Novel method for rapid monitoring of OPFRs by LLE and GC-MS as a tool for assessing biodegradation: validation and applicability.

Organophosphate flame retardants (OPFRs) are high-production volume chemicals widely present in environmental compartments. The presence of water-soluble OPFRs (tri-n-butyl phosphate (TnBP), tris(2-butoxyethyl) phosphate (TBEP), tris(2-chloroethyl) phosphate (TCEP), tris(2-chloroisopropyl) phosphate (TCPP), and triethyl phosphate (TEP)) in water compartments evidences the struggle of conventional wastewater treatment plants (WWTPs) to effectively eliminate these toxic compounds. This study reports for the first time the use of white-rot fungi as a promising alternative for the removal of these OPFRs. To accomplish this, a simple and cost-efficient quantification method for rapid monitoring of these contaminants' concentrations by GC-MS while accounting for matrix effects was developed. The method proved to be valid and reliable for all the tested parameters. Sample stability was examined under various storage conditions, showing the original samples to be stable after 60 days of freezing, while post-extraction storage techniques were also effective. Finally, a screening of fungal degraders while assessing the influence of the glucose regime on OPFR removal was performed. Longer chain organophosphate flame retardants, TBP and TBEP, could be easily and completely removed by the fungus Ganoderma lucidum after only 4 days. This fungus also stood out as the sole organism capable of partially degrading TCEP (35% removal). The other chlorinated compound, TCPP, was more easily degraded and 70% of its main isomer was removed by T. versicolor. However, chlorinated compounds were only partially degraded under nutrient-limiting conditions. TEP was either not degraded or poorly degraded, and it is likely that it is a transformation product from another OPFR's degradation. These results suggest that degradation of chlorinated compounds is dependent on the concentration of the main carbon source and that more polar OPFRs are less susceptible to degradation, given that they are less accessible to radical removal by fungi. Overall, the findings of the present study pave the way for further planned research and a potential application for the degradation of these contaminants in real wastewaters.

Fungal degradation of selected medium to highly polar pesticides by Trametes versicolor: kinetics, biodegradation pathways, and ecotoxicity of treated waters.

The massive use of pesticides represents one of the main causes of environmental deterioration, as they have adverse effects on non-target organisms. Thus, the development of technologies capable of reducing their release into the environment is urgently needed. This study reports for the first time the white-rot fungus Trametes versicolor as an alternative towards the degradation of medium to highly polar pesticides such as the organophosphate malathion, and the neonicotinoids acetamiprid and imidacloprid. Specifically, T. versicolor could completely remove 1 mg/L of malathion in an Erlenmeyer flask within 48 h, while experiments of acetamiprid and imidacloprid (4 mg/L), conducted in air-pulse fluidized bioreactors, resulted in degradation percentages of 20% and 64.7%, respectively, after 7 days of operation. Enzymatic exploration studies revealed that the cytochrome P450 system, instead of the extracellular enzyme laccase, is involved in the degradation of acetamiprid and imidacloprid. The degradation pathways were proposed based on the main transformation products (TPs) formed in the solutions: seven in the case of malathion, and two and one in the case of imidacloprid and acetamiprid, respectively. Although the TPs identified were predicted to be less toxic than the investigated pesticides, the toxicity of the individual solutions slightly increased throughout the degradation process, according to the Microtox assay. However, the solution toxicity was always below the threshold established in the local regulation. Although additional research is needed to implement this treatment at a pilot plant scale, this work highlights the potential of T. versicolor to bio-remediate pesticide-contaminated waters.

Process intensification at the expression system level for the production of 1-phosphate aldolase in antibiotic-free E. coli fed-batch cultures.

To successfully design expression systems for industrial biotechnology and biopharmaceutical applications; plasmid stability, efficient synthesis of the desired product and the use of selection markers acceptable to regulatory bodies are of utmost importance. In this work we demonstrate the application of a set of IPTG-inducible protein expression systems -- harboring different features namely, antibiotic vs auxotrophy marker; two-plasmids vs single plasmid expression system; expression levels of the repressor protein (LacI) and the auxotrophic marker (glyA) -- in high-cell density cultures to evaluate their suitability in bioprocess conditions that resemble industrial settings. Results revealed that the first generation of engineered strain showed a 50% reduction in the production of the model recombinant protein fuculose-1-phosphate aldolase (FucA) compared to the reference system from QIAGEN. The over-transcription of glyA was found to be a major factor responsible for the metabolic burden. The second- and third-generation of expression systems presented an increase in FucA production and advantageous features. In particular, the third-generation expression system is antibiotic-free, autotrophy-selection based and single-plasmid and, is capable to produce FucA at similar levels compared to the original commercial expression system. These new tools open new avenues for high-yield and robust expression of recombinant proteins in E. coli.

Comparison between two reactors using Trametes versicolor for agricultural wastewater treatment under non-sterile condition in sequencing batch mode.

Agricultural wastewater is a major source of herbicides, which pose environmental and health concerns owing to their substantial use and poor elimination rate in conventional wastewater treatment plants. White-rot fungi are versatile in degrading xenobiotics; however, the key problem encountered with their application in actual scenarios is competition with indigenous microorganisms, mainly bacteria. To address this barrier, two different strategies were implemented in the present study. One strategy was to set up a trickle bed with Trametes versicolor immobilized on pine wood, and another strategy was to employ a T. versicolor-pelleted, fluidized-bed reactor to remove diuron and bentazon from actual wastewater under non-sterile conditions. The residence time in the trickle bed was estimated using three methodologies. With 10 batches of a 3-day cycle operation, although the trickle-bed reactor possessed a shorter contact time (8.5 h per cycle) and lower laccase activity compared with those of the fluidized-bed reactor, it demonstrated a higher removal yield and lower bacterial counts. In addition, the utilization of pine wood as a carrier obviously reduced the cost since no additional nutrients were required. Hence, after evaluating all advantages and limitations of both bioreactors, for the purpose of treating over the long term and scaling up, a trickle-bed reactor is the preferred choice.

Influence of process variables in a continuous treatment of non-sterile hospital wastewater by Trametes versicolor and novel method for inoculum production.

Micropollutants such as pharmaceutical active compounds, present at high concentration in hospital wastewater (HWW), pose both environmental and human health challenges. Fungal reactors can effectively remove such contaminants and produce non-toxic effluents, but their ability to operate for a long period of time is yet to be demonstrated in real hospital wastewater. Several process variables need to be studied beforehand. Here, variables: pellet size, aeration and carbon-to-nitrogen ratio are studied in continuous operations with real HWW. Moreover, a novel strategy for inoculum production that could reduce economical and operational costs is proposed and tested. Optimum pellet size was found to be 2 mm and an aeration of 0.8 L min-1 was needed to maintain fungal viability. The carbon-to-nitrogen ratio of 7.5 was selected and the pellet production time was reduced from 6 to 3 days. The novel low-cost inoculum preparation produced pellets with the same characteristics as the traditionally prepared ones.

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.

OPFR removal by white rot fungi: screening of removers and approach to the removal mechanism.

The persistent presence of organophosphate flame retardants (OPFRs) in wastewater (WW) effluents raises significant environmental and health concerns, highlighting the limitations of conventional treatments for their remotion. Fungi, especially white rot fungi (WRF), offer a promising alternative for OPFR removal. This study sought to identify fungal candidates (from a selection of four WRF and two Ascomycota fungi) capable of effectively removing five frequently detected OPFRs in WW: tributyl phosphate (TnBP), tributoxy ethyl phosphate (TBEP), trichloroethyl phosphate (TCEP), trichloro propyl phosphate (TCPP) and triethyl phosphate (TEP). The objective was to develop a co-culture approach for WW treatment, while also addressing the utilization of less assimilable carbon sources present in WW. Research was conducted on carbon source uptake and OPFR removal by all fungal candidates, while the top degraders were analyzed for biomass sorption contribution. Additionally, the enzymatic systems involved in OPFR degradation were identified, along with toxicity of samples after fungal contact. Acetate (1.4 g·L-1), simulating less assimilable organic matter in the carbon source uptake study, was eliminated by all tested fungi in 4 days. However, during the initial screening where the removal of four OPFRs (excluding TCPP) was tested, WRF outperformed Ascomycota fungi. Ganoderma lucidum and Trametes versicolor removed over 90% of TnBP and TBEP within 4 days, with Pleorotus ostreatus and Pycnoporus sanguineus also displaying effective removal. TCEP removal was challenging, with only G. lucidum achieving partial removal (47%). A subsequent screening with selected WRF and the addition of TCPP revealed TCPP's greater susceptibility to degradation compared to TCEP, with T. versicolor exhibiting the highest removal efficiency (77%). This observation, plus the poor degradation of TEP by all fungal candidates suggests that polarity of an OPFR inversely correlates with its susceptibility to fungal degradation. Sorption studies confirmed the ability of top-performing fungi of each selected OPFR to predominantly degrade them. Enzymatic system tests identified the CYP450 intracellular system responsible for OPFR degradation, so reactions of hydroxylation, dealkylation and dehalogenation are possibly involved in the degradation pathway. Finally, toxicity tests revealed transformation products obtained by fungal degradation to be more toxic than the parent compounds, emphasizing the need to identify them and their toxicity contributions. Overall, this study provides valuable insights into OPFR degradation by WRF, with implications for future WW treatment using mixed consortia, emphasizing the importance of reducing generated toxicity.

Direct measurements of IPTG enable analysis of the induction behavior of E. coli in high cell density cultures.

BACKGROUND: The E. coli lac operon and its components have been studied for decades, and lac-derived systems are widely used for recombinant protein production. However, lac operon dynamics and induction behavior remain the paradigm of gene regulation. Recently, an HPLC-MS-based method to quantify IPTG in the medium and inside the biomass has been established, and this tool may be useful to uncover the lack of knowledge and allow optimization of biotechnological processes. RESULTS: The results obtained from the study of IPTG distribution profiles in fed-batch, high cell density cultures allowed discrimination between two different depletion patterns of an inducer from the medium to the biomass in E. coli-expressing rhamnulose-1-phosphate aldolase (RhuA). Moreover, we could demonstrate that active transport mediates the uptake of this gratuitous inducer. Additionally, we could study the induction behaviors of this expression system by taking into account the biomass concentration at the induction time. CONCLUSIONS: In the bistable range, partial induction occurred, which led to intermediate levels of RhuA activity. There was a direct relationship between the initial inducer concentrations and the initial inducer transport rate together with the specific activity. A majority of the inducer remains in the medium to reach equilibrium with the intracellular level. The intracellular inducer accumulation was a further evidence of bistability of the lac operon.

Bioaugmentation of sewage sludge with Trametes versicolor in solid-phase biopiles produces degradation of pharmaceuticals and affects microbial communities.

The use of sludge (biosolids) in land application may contribute to the spread of organic micropollutants as wastewater treatments do not completely remove these compounds. Therefore, the development of alternative strategies for sludge treatment is a matter of recent concern. The elimination of pharmaceuticals at pre-existent concentrations from sewage sludge was assessed, for the first time, in nonsterile biopiles by means of fungal bioaugmentation with Trametes versicolor (BTV-systems) and compared with the effect of autochthonous microbiota (NB-systems). The competition between the autochthonous fungal/bacterial communities and T. versicolor was studied using denaturing gradient gel electrophoresis (DGGE) and the cloning/sequencing approach. An inhibitory effect exerted by T. versicolor over bacterial populations was suggested. However, after 21 days, T. versicolor was no longer the main taxon in the fungal communities. The elimination profiles revealed an enhanced removal of atorvastatin-diclofenac-hydrochlorothiazide (during the whole treatment) and ranitidine-fenofibrate (at short periods) in the BTV biopiles in respect to NB biopiles, coincident with the presence of the fungus. For ibuprofen-clarithromycin-furosemide, the elimination profiles were similar irrespective of the system, and with carbamazepine no significant degradation was obtained. The results suggest that a fungal treatment with T. versicolor could be a promising process for the remediation of some pharmaceuticals in complex matrices such as biosolids.

Optimisation of the operational conditions of trichloroethylene degradation using Trametes versicolor under quinone redox cycling conditions using central composite design methodology.

Extracellular radicals produced by Trametes versicolor under quinone redox cycling conditions can degrade a large variety of pollutant compounds, including trichloroethylene (TCE). This study investigated the effect of the agitation speed and the gas-liquid phase volume ratio on TCE degradation using central composite design (CCD) methodology for a future scale-up to a reactor system. The agitation speed ranged from 90 to 200 rpm, and the volume ratio ranged from 0.5 to 4.4. The results demonstrated the important and positive effect of the agitation speed and an interaction between the two factors on TCE degradation. Although the volume ratio did not have a significant effect if the agitation speed value was between 160 and 200 rpm, at lower speed values, the specific pollutant degradation was clearly more extensive at low volume ratios than at high volume ratios. The fitted response surface was validated by performing an experiment using the parameter combination in the model that maximised TCE degradation. The results of the experiments carried out using different biomass concentrations demonstrated that the biomass concentration had a positive effect on pollutant degradation if the amount of biomass present was lower than 1.6 g dry weight l(-1). The results show that the maximum TCE degradation was obtained at the highest speed (200 rpm), gas-liquid phase volume ratio (4.4), and a biomass concentration of 1.6 g dry weight l(-1).

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.

Cloning, expression, and one-step purification/immobilization of two carbohydrate-binding module-tagged alcohol dehydrogenases.

BACKGROUND: The feasibility of biochemical transformation processes is usually greatly dependent on biocatalysts cost. Therefore, immobilizing and reusing biocatalysts is an approach to be considered to bring biotransformations closer to industrial feasibility, since it does not only allow to reuse enzymes but can also improve their stability towards several reaction conditions. Carbohydrate-Binding Modules (CBM) are well-described domains involved in substrate binding which have been already used as purification tags. RESULTS: In this work, two different Carbohydrate-Binding Modules (CBM3 and CBM9) have been successfully fused to an alcohol dehydrogenase from Saccharomyces cerevisiae, which has been produced in bench-scale reactor using an auxotrophic M15-derived E. coli strain, following a fed-batch strategy with antibiotic-free medium. Around 40 mg·g- 1 DCW of both fusion proteins were produced, with a specific activity of > 65 AU·mg- 1. Overexpressed proteins were bound to a low-cost and highly selective cellulosic support by one-step immobilization/purification process at > 98% yield, retaining about a 90% of initial activity. Finally, the same support was also used for protein purification, aiming to establish an alternative to metal affinity chromatography, by which CBM9 tag proved to be useful, with a recovery yield of > 97% and 5-fold increased purity grade. CONCLUSION: CBM domains were proved to be suitable for one-step immobilization/purification process, retaining almost total activity offered. However, purification process was only successful with CBM9.

Expression of metallocarboxypeptidase inhibitors in Escherichia coli: effect of cysteine content and protein size in the secretory production of disulfide-bridged proteins.

Metallocarboxypeptidase inhibitors are proteins with possible applications in biomedicine given their properties as anticoagulant and antitumoral factors. They are small, eukaryotic polypeptides comprising several disulfide bridges, which makes them hard to express in inexpensive bacterial hosts. In this work, three of them were produced in high-cell-density cultures of Escherichia coli: PCI (39 residues and three bridges), LCI (66 residues and four bridges) and TCI (75 residues and six bridges). The genes coding for the mentioned inhibitors were cloned in an arabinose-inducible plasmid fused to the signal peptide of DsbA in order to have them secreted and grant the formation of the bridges. The trigger-factor defective strain KTD101 was used as the expression host. The resulting recombinant strains were cultured in fed-batch mode employing minimal media and an exponential feed profile, keeping the specific growth rate at µ = 0.1 h(-1) by limitation of the fed carbon source (glycerol). Between 380 and 540 mg l(-1) of active inhibitors were obtained in both the periplasmic extracts and extracellular media of the cultures. Later on, excretion was enhanced using a cell permeabilization treatment, allowing the recovery of over 80% of the products from the extracellular fraction. Protein yields were found to be inversely proportional to cysteine content of the inhibitor, whereas protein excretion rates were inversely proportional to the protein size. Overall, these results offer insight into the secretory production of active disulfide-bridged proteins in high-cell-density cultures of E. coli.

Synthesis of a precursor of D-fagomine by immobilized fructose-6-phosphate aldolase.

Fructose-6-phosphate aldolase (FSA) is an important enzyme for the C-C bond-forming reactions in organic synthesis. The present work is focused on the synthesis of a precursor of D-fagomine catalyzed by a mutant FSA. The biocatalyst has been immobilized onto several supports: magnetic nanoparticle clusters (mNC), cobalt-chelated agarose (Co-IDA), amino-functionalized agarose (MANA-agarose) and glyoxal-agarose, obtaining a 29.0%, 93.8%, 89.7% and 53.9% of retained activity, respectively. Glyoxal-agarose FSA derivative stood up as the best option for the synthesis of the precursor of D-fagomine due to the high reaction rate, conversion, yield and operational stability achieved. FSA immobilized in glyoxal-agarose could be reused up to 6 reaction cycles reaching a 4-fold improvement in biocatalyst yield compared to the non-immobilized enzyme.

Prospects on coupling UV/H2O2 with activated sludge or a fungal treatment for the removal of pharmaceutically active compounds in real hospital wastewater.

Conventional active sludge (AS) process at municipal centralized wastewater treatment facilities may exhibit little pharmaceuticals (PhACs) removal efficiencies when treating hospital wastewater (HWW). Therefore, a dedicated efficient wastewater treatment at the source point is recommended. In this sense, advanced oxidation processes (AOPs) and fungal treatment (FG) have evidenced promising results in degrading PhACs. The coupling of the AOP based on UV/H2O2 treatment with biological treatment (AS or FG) treating a real non-sterile HWW, was evaluated in this work. In addition, a coagulation-flocculation pretreatment was applied to improve the efficiency of all approaches. Twenty-two PhACs were detected in raw HWW, which were effectively removed (93-95%) with the combination of any of the biological treatment followed by UV/H2O2 treatment. Similar removal results (94%) were obtained when placing UV/H2O2 treatment before FG, while a lower removal (83%) was obtained in the combination of UV/H2O2 followed by AS. However, the latest was the only treatment combination that achieved a decrease in the toxicity of water. Moreover, deconjugation of conjugated PhACs has been suggested for ofloxacin and lorazepam after AS treatment, and for ketoprofen after fungal treatment. Monitoring of carbamazepine and its transformation products along the treatment allowed to identify the same carbamazepine degradation pathway in UV/H2O2 and AS treatments, unlike fungal treatment, which followed another degradation route.

Remediation of bentazone contaminated water by Trametes versicolor: Characterization, identification of transformation products, and implementation in a trickle-bed reactor under non-sterile conditions.

Bentazone, an herbicide widely applied in rice and cereal crops, is widespread in the aquatic environment. This study evaluated the capacity of Trametes versicolor to remove bentazone from water. The fungus was able to completely remove bentazone after three days at Erlenmeyer-scale incubation. Both laccase and cytochrome P450 enzymatic systems were involved in bentazone degradation. A total of 19 transformation products (TPs) were identified to be formed during the process. The reactions involved in their formation included hydroxylations, oxidations, methylations, N-nitrosation, and dimerization. A laccase mediated radical mechanism was proposed for TP formation. In light of the results obtained at the Erlenmeyer scale, a trickle-bed reactor with T. versicolor immobilized on pine wood chips was set up to evaluate its stability during bentazone removal under non-sterile conditions. After 30 days of sequencing batch operation, an average bentazone removal of 48% was obtained, with a considerable contribution of adsorption onto the lignocellulosic support material. Bacterial contamination, which is the bottleneck in the implementation of fungal bioreactors, was successfully addressed by this particular system according to its maintained performance. This research is a pioneering step forward to the implementation of fungal bioremediation on a real scale.

Development and validation of a liquid chromatography-mass spectrometry assay for the quantitation of IPTG in E. Coli fed-batch cultures.

IPTG (Isopropyl-beta-d-1-thiolgalactopyranoside) is a gratuitous inducer commonly used for the overexpression of heterologous recombinant proteins in Escherichia coli. A reliable method has been developed for the determination of IPTG in E.coli fed-batch fermentation samples with a minimal sample treatment. Analysis was performed in single ion monitoring positive mode using ESI source. The extracted ion was 261 m/z and the retention time of IPTG was 12.4 min with a total run time in samples of 30 min. The flow was directed to mass spectrometer 11 min after the start of the run and diverted from mass spectrometer after 14.5 min in order to avoid interference of salts and other metabolites. The assay was validated for medium and intracellular matrices and linear calibration curves of 3 orders of magnitude were obtained (R(2) >or= 0.99). Quality control samples were analyzed and showed precision and accuracy within the limits according to FDA Guidelines for analytical method validation. Recovery for both matrices was between 95.8 and 113.5%. The limit of detection (LOD) was set at 0.02 microM being the 0.1% of the lowest IPTG concentration used for induction of recombinant protein overexpression. The developed procedure has been applied to determine the IPTG distribution profiles in medium and intracellular samples in high cell density induced cultures for the production of the recombinant protein rhamnulose-1-phosphate aldolase (RhuA).

Exploring the degradation capability of Trametes versicolor on selected hydrophobic pesticides through setting sights simultaneously on culture broth and biological matrix.

Pesticides introduced inadvertently or deliberately into environment by global agricultural practices have caused growing public concern, therefore the search of approaches for elimination of such xenobiotics should be motivated. The degradation of hydrophobic pesticides including chlorpyrifos, dicofol and cypermethrin were assayed with the white-rot fungus Trametes versicolor. Experiments were set at realistic concentration as 5 µg L-1, and both culture medium and biologic matrix were analyzed for pollutants residues. Results showed that the first step was due to a fast adsorption, which also played an important role, accounting for more than 90% removal in average. Then mass balances proposal evidenced the biodegradation of the adsorbed pollutants, demonstrating efficient depletion as 94.7%, 87.9% and 93.1%, respectively. Additionally, the related degradation metabolites were identified using ultra performance liquid chromatography coupled to high resolution mass spectrometry. Two compounds, namely O,O-diethyl thiophosphate and diethyl phosphate were detected as transformation products of chlorpyrifos, whereas dicofol was degraded into benzaldehyde that is first time to be reported. It also confirms the degradation capability of T. versicolor. Our results suggest that T. versicolor is a potential microorganism for bioremediation of hydrophobic pesticide contaminated environments.

The removal of diuron from agricultural wastewaters by Trametes versicolor immobilized on pinewood in simple channel reactors.

The presence of pesticides in agricultural wastewater entails harmful risks to both the environment and public health. In this study, two channel-type bioreactors with Trametes versicolor immobilized on pinewood chips were evaluated in terms of the removal efficiency of diuron from agricultural wastewater under non-sterile conditions. First, both single and successive sorption processes of diuron on pinewood chips were evaluated. The Freundlich model showed the best correlation in the sorption isotherm study (R2 = 0.993; Δq = 5.245), but according to repeated sorption experiments, the Langmuir model (R2 = 0.993; Δq = 5.757) was considered more representative. Equilibrium was reached after approximately 48 h, and the Elovich kinetic model gave the best fit with the experimental data. A packed-bed channel bioreactor (PBCB) was found to be a remarkable alternative able to remove up to 94% diuron from agricultural wastewater during 35 d. However, periodic manual mixing was required to guarantee an aerobic process, and a rotating drum bioreactor (RDB) was subsequently proposed as an enhanced version. The RDB removed up to 61% diuron during 16 d using almost 7 times lower wood dose (152 g wood·L-1) than in the PBCB (1000 g wood·L-1).

Fungal bioremediation of diuron-contaminated waters: Evaluation of its degradation and the effect of amendable factors on its removal in a trickle-bed reactor under non-sterile conditions.

The occurrence of the extensively used herbicide diuron in the environment poses a severe threat to the ecosystem and human health. Four different ligninolytic fungi were studied as biodegradation candidates for the removal of diuron. Among them, T. versicolor was the most effective species, degrading rapidly not only diuron (83%) but also the major metabolite 3,4-dichloroaniline (100%), after 7-day incubation. During diuron degradation, five transformation products (TPs) were found to be formed and the structures for three of them are tentatively proposed. According to the identified TPs, a hydroxylated intermediate 3-(3,4-dichlorophenyl)-1-hydroxymethyl-1-methylurea (DCPHMU) was further metabolized into the N-dealkylated compounds 3-(3,4-dichlorophenyl)-1-methylurea (DCPMU) and 3,4-dichlorophenylurea (DCPU). The discovery of DCPHMU suggests a relevant role of hydroxylation for subsequent N-demethylation, helping to better understand the main reaction mechanisms of diuron detoxification. Experiments also evidenced that degradation reactions may occur intracellularly and be catalyzed by the cytochrome P450 system. A response surface method, established by central composite design, assisted in evaluating the effect of operational variables in a trickle-bed bioreactor immobilized with T. versicolor on diuron removal. The best performance was obtained at low recycling ratios and influent flow rates. Furthermore, results indicate that the contact time between the contaminant and immobilized fungi plays a crucial role in diuron removal. This study represents a pioneering step forward amid techniques for bioremediation of pesticides-contaminated waters using fungal reactors at a real scale.