Biodeterioration of Compost-Pretreated Polyvinyl Chloride Films by Microorganisms Isolated From Weathered Plastics.

Polyvinyl chloride (PVC) is a petroleum-based plastic used in various applications, polluting the environment because of its recalcitrance, large content of additives, and the presence of halogen. In our case study, a new, two-stage biodegradation technology that combined composting process used for PVC pretreatment with a subsequent PVC attack by newly-isolated fungal and bacterial strains under SSF conditions was used for biodegradation of commercial PVC films. The novelty consisted in a combined effect of the two biodegradation processes and the use for augmentation of microbial strains isolated from plastic-polluted environments. First, the ability of the newly-isolated strains to deteriorate PVC was tested in individual, liquid-medium- and SSF cultures. Higher mass-reductions of PVC films were obtained in the former cultures, probably due to a better mass transfer in liquid phase. Using the two-stage biodegradation technology the highest cumulative mass-reductions of 29.3 and 33.2% of PVC films were obtained after 110 days with Trichoderma hamatum and Bacillus amyloliquefaciens applied in the second stage in the SSF culture, respectively. However, FTIR analysis showed that the mass-reductions obtained represented removal of significant amounts of additives but the PVC polymer chain was not degraded.

Fungal pretreatment parameters for improving methane generation from anaerobic digestion of corn silage.

Corn silage was treated by white rot fungi (WRF) to investigate the effect of pretreatment on material's ability to produce methane in anaerobic digestion (AD). The selective fungi Pleurotus ostreatus and Dichomitus squalens promoted biogas generation, whereas the non-selective Trametes versicolor and Irpex lacteus had negative effect. Cumulative methane production after 10-day pretreatment with P. ostreatus at 28 °C rose 1.55-fold. The longer pretreatments of 30 and 60-days had smaller effect. When the pretreatment with P. ostreatus was carried out at 40 °C a high H2S release affected the AD process. Effect of WRF action dependent on the type of corn silage. With typical corn silage, the lignin depolymerisation raised the methane generation from 0.301 to 0.465 m3kgVS-1. In contrast, extensive decomposition of hemicellulose in hybrid corn silage deteriorated the effect of pretreatment on methane production.

Killing Effect of Bacillus Velezensis FZB42 on a Xanthomonas Campestris pv. Campestris (Xcc) Strain Newly Isolated from Cabbage Brassica Oleracea Convar. Capitata (L.): A Metabolomic Study.

The potential use of Bacillus velezensis FZB42 for biological control of various phytopathogens has been documented over the past few years, but its antagonistic interactions with xanthomonads has not been studied in detail. Novel aspects in this study consist of close observation of the death of Xanthomonas campestris pv. campestris cells in a co-culture with B. velezensis FZB42, and quantification of lipopeptides and a siderophore, bacillibactin, involved in the killing process. A new robust Xcc-SU isolate tolerating high concentrations of ferric ions was used. In a co-culture with the antagonist, the population of Xcc-SU was entirely destroyed within 24-48 h, depending on the number of antagonist cells used for inoculation. No inhibitory effect of Xcc-SU on B. velezensis was observed. Bacillibactin and lipopeptides (surfactin, fengycin, and bacillomycin) were present in the co-culture and the monoculture of B. velezensis. Except for bacillibactin, the maximum contents of lipopeptides were higher in the antagonist monoculture compared with the co-culture. Scanning electron microscopy showed that the death of Xcc-SU bacteria in co-culture was caused by cell lysis, leading to an enhanced occurrence of distorted cells and cell ghosts. Analysis by mass spectrometry showed four significant compounds, bacillibactin, surfactin, fengycin, and bacillomycin D amongst a total of 24 different forms detected in the co-culture supernatant: Different forms of surfactin and fengycin with variations in their side-chain length were also detected. These results demonstrate the ability of B. velezensis FZB42 to act as a potent antagonistic strain against Xcc.

Effect of Agroecological Conditions on Biologically Active Compounds and Metabolome in Carrot.

Carrot serves as a source of health-beneficial phytochemicals for human diet whose content is affected by agroecological conditions. The effect of conventional, integrated and organic farming on ascorbic acid (AA) and α,ß-carotene levels of new carrot cultivars Cortina F1 and Afalon F1 was investigated and their metabolomic profiles were measured by direct analysis in real time ion source coupled with a high-resolution mass spectrometer (DART-HRMS). Cortina and Afalon exhibited high levels of AA and total carotenes under all agroecological conditions tested that fluctuated in broad ranges of 215-539 and 173-456 mg AA.kg-1 dry biomass and 1069-2165 and 1683-2165 mg carotene.kg-1 dry biomass, respectively. The ratio of ß- to α-carotene in both cultivars was about 1.3. The most important variable for the PCA and the partial least squares discriminant analysis (PLS-DA) models for ethyl acetate extracts measured in positive and negative ionization mode was 6-methoxymellein (6-MM). Total carotene content and 6-MM levels were higher in the organic carrot compared to the conventional one and were correlated with a higher level of spontaneous infection. Other important compounds identified were sitosterol, hexose and various organic acids including antioxidant ferulic and coumaric acids. The findings allow comparison of metabolomic profiles and the AA and carotene contents of both cultivars with those of other commercially used carrots.

Gene family expansions and transcriptome signatures uncover fungal adaptations to wood decay.

Because they comprise some of the most efficient wood-decayers, Polyporales fungi impact carbon cycling in forest environment. Despite continuous discoveries on the enzymatic machinery involved in wood decomposition, the vision on their evolutionary adaptation to wood decay and genome diversity remains incomplete. We combined the genome sequence information from 50 Polyporales species, including 26 newly sequenced genomes and sought for genomic and functional adaptations to wood decay through the analysis of genome composition and transcriptome responses to different carbon sources. The genomes of Polyporales from different phylogenetic clades showed poor conservation in macrosynteny, indicative of genome rearrangements. We observed different gene family expansion/contraction histories for plant cell wall degrading enzymes in core polyporoids and phlebioids and captured expansions for genes involved in signalling and regulation in the lineages of white rotters. Furthermore, we identified conserved cupredoxins, thaumatin-like proteins and lytic polysaccharide monooxygenases with a yet uncharacterized appended module as new candidate players in wood decomposition. Given the current need for enzymatic toolkits dedicated to the transformation of renewable carbon sources, the observed genomic diversity among Polyporales strengthens the relevance of mining Polyporales biodiversity to understand the molecular mechanisms of wood decay.

Biodegradation of Reactive Orange 16 azo dye by simultaneous action of Pleurotus ostreatus and the yeast Candida zeylanoides.

The purpose was to investigate a simultaneous biodegradation of the recalcitrant monoazo dye Reactive Orange 16 (RO16) in a mixed culture consisting of a biofilm of Pleurotus ostreatus-colonizing polyamide carrier and a suspension of the yeast Candida zeylanoides to see their biological interactions and possible synergistic action during degradation. Decolorization in the mixed culture was more effective than in the fungal monoculture, the respective decolorizations reaching 87.5% and 70% on day 11. The proliferation of yeast was reduced compared with the C. zeylanoides monoculture but enabled the yeast to participate in decolorization. The interaction of P. ostreatus with the yeast resulted in a gradual decrease of fungal manganese-dependent peroxidase (MnP) and laccase activities. Gas chromatography-mass spectrometry (GC-MS) analysis of the degradation products brought evidence that P. ostreatus split the dye molecule asymmetrically to provide 4-(ethenylsulfonyl) benzene whose concentration was much decreased in the mixed culture suggesting its increased metabolization in the presence of the yeast. In contrast, C. zeylanoides split the azo bond symmetrically producing the metabolites 4-(ethenylsulfonyl) aniline and α-hydroxybenzenepropanoic acid. Those metabolites were rapidly degraded in the mixed culture. A novel aspect is represented by the evidence of a mutual cooperative action of the fungal and yeast microorganisms in the mixed culture resulting in rapid decolorization and degradation of the dye.

Metabolic profiling of Fusarium oxysporum f. sp. conglutinans race 2 in dual cultures with biocontrol agents Bacillus amyloliquefaciens, Pseudomonas aeruginosa, and Trichoderma harzianum.

There are increasing efforts to identify biocontrol-active microbial metabolites in order to improve strategies for biocontrol of phytopathogens. In this work, Fusarium oxysporum f. sp. conglutinans was confronted with three different biocontrol agents: Trichoderma harzianum, Bacillus amyloliquefaciens, and Pseudomonas aeruginosa in dual culture bioassays. Metabolites produced during the microbial interactions were screened by a matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). T. harzianum exhibited the strongest inhibition of growth of F. oxysporum resulting in overlay of the pathogen colony with its mycelium. Recorded metabolite profiles suggested a direct attack of F. oxysporum mycelium by T. harzianum and B. amyloliquefaciens by means of membrane-attacking peptaibols and a set of antimicrobial lipopeptides and siderophores, respectively. The direct mode of the biocontrol activity of T. harzianum and B. amyloliquefaciens corresponded to their ability to suppress F. oxysporum production of mycotoxin beauvericin suggesting that this ability is not specific only for Trichoderma species. In the case of P. aeruginosa, siderophores pyoverdine E/D and two rhamnolipids were produced as major bacterial metabolites; the rhamnolipid production was blocked by F. oxysporum. The results showed that this type of biocontrol activity was the least effective against F. oxysporum. The effective application of MALDI-MS profiling to the screening of nonvolatile microbial metabolites produced during the interaction of the phytopathogen and the biocontrol microorganisms was demonstrated.

Ascorbic Acid and Glucosinolate Levels in New Czech Cabbage Cultivars: Effect of Production System and Fungal Infection.

Nutritional value and disease-preventive effects of cabbage are well-known. Levels of the antioxidant compounds ascorbic acid (AA) and glucosinolates (GSL) in new Czech cabbage cultivars were determined in the context of different production systems. The contents of AA and GSLs in cabbage biomass were determined by HPLC. Individual GSLs were identified according to their exact masses with sinigrin used as the external standard. Artificial infection with A. brassicicola generally raised the AA levels. The major GSLs (≥10 mg kg-1) were glucobrassicin, sinigrin, and glucoiberin. Indole and aliphatic GSLs were present, but no aromatic ones were detected. Ecological growth conditions and the artificial fungal infection increased the total content of GSLs and, also, of the methoxylated indole GSLs. Sulforaphane, iberin, indole-3-carbinol, and ascorbigen resulting from the hydrolysis of GSLs were found in both cultivars. The amounts and profiles of GSLs present in the two Czech cultivars demonstrated their good nutritional value. The decomposition products sulforaphane, iberin, indole-3-carbinol, and ascorbigen detected improve its health-promoting qualities and represent a suitable component of the human diet.

Antibiotic Resistance in Czech Urban Wastewater Treatment Plants: Microbial and Molecular Genetic Characterization.

Quantitative changes in antibiotic resistance genes (ARGs) were investigated in six urban wastewater treatment plants (WWTPs) treating municipal and industrial wastewaters. In a selected WWTP, the fate of ARGs was studied in a 1-year time interval and in two phases of wastewater treatment process. Nine ARGs (tetW, tetO, tetA, tetB, tetM, blaTEM, ermB, sul1, and intl1) were quantified in total and their relative abundance assessed by ARG copies/16SrRNA copies. From the tetracycline resistance genes, tetW was the only one detected in all sampled WWTPs. Its relative abundance in the nitrification tank of WWTP5 was found stable during the 1-year period, but was lowered by secondary sedimentation processes in the wastewater treatment down to 24% compared to the nitrification tank. Bacterial isolates showing high tetracycline resistance (minimal inhibition concentrations >100 µg/mL) were identified as members of Acinetobacter, Klebsiella, Citrobacter, Bacillus, and Enterobacter genera. Dynamic shifts in the relative abundance of ermB and sul1 were also demonstrated in wastewater samples from WWTP5.

Bioreactors based on immobilized fungi: bioremediation under non-sterile conditions.

White-rot fungi are renowned for their remarkable potential to degrade a wide range of organic pollutants. They are applicable in standard bioreactors offering both the use of the continuous mode of action and easy upscaling of the biodegradation process. The recent advance in this field consisted in the use of various fungi and different types of reactors in the treatment of real wastewaters. Most degradation studies involving white-rot fungi carried out so far used controlled, aseptic conditions. However, during bioremediation of real wastewaters, the degradation capacity of the fungi would be significantly affected by autochthonous microorganisms. Consequently, for the development of sustainable bioremediation technologies, it is important to understand the mechanisms involved in the intermicrobial interactions occurring during the bioremediation process. This review summarizes recent applications of white-rot fungi to biodegradation of recalcitrant organopollutants under non-sterile conditions describing the invading microorganism(s) and the way how they affect the stability and degradation efficiency of the fungal bioreactor cultures. In addition, studies where fungal cultures were exposed to defined microbial stress are also reported documenting the effect and mechanisms of microbial interactions. Advanced OMICs techniques, specifically the genomics and metabolomics analyses, are suggested to help in identification of the invading microorganisms and in discovery of mechanisms taking part in the interspecific interactions.

Effect of yeasts on biodegradation potential of immobilized cultures of white rot fungi.

The aim was to investigate the effect of yeast organisms on the degradation process by immobilized cultures of ligninolytic fungi. Immobilization was accomplished by 7-day colonization of polyamide mesh with mycelial fragments. Irpex lacteus decolorized >90% of the initial concentration of 150mgl-1 of anthraquinone Remazol Brilliant Blue R dye in three subsequent decolorization cycles and the degradation capacity was not negatively affected by the presence of 106Saccharomyces cerevisiae cells per ml in the mixed culture. The yeast was not able to degrade the dye. I. lacteus biofilm was also resistant to bacterial infection with E. coli. Inoculation of the yeast to pre-formed I. lacteus biofilm culture resulted in a reduction of fungal biomass by 27%. Levels of LiP, MnP and laccase of I. lacteus were not much influenced by S. cerevisiae or E. coli. Similar resilience of P. ostreatus biofilms was observed after exposure to yeast Issatchenkia occidentalis when the fungal degradation capacity measured with Reactive Orange 16 azo dye was maintained over two decolorization cycles. I. occidentalis did not degrade the dye under the conditions used. Formation of densely packed fungal biofilms with abundant extracellular polysaccharide was not impeded by the yeast. Increase of MnP and laccase levels attributable to the presence of I. occidentalis was observed.

Effect of bacteria on the degradation ability of Pleurotus ostreatus.

White-rot fungi are efficient degraders of lignin whose extracellular enzymes have a potential to degrade organopollutants. In natural conditions these fungi enter into interactions with other organisms, which may affect their biodegradation capacity. The aim was to investigate the ability of Pleurotus ostreatus to form stable biofilms and to test the capacity of the fungus to degrade Remazol Brilliant Blue R in mixed cultures with bacteria. Bacterial counts were determined to see the behavior of the bacterium in the mixed culture with the fungus. In axenic conditions, the homogenized fungal mycelium was able to form an active biofilm which quickly degraded the dye. The addition of Pseudomonas fluorescens or Bacillus licheniformis bacteria at 106CFU·mL-1 did not affect the decolorization rate by 7-d-old fungal biofilms where the decolorization rate reached 90%. In contrast, when fragments of the fungal mycelium were used for inoculation to pre-formed biofilm of P. fluorescens, the biofilm was allowed to develop for one week's time, no decolorization of RBBR was observed and low activities of MnP and laccase were detected. The use of agar disks covered with fungal mycelium for the inoculation to pre-formed biofilm of P. fluorescens resulted in a fully developed biofilm that decolorized RBBR with similar efficiency as the pure P. ostreatus. The difference between the agar-disk- and homogenized-mycelium inoculated fungal biofilms was corroborated by the measurement of total fungal biofilm biomass that was 6-fold lower in the latter biofilm. Capability of the fungus to overcome the competition of the bacterial biofilm thus depended on the type of fungal growth centres, where intact hyphae were superior to the fragments of mycelium. A similar effect was not observed with the biofilms of B. licheniformis where the bacterial growth was less massive. The ability of P. ostreatus biofilms to resist massive bacterial stress was demonstrated.

Immobilization of Irpex lacteus to liquid-core alginate beads and their application to degradation of pollutants.

White rot fungi (WRF) are applicable to biodegradation of recalcitrant pollutants. However, excessive biomass growth typical for WRF cultivation can hinder their large scale applications. Therefore, immobilization of Irpex lacteus to liquid-core alginate beads restricting excessive mycelium growth and simultaneously keeping high degradation rate of pollutants was tested. Effective diffusivities of dyes to the beads varied from (2.98 ± 0.69) × 10-10 to (10.27 ± 2.60) × 10-10 m2/s. Remazol Brilliant Blue R (RBBR), Reactive Orange 16 (RO16), and Naphthol Blue Black (NBB) were used as model dyes. The immobilized fungus decolorized model dyes when applied both in microwell plates and in fluidized bed reactors. Using the microwell plates, the apparent reaction rate constants ranged from (2.06 ± 0.11) × 10-2 to (11.06 ± 0.27) × 10-2 1/h, depending on the dye used and its initial concentration. High initial concentrations negatively affected the dye decolorization rate. No fungal growth outside the beads was observed in fluidized bed reactors and thus no operational problems linked to an excessive biomass growth occurred. When RBBR was decolorized in subsequent batches in the fluidized bed reactor, the apparent reaction rate constant increased from (11.63 ± 0.35) × 10-2 to (29.26 ± 7.19) × 10-2 1/h.

Mutual interactions of Pleurotus ostreatus with bacteria of activated sludge in solid-bed bioreactors.

White rot fungi are well known for their ability to degrade xenobiotics in pure cultures but few studies focus on their performance under bacterial stress in real wastewaters. This study investigated mutual interactions in co-cultures of Pleurotus ostreatus and activated sludge microbes in batch reactors and different culture media. Under the bacterial stress an increase in the dye decolorization efficiency (95 vs. 77.1 %) and a 2-fold elevated laccase activity (156.7 vs. 78.4 Ul(-1)) were observed in fungal-bacterial cultures compared to pure P. ostreatus despite a limited growth of bacteria in mixed cultures. According to 16S-rDNA analyses, P. ostreatus was able to alter the structure of bacterial communities. In malt extract-glucose medium the fungus inhibited growth of planktonic bacteria and prevented shifts in bacterial utilization of potential C-sources. A model bacterium, Rhodococcus erythropolis responded to fungal metabolites by down regulation of uridylate kinase and acetyl-CoA synthetase.

Biodegradation and detoxification potential of rotating biological contactor (RBC) with Irpex lacteus for remediation of dye-containing wastewater.

Use of fungal organisms in rotating biological contactors (RBC) for bioremediation of liquid industrial wastes has so far been limited in spite of their significant biodegradation potential. The purpose was to investigate the power of RBC using Irpex lacteus for decolorization and detoxification of industrial dyes and dyeing textile liquors. Recalcitrant dye Methylene Blue (150 mg L(-1)) was decolorized within 70 days, its mutagenicity removed, and the biological toxicity decreased more than 10-fold. I. lacteus biofilm in the RBC completely decolorized within 26 and 47 days dyeing liquors containing disperse or reactive dyes adjusted to pH4.5 and 5-fold diluted with the growth medium, respectively. Their respective biological toxicity values were reduced 10- to 10(4)-fold in dependence of the test used. A battery of toxicity tests comprising Vibrio fisheri, Lemna minor and Sinapis alba was efficient to monitor the toxicity of textile dyes and wastewaters. Strong decolorization and detoxification power of RBC using I. lacteus biofilms was demonstrated.

The use of the fungus Dichomitus squalens for degradation in rotating biological contactor conditions.

Biodegradation potential of Dichomitus squalens in biofilm cultures and rotating biological contactor (RBC) was investigated. The fungus formed thick biofilms on inert and lignocellulosic supports and exhibited stable activities of laccase and manganese peroxidase to reach 40-62 and 25-32% decolorization of anthraquinone Remazol Brilliant Blue R and heterocyclic phthalocyanine dyes, respectively. The decolorization ceased when glucose concentration dropped to 1 mmol l(-1). In RBC reactor, respective decolorizations of Remazol Brilliant Blue R and heterocyclic Methylene Blue and Azure B dyes (50 mg l(-1)) attained 99%, 93%, and 59% within 7, 40 and 200 h. The fungus exhibited tolerance to coliform and non-coliform bacteria on rich organic media, the inhibition occurred only on media containing tryptone and NaCl. The degradation efficiency in RBC reactor, capability to decolorize a wide range of dye structures and tolerance to bacterial stress make D. squalens an organism applicable to remediation of textile wastewaters.

Potential of combined fungal and bacterial treatment for color removal in textile wastewater.

Low efficiency of dye removal by mixed bacterial communities and high rates of dye decolorization by white-rot fungi suggest a combination of both processes to be an option of treatment of textile wastewaters containing dyes and high concentrations of organics. Bacteria were able to remove mono-azo dye but not other chemically different dyes whereas decolorization rates using Irpex lacteus mostly exceeded 90% within less than one week irrespective of dye structure. Decolorization rates for industrial textile wastewaters containing 2-3 different dyes by fungal trickling filters (FTF) attained 91%, 86%, 35% within 5-12 d. Sequential two-step application of FTF and bacterial reactors resulted in efficient decolorization in 1st step (various single dyes, 94-99% within 5 d; wastewater I, 90% within 7 d) and TOC reduction of 95-97% in the two steps. Large potential of combined use of white-rot fungi and traditional bacterial treatment systems for bioremediation of textile wastewaters was demonstrated.

The implication of Dichomitus squalens laccase isoenzymes in dye decolorization by immobilized fungal cultures.

The study focuses on the production of ligninolytic enzymes and dye degradation capacity of Dichomitus squalens immobilized on polyurethane foam (PUF) or pine wood (PW) in a fixed bed reactor at a laboratory scale (working volume of 0.6l). Immobilization of fungal cultures on pine wood improved eminently laccase production in comparison to the liquid cultures. Immobilized D. squalens was able to decolorize an anthraquinone dye Remazol Brilliant Blue R and an azo dye Reactive Orange 16, however, only a limited decolorization of Copper(II)phthalocyanine dye was observed in both types of reactor cultures. The involvement of a laccase activity in dye decolorization was suggested. Further, two different chromatographical forms of laccases, Lc1 and Lc2, were isolated from PW cultures of D. squalens using a fast, two step FPLC method. Enzymes revealed identical molecular masses of 68 kDa (estimated by SDS-PAGE) and similar pI's, however, they differed in their catalytic properties such as pH dependence of the activity and ABTS oxidation rates. In this study, we demonstrated different dye decolorization capacities of Lc1 and Lc2 as well.

Ecotoxicity monitoring of hydrocarbon-contaminated soil during bioremediation: a case study.

The ecotoxicity of hydrocarbon-contaminated soil originating from a brownfield site was evaluated during a 17-month biodegradation pilot test. The initial concentration of total petroleum hydrocarbons (TPHs) in the soil was 6380 microg/g dry weight. An amount of 200 kg soil was inoculated with 1.5 L of the bacterial preparation GEM-100 containing Pseudomonas sp. and Acinetobacter sp. strains (5.3 x 10(10) CFU.mL(-1)) adapted to diesel fuel. The concentration of TPHs in the soil decreased by 65.5% after bioremediation. Different organisms such as the bacterium Vibrio fischeri, terrestrial plants Sinapis alba, Lactuca sativa, and Hordeum vulgare, the water plant Lemna minor, the earthworm Eisenia fetida, and the crustacean Heterocypris incongruens were used for ecotoxicity evaluation. The highest toxicity was detected in the first period of bioremediation. However, certain toxic effects were detectable during the whole bioremediation process. The contact tests with plants, earthworms, and crustaceans were the most sensitive of all of the bioassays. Therefore, the contact tests performed directly on soil samples were shown to be a better tool for ecotoxicity evaluation of hydrocarbon-contaminated soil than the tests performed on soil elutriates. The ecotoxicity measured by the responses of the tests did not always correlate with the decrease in TPH concentrations in the soil during bioremediation.

Reduction in the mutagenicity of synthetic dyes by successive treatment with activated sludge and the ligninolytic fungus, Irpex lacteus.

Synthetic dyes are released in wastewater from textile manufacturing plants, and many of these dyes are genotoxic. In the present study, the mutagenicity of azo, anthraquinone, and triphenyl methane dyes was investigated before and after successive biodegradation with activated sludge and the ligninolytic fungus, Irpex lacteus. Two biodegradation systems were used to reduce the genotoxicity of dyes that were not efficiently inactivated by activated sludge alone. Mutagenicity was monitored with the Salmonella reversion assay conducted with the base-pair substitution detector strains, TA100 and YG1042, and the frame-shift detector strains, TA98 and YG1041, with and without rat liver S9. All dyes except for Congo Red (CR) were mutagenic with S9 activation. Assays conducted with the dyes indicated that only the azo dye Reactive Orange 16 (RO16) was mutagenic in both TA98 and TA100. Methyl Red and Disperse Blue 3 (DB3) were mutagenic in TA98, YG1041 and YG1042, while Reactive Black 5 was mutagenic in YG1041 and YG1042. Remazol Brilliant Blue R (RBBR), Crystal violet (CV) and Bromophenol Blue (BPB) were mutagenic only in TA98, but the toxicity of the latter two dyes complicated the evaluation of their mutagenicity. CR was not mutagenic in any of the tester strains. Biodegradation studies conducted with RO16 and DB3 indicated that the two-step biodegradation process reduced the mutagenic potential of RO16 and DB3 to a greater extent than activated sludge alone; the mutagenicity of the two dyes was reduced by 95.2% and 77.8%, respectively, by the two-step process. These data indicate that the combined biodegradation process may be useful for reducing the mutagenicity associated with wastewater from textile factories that contain recalcitrant dyes.