Gene cloning system for sulfonamide-mineralizing Microbacterium sp. strain BR1.

The wide application of sulfonamide (SA) antibiotics in human and veterinary medicine contributes to the accumulation of these antibiotics in the environment and the corresponding onset of antibiotic resistance among bacteria. Microbacterium sp. BR1 is capable of mineralizing sulfamethoxazole and other SAs via a novel mechanism. The genetic basis of SA elimination by BR1 remains unknown. Development of an efficient plasmid transfer protocol for Microbacterium sp. BR1 is highly desirable, as it would open the door to genetic analysis and manipulation of its genome. Here we report that intergeneric Escherichia coli-Microbacterium spp. BR1 conjugation is an efficient way to introduce various plasmids into BR1. The generated transconjugants were stable in the presence of antibiotics and the plasmids showed no signs of rearrangements. Nevertheless, the plasmids were rapidly lost in the absence of selection. We also show that the cumate-inducible beta-glucuronidase reporter gene functions in BR1 and is strictly regulated. Our results set the working ground for further genetic manipulations of BR1, such as the overexpression of sulfonamide degradation genes or the selection of strong microbacterial promoters.

Role of biotransformation, sorption and mineralization of (14)C-labelled sulfamethoxazole under different redox conditions.

(14)C-sulfamethoxazole biotransformation, sorption and mineralization was studied with heterotrophic and autotrophic biomass under aerobic and anoxic conditions, as well as with anaerobic biomass. The (14)C-radiolabelled residues distribution in the solid, liquid and gas phases was closely monitored along a total incubation time of 190 h. Biotransformation was the main removal mechanism, mineralization and sorption remaining below 5% in all the cases, although the presence of a carbon source exerted a positive effect on the mineralization rate by the aerobic heterotrophic bacteria. In fact, an influence of the type of primary substrate and the redox potential was observed in all cases on the biotransformation and mineralization rates, since an enhancement of the removal rate was observed when an external carbon source was used as a primary substrate under aerobic conditions, while a negligible effect was observed under nitrifying conditions. In the liquid phases collected from all assays, up to three additional peaks corresponding to (14)C-radiolabelled residues were detected. The highest concentration was observed under anaerobic conditions, where two radioactive metabolites were detected representing each around 15% of the total applied radioactivity after 180 h incubation. One of the metabolites detected under anoxic and anaerobic conditions, is probably resulting from ring cleavage of the isoxazole ring.

Assessing the use of nanoimmobilized laccases to remove micropollutants from wastewater.

Enzymes immobilization is a useful way to allow enzyme reuse and increase their stability. A high redox potential laccase from Trametes versicolor (TvL) and a low redox potential, but commercially available low-cost laccase from Myceliophthora thermophila (MtL), were successfully immobilized and co-immobilized onto fumed silica nanoparticles (fsNP). Enzyme loads of 1.78 ± 0.07, 0.69 ± 0.03, and 1.10 ± 0.01 U/mg fsNP were attained for the optimal doses of TvL, MtL, and co-immobilized laccases, respectively. In general, the laccase-fsNP conjugates showed a higher resistance against an acidic pH value (i.e., pH 3), and a higher storage stability than free enzymes. In addition, immobilized enzymes exhibited a superior long-term stability than free laccases when incubated in a secondary effluent from a municipal wastewater treatment plant (WWTP). For instance, the residual activity after 2 weeks for the co-immobilized laccases and the mixture of free laccases were 40.2 ± 2.5% and 16.8 ± 1.0%, respectively. The ability of the laccase-fsNP to remove a mixture of (14)C-bisphenol A (BPA) and (14)C-sodium diclofenac (DCF) from spiked secondary effluents was assessed in batch experiments. The catalytic efficiency was highly dependent on both the microbial source and state of the biocatalyst. The high redox potential TvL in free form attained a four-fold higher percentage of BPA transformation than the free MtL. Compared to free laccases, immobilized enzymes led to much slower rates of BPA transformation. For instance, after 24 h, the percentages of BPA transformation by 1000 U/L of a mixture of free laccases or co-immobilized enzymes were 67.8 ± 5.2 and 27.0 ± 3.9%, respectively. Nevertheless, the use of 8000 U/L of co-immobilized laccase led to a nearly complete removal of BPA, despite the unfavorable conditions for laccase catalysis (pH ~ 8.4). DCF transformation was not observed for any of the enzymatic systems, showing that this compound is highly recalcitrant toward laccase oxidation under realistic conditions.

The degradation of alkylphenols by Sphingomonas sp. strain TTNP3 - a review on seven years of research.

Over the past seven years, we have been working with Sphingomonas sp. strain TTNP3, a bacterium capable of growing on numerous alkylphenolic compounds as a source of carbon and energy. We succeeded in elucidating an unusual pathway involving an attack at the quaternary alpha-carbon atom of the substrate, a position previously thought to be highly resistant to biodegradation. Combining analytical and bioanalytical methods, a good understanding of the reaction mechanisms, the enzymes catalysing them and the organization of the genes encoding them could be gained. First studies on the use of Sphingomonas sp. strain TTNP3 in wastewater treatment have been performed revealing promising results.

Identification of the flavin monooxygenase responsible for ipso substitution of alkyl and alkoxyphenols in Sphingomonas sp. TTNP3 and Sphingobium xenophagum Bayram.

We previously showed that opdA from Sphingomonas sp. PWE1 encodes a putative flavin monooxygenase capable of transforming octylphenol (OP) via type II ipso substitution. Here, we demonstrate that an opdA homolog is responsible for OP and related alkyl/alkoxyphenol degradation in the nonylphenol degrader Sphingomonas sp. TTNP3. PCR and Southern blot analyses revealed that TTNP3 contained an opdA homolog, while a TTNP3 derivative unable to grow on nonylphenol (TTNP3d) did not. OpdA expression was confirmed in wild-type TTNP3 via two dimensional gel electrophoresis. Activity was restored to TTNP3d following complementation with opdA. Sequence analysis of an opdA homolog from another nonylphenol degrader, Sphingobium xenophagum Bayram, revealed that the predicted protein sequences from PWE1 and Bayram were identical, but differed from TTNP3 by four amino acids. In order to assess differences, we heterologously expressed the two unique opdA homologs and compared their effect on the disappearance of five alkyl/alkoxyphenol substrates and subsequent appearance of hydroquinone. For all substrates, except OP, the levels of substrate disappearance and hydroquinone appearance were significantly lower in cultures expressing odpA (TTNP3) than those expressing opdA (PWE1/Bayram). These differences in substrate specificity were consistent with an in silico model which predicted that two of the amino acid differences between odpA (TTNP3) and opdA (PWE1/Bayram) lay in a putative substrate binding pocket. While these strains are known to use the same type II ipso substitution mechanism for alkylphenol degradation, this work provides the first preliminary evidence that opdA homologs also encode the type I ipso substitution activity responsible for the degradation of alkoxyphenols.

The fate of 14C-radiolabelled diclofenac and 4'-hydroxydiclofenac in membrane bioreactor treatment of wastewater.

This study aimed at enhancing knowledge on the fate of diclofenac (DF), together with its main human metabolite 4'-hydroxydiclofenac (4'OHDF), during wastewater treatment by using a laboratory-scale membrane bioreactor (MBR). The reactor was fed continuously with non-radiolabelled diclofenac for a one month period prior to a single pulse of a 14C-radiolabelled solution of DF and 4'OHDF. The solution spike contained approximately 25% 4'OHDF and 65% DF, which corresponds to the ratio observed in municipal sewage, as well as traces of two other metabolites. The radioactivity was monitored for a total of twelve days in the various output streams. The calculation of the complete mass balance in the system demonstrated that the major part of the radioactivity left the reactor with the permeate (88.7%), while 2.1% was recovered in the excess sludge. Negligible amounts were recovered in the off-gas traps and on the membranes. Chromatographic analyses of effluent samples, by means of HPLC-MS coupled in parallel to a radiodetector, displayed a different pattern than the one of the spiked solution. It showed the occurrence of three additional metabolites.

Impact of sewage sludge conditioning and dewatering on the fate of nonylphenol in sludge-amended soils.

The fate of (14)C-labelled p353-nonylphenol (NP) in soils amended with differently treated sludges originating from the same precursor sludge was assessed. The effects of commonly applied conditioning and dewatering techniques were investigated. Nonylphenol was degraded considerably faster in soils amended with liquid sludge, while a significant portion of it remained intact and extractable by organic solvents when sludge had been centrifuged before soil amendment. Mineralization was reduced or even inhibited when freeze-thaw or lime conditioning was applied, respectively. Flocculation by an acrylamide-based cationic polymer led to the formation of a nitro-addition product of nonylphenol in soil, as well to decreased mineralization rates after prolonged incubation times. Possible mechanisms underlying the observations are suggested and discussed.

The role of sludge conditioning and dewatering in the fate of nonylphenol in sludge-amended soils.

One of the main concerns associated with the recycling of biosolids to arable land is their contamination by organic pollutants, like endocrine disruptors. Conditioning and dewatering are usually the last steps of the sewage sludge treatment, before its further utilization. The choice of the specific conditioning/dewatering method may have an effect, not only on the amount of residues in the biosolids, but also on the fate of these compounds in amended soils. Anaerobically digested wastewater sludge was conditioned at lab-scale by means of physical and chemical methods and subsequently dewatered by centrifugation. The produced biosolids plus non-conditioned and non-dewatered sludges were amended separately to soil and spiked with 14C radiolabelled single isomer of nonylphenol. The persistence and leaching potential of nonylphenol after an incubation period of three months were correlated to the sludge treatment method. In comparison to non-conditioned sludge, 54% and 72% higher amount of pollutant residues were extractable when freeze-thawed and limed sludge, respectively, were used. Conditioning of sludge with cationic polymer decreased the leaching potential of nonylphenol in sludge-amended soils, while liming increased it. Fractions of the model compound recovered as extractable and bound residues were analyzed in order to interpret nonylphenol fate.

Degradation pathway of bisphenol A: does ipso substitution apply to phenols containing a quaternary alpha-carbon structure in the para position?

The degradation of bisphenol A and nonylphenol involves the unusual rearrangement of stable carbon-carbon bonds. Some nonylphenol isomers and bisphenol A possess a quaternary alpha-carbon atom as a common structural feature. The degradation of nonylphenol in Sphingomonas sp. strain TTNP3 occurs via a type II ipso substitution with the presence of a quaternary alpha-carbon as a prerequisite. We report here a new degradation pathway of bisphenol A. Consequent to the hydroxylation at position C-4, according to a type II ipso substitution mechanism, the C-C bond between the phenolic moiety and the isopropyl group of bisphenol A is broken. Besides the formation of hydroquinone and 4-(2-hydroxypropan-2-yl)phenol as the main metabolites, further compounds resulting from molecular rearrangements consistent with a carbocationic intermediate were identified. Assays with resting cells or cell extracts of Sphingomonas sp. strain TTNP3 under an (18)O(2) atmosphere were performed. One atom of (18)O(2) was present in hydroquinone, resulting from the monooxygenation of bisphenol A and nonylphenol. The monooxygenase activity was dependent on both NADPH and flavin adenine dinucleotide. Various cytochrome P450 inhibitors had identical inhibition effects on the conversion of both xenobiotics. Using a mutant of Sphingomonas sp. strain TTNP3, which is defective for growth on nonylphenol, we demonstrated that the reaction is catalyzed by the same enzymatic system. In conclusion, the degradation of bisphenol A and nonylphenol is initiated by the same monooxygenase, which may also lead to ipso substitution in other xenobiotics containing phenol with a quaternary alpha-carbon.

Contribution to the Detection and Identification of Oxidation Metabolites of Nonylphenol in Sphingomonas sp. strain TTNP3.

Sphingomonas sp. strain TTNP3 has been previously described as a bacterium that is capable of degrading the technical mixture of nonylphenol (NP) isomers and also the 4(3',5'-dimethyl-3'-heptyl)-phenol single isomer of NP. Until recently, 3,5-dimethyl-3-heptanol was the only reported metabolite of 4(3',5'-dimethyl-3'-heptyl)-phenol. A short time ago, the detection of an intracellular metabolite resulting from the oxidation of 4(3',5'-dimethyl-3'-heptyl)-phenol which was identified as 2(3,5-dimethyl-3-heptyl)-benzenediol has been reported. A decisive element for this identification was the occurrence of some slight differences with the two most probable metabolites i.e. 4(3',5'-dimethyl-3'-heptyl)-resorcinol and 4(3',5'-dimethyl-3'-heptyl)-catechol. These facts led us to hypothesise some NIH shift mechanisms explaining the formation of 2(3',5'-dimethyl-3'-heptyl)-benzenediol. In the present work, we describe the steps that led to the detection of these metabolites in the intracellular fraction of Sphingomonas sp. strain TTNP3. The formation of analogous intracellular metabolites resulting from the degradation of the technical mixture of NP is reported. To further elucidate these degradation products, studies were carried out with cells grown with 4(3',5'-dimethyl-3'-heptyl)-phenol as sole carbon source. The description of the syntheses of reference compounds, i.e. 4(3',5'-dimethyl-3'-heptyl)-resorcinol and 4(3',5'-dimethyl-3'-heptyl)-catechol and their comparative analyses with the intermediates of the degradation of 4(3',5'-dimethyl-3'-heptyl)-phenol are presented.

Microbial degradation of nonylphenol and other alkylphenols--our evolving view.

Because the endocrine disrupting effects of nonylphenol (NP) and octylphenol became evident, the degradation of long-chain alkylphenols (AP) by microorganisms was intensively studied. Most NP-degrading bacteria belong to the sphingomonads and closely related genera, while NP metabolism is not restricted to defined fungal taxa. Growth on NP and its mineralization was demonstrated for bacterial isolates, whereas ultimate degradation by fungi still remains unclear. While both bacterial and fungal degradation of short-chain AP, such as cresols, and the bacterial degradation of long-chain branched AP involves aromatic ring hydroxylation, alkyl chain oxidation and the formation of phenolic polymers seem to be preferential elimination pathways of long-chain branched AP in fungi, whereby both intracellular and extracellular oxidative enzymes may be involved. The degradation of NP by sphingomonads does not proceed via the common degradation mechanisms reported for short-chain AP, rather, via an unusual ipso-substitution mechanism. This fact underlies the peculiarity of long-chain AP such as NP isomers, which possess highly branched alkyl groups mostly containing a quaternary alpha-carbon. In addition to physicochemical parameters influencing degradation rates, this structural characteristic confers to branched isomers of NP a biodegradability different to that of the widely used linear isomer of NP. Potential biotechnological applications for the removal of AP from contaminated media and the difficulties of analysis and application inherent to the hydrophobic NP, in particular, are also discussed. The combination of bacteria and fungi, attacking NP at both the phenolic and alkylic moiety, represents a promising perspective.

The degradation of alpha-quaternary nonylphenol isomers by Sphingomonas sp. strain TTNP3 involves a type II ipso-substitution mechanism.

The degradation of radiolabeled 4(3',5'-dimethyl-3'-heptyl)-phenol [nonylphenol (NP)] was tested with resting cells of Sphingomonas sp. strain TTNP3. Concomitantly to the degradation of NP, a metabolite identified as hydroquinone transiently accumulated and short-chain organic acids were then produced at the expense of hydroquinone. Two other radiolabeled isomers of NP, 4(2',6'-dimethyl-2'-heptyl)-phenol and 4(3',6'-dimethyl-3'-heptyl)-phenol, were synthesized. In parallel experiments, the 4(2',6'-dimethyl-2'-heptyl)-phenol was degraded more slowly than the other isomers of NP by strain TTNP3, possibly because of effects of the side-chain structure on the kinetics of degradation. Alkylbenzenediol and alkoxyphenol derivatives identified as metabolites during previous studies were synthesized and tested as substrates. The derivatives were not degraded, which indicated that the mineralization of NP does not proceed via alkoxyphenol as the principal intermediate. The results obtained led to the elucidation of the degradation pathway of NP isomers with a quaternary alpha-carbon. The proposed mechanism is a type II ipso substitution, leading to hydroquinone and nonanol as the main metabolites and to the dead-end metabolites alkylbenzenediol or alkoxyphenol, depending on the substitution at the alpha-carbon of the carbocationic intermediate formed.

Identification of isomeric 4-nonylphenol structures by gas chromatography-tandem mass spectrometry combined with cluster analysis.

The endocrine-disrupting effect of 4-nonylphenols (NP) formed from industrial detergents such as nonylphenol polyethoxylates is well known today. The technical mixture contains a great variety of 4-iso-nonylphenol isomers having different endocrine-disrupting activities. Currently used gas chromatography-mass spectrometry (GC-MS) protocols allow the detection of about 20 peaks, mostly co-eluting isomers. In the present study, Product Ion mass spectrometry obtained by ion trap technology enhanced the selectivity in NP detection resulting in improved gas chromatographic resolution as well as structure assignment of the isomers. The structure proposals of 4-nonylphenol isomers given were derived from GC-MS-MS data processed by multivariate statistics. The cluster analysis allowed the classification of NP due to common structural features that were reflected in the mass spectra. The fragmentation pathways of three reference NP isomers, 4-(1-ethyl-1,4-dimethylpentyl)phenol (NP1), 4-(1,1,5-trimethylhexyl)phenol (NP2) and 4-(1-ethyl-1,3-dimethylpentyl)phenol (NP3), were investigated in more detail. They served as model compounds to aid the interpretation of spectra from unknown NP isomers. Structures of two groups of isomers, characterized by alpha-ethyl as well as alpha-propyl substituents, could be proposed.

Degradation of a nonylphenol single isomer by Sphingomonas sp. strain TTNP3 leads to a hydroxylation-induced migration product.

Sphingomonas sp. strain TTNP3 degrades 4(3',5'-dimethyl-3'-heptyl)-phenol and unidentified metabolites that were described previously. The chromatographic analyses of the synthesized reference compound and the metabolites led to their identification as 2(3',5'-dimethyl-3'-heptyl)-1,4-benzenediol. This finding indicates that the nonylphenol metabolism of this bacterium involves unconventional degradation pathways where an NIH shift mechanism occurs.

Behaviour of endocrine disrupting chemicals during the treatment of municipal sewage sludge.

Agricultural application of municipal sewage sludge has been emotionally discussed in the last decades, because the latter contains endocrine disrupting chemicals (EDCs) and other organic micropollutants with unknown fate and risk potential. Bisphenol A (BPA) was chosen as a model substance to investigate the influence of sludge conditioning on the end-concentration of EDCs in sludge. Adsorption studies with radioactive-labelled BPA showed that more than 75% BPA in anaerobically digested sludge is bound to solids (log Kd = 2.09-2.30; log Koc = 2.72-3.11). Sludge conditioning with polymer or iron (III) chloride alone had no influence on the adsorption of BPA. After conditioning with iron (III) chloride and calcium hydroxide desorption of BPA took place. Apparently, it occurred due to the deprotonation of BPA (pKa= 10.3) as the pH-value reached 12.4 during the process. The same behaviour is expected for other phenolic EDCs with similar pKa (nonylphenol, 17beta-estradiol, estron, estriol, 17alpha-ethinylestradiol). This study shows high affinity of BPA to the anaerobically digested sludge and importance of conditioning in the elimination of EDCs during the sludge treatment. Addition of polymer is favourable in the case of sludge incineration. Conditioning with iron (III) chloride and calcium hydroxide shows advantages for the use of sludge as fertiliser.

Microbial degradation of a single branched isomer of nonylphenol by Sphingomonas TTNP3.

The endocrine disrupting chemical nonylphenol (NP) is a technical product which consists of a complex mixture of nonylphenols with different alkyl side-chain isomers. Since the bio-degradation of each NP isomer may lead to its own range of metabolites, the isolation and identification of transformation products is very difficult. In order to overcome this difficulty, the nonylphenol isomer 4(3',5'-dimethyl-3'-heptyl)-phenol (p353NP) was synthesized, and its degradation by an axenic culture of Sphingomonas TTNP3 was investigated with [ring-U-14C]-labelled and non-labelled p353NP including a time-course study. Radioactive mass balancing resulted in different polar soluble fractions, in insoluble radioactivity associated with biomass, and volatile radioactivity in the form of the mineralization product 14CO2. In the extracellular media, the presence of nonanol corresponding to the nonyl chain of the NP isomer was confirmed and its concentration was determined during the course of fermentation. No other radioactive compounds were detected beside the parent isomer. Radioactive metabolites were only found in the intracellular fraction of S. TTNP3.

Bioavailability of a nonylphenol isomer in dependence on the association to dissolved humic substances.

Humic substances are important environmental components since they represent a very large part of organic compounds on earth. According to many reports, dissolved humic substances are a determinant parameter for the bioavailability of xenobiotic compounds. For the present bioavailability studies, two kinds of dissolved humic substances, a commercially available humic acid and fulvic acids isolated from peat were used. As the relevant xenobiotic, a defined branched nonylphenol isomer, 4(3',5'-dimethyl-3'-heptyl)-phenol (p353NP) was synthesised according to Friedel-Crafts alkylation. Equilibrium dialysis studies were implemented in order to investigate the association between 14C-labelled p353NP and dissolved humic substances. The biodegradability in the presence of dissolved humic substances was examined in experiments with the nonylphenol degrading bacterium strain Sphingomonas TTNP3 and with p353NP as sole carbon source. The results showed that p353NP-humic acid associates were formed in high amounts, whereas no adducts with fulvic acids occurred. In the degradation studies with Sphingomonas TTNP3, no effects of dissolved humic substances on the bioavailability of p353NP could be observed. It was assumed that the association between nonylphenol and humic acids occurs rapidly and is reversible. Thus, the formation of "labile" complexes did not influence biodegradation rates, which were quite low.

Degradation of the radioactive and non-labelled branched 4(3',5'-dimethyl 3'-heptyl)-phenol nonylphenol isomer by sphingomonas TTNP3.

The degradation of the 4(3',5'-dimethyl-3'-heptyl)-phenol (p353NP) nonylphenol isomer in cultures of Sphingomonas TTNP3 supplemented with the technical mixture of nonylphenol was first assessed. Then the radioactive and non-labelled form of these diastereomers were both synthesised. The radioactive isomers were synthesised using [ring-U-14C]-labelled phenol and 3,5-dimethyl-3-heptanol by Friedel and Crafts alkylation. The time-course of degradation was performed with and without 14C-p353NP; balancing of radioactivity was calculated from different soluble fractions (organic, aqueous), bacterial biomass, and 14CO2 evolved as mineralization product. The noticeable portion of 14C bound to biomass showed that at least the aromatic ring of 14C-p353NP was degraded and served as energy source and probably as carbon source for bacterial growth. In addition, the appearance of 3,5-dimethyl-3-heptanol, the nonanol corresponding with the side-chain of p353NP, was demonstrated in the bacterial media, and its concentration determined during the course of fermentation. Besides the parent 14C-p353NP, no other radioactive compounds, i.e. metabolites of 14C-p353NP were detected in the media.

Intracellular pH determination of pristinamycin-producing Streptomyces pristinaespiralis by image analysis.

Intracellular pH (pH(i)) is an essential parameter in the regulation of intracellular processes. Thus, its measurement might provide clues regarding the physiological state of cells cultivated in vitro. pH(i) of the filamentous, pristinamycin-producing Streptomyces pristinaespiralis was determined by epifluorescence microscopy and image analysis using the pH-sensitive fluorescent probe BCECF-AM [2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, acetoxymethyl ester]. Staining cell culture samples (OD(660)=1) of S. pristinaespiralis with 20 microM BCECF at 28 degrees C for 30 min yielded a green/red fluorescence ratio (R:(527/600)) that correlated with the pH(i) of the cells for values ranging from 6.5 to 8.5. When S. pristinaespiralis was cultivated in pristinamycin-producing conditions (in batch mode, with a constant external pH of 6.8), the measured pH(i) varied between 6.3 and 8.7. In fact, pH(i) correlated with the excretion of pristinamycins and glucose consumption during the production process.