Biological removal processes in aerobic granular sludge exposed to diclofenac.

Diclofenac is a worldwide consumed drug included in the watch list of substances to be monitored according to the European Union Water Framework Directive (Directive 2013/39/EU). Aerobic granular sludge sequencing batch reactors (AGS-SBR) are increasingly used for wastewater treatment but there is scant information on the fate and effect of micropollutants to nutrient removal processes. An AGS-SBR fed with synthetic wastewater containing diclofenac was bioaugmented with a diclofenac degrading bacterial strain and performance and microbial community dynamics was analysed. Chemical oxygen demand, phosphate and ammonia removal were not affected by the micropollutant at 0.03 mM (9.54 mg L-1). The AGS was able to retain the degrading strain, which was detected in the sludge throughout after augmentation. Nevertheless, besides some adsorption to the biomass, diclofenac was not degraded by the augmented sludge given the short operating cycles and even if batch degradation assays confirmed that the bioaugmented AGS was able to biodegrade the compound. The exposure to the pharmaceutical affected the microbial community of the sludge, separating the two first phases of reactor operation (acclimatization and granulation) from subsequent phases. The AGS was able to keep the bioaugmented strain and to maintain the main functions of nutrient removal even through the long exposure to the pharmaceutical, but combined strategies are needed to reduce the spread of micropollutants in the environment.

Simultaneous nitrification and phosphate removal by bioaugmented aerobic granules treating a fluoroorganic compound.

The presence of toxic compounds in wastewater can cause problems for organic matter and nutrient removal. In this study, the long-term effect of a model xenobiotic, 2-fluorophenol (2-FP), on ammonia-oxidizing bacteria (AOB), nitrite oxidizing bacteria (NOB) and phosphate accumulating organisms (PAO) in aerobic granular sludge was investigated. Phosphate (P) and ammonium (N) removal efficiencies were high (>93%) and, after bioaugmentation with 2-FP degrading strain FP1, 2-FP was completely degraded. Neither N nor P removal were affected by 50 mg L-1 of 2-FP in the feed stream. Changes in the aerobic granule bacterial communities were followed. Numerical analysis of the denaturing gradient gel electrophoresis (DGGE) profiles showed low diversity for the ammonia monooxygenase (amoA) gene with an even distribution of species. PAOs, including denitrifying PAO (dPAO), and AOB were present in the 2-FP degrading granules, although dPAO population decreased throughout the 444 days reactor operation. The results demonstrated that the aerobic granules bioaugmented with FP1 strain successfully removed N, P and 2-FP simultaneously.

Carbamazepine is degraded by the bacterial strain Labrys portucalensis F11.

The occurrence of pharmaceuticals in the environment is a topic of concern. Carbamazepine (CBZ) is a widespread antiepileptic drug and due to its physical-chemical characteristics minimal removal is achieved in conventional water treatments, and thus has been suggested as a molecular marker of wastewater contamination in surface water and groundwater. The present study reports the biotransformation of CBZ by the bacterial strain Labrys portucalensis F11. When supplied as a sole carbon source, a 95.4% biotransformation of 42.69 µM CBZ was achieved in 30 days. In co-metabolism with acetate, complete biotransformation was attained at a faster rate. Following a target approach, the detection and identification of 14 intermediary metabolites was achieved through UPLC-QTOF/MS/MS. Biotransformation of CBZ by the bacterial strain is mostly based on oxidation, loss of -CHNO group and ketone formation reactions; a biotransformation pathway with two routes is proposed. The toxicity of untreated and treated CBZ solutions was assessed using Vibrio Fischeri and Lepidium sativum acute toxicity tests and Toxi-Chromo Test. The presence of CBZ and/or its degradations products in solution resulted in moderate toxic effect on Vibrio Fischeri, whereas the other organisms were not affected. To the best of our knowledge this is the first report that proposes the metabolic degradation pathway of CBZ by a single bacterial strain.

Biodegradation of Diclofenac by the bacterial strain Labrys portucalensis F11.

Diclofenac (DCF) is a widely used non-steroidal anti-inflammatory pharmaceutical which is detected in the environment at concentrations which can pose a threat to living organisms. In this study, biodegradation of DCF was assessed using the bacterial strain Labrys portucalensis F11. Biotransformation of 70% of DCF (1.7-34 µM), supplied as the sole carbon source, was achieved in 30 days. Complete degradation was reached via co-metabolism with acetate, over a period of 6 days for 1.7 µM and 25 days for 34 µM of DCF. The detection and identification of biodegradation intermediates was performed by UPLC-QTOF/MS/MS. The chemical structure of 12 metabolites is proposed. DCF degradation by strain F11 proceeds mainly by hydroxylation reactions; the formation of benzoquinone imine species seems to be a central step in the degradation pathway. Moreover, this is the first report that identified conjugated metabolites, resulting from sulfation reactions of DCF by bacteria. Stoichiometric liberation of chlorine and no detection of metabolites at the end of the experiments are strong indications of complete degradation of DCF by strain F11. To the best of our knowledge this is the first report that points to complete degradation of DCF by a single bacterial strain isolated from the environment.

Characterization of the bacterial communities of aerobic granules in a 2-fluorophenol degrading process.

Aerobic granular sludge constitutes a novel technology for wastewater treatment. This study focused on the effect of 2-fluorophenol (2-FP) shock loadings on the microbial community diversity present in aerobic granules before and after inoculation with a bacterial strain able to degrade 2-FP, Rhodococcus sp. strain FP1. After bioaugmentation, apart from strain FP1, five culturable bacteria were isolated from the 2-FP degrading granules, belonging to the following genera: Serratia, Chryseobacterium, Xanthomonas, Pimelobacter and Rhodococcus. The latter two isolates are able to degrade 2-FP. Changes in the aerobic granules' bacterial communities related to 2-FP shock loadings were examined using denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene pool. Numerical analysis of the DGGE profiles showed high diversity with an even distribution of species. Based on cluster analysis of the DGGE profiles, the bacterial communities present in the aerobic granules changes were related to the sampling time and the 2-FP concentration fed.

Bacterial community dynamics in a rotating biological contactor treating 2-fluorophenol-containing wastewater.

One of the main factors affecting the performance of rotating biological contactors (RBC) is the biofilm characteristics. Therefore, a deep understanding of the microbial population dynamics and structure of the biofilm is mandatory if optimization of organic matter and nutrients removal is targeted. This study focused on the effects of organic shock loads of 2-fluorophenol (2-FP) on the microbial diversity present in an RBC biofilm. The RBC was seeded with activated sludge from a conventional wastewater treatment plant and was operated during 496 days. During the first 126 days, the RBC was subjected to intermittent 2-FP shocks of 25 mg l(-1) and no degradation occurred. Therefore, the reactor was subsequently augmented with a 2-FP-degrading strain (FP1). Afterwards, the RBC had a stable performance when subjected to 2-FP shocks up to 50 mg l(-1) and to a starvation period, as indicated by removal of the compound. Denaturing gradient gel electrophoresis (DGGE) revealed large shifts in microbial communities present in the first and fifth stages, although no clear relation between the sample collection time and spatial factor was found. Phylogenetic affiliation of some predominant members was assessed by direct sequencing of correspondent DGGE bands. Affiliations to α-, ß- and δ-Proteobacteria were found. Several bacterial strains isolated from the reactor showed capacity for 2-FP degradation. Strain FP1 was successfully recovered from the biofilm by plating and by DGGE, reinforcing that bioaugmentation was successfully achieved.

Performance of an aerobic granular sequencing batch reactor fed with wastewaters contaminated with Zn2+.

The main aim of this study was to investigate the performance of an aerobic granular sludge sequencing batch reactor (AGS-SBR) receiving water streams supplied with different loads of Zn(2+) (50 and 100 mg L(-1)) during an operation of 866 cycles (ca. 109 days). When the metal was not fed, chemical oxygen demand (COD), PO4(3-) and NH4(+) were efficiently removed, with efficiencies of 56, 23 and 72% respectively. DGGE profiles showed that Zn(2+) supply negatively affected the bacterial diversity and community structure of the granules. Consequently, the shock loadings with Zn(2+), particularly at the higher levels (100 mg L(-1)), affected the nutrient removal in the AGS-SBR, although the reactor still generally complied with admissible legal values concerning organic matter, nitrogen and Zn. Simultaneous removal of PO4(3-) and TSS in such conditions needs further refining but the application of aerobic granular SBR in the treatment of Zn(2+) contaminated wastewaters seems viable.

Isolation and characterization of a Rhodococcus strain able to degrade 2-fluorophenol.

A pure bacterial culture able to utilize 2-fluorophenol (2-FP) as sole carbon and energy source was isolated by selective enrichment from sediments collected from a contaminated site in Northern Portugal. 16S rRNA gene analysis showed that the organism (strain FP1) belongs to the genus Rhodococcus. When grown aerobically on 2-FP, growth kinetics of strain FP1 followed the Luong model. An inhibitory effect of increasing 2-FP concentrations was observed with no growth occurring at 2-FP levels higher than ca. 4 mM. Rhodococcus strain FP1 was able to degrade a range of other organofluorine compounds, including 2-fluorobenzoate, 3-fluorobenzoate, 4-fluorobenzoate, 3-fluorophenol, 4-fluorophenol, 3-fluorocatechol, and 4-fluorocatechol, as well as chlorinated compounds such as 2-chlorophenol and 4-chlorophenol. Experiments with cell-free extracts and partially purified enzymes indicated that the first step of 2-fluorophenol metabolism was conversion to 3-fluorocatechol, suggesting an unusual pathway for fluoroaromatic metabolism. To our knowledge, this is the first time that utilization of 2-FP as a growth substrate by a pure bacterial culture is reported.

2-fluorophenol degradation by aerobic granular sludge in a sequencing batch reactor.

Aerobic granular sludge is extremely promising for the treatment of effluents containing toxic compounds, and it can economically compete with conventional activated sludge systems. A laboratory scale granular sequencing batch reactor (SBR) was established and operated during 444 days for the treatment of an aqueous stream containing a toxic compound, 2-fluorophenol (2-FP), in successive phases. Initially during ca. 3 months, the SBR was intermittently fed with 0.22 mM of 2-FP added to an acetate containing medium. No biodegradation of the target compound was observed. Bioaugmentation with a specialized bacterial strain able to degrade 2-FP was subsequently performed. The reactor was thereafter continuously fed with 0.22 and 0.44 mM of 2-FP and with 5.9 mM of acetate (used as co-substrate), for 15 months. Full degradation of the compound was reached with a stoichiometric fluoride release. The 2-FP degrading strain was successfully retained by aerobic granules, as shown through the recovering of the strain from the granular sludge at the end of the experiment. Overall, the granular SBR has shown to be robust, exhibiting a high performance after bioaugmentation with the 2-FP degrading strain. This study corroborates the fact that bioaugmentation is often needed in cases where biodegradation of highly recalcitrant compounds is targeted.

Bioaugmentation of a rotating biological contactor for degradation of 2-fluorophenol.

The performance of a laboratory scale rotating biological contactor (RBC) towards shock loadings of 2-fluorophenol (2-FP) was investigated. During a period of ca. 2 months organic shock loadings of 25 mg L⁻¹ of 2-FP were applied to the RBC. As no biodegradation of 2-FP was observed, bioaugmentation of the RBC with a 2-FP degrading strain was carried out and, along ca. 6 months, organic shock loadings within a range of 25-200 mg L⁻¹ of 2-FP were applied. Complete biodegradation of 50 mg L⁻¹ of 2-FP was observed during operation of the reactor. The RBC showed to be robust towards starvation periods, as after ca. 1month of non-supply of the target compound, the reactor resumed 2-FP degradation. The inoculated strain was retained within the biofilm in the disks, as the 2-FP degrading strain was recovered from the biofilm by the end of the experiment, thus bioaugmentation was successfully achieved.