removal in a fungal treatment of real wastewater were identified in batch bioreactor treating reverse osmosis concentrate (ROC) from urban wastewater treatment plant (WWTP). We found that degradation of PhACs by Trametes versicolor was enhanced by addition of external nutrients (global removal of 44%). Moreover, our results point out that high aeration might be involved in the increase in the concentration of some PhACs. In fact, conjugation and deconjugation processes (among others) affect the removal assessment of emerging contaminants when working with real concentrations in comparison to experiments with spiked samples. Moreover, factors that could affect the quantification of micropollutants at lab-scale experiments were studied.

Degradation of pharmaceuticals from membrane biological reactor sludge with Trametes versicolor.

Emerging contaminants are a wide group of chemical products that are found at low concentrations in the environment. These contaminants can be either natural, e.g., estrogens, or synthetics, such as pesticides and pharmaceuticals, which can enter the environment through the water and sludge from wastewater treatment plants (WWTP). The growth of Trametes versicolor on membrane biological reactor (MBR) sludge in bioslurry systems at the Erlenmeyer scale was assessed and its capacity for removing pharmaceutical and personal care products (PPCPs) was evaluated. The ability of the fungus to remove hydrochlorothiazide (HZT) from liquid media cultures was initially assessed. Consequently, different bioslurry media (complete nutrient, glucose and no-nutrient addition) and conditions (sterile and non-sterile) were tested, and the removal of spiked HZT was monitored under each condition. The highest spiked HZT removal was assessed under non-sterile conditions without nutrient addition (93.2%). Finally, the removal assessment of a broad set of pharmaceuticals was performed in non-spiked bioslurry. Under non-sterile conditions, the fungus was able to completely degrade 12 out of the 28 drugs initially detected in the MBR sludge, achieving an overall degradation of 66.9%. Subsequent microbial analysis showed that the microbial diversity increased after 15 days of treatment, but there was still some T. versicolor in the bioslurry. Results showed that T. versicolor can be used to remove PPCPs in bioslurry systems under non-sterile conditions, without extra nutrients in the media, and in matrices as complex as an MBR sludge.

Use of stable isotope probing to assess the fate of emerging contaminants degraded by white-rot fungus.

The widespread of emerging contaminants in the environment and their potential impact on humans is a matter of concern. White-rot fungi are cosmopolitan organisms able to remove a wide range of pharmaceuticals and personal care products (PPCP) through cometabolism (i.e. laccases and peroxidases) or detoxification mechanisms (i.e. cytochrome P450 system). However, the use of PPCP as carbon source for these organisms is largely unexplored. Here, we used carbon stable isotope tracer experiments to assess the fate of anti-inflammatory diclofenac (DCF) and UV filter benzophenone-3 (BP3) during degradation by Trametes versicolor. The comparison between carbon isotopic composition of emitted carbon dioxide from 13C-labelled DCF ([acetophenyl ring-13C6]-DCF) and 13C-BP3 ([phenyl-13C6]-BP3) versus their 12C-homologue compounds showed mineralization of about 45% and 10% of the 13C contained in their respective molecules after 9 days of incubation. The carbon isotopic composition of the bulk biomass and the application of amino acid-stable isotope probing (SIP) allowed distinguishing between incorporation of 13C from BP3 into amino acids, which implies the use of this emerging contaminant as carbon source, and major intracellular accumulation of 13C from DCF without implying the transformation of its labelled phenyl ring into anabolic products. A mass balance of 13C in different compartments over time provided a comprehensive picture of the fate of DCF and BP3 across their different transformation processes. This is the first report assessing biodegradation of PPCP by SIP techniques and the use of emerging contaminants as carbon source for amino acid biosynthesis.

Evaluation of fungal- and photo-degradation as potential treatments for the removal of sunscreens BP3 and BP1.

Photodecomposition might be regarded as one of the most important abiotic factors affecting the fate of UV absorbing compounds in the environment and photocatalysis has been suggested as an effective method to degrade organic pollutants. However, UV filters transformation appears to be a complex process, barely addressed to date. The white rot fungus Trametes versicolor is considered as a promising alternative to conventional aerobic bacterial degradation, as it is able to metabolise a wide range of xenobiotics. This study focused on both degradation processes of two widely used UV filters, benzophenone-3 (BP3) and benzophenone-1 (BP1). Fungal treatment resulted in the degradation of more than 99% for both sunscreens in less than 24 h, whereas photodegradation was very inefficient, especially for BP3, which remained unaltered upon 24 h of simulated sunlight irradiation. Analysis of metabolic compounds generated showed BP1 as a minor by-product of BP3 degradation by T. versicolor while the main intermediate metabolites were glycoconjugate derivatives. BP1 and BP3 showed a weak, but significant estrogenic activity (EC50 values of 0.058 mg/L and 12.5 mg/L, respectively) when tested by recombinant yeast assay (RYA), being BP1 200-folds more estrogenic than BP3. Estrogenic activity was eliminated during T. versicolor degradation of both compounds, showing that none of the resulting metabolites possessed significant estrogenic activity at the concentrations produced. These results demonstrate the suitability of this method to degrade both sunscreen agents and to eliminate estrogenic activity.