Catarina L. Amorim

Degradation of fluoroquinolone antibiotics and identification of metabolites/transformation products by liquid chromatography–tandem mass spectrometry

Antibiotics are a therapeutic class widely found in environmental matrices and extensively studied due to its persistence and implications for multi-resistant bacteria development. This work presents an integrated approach of analytical multi-techniques on assessing biodegradation of fluorinated antibiotics at a laboratory-scale microcosmos to follow removal and formation of intermediate compounds. Degradation of four fluoroquinolone antibiotics, namely Ofloxacin (OFL), Norfloxacin (NOR), Ciprofloxacin (CPF) and Moxifloxacin (MOX), at 10 mg L(-1) using a mixed bacterial culture, was assessed for 60 days. The assays were followed by a developed and validated analytical method of LC with fluorescence detection (LC-FD) using a Luna Pentafluorophenyl (2) 3 μm column. The validated method demonstrated good selectivity, linearity (r(2)>0.999), intra-day and inter-day precisions (RSD<2.74%) and accuracy. The quantification limits were 5 μg L(-1) for OFL, NOR and CPF and 20 μg L(-1) for MOX. The optimized conditions allowed picturing metabolites/transformation products formation and accumulation during the process, stating an incomplete mineralization, also shown by fluoride release. OFL and MOX presented the highest (98.3%) and the lowest (80.5%) extent of degradation after 19 days of assay, respectively. A representative number of samples was selected and analyzed by LC-MS/MS with triple quadrupole and the molecular formulas were confirmed by a quadruple time of flight analyzer (QqTOF). Most of the intermediates were already described as biodegradation and/or photodegradation products in different conditions; however unknown metabolites were also identified. The microbial consortium, even when exposed to high levels of FQ, presented high percentages of degradation, never reported before for these compounds.

Removal of fluoxetine and its effects in the performance of an aerobic granular sludge sequential batch reactor

Fluoxetine (FLX) is a chiral fluorinated pharmaceutical mainly indicated for treatment of depression and is one of the most distributed drugs. There is a clear evidence of environmental contamination with this drug. Aerobic granular sludge sequencing batch reactors constitute a promising technology for wastewater treatment; however the removal of carbon and nutrients can be affected by micropollutants. In this study, the fate and effect of FLX on reactor performance and on microbial population were investigated. FLX adsorption/desorption to the aerobic granules was observed. FLX shock loads (≤4μM) did not show a significant effect on the COD removal. Ammonium removal efficiency decreased in the beginning of first shock load, but after 20 days, ammonia oxidizing bacteria became adapted. The nitrite concentration in the effluent was practically null indicating that nitrite oxidizing bacteria was not inhibited, whereas, nitrate was accumulated in the effluent, indicating that denitrification was affected. Phosphate removal was affected at the beginning showing a gradual adaptation, and the effluent concentration was <0.04mM after 70 days. A shift in microbial community occurred probably due to FLX exposure, which induced adaptation/restructuration of the microbial population. This contributed to the robustness of the reactor, which was able to adapt to the FLX load.

Bioaugmentation for treating transient 4-fluorocinnamic acid shock loads in a rotating biological contactor

A rotating biological contactor (RBC) was used to treat shock loadings of 4-fluorocinnamic acid (4-FCA). Intermittent 4-FCA shocks of 35 mg L(-1) were applied (ca. 3 months) with only limited mineralization occurring and accumulation of 4-fluorobenzoate (4-FBA) as an intermediate. After bioaugmentation with a degrading bacterium the RBC was able to deal with 4-FCA intermittent loading of 80 mg L(-1) however, a gradual decline in RBC performance occurred, leading to 4-FBA accumulation. The degrading strain was recovered from the biofilm during 2 months but intermittent feeding may have led to diminishing strain numbers. Distinct bacterial communities in the 1st and the 5th and 10th stages of the RBC were revealed by denaturating gradient gel electrophoresis. Several isolates retrieved from the RBC transformed 4-FCA into 4-FBA but only two strains mineralized the compound. Bioaugmentation allowed removal of the fluorinated compound however intermittent feeding may have compromised the bioreactor efficiency.

MALDI-TOF MS for the Identification of Cultivable Organic-Degrading Bacteria in Contaminated Groundwater near Unconventional Natural Gas Extraction Sites

Groundwater quality and quantity is of extreme importance as it is a source of drinking water in the United States. One major concern has emerged due to the possible contamination of groundwater from unconventional oil and natural gas extraction activities. Recent studies have been performed to understand if these activities are causing groundwater contamination, particularly with respect to exogenous hydrocarbons and volatile organic compounds. The impact of contaminants on microbial ecology is an area to be explored as alternatives for water treatment are necessary. In this work, we identified cultivable organic-degrading bacteria in groundwater in close proximity to unconventional natural gas extraction. Pseudomonas stutzeri and Acinetobacter haemolyticus were identified using matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry (MALDI-TOF MS), which proved to be a simple, fast, and reliable method. Additionally, the potential use of the identified bacteria in water and/or wastewater bioremediation was studied by determining the ability of these microorganisms to degrade toluene and chloroform. In fact, these bacteria can be potentially applied for in situ bioremediation of contaminated water and wastewater treatment, as they were able to degrade both compounds.

Biological treatment of a contaminated gaseous emission from a leather industry in a suspended-growth bioreactor.

A suspended-growth bioreactor (SGB) was operated for the treatment of a gaseous stream mimicking emissions generated at a leather industrial company. The main volatile organic compounds (VOCs) present in the gaseous stream consisted of 1-methoxy-2-propanol, 2,6-dimethyl-4-heptanone, 2-butoxyethanol, toluene and butylacetate. A microbial consortium able to degrade these VOCs was successfully enriched. A laboratory-scale SGB was established and operated for 210-d with an 8h cycle period and with shutdowns at weekends. Along this period, the SGB was exposed to organic loads (OL) between 6.5 and 2.3 x 10(2) g h(-1) m(-3). Most of the compounds were not detected at the outlet of the SGB. The highest total VOC removal efficiency (RE) (ca 99%) was observed when an OL of 1.6 x 10(2) g h(-1) m(-3) was fed to the SGB. The maximum total VOC elimination capacity (1.8 x 10(2) g h(-1) m(-3)) was achieved when the OL applied to the SGB was 2.3 x 10(2) g h(-1) m(-3). For all the operating conditions, the SGB showed high levels of degradation of toluene and butylacetate (RE approximately equal to 100%). This study also revealed that recirculation of the gaseous effluent improved the performance of the SGB. Overall, the SGB was shown to be robust, showing high performance after night and weekend shutdown periods.

Bacterial community dynamics within an aerobic granular sludge reactor treating wastewater loaded with pharmaceuticals

Pharmaceuticals are micropollutants often present in wastewater treatment systems. In this study, the potential impact of such micropollutants on the bacterial population within aerobic granular sludge (AGS) bioreactor was investigated. The AGS bacterial community structure and composition were accessed combining DGGE fingerprinting and barcoded pyrosequencing analysis. Both revealed the existence of a dynamic bacterial community, independently of the pharmaceuticals presence. The AGS microbiome at both phylum and class levels varied over time and, after stopping pharmaceuticals feeding, the bacterial community did not return to its initial composition. Nevertheless, most of the assigned OTUs were present throughout the different operational phases. This core microbiome, represented by over 72% of the total sequences in each phase, probably played an important role in biological removal processes, avoiding their failure during the disturbance period. Quantitative-PCR revealed that pharmaceuticals load led to gradual changes on the abundance of ammonia-oxidizing bacteria (AOB), nitrite-oxidizing bacteria (NOB) and polyphosphate-accumulating organisms (PAO) but their persistence during that phase demonstrated the resilience of such bacterial groups. AGS microbiome changed over time but a core community was maintained, probably ensuring the accomplishment of the main biological removal processes.

Simultaneous partial nitrification and 2-fluorophenol biodegradation with aerobic granular biomass: reactor performance and microbial communities

An aerobic granular bioreactor was operated for over 4months, treating a synthetic wastewater with a high ammonium content (100mgNL-1). The inoculum was collected from a bioreactor performing simultaneous partial nitrification and aromatic compounds biodegradation. From day-56 onwards, 2-fluorophenol (2-FP) (12.4mgL-1) was added to the feeding wastewater and the system was bioaugmented with a 2-FP degrading bacteria (Rhodococcus sp. FP1). By the end of operation, complete 2-FP biodegradation and partial nitrification were simultaneously achieved. Aerobic granules remained stable over time. During the 2-FP loading, a shift in the community structure occurred, coinciding with the improvement of 2-FP degradation. DGGE analysis did not allow to infer on the bioaugmented strain presence but pyrosequencing analysis detected Rhodococcus genus by the end of operation. Together with other potential phenolic-degraders within granules, these microorganisms were probably responsible for 2-FP degradation.

Development of a low pressure chromatographic flow system for monitoring the biodegradation of ofloxacin and ciprofloxacin

In this work, we propose a simple low pressure chromatography method with a high throughput for monitoring the biodegradation of fluoroquinolones. Fluoroquinolones are a class of antibiotics that have been accumulating in the environment as a consequence of their release from different sources, namely hospital waste. It has been found that wastewater treatment plants are not able to completely remove this type of micro-pollutants and so, alternative solutions are necessary. Some biodegradation studies have been done but the HPLC-based methodologies used to monitor the degradation process usually require high cost instrumentation such as analytical columns and detectors. For this reason, a 1 cm monolithic column was coupled to a flow injection system and used for the simultaneous quantification of ofloxacin and ciprofloxacin with UV detection at 295 and 275 nm, respectively. LODs of 0.5 and 0.29 mg L−1 for ofloxacin and ciprofloxacin were obtained, respectively, with repeatability within the range of 2–10%. The developed method was successfully applied to monitor the biodegradation of ofloxacin and ciprofloxacin by the strain Labrys portucalensis F11. The results proved that the low pressure chromatography method is a simpler, cheaper, and faster alternative to monitor biodegradation studies.