Bioremediation of coastal aquaculture effluents spiked with florfenicol using microalgae-based granular sludge - a promising solution for recirculating aquaculture systems.

Aquaculture is a crucial industry in the agri-food sector, but it is linked to serious environmental problems. There is a need for efficient treatment systems that allow water recirculation to mitigate pollution and water scarcity. This work aimed to evaluate the self-granulation process of a microalgae-based consortium and its capacity to bioremediate coastal aquaculture streams that sporadically contain the antibiotic florfenicol (FF). A photo-sequencing batch reactor was inoculated with an autochthonous phototrophic microbial consortium and was fed with wastewater mimicking coastal aquaculture streams. A rapid granulation process occurred within ca. 21 days, accompanied by a substantially increase of extracellular polymeric substances in the biomass. The developed microalgae-based granules exhibited high and stable organic carbon removal (83-100%). Sporadically wastewater contained FF which was partially removed (ca. 5.5-11.4%) from the effluent. In periods of FF load, the ammonium removal slightly decreased (from 100 to ca. 70%), recovering 2 days after FF feeding ceased. A high-chemical quality effluent was obtained, complying with ammonium, nitrite, and nitrate concentrations for water recirculation within a coastal aquaculture farm, even during FF feeding periods. Members belonging to the Chloroidium genus were predominant in the reactor inoculum (ca. 99%) but were replaced from day-22 onwards by an unidentified microalga from the phylum Chlorophyta (>61%). A bacterial community proliferated in the granules after reactor inoculation, whose composition varied in response to feeding conditions. Bacteria from the Muricauda and Filomicrobium genera, Rhizobiaceae, Balneolaceae, and Parvularculaceae families, thrived upon FF feeding. This study demonstrates the robustness of microalgae-based granular systems for aquaculture effluent bioremediation, even during periods of FF loading, highlighting their potential as a feasible and compact solution in recirculation aquaculture systems.

Valorization of Brewery Waste through Polyhydroxyalkanoates Production Supported by a Metabolic Specialized Microbiome.

Raw brewers' spent grain (BSG), a by-product of beer production and produced at a large scale, presents a composition that has been shown to have potential as feedstock for several biological processes, such as polyhydroxyalkanoates (PHAs) production. Although the high interest in the PHA production from waste, the bioconversion of BSG into PHA using microbial mixed cultures (MMC) has not yet been explored. This study explored the feasibility to produce PHA from BSG through the enrichment of a mixed microbial culture in PHA-storing organisms. The increase in organic loading rate (OLR) was shown to have only a slight influence on the process performance, although a high selectivity in PHA-storing microorganisms accumulation was reached. The culture was enriched on various PHA-storing microorganisms, such as bacteria belonging to the Meganema, Carnobacterium, Leucobacter, and Paracocccus genera. The enrichment process led to specialization of the microbiome, but the high diversity in PHA-storing microorganisms could have contributed to the process stability and efficiency, allowing for achieving a maximum PHA content of 35.2 ± 5.5 wt.% (VSS basis) and a yield of 0.61 ± 0.09 CmmolPHA/CmmolVFA in the accumulation assays. Overall, the production of PHA from fermented BSG is a feasible process confirming the valorization potential of the feedstock through the production of added-value products.

Microalgae-bacterial biomass outperforms PN-anammox biomass for oxygen saving in continuous-flow granular reactors facing extremely low-strength freshwater aquaculture streams.

The dissolved oxygen (DO) concentration in water streams is one of the most important and critical quality parameters in aquaculture farms. The main objective of this study was to evaluate the potential of two Continuous Flow Granular Reactors, one based on Partial Nitrification-Anammox biomass (Aquammox CFGR) and the other on Microalgae-Bacteria biomass (AquaMab CFGR), for improving dissolved oxygen availability in the recirculation aquaculture systems (RAS). Both reactors treated the extremely low-strength effluents from a freshwater trout farm (1.39 mg NH4+-N/L and 7.7 mg TOC/L). The Aquammox CFGR, removed up to 68% and 100% of ammonium and nitrite, respectively, but the DO concentration in the effluent was below 1 mg O2/L while the anammox activity was not maintained. In the AquaMab CFGR, bioaugmentation of aerobic granules with microalgae was attained, producing an effluent with DO concentrations up to 9 mg O2/L and removed up to 77% and 80% of ammonium and nitrite, respectively, which is expected to reduce the aeration costs in fish farms.

Quantitative image analysis as a robust tool to assess effluent quality from an aerobic granular sludge system treating industrial wastewater.

Quantitative image analysis (QIA) is a simple and automated method for process monitoring, complementary to chemical analysis, that when coupled to mathematical modelling allows associating changes in the biomass to several operational parameters. The majority of the research regarding the use of QIA has been carried out using synthetic wastewater and applied to activated sludge systems, while there is still a lack of knowledge regarding the application of QIA in the monitoring of aerobic granular sludge (AGS) systems. In this work, chemical oxygen demand (COD), ammonium (N-NH4+), nitrite (N-NO2-), nitrate (N-NO3-), salinity (Cl-), and total suspended solids (TSS) levels present in the effluent of an AGS system treating fish canning wastewater were successfully associated to QIA data, from both suspended and granular biomass fractions by partial least squares models. The correlation between physical-chemical parameters and QIA data allowed obtaining good assessment results for COD (R2 of 0.94), N-NH4+ (R2 of 0.98), N-NO2- (R2 of 0.96), N-NO3- (R2 of 0.95), Cl- (R2 of 0.98), and TSS (R2 of 0.94). While the COD and N-NO2- assessment models were mostly correlated to the granular fraction QIA data, the suspended fraction was highly relevant for N-NH4+ assessment. The N-NO3-, Cl- and TSS assessment benefited from the use of both biomass fractions (suspended and granular) QIA data, indicating the importance of the balance between the suspended and granular fractions in AGS systems and its analysis. This study provides a complementary approach to assess effluent quality parameters which can improve wastewater treatment plants monitoring and control, with a more cost-effective and environmentally friendly procedure, while avoiding daily physical-chemical analysis.

Cultivable microalgae diversity from a freshwater aquaculture filtering system and its potential for polishing aquaculture-derived water streams.

AIMS: Microalgae are ubiquitous in aquatic environments, including aquaculture farms, but few studies have delved into their phytoplankton diversity and bioremediation potential. In this study, the cultivable phytoplankton of a rainbow trout freshwater aquaculture farm was isolated, phylogenetically analysed and used to assemble a consortium to polish an aquaculture-derived effluent, with low concentrations of ammonium, nitrite and nitrate. METHODS AND RESULTS: Through standard plating in different selective media, a total of 15 microalgae strains were isolated from sludge from a rotary drum filtering system which removes suspended solids from the water exiting the facility. Based on 18S rRNA gene sequences, isolates were assigned to nine different genera of the Chlorophyta phylum: Asterarcys, Chlorella, Chloroccocum, Chlorosarcinopsis, Coelastrella, Desmodesmus, Micractinium, Parachlorella and Scenedesmus. Species from most of these genera are known to inhabit freshwater systems in Galicia and continental Spain, but the Coelastrella, Asterarcys or Parachlorella genera are not usually present in freshwater streams. In an onsite integrative approach, the capacity of a consortium of native microalgae isolates to grow on aquaculture-derived effluents and its nutrient removal capacity were assessed using a raceway pond. After 7 days, removal efficiencies of approximately 99%, 92% and 49% for ammonium, nitrite and nitrate, respectively, were achieved concomitantly with a microalgae biomass increase of ca. 17%. CONCLUSIONS: Sludge from the aquaculture filtering system presents a high diversity of microalgae species from the Chlorophyta phylum, whose application in a consortial approach revealed to be efficient to polish aquaculture-derived effluents with low nutrient content. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of native microalgae consortia from aquaculture systems can contribute to the development of efficient treatment systems for low-nutrient wastewater, avoiding nutrients release to the environment and promoting water recirculation. This may further strengthen the use of phycoremediation at the industrial scale, as an environment-friendly strategy.

Wastewater Valorization: Practice around the World at Pilot- and Full-Scale.

Over the last few years, wastewater treatment plants (WWTPs) have been rebranded as water resource recovery facilities (WRRFs), which recognize the resource recovery potential that exists in wastewater streams. WRRFs contribute to a circular economy by not only producing clean water but by recovering valuable resources such as nutrients, energy, and other bio-based materials. To this aim, huge efforts in technological progress have been made to valorize sewage and sewage sludge, transforming them into valuable resources. This review summarizes some of the widely used and effective strategies applied at pilot- and full-scale settings in order to valorize the wastewater treatment process. An overview of the different technologies applied in the water and sludge line is presented, covering a broad range of resources, i.e., water, biomass, energy, nutrients, volatile fatty acids (VFA), polyhydroxyalkanoates (PHA), and exopolymeric substances (EPS). Moreover, guidelines and regulations around the world related to water reuse and resource valorization are reviewed.

Sequencing versus continuous granular sludge reactor for the treatment of freshwater aquaculture effluents.

Ammonium and nitrite levels in water are crucial for fish health preservation and growth maintenance in freshwater aquaculture farms, limiting water recirculation. The aim of the present work was the evaluation and comparison of two granular sludge reactors which were operated to treat freshwater aquaculture streams at laboratory-scale: an Aerobic Granular Sludge - Sequencing Batch Reactor (AGS-SBR) and a Continuous Flow Granular Reactor (CFGR). Both units were fed with a synthetic medium mimicking an aquaculture recycling water (1.9-2.9 mg N/L), with low carbon content, and operational temperature varied between 17 and 25 °C. The AGS-SBR, inoculated with mature granules from a full-scale wastewater treatment plant, achieved high carbon and ammonium removal during the 157 operational days. Even at low hydraulic retention time (HRT), varying from 474 to 237 min, ammonium removal efficiencies of approximately 87-100% were observed, with an ammonium removal rate of approximately 14.5 mg NH4+-N/(L⋅d). Partial biomass washout occurred due to the extremely low carbon and nitrogen concentrations in the feeding, which could only support the growth of a small portion of bacteria, but no major changes on the reactor removal performance were observed. The CFGR was inoculated with activated sludge and operated for 98 days. Biomass granulation occurred in 7 days, improving the settling properties due to a high up-flow velocity of 11 m/h and an applied HRT of 5 min. The reactor presented mature granules after 32 days, achieving an average diameter of 1.9 mm at day 63. The CFGR ammonium removal efficiencies were of approximately 10-20%, with ammonium removal rates of 90.0 mg NH4+-N/(L⋅d). The main biological processes taking place in the AGS-SBR were nitrification and heterotrophic growth, while in the CFGR the ammonium removal occurred only by heterotrophic assimilation, with the reactor also presenting complete and partial denitrification, which caused nitrite production. Comparing both systems, the CFGR achieved 6 times higher ammonium removal rates than the AGS-SBR, being suitable for treating extremely high flows. On the other hand, the AGS-SBR removed almost 100% of ammonium content in the wastewater, discharging a better quality effluent, less toxic for the fish but treated lower flows.

Recovered granular sludge extracellular polymeric substances as carrier for bioaugmentation of granular sludge reactor.

An increasing amount of industrial chemicals are being released into wastewater collection systems and indigenous microbial communities in treatment plants are not always effective for their removal. In this work, extracellular polymeric substances (EPS) recovered from aerobic granular sludge (AGS) were used as a natural carrier to immobilize a specific microbial strain, Rhodococcus sp. FP1, able to degrade 2-fluorophenol (2-FP). The produced EPS granules exhibited a 2-FP degrading ability of 100% in batch assays, retaining their original activity after up to 2-months storage. Furthermore, EPS granules were added to an AGS reactor intermittently fed with saline wastewater containing 2-FP. Degradation of 2-FP and stoichiometric fluorine release occurred 8 and 35 days after bioaugmentation, respectively. Chemical oxygen demand removal was not significantly impaired by 2-FP or salinity loads. Nutrients removal was impaired by 2-FP load, but after bioaugmentation, the phosphate and ammonium removal efficiency improved from 14 to 46% and from 25 to 42%, respectively. After 2-FP feeding ceased, at low/moderate salinity (0.6-6.0 g L-1 NaCl), ammonium removal was completely restored, and phosphate removal efficiency increased. After bioaugmentation, 11 bacteria isolated from AGS were able to degrade 2-FP, indicating that horizontal gene transfer could have occurred in the reactor. The improvement of bioreactor performance after bioaugmentation with EPS immobilized bacteria and the maintenance of cell viability through storage are the main advantages of the use of this natural microbial carrier for bioaugmentation, which can benefit wastewater treatment processes.

Long-term stability of a non-adapted aerobic granular sludge process treating fish canning wastewater associated to EPS producers in the core microbiome.

The tolerance of aerobic granular sludge (AGS) to variable wastewater composition is perceived as one of its greatest advantages compared to other aerobic processes. However, research studies select optimal operational conditions for evaluating AGS performance, such as the use of pre-adapted biomass and the control of wastewater composition. In this study, non-adapted granular sludge was used to treat fish canning wastewater presenting highly variable organic, nutrient and salt levels over a period of ca. 8 months. Despite salt levels up to 14 g NaCl L-1, the organic loading rate (OLR) was found to be the main factor driving AGS performance. Throughout the first months of operation, the OLR was generally lower than 1.2 kg COD m-3 day-1, resulting in stable nitrification and low COD and phosphorous levels at the outlet. An increase in OLR up to 2.3 kg COD m-3 day-1 disturbed nitrification and COD and phosphate removal, but a decrease to average values between 1 and 1.6 kg COD m-3 day-1 led to resuming of those processes. Most of the bacteria present in the AGS core microbiome were associated to extracellular polymeric substances (EPS) production, such as Thauera and Paracoccus, which increased during the higher OLR period. Ammonium-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) species were detected in AGS biomass; while AOB were identified throughout the operation, NOB were no further identified after the period of increased OLR. Different polyphosphate-accumulating organisms (PAOs) were detected along the process: CandidatusAccumulibacter, Tetrasphaera and Gemmatimonas. A non-adapted granular sludge was able to treat the fish canning wastewater and to tolerate salinity fluctuations up to 14 g L-1. Overall, a high microbial diversity associated to EPS producers allowed to preserve bacterial groups responsible for nutrients removal, contributing to the adaptation and long-term stability of the AGS system.

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.

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.

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.

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.

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.

Performance of aerobic granular sludge in a sequencing batch bioreactor exposed to ofloxacin, norfloxacin and ciprofloxacin.

A granular sludge sequencing batch reactor (SBR) was operated for 340 days for treating a synthetic wastewater containing fluoroquinolones (FQs), namely ofloxacin, norfloxacin and ciprofloxacin. The SBR was intermittently fed with FQs, at concentrations of 9 and 32 µM. No evidence of FQ biodegradation was observed but the pharmaceutical compounds adsorbed to the aerobic granular sludge, being gradually released into the medium in successive cycles after stopping the FQ feeding. Overall COD removal was not affected during the shock loadings. Activity of ammonia oxidizing bacteria and nitrite oxidizing bacteria did not seem to be inhibited by the presence of FQs (maximum of 0.03 and 0.01 mM for ammonium and nitrite in the effluent, respectively). However, during the FQs feeding, nitrate accumulation up to 1.7 mM was observed at the effluent suggesting that denitrification was inhibited. The activity of phosphate accumulating organisms was affected, as indicated by the decrease of P removal capacity during the aerobic phase. Exposure to the FQs also promoted disintegration of the granules leading to an increase of the effluent solid content, nevertheless the solid content at the bioreactor effluent returned to normal levels within ca. 1 month after removing the FQs in the feed allowing recovery of the bedvolume. Denaturing gradient gel electrophoresis revealed a dynamic bacterial community with gradual changes due to FQs exposure. Bacterial isolates retrieved from the granules predominantly belonged to α- and γ-branch of the Proteobacteria phylum. The capacity of the system to return to its initial conditions after withdrawal of the FQ compounds in the inlet stream, reinforced its robustness to deal with wastewaters containing organic pollutants.

Biodegradation of ofloxacin, norfloxacin, and ciprofloxacin as single and mixed substrates by Labrys portucalensis F11.

Fluoroquinolone (FQ) antibiotics are extensively used both in human and veterinary medicine, and their accumulation in the environment is causing an increasing concern. In this study, the biodegradation of the three most worldwide used FQs, namely ofloxacin, norfloxacin, and ciprofloxacin, by the fluoroorganic-degrading strain Labrys portucalensis F11 was assessed. Degradation occurred when the FQs were supplied individually or as mixture in the culture medium, in the presence of an easily degradable carbon source. Consumption of individual FQs was achieved at different extents depending on its initial concentration, ranging from 0.8 to 30 µM. For the lowest concentration, total uptake of each FQ was observed but stoichiometric fluoride release was not achieved. Intermediate compounds were detected and identified by LC-MS/MS with a quadrupole time of flight detector analyzer. Biotransformation of FQs by L. portucalensis mainly occurred through a cleavage of the piperazine ring and displacement of the fluorine substituent allowing the formation of intermediates with less antibacterial potency. FQ-degrading microorganisms could be useful for application in bioaugmentation processes towards more efficient removal of contaminants in wastewater treatment plants.

Mineralization of 4-fluorocinnamic acid by a Rhodococcus strain.

A bacterial strain capable of aerobic degradation of 4-fluorocinnamic acid (4-FCA) as the sole source of carbon and energy was isolated from a biofilm reactor operating for the treatment of 2-fluorophenol. The organism, designated as strain S2, was identified by 16S rRNA gene analysis as a member of the genus Rhodococcus. Strain S2 was able to mineralize 4-FCA as sole carbon and energy source. In the presence of a conventional carbon source (sodium acetate [SA]), growth rate of strain S2 was enhanced from 0.04 to 0.14 h(-1) when the culture medium was fed with 0.5 mM of 4-FCA, and the time for complete removal of 4-FCA decreased from 216 to 50 h. When grown in SA-supplemented medium, 4-FCA concentrations up to 1 mM did not affect the length of the lag phase, and for 4-FCA concentrations up to 3 mM, strain S2 was able to completely remove the target fluorinated compound. 4-Fluorobenzoate (4-FBA) was transiently formed in the culture medium, reaching concentrations up to 1.7 mM when the cultures were supplemented with 3.5 mM of 4-FCA. Trans,trans-muconate was also transiently formed as a metabolic intermediate. Compounds with molecular mass compatible with 3-carboxymuconate and 3-oxoadipate were also detected in the culture medium. Strain S2 was able to mineralize a range of other haloorganic compounds, including 2-fluorophenol, to which the biofilm reactor had been exposed. To our knowledge, this is the first time that mineralization of 4-FCA as the sole carbon source by a single bacterial culture is reported.

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.

Effect of the metals iron, copper and silver on fluorobenzene biodegradation by Labrys portucalensis.

Organic and metallic pollutants are ubiquitous in the environment. Many metals are reported to be toxic to microorganisms and to inhibit biodegradation. The effect of the metals iron, copper and silver on the metabolism of Labrys portucalensis F11 and on fluorobenzene (FB) biodegradation was examined. The results indicate that the addition of 1 mM of Fe(2+) to the culture medium has a positive effect on bacterial growth and has no impact in the biodegradation of 1 and 2 mM of FB. The presence of 1 mM of Cu(2+) was found to strongly inhibit the growth of F11 cultures and to reduce the biodegradation of 1 and 2 mM of FB to ca. 50 %, with 80 % of stoichiometrically expected fluoride released. In the experiments with resting cells, the FB degraded (from 2 mM supplied) was reduced ca. 20 % whereas the fluoride released was reduced to 45 % of that stoichiometrically expected. Ag(+) was the most potent inhibitor of FB degradation. In experiments with growing cells, the addition of 1 mM of Ag(+) to the culture medium containing 1 and 2 mM of FB resulted in no fluoride release, whereas FB degradation was only one third of that observed in control cultures. In the experiments with resting cells, the addition of Ag(+) resulted in 25 % reduction in substrate degradation and fluoride release was only 20 % of that stoichiometrically expected. The accumulation of catechol and 4-fluorocatechol in cultures supplemented with Cu(2+) or Ag(+) suggest inhibition of the key enzyme of FB metabolism-catechol 1,2-dioxygenase.

Degradation of difluorobenzenes by the wild strain Labrys portucalensis.

This study focuses on the biodegradation of difluorobenzenes (DFBs), compounds commonly used as intermediates in the industrial synthesis of various pharmaceutical and agricultural chemicals. A previously isolated microbial strain (strain F11), identified as Labrys portucalensis, able to degrade fluorobenzene (FB) as sole carbon and energy source, was tested for its capability to degrade 1,2-, 1,3- and 1,4-DFB in batch cultures. Strain F11 could use 1,3-DFB as a sole carbon and energy source, with quantitative release of fluoride, but 1,4-DFB was only degraded and defluorinated when FB was supplied simultaneously. Growth of strain F11 with 0.5 mM of 1,3-DFB led to stoichiometric release of fluoride ion. The same result was obtained in cultures fed with 1 mM of 1,3-DFB or 0.5 mM of 1,4-DFB, in the presence of 1 mM of FB. No growth occurred with 1,2-DFB as substrate, and degradation of FB was inhibited when supplied simultaneously with 1,2-DFB. To our knowledge, this is the first time biodegradation of 1,3-DFB as a sole carbon and energy source, and cometabolic degradation of 1,4-DFB, by a single bacterium, is reported.