Bioconversion of glycerol into polyhydroxyalkanoates through an atypical metabolism shift using Priestia megaterium during fermentation processes: A statistical analysis of carbon and nitrogen source concentrations.

Polyhydroxyalkanoates (PHA) are bioplastics which are well known as intracellular energy storage compounds and are produced in a large number of prokaryotic species. These bio-based inclusions are biodegradable, biocompatible and environmental friendly. Industrial production of, short chain and medium chain length PHA, involves the use of microorganisms and their enzymes. Priestia megaterium previously known as Bacillus megaterium is a well-recognized bacterium for producing short chain length PHA. This study focuses to characterize this bacterium for the production of medium chain length PHA, and a novel blend of both types of monomers having enhanced properties and versatile applications. Statistical analyses and simulations were used to demonstrate that cell dry weight can be derived as a function of OD600 and PHA content. Optimization of growth conditions resulted in the maximum PHA production as: 0. 05 g. g-x. H-1, where the rate of PHA production was 0.28 g L-1. H-1 and PHA concentration was 4.94 g. L-1. This study also demonstrated FTIR to be a semi quantitative tool for PHA production. Moreover, conversion of scl-PHA to mcl-PHA with reference to time intermissions using GC-FID are shown.

New model development for qualitative and quantitative analysis of microbial polyhydroxyalkanoates: A comparison of Fourier Transform Infrared Spectroscopy with Gas Chromatography.

FT-IR spectroscopy is a non-destructive testing technique that requires minimal sample preparation time and allows the rapid characterization of structural features of complex, polymeric material. This technique has been frequently used in the past for the detection of PHA (Polyhydroxyalkanoates) but rarely for their quantification. In this work, by using cluster and discriminant statistical analysis of FT-IR data, different models are proposed for rapid identification of PHA monomers produced under different growth conditions by bacterial strains, and for their semi quantification. The results on the ability to produce large amounts of PHA (of 21 strains) in different environmental conditions of medium, substrates and deficiency of nutrients are presented. The spectral data analysis gives qualitative and semi quantitative information about the PHA produced in the samples. Models are proposed to test a large number of cultural conditions of strains and substrates in the field of screening and for identifying best conditions of PHA production in lab scale bioreactor and on industrial scale.

Medium chain length polyhydroxyalkanoates biosynthesis in Pseudomonas putida mt-2 is enhanced by co-metabolism of glycerol/octanoate or fatty acids mixtures.

The synthesis of medium chain length polyhydroxyalkanoates (mcl-PHAs) by Pseudomonas putida mt-2 was investigated under nitrogen-rich then deficient conditions with glycerol/octanoate or long-chain fatty acids (LCFAs) as carbon sources. When mixed, glycerol and octanoate were co-assimilated regardless of nitrogen availability but provided that glycerol uptake has been already triggered under non-limiting nutrient conditions. This concomitant consumption allowed to enhance mcl-PHAs accumulation (up to 57% of cell dry weight (CDW)) under both non-limiting and nitrogen deficient conditions. Octanoate then mostly drove anabolism of the polyester with 3-hydroxyoctanoate (3HO) synthesized as the main monomer (83%). If the preferred PHA precursor octanoate was supplied, glycerol was mainly involved in cell growth and/or maintenance but very little in PHA production even under nitrogen starvation. P. putida cells accumulated higher amounts of mcl-PHAs when grown on mixtures of LCFAs compared to LCFAs supplied as single substrate (25% and 9% of CDW, respectively). However, only a weak enrichment of the polyester was observed after transfer of cells in a fresh nitrogen-free medium containing the same combination of LCFAs. Some typical units within the polyester were related to the LCFAs ratio supplied in the medium indicating that tailor-made monomers could be synthesized.

Comet assay with gill cells of Mytilus galloprovincialis end point tools for biomonitoring of water antibiotic contamination: Biological treatment is a reliable process for detoxification.

This article investigates the ability of Pseudomonas peli to treat industrial pharmaceuticals wastewater (PW). Liquid chromatography-mass spectrometry (MS)/MS analysis revealed the presence, in this PW, of a variety of antibiotics such as sulfathiazole, sulfamoxole, norfloxacine, cloxacilline, doxycycline, and cefquinome.P. peli was very effective to be grown in PW and inducts a remarkable increase in chemical oxygen demand and biochemical oxygen demand (140.31 and 148.51%, respectively). On the other hand, genotoxicity of the studied effluent, before and after 24 h of shaking incubation with P. peli, was evaluated in vivo in the Mediterranean wild mussels Mytilus galloprovincialis using comet assay for quantification of DNA fragmentation. Results show that PW exhibited a statistically significant (p< 0.001) genotoxic effect in a dose-dependent manner; indeed, the percentage of genotoxicity was 122.6 and 49.5% after exposure to 0.66 ml/kg body weight (b.w.); 0.33 ml/kg b.w. of PW, respectively. However, genotoxicity decreased strongly when tested with the PW obtained after incubation with P. peli We can conclude that using comet assay genotoxicity end points are useful tools to biomonitor the physicochemical and biological quality of water. Also, it could be concluded that P. peli can treat and detoxify the studied PW.

Cytotoxic effect of chlorpyrifos ethyl and its degradation derivatives by Pseudomonas peli strain isolated from the Oued Hamdoun River (Tunisia).

A bacterium was isolated from the river of Oued Hamdoun (Tunisia), and its phenotypic features, physiological and chemotaxonomic characteristics and phylogenetic analysis of 16S ribosomal RNA sequence revealed it as Pseudomonas peli (P. peli). Chlorpyrifos ethyl (CP) was used as the sole source of carbon and energy by P. peli, and it was cometabolised in the presence of glucose. CP was completely degraded by P. peli after 96 h of shake incubation. High-performance liquid chromatography analysis indicated that the biodegradation kinetics was not affected by the addition of glucose into the culture medium. In the present study, only transient accumulation of one major no-identified product was observed after 48 h of incubation, with no other persistent metabolites detected. Cytotoxicity of CP, before and after biodegradation with P. peli, was evaluated in vitro using the MTT-colorimetric assay against three human cancer cell lines (A549, lung cell carcinoma, HT29, colon adenocarcinoma and MCF7, breast adenocarcinoma). CP reduced viability of all human cell lines in a dose-dependent manner. Its activity was very remarkable against A549 cell line. However, cytotoxicity strongly decreased in CP obtained after incubation with P. peli Hence, we conclude that when incubated under appropriate conditions,P. peli has a metabolism that completely detoxifies CP.

Impact of carbon source and variable nitrogen conditions on bacterial biosynthesis of polyhydroxyalkanoates: evidence of an atypical metabolism in Bacillus megaterium DSM 509.

Twenty bacterial strains were examined on their ability to produce polyhydroxyalkanoates (PHA) from different carbon sources under rich and depleted nitrogen conditions. Preliminary experiments with glucose as sole carbon source allowed to select PHA producing bacteria using FTIR spectroscopy. They were further tested with eight additional carbon substrates including organic, fatty acids or sugars. PHA content and monomer composition of four chosen strains (Pseudomonas putida mt-2, Bacillus megaterium DSM 90 and DSM 509, Corynebacterium glutamicum DSM 20137) were assessed by gas chromatography techniques for two cultural conditions: during growth phase on a mineral medium (MM) and after transfer of cells on a fresh MM without nitrogen (MM-N). For several carbon substrates, substantial amounts of PHA (up to 53% of the cell dry weight: CDW) were already obtained in MM for C. glutamicum DSM 20137 and the two B. megaterium strains; after transfer in MM-N, PHA contents remained constant except for B. megaterium DSM 509 where PHA production increased whatever the carbon source. P. putida mt-2 synthesized PHA under deprived nitrogen conditions. Highest PHA accumulation reached 48 and 77% of CDW with octanoic acid as substrate in B. megaterium DSM 90 and P. putida mt-2, respectively. Surprisingly, an atypical metabolic shift was observed for B. megaterium DSM 509 cultivated with nearly all unrelated carbon sources: whereas short chain length PHA (scl-PHA) were synthesized in MM, medium chain length PHA (mcl-PHA) were produced after transfer of cells into MM-N supplemented with the same substrate.

Decolorization does not always mean detoxification: case study of a newly isolated Pseudomonas peli for decolorization of textile wastewater.

The textile industry is a favor to the Tunisian economy by offering several job positions. However, it's not environmentally friendly. In fact, textile industries discharge high volumes of wastewater which contain several toxic pollutants such as dyes, fixator, and whiteness. In our study, Pseudomonas peli, isolated and characterized from Oued Hamdoun (center of Tunisia), was found able to decolorize textile effluent about 81 % after 24 h shaking incubation. On the other hand, the in vitro antiproliferative effects of the untreated and treated effluent was evaluated by their potential cytotoxic activity using the MTT colorimetric method against three human cancer cell lines (A549, lung cell carcinoma; HT29, colon adenocarcinoma; and MCF7, breast adenocarcinoma). Results showed that intact textile effluent and its content azo dyes didn't inhibit the proliferation of all tested cell lines. However, the cytotoxic effect was remarkable when we tested effluent obtained after treatment by P. peli in a dose-dependent manner. This activity was attributed to the presence, in our treated effluent, of some azo products of dyes which are responsible for inhibition of human cell lines proliferation. Thus, the use of this strain for testing on the industrial scale seems impossible and disadvantageous.

Human cell death in relation to DNA damage after exposure to the untreated and biologically treated pharmaceutical wastewater.

Among all the pharmaceutical drugs that contaminate the environment, antibiotics occupy an important place due to their high consumption rates in both veterinary and human medicine. The present study examined the ability of Pseudomonas putida to grow on the antibiotic wastewater, currently expanding in Tunisia, containing amoxicillin and cefadroxil. P. putida was very efficient to grow quickly in pharmaceutical wastewater (PW) and in reducing the total dissolved solids to 80.1 %. Cytotoxicity of PW, before and after biodegradation with P. putida mt-2, was evaluated in vitro, using the MTT assay, against four human tumor cell lines such as A549 (lung cell carcinoma), HCT15 (colon cell carcinoma), MCF7 (breast adenocarcinoma), and U373 (glioma cell carcinoma). The PW reduced all human cell lines viability in a dose-dependent manner. This activity was very remarkable against U373 cell line. For this reason, we have tested the genotoxicity of PW using comet assay for quantification of DNA fragmentation. In fact, PW has statistically significant (p<0.001) influence on DNA. Indeed, the percentage of genotoxicity was 66.87 and 87.5 %, after 24 and 48 h of treatment, respectively. However, cytotoxicity and genotoxicity decreased strongly when tested the PW obtained after incubation with P. putida mt-2. Our results indicate that P. putida is a promising and improved alternative to treating industrial-scale effluent compared to current chemical treatment procedures used by the industrials.

Bioremediation of industrial pharmaceutical drugs.

Recently, attention has been drawn toward the occurrence of pharmaceuticals in the environment. In recent years, many reports have been made on the occurrence of the large, differentiated group of pharmaceuticals in wastewater (PW), surface water, ground water, and in soil. The pharmaceutical sector is currently expanding in Tunisia, with more than 34 industries. The aim of this work was to evaluate the ability of Pseudomonas putida mt-2 to treat PW. P. putida was very efficient in reducing chemical oxygen demand (COD), total dissolved solids (TDS), and turbidity of solution (85.5, 89.1, and 81.5%, respectively). Genotoxicity of effluent, before and after biodegradation, was evaluated in vivo in mouse bone marrow by assessing the percentage of cells bearing different chromosome aberrations. Results indicated that PW showed a significant ability to induce DNA damage. In addition, PW induced a remarkable lipid peroxidation (LPO) effect, however, activities of both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) were unchanged when treated with PW, compared to nontreated PW. This toxicity was imputed to the presence of pharmaceutical compounds in wastewater. However, chromosome aberration, as well as LPO of PW, were significantly reduced after bioremediation. Thus, the use of this strain for testing on the industrial scale seems possible and advantageous.

Degradation and detoxification of acid orange 52 by Pseudomonas putida mt-2: a laboratory study.

INTRODUCTION: Acid orange 52 (AO52), extensively used in textile industries, was decolorized by Pseudomonas putida mt-2. AO52 azoreduction products such as N,N'-dimethyl-p-phenylenediamine (DMPD) and 4-aminobenzenesulfonic acid (4-ABS), were identified in the static degradation mixture. These amines were identified only in media of static incubation, which is consistent with their biotransformation under shaken incubation (aerobic conditions). MATERIALS AND METHODS: Tests with azo products were carried out, and whole cells were found able to easily degrade DMPD contrary to 4-ABS. However, this last could be attacked by cell extract, and an oxygen uptake was observed during the reaction. RESULTS: Degradation of DMPD by entire cells led to the formation of catechol. These results show that P. putida was able to decolorize AO52 and metabolize its derivative amines. In addition, the ability of tested compounds was evaluated in vitro to reduce human plasma butyrylcholinesterase (BuChE) activity. CONCLUSION: Azoreduction products seem to be responsible for BuChE inhibition activity observed in static biodegradation extract. However, toxicity of AO52 completely disappears after shaken incubation with P. putida, suggesting that bacterium has a catabolism which enables it to completely degrade AO52 and especially, to detoxify the dye mixture.

Alteration of in vitro and acute in vivo toxicity of textile dyeing wastewater after chemical and biological remediation.

INTRODUCTION: Textile industry is one of the most common and essential sectors in Tunisia. However, the treatment of textile effluents becomes a university because of their toxic impacts on waters, soils, flora, and fauna. MATERIALS AND METHODS: The aim of this work was to evaluate the ability of Pseudomonas putida mt-2 to decolorize a textile wastewater and to compare the biologic decolorization process to the chemical one currently used by the textile industry. RESULTS: P. putida exhibited a high decolorizing capacity of the studied effluent, compared to the coagulation-flocculation method with decolorization percentage of 86% and 34.5%, respectively. Genotoxicity of the studied effluent, before and after decolorization by P. putida mt-2, was evaluated in vitro, using the SOS chromotest, and in vivo, in mouse bone marrow, by assessing the percentage of cells bearing different chromosome aberrations compared to not treated mice. In addition, textile effluent statistically significant influenced acetylcholinesterase and butyrylcholinesterase activities and lipid peroxidation (p < 0.01) when compared to not-treated mice. Coagulation-flocculation treatment process used by industry was revealed to be ineffective. Indeed toxicities persisted after treatment and the effluent did not show any statistically significant decrease in toxicities compared to non-treated effluent. Our results indicate that P. putida is a promising and improved alternative to treating industrial scale effluent compared to current chemical decolorization procedures used by the Tunisian textile industry.

Acid violet 7 and its biodegradation products induce chromosome aberrations, lipid peroxidation, and cholinesterase inhibition in mouse bone marrow.

INTRODUCTION: Acid violet 7 (AV7), mostly used in food, paper, cosmetic, and especially in textile industries, was degraded by Pseudomonas putida mt-2 at concentrations up to 200 mg/l. MATERIALS AND METHODS: In this study, toxicity of AV7, before and after biodegradation, was evaluated in vivo, in mouse bone marrow, by assessing the percentage of cells bearing different chromosome aberrations, membrane lipid peroxidation, and acetylcholinesterasic activity inhibition. The studies included same conditions for animal treatment, corresponding to increasing doses by intraperitoneal (ip) injection. RESULTS: Results indicated that AV7 showed a significant ability to induce chromosome aberrations, lipid peroxidation, and acetylcholinesterase inhibitory effect. The toxicity of AV7 increased significantly after static biodegradation with P. putida mt-2 and totally disappeared after shaken incubation. In addition, the toxicity generated by the pure azo dye and the corresponding azoreduction metabolites (4'-aminoacetanilide (4'-AA) and 5-acetamido-2-amino-1-hydroxy-3,6-naphtalene disulfonic acid (5-ANDS)) were compared. 4'-AA and 5-ANDS would be responsible of static biodegradation medium toxicity. The present study demonstrates that P. putida mt-2, incubated under aerobic condition, has a catabolism which enables it to degrade AV7, and especially to completely detoxify the dye mixture.

Mutagenicity and genotoxicity of acid yellow 17 and its biodegradation products.

Acid yellow 17 (AY17), a very important commercial azo dye used in the textile industry, was degraded by Pseudomonas putida mt-2 at a concentration of up to 200 mg/L. High-performance liquid chromatography analysis of the biodegradation media revealed the presence of 4-aminobenzensulfonic acid (4-ABS) derived from AY17 azoreduction, which attests the expression of an azoreductase by this bacterium. This amine was identified only in the medium of static incubation, which is consistent with its biotransformation under shaken incubation (i.e., aerobic conditions). The mutagenicity of AY17 and its biodegradation products was evaluated by using Salmonella typhimurium TA102 and TA104. No mutagenicity was observed in the presence or absence of a metabolic activation system (S9). In addition, the ability of tested compounds to induce DNA damage in vitro with the DNA strand scission assay was evaluated. Results showed that only static decolorization culture of AY17 showed a significant ability to induce the pKS plasmid DNA opening. The present study showed that P. putida mt-2, cultivated under aerobic conditions, was able to decolorize, and especially to detoxify, AY17.

Genotoxic and antibutyrylcholinesterasic activities of acid violet 7 and its biodegradation products.

Acid violet 7, a sulfonated azo dye was degraded by Pseudomonas putida mt-2 in mineral medium at concentrations up to 200 mg/L. The genotoxicity of AV7 and its biodegradation extracts was evaluated by using the DNA-strand scission assay. No genotoxicity was observed, even with or without exposition to UV irradiation, for biodegradation under shaking conditions, but increased significantly after biodegradation under static conditions. In addition, the ability of tested compounds to reduce human plasma butyrylcholinesterase (BuChE) activity was evaluated in vitro. Genotoxicity and anti-BuChE activity generated by the azoreduction products [4'-aminoacetanilid (4'-AA) and 5-acetamido-2-amino-1-hydroxy-3,6-naphtalene disulfonic acid (5-ANDS)] were assessed and compared with that of the parent unsubstituted amines. 4'-AA exhibited a strong genotoxicity, which was imputed to the presence of the acetoxy (COCH3) substituent on the aromatic amine; however, the presence of sulphonic groups in 5-ANDS seems to be responsible for its BuChE inhibition activity. The present study demonstrates that P. putida mt-2, incubated under aerobic conditions, has a catabolism that enables it to degrade AV7 and, especially, to detoxify the dye mixtures.

In vitro mutagenicity of Acid Violet 7 and its degradation products by Pseudomonas putida mt-2: Correlation with chemical structures.

Acid Violet 7 (AV7), a very important commercial azo dye used in the textile, food, paper and cosmetic industries, was degraded by Pseudomonas putida mt-2 at a concentration up to 200mg/l. HPLC analysis of the biodegradation media revealed the presence of either 4'-aminoacetanilide (4'-AA) or 5-acetamido-2-amino-1-hydroxy-3,6-naphthalene disulfonic acid (5-ANDS) deriving from AV7 azoreduction which attests the expression of an azoreductase by this bacterium. These amines were identified only in media of static incubation, which is consistent with their biotransformation under shaken incubation (aerobic conditions). Pure azo dye, pure azoreduction products and total lyophilized biodegradation extracts were assayed for their mutagenic properties using Ames test. Mutagenicity of AV7 even with or without the S9 metabolizing system increased significantly after static biodegradation and totally disappeared after shaken incubation. In addition, mutagenicity of pure azo reduction products of AV7 was assessed and compared with that of the parent unsubstituted amines. 4'-AA exhibited a strong mutagenicity which was imputed to the presence of the acetoxy (COCH(3)) substituent on the aromatic amine; however, the presence of sulphonic groups in 5-ANDS limited its mutagenicity.

In vitro study of DNA damage induced by acid orange 52 and its biodegradation derivatives.

Mutagenicity of acid orange 52 (AO52) and its degradation products by Pseudomonas putida mt-2 was evaluated with the use of Salmonella Typhimurium TA102 and TA104 with and without the metabolic activation system (S9). No mutagenicity was observed in the absence of S9 and in the presence of S9 for biodegradation under shaking conditions, but it increased significantly in the presence of S9 after biodegradation under static conditions. In addition, the ability of tested compounds to induce DNA damage in vitro was evaluated with the DNA strand scission assay. The toxicity generated by the pure azo dye and the corresponding azoreduction products (4-aminobenzenesulfonic acid and N,N'-dimethyl-p-phenylenediamine) were compared. We suggest that the mutagenicity mechanism of these molecules occurs through free radical generation processes. In this study, we demonstrate that P. putida mt-2 incubated under aerobic conditions undergoes catabolism that enables it to degrade AO52 completely and, especially, to detoxify the dye mixtures.

Acclimated biomass that degrades Sulfonated Naphthalene Formaldehyde Condensate.

A number of aerobic species were isolated from textile industry activated sludge wastewater. The bacterial consortium was acclimated during seven days before testing its capacity of Sulfonated Naphthalene-Formaldehyde Condensate (SNFC) recalcitrant compound degradation. SNFC's degradation was evaluated by using different techniques including: vapour pressure osmometry, spectroscopy UV-Visible and Chemical Oxygen Demand (COD). The degradation of SNFC by acclimated bacterial consortium was determined by monitoring the decrease of absorbance and of COD at wavelength 288 nm. We were able to deduce that biodegradation of SNFC involves two steps: cleavage of CH2 bridges and the degradation of the aromatic nuclei. The bacteria species community that was able to degrade SNFC consisted of aerobic Gram-negative rods belonging to the Pseudomonadaceae family. The strains were identified as Bukholderia cepacia, Brevundimonas vesicularis, Pseudomonas stutzeri, Ralostonia picketti, Shewanella putrefaciens, Sphingomonas paucimobilis and Agrobacterium radiobacter.

Physiological states and energetic adaptation during growth of Pseudomonas putida mt-2 on glucose.

Kinetic study of growth of Pseudomonas putida mt-2 was investigated in batch culture under aerobic conditions, on glucose as initial carbon and energy source. Cell growth was continuous and three phases were found regarding accumulation of intermediates: (1) glucose was largely converted to gluconate and 2-ketogluconate, (2) then gluconate was converted to 2-ketogluconate and (3) the latter was consumed after gluconate depletion. Examination of growth kinetics and yields showed that glucose flux was mainly oriented to oxidation reduction in the periplasm and less towards biosynthesis. Values of respiratory quotient and of CO2/biomass and O2/biomass yields were characteristic of each phase. Main enzymatic activities involved in the use of these substrates were always detected meaning that concomitant assimilation is possible. However the levels of these activities varied during growth. Membrane conversions seem to have a significant energetic contribution explaining the higher specific growth rate obtained in glucose phase compared to gluconate and 2-ketogluconate ones. This is also noticeable through the evolution of the yields Y(O2)/X and Y(CO2)/X. Although the three convergent pathways are operational and can be genetically controlled, the progression of the culture in successive phases highlights an overall level of regulation in response to the energetic needs.

Comparative study of Cyperus rotundus essential oil by a modified GC/MS analysis method. Evaluation of its antioxidant, cytotoxic, and apoptotic effects.

Gas chromatography coupled with mass spectrometry (GC/MS), using both electron impact (EI) and chemical ionization (CI) detection modes on apolar and polar stationary phases, led to the determination of the volatile composition of the essential oil obtained from tubers of Cyperus rotundus (Cyperaceae). In this study, more than 33 compounds were identified and then compared with the results obtained in our previous work. Cyperene, alpha-cyperone, isolongifolen-5-one, rotundene, and cyperorotundene were the principal compounds comprising 62% of the oil. An in vitro cytotoxicity assay with MTT indicated that this oil was very effective against L1210 leukaemia cells line. This result correlates with significantly increased apoptotic DNA fragmentation. The oxidative effects of the essential oil were evaluated using the 1,1-diphenyl-2-picrylhydrazyl (DPPH), xanthine/xanthine oxidase assays, and the scavenging of superoxide radical assay generated by photo-reduction of riboflavin. The antimutagenic activity of essential oil has been examined by following the inhibition of H(2)O(2) UV photolysis which induced strand-break formation in pBS plasmid DNA scission assay. Based on all these results, it is concluded that C. rotundus essential-oil composition established by GC/MS analysis, in EI- and CI-MS modes, presents a variety of a chemical composition we were not able to detect with only GC/MS analysis in our previous work. This essential oil exhibited antioxidant, cytotoxic, and apoptotic properties.

Evaluation of genotoxicity and pro-oxidant effect of the azo dyes: acids yellow 17, violet 7 and orange 52, and of their degradation products by Pseudomonas putida mt-2.

Acids yellow 17, violet 7 and orange 52, very important commercial azo dyes used in the textile, food, paper and cosmetic industries, were degraded by Pseudomonas putida mt-2 at concentrations up to 100mg/l. The culture media was completely decolorized under static incubation for 60 h, this faster than under continuous shaking incubation. SOS chromotest using Escherichia coli PQ37, with and without metabolic activation (S-9 preparations), was used to assess genotoxicity potential of these dyes before and after biodegradation. None of these dyes or their metabolites was found to be genotoxic in the absence of "Araclor-Induced rat liver microsome" preparations (S-9). However, in presence of the preparation S-9, the genotoxicity of the biodegradation products was highlighted. Metabolites resulting from static cultures were more genotoxic than those obtained in shaken conditions. In addition to genotoxic effects, metabolites have shown a significant ability to induce the formation of superoxide free radical anion (O(2)(*-)). The toxicities generated by the pure azo dyes and the pure azo-reduction products (sulfanilic acid, N,N'-dimethyl-p-phenylenediamine and 4'-aminoacetanilid) were compared. These results suggest that P. putida mt-2 degrades the studied azo dyes in two steps: an azo-reduction followed by an oxygen-dependent metabolization. Some of the derived metabolites would be responsible of genotoxicity and metabolic toxicity.