Role of IncP-1ß plasmids pWDL7::rfp and pNB8c in chloroaniline catabolism as determined by genomic and functional analyses.

Broad-host-range catabolic plasmids play an important role in bacterial degradation of man-made compounds. To gain insight into the role of these plasmids in chloroaniline degradation, we determined the first complete nucleotide sequences of an IncP-1 chloroaniline degradation plasmid, pWDL7::rfp and its close relative pNB8c, as well as the expression pattern, function, and bioaugmentation potential of the putative 3-chloroaniline (3-CA) oxidation genes. Based on phylogenetic analysis of backbone proteins, both plasmids are members of a distinct clade within the IncP-1ß subgroup. The plasmids are almost identical, but whereas pWDL7::rfp carries a duplicate inverted catabolic transposon, Tn6063, containing a putative 3-CA oxidation gene cluster, dcaQTA1A2BR, pNB8c contains only a single copy of the transposon. No genes for an aromatic ring cleavage pathway were detected on either plasmid, suggesting that only the upper 3-CA degradation pathway was present. The dcaA1A2B gene products expressed from a high-copy-number vector were shown to convert 3-CA to 4-chlorocatechol in Escherichia coli. Slight differences in the dca promoter region between the plasmids and lack of induction of transcription of the pNB8c dca genes by 3-CA may explain previous findings that pNB8C does not confer 3-CA transformation. Bioaugmentation of activated sludge with pWDL7::rfp accelerated removal of 3-CA, but only in the presence of an additional carbon source. Successful bioaugmentation requires complementation of the upper pathway genes with chlorocatechol cleavage genes in indigenous bacteria. The genome sequences of these plasmids thus help explain the molecular basis of their catabolic activities.

DNA rearrangement has occurred in the carbazole-degradative plasmid pCAR1 and the chromosome of its unsuitable host, Pseudomonas fluorescens Pf0-1.

The carbazole-degradative plasmid pCAR1 carries the class II transposon Tn4676, which contains the car and ant genes, essential for conversion of carbazole into anthranilate, and anthranilate into catechol, respectively. In our previous study, DNA rearrangements in pCAR1 were frequently detected in the host Pseudomonas fluorescens Pf0-1 in the presence of carbazole, resulting in the improvement of host survivability. Several Pf0-1 mutants harbouring pCAR1 were isolated, and deletion of DNA in the plasmid ant gene was found. Here, we compared genome sequences of the parent strain Pf0-1L(pCAR1::rfp) and one of its mutants, 5EP83, to assess whether other DNA rearrangements occurred in either the plasmid or the host chromosome. We found transposition of Tn4676 into the 5EP83 chromosome. In addition, ISPre1 had transposed into the car gene intergenic region on the pCAR1-derivative plasmid of 5EP83, which inhibited car transcription. As a result of these transpositions, 5EP83 was able to metabolize carbazole due to the Tn4676 on its chromosome, although the car genes on its plasmid were non-functional. We also found that one copy of duplicate carAa genes had been deleted, and that ISPre4 had transposed into both the host chromosome and the plasmid. Our findings suggest that Pf0-1 harbouring pCAR1 is subjected to DNA rearrangements not only on the plasmid but also on its chromosome in the presence of carbazole.

Effect of vitamin E supplementation with exercise on cognitive functions and total antioxidant capacity in older people.

OBJECTIVE: We investigated the effects of six months vitamin E administration on cognition evaluated by event-related potentials in exercising older subjects. DESIGN: Randomised controlled trial. SETTING: Retirement home in Antalya, Turkey. PARTICIPANTS: Fifty-seven adults aged 60-85 years were randomly assigned to one of four groups: sedentary control (C), vitamin E (V), exercise training (E) and vitamin E under training (EV). INTERVENTION: V and EV groups were received vitamin E at a dose of 900 IU/day P.O. for 6 months. Trained groups were subjected to walking exercise involved 3 sessions per week for 6 months. Walking duration was gradually increased during 8 weeks, and stayed constant until the end of training period. Participants were begun walking at % 70 heart rate reserve for 20 min/day at the first two weeks, and walking duration was increased by 5 minutes/day of each week until subjects were reached a level of 50 min/day by week 8. MEASUREMENTS: Plasma vitamin E concentration, total antioxidant capacity and two parameters of event-related potentials namely P3 latency and amplitude were performed on all study groups both before and after training. RESULTS: Significant improvement in P3 latency was found in exercising groups. However, no significant differences were found between vitamin and other groups for P3 latency. Amplitude measurements were found unaltered among all groups. CONCLUSION: We concluded that although six months training results improvement in P3 latency, vitamin E supplementation does not affect cognitive function evaluated by event-related potentials in older subjects.

Culture independent detection of Sphingomonas sp. EPA 505 related strains in soils contaminated with polycyclic aromatic hydrocarbons (PAHs).

The Sphingomonas genus hosts many interesting pollutant-degrading strains. Sphingomonas sp. EPA505 is the best studied polycyclic aromatic hydrocarbon (PAH)-degrading Sphingomonas strain. Based on 16S rRNA gene sequence analysis, Sphingomonas sp. strain EPA505 forms a separate branch in the Sphingomonas phylogenetic tree grouping exclusively PAH-degrading isolates. For specific PCR detection and monitoring of Sphingomonas sp. EPA505 and related strains in PAH-contaminated soils, a new 16S rRNA gene-based primer set was designed. The new primer set was shown to be highly selective for Sphingomonas sp. strain EPA505 as it only amplified DNA from strain EPA505 and not from other tested Sphingomonas strains or soil bacteria not belonging to the Sphingomonas genus. Using DNA extracts of a variety of inoculated PAH-contaminated soils, the primer pair was able to detect EPA505 in concentrations as low as 10(2) cells per gram of soil. Applying the new primer set, 16S rRNA gene fragments which were 99-100% similar to the corresponding gene of strain EPA505 were amplified from four of five PAH-contaminated soils. On the other hand, no PCR products were obtained from any of five tested uncontaminated soils. The preferential presence of EPA505 related Sphingomonas strains in PAH-contaminated soils with very different contamination profiles and different origin suggests an important role of this type of Sphingomonas in the natural Sphingomonas community colonizing PAH-contaminated sites.

The complete sequences of plasmids pB2 and pB3 provide evidence for a recent ancestor of the IncP-1beta group without any accessory genes.

The nucleotide sequences of the broad-host-range antibiotic resistance plasmids pB2 (61 kb) and pB3 (56 kb), which were isolated from a wastewater treatment plant, were determined and analysed. Both have a nearly identical IncP-1beta backbone, which diverged early from the sequenced IncP-1beta plasmids R751, pB10, pJP4, pADP1 and pUO1. In contrast to the latter plasmids, the pB2 and pB3 backbone does not seem to have undergone any deletions. The complete partition gene parA is located downstream of the mating pair formation (trb) module. A 14.4 kb or 19.0 kb mobile genetic element is present between traC and parA of pB3 and pB2, respectively. This region is typical for insertions in IncP-1beta plasmids, but the insertion site is unique. Both elements differ only by a duplication in pB2 of a tetA(C)-tetR-tnpA(IS26) fragment. The 5 bp target site duplication and the 26 bp inverted repeats flanking the mobile genetic elements are still intact, indicating that the insertion occurred recently. The element consists of three nested transposable elements: (i) a relict of a Tn402-like transposon with a gene for a new class D beta-lactamase (bla(NPS-2)); (ii) within that, another Tn402-like element with a class 1 integron harbouring the gene cassettes cmlA1 for a chloramphenicol efflux protein and aadA2 encoding a streptomycin/spectinomycin adenylyltransferase, and a copy of IS6100; (iii) into the integrase gene intI1 a tetracycline resistance module tetA(C)-tetR flanked by copies of IS26 is inserted. Interestingly, in contrast to all other IncP-1beta plasmids analysed so far, the oriV region between trfA and klcA is not interrupted by accessory genes, and there is no indication that previously inserted accessory genes have subsequently been deleted. The genes kluAB are also missing in that region and should thus be considered acquired genes. These findings, together with the fact that IncP-1beta plasmids acquired accessory elements at various positions in the backbone, suggest that IncP-1beta plasmids without any accessory genes exist in microbial communities. They must occasionally acquire accessory genes by transposition events, resulting in those plasmids that have been found based on selectable phenotypic traits.

The 64 508 bp IncP-1beta antibiotic multiresistance plasmid pB10 isolated from a waste-water treatment plant provides evidence for recombination between members of different branches of the IncP-1beta group.

The complete 64508 bp nucleotide sequence of the IncP-1beta antibiotic-resistance plasmid pB10, which was isolated from a waste-water treatment plant in Germany and mediates resistance against the antimicrobial agents amoxicillin, streptomycin, sulfonamides and tetracycline and against mercury ions, was determined and analysed. A typical class 1 integron with completely conserved 5' and 3' segments is inserted between the tra and trb regions. The two mobile gene cassettes of this integron encode a beta-lactamase of the oxacillin-hydrolysing type (Oxa-2) and a gene product of unknown function (OrfE-like), respectively. The pB10-specific gene load present between the replication module (trfA1) and the origin of vegetative replication (oriV) is composed of four class II (Tn3 family) transposable elements: (i). a Tn501-like mercury-resistance (mer) transposon downstream of the trfA1 gene, (ii). a truncated derivative of the widespread streptomycin-resistance transposon Tn5393c, (iii). the insertion sequence element IS1071 and (iv). a Tn1721-like transposon that contains the tetracycline-resistance genes tetA and tetR. A very similar Tn501-like mer transposon is present in the same target site of the IncP-1beta degradative plasmid pJP4 and the IncP-1beta resistance plasmid R906, suggesting that pB10, R906 and pJP4 are derivatives of a common ancestor. Interestingly, large parts of the predicted pB10 restriction map, except for the tetracycline-resistance determinant, are identical to that of R906. It thus appears that plasmid pB10 acquired as many as five resistance genes via three transposons and one integron, which it may rapidly spread among bacterial populations given its high promiscuity. Comparison of the pB10 backbone DNA sequences with those of other sequenced IncP-1beta plasmids reveals a mosaic structure. While the conjugative transfer modules (trb and tra regions) and the replication module are very closely related to the corresponding segments of the IncP-1beta resistance plasmid R751 and even more similar to the IncP-1beta degradative plasmids pTSA and pADP-1, the stable inheritance operons klcAB-korC and kleAEF are most similar to those of the IncP-1beta resistance plasmid pB4, and clearly less similar to the other IncP-1beta plasmids. This suggests that IncP-1beta plasmids can undergo recombination in the environment, which may enhance plasmid diversity and bacterial adaptability.

Linking floc structure and settling properties to activated sludge population dynamics in an SBR.

Over a period of 227 days properties of activated sludge grown in an sequencing batch reactor (SBR) operated under stable conditions were analyzed. Settling properties (sludge volume index (SVI)) of the activated sludge were compared with on-line measurements of floc size and size distribution obtained by using a laser light scattering technique (Malvern Mastersizer/S, Malvern, UK), and with measurements of microbial community dynamics analyzed by denaturing gradient gel electrophoresis (DGGE) patterns of 16S rRNA genes. In addition, microscopical observations were used to confirm the results. Three distinct stages in the SBR evolution were observed. In the first stage the structural floc properties showed predominant presence of floc-forming bacteria in the activated sludge. A good correlation between floc size, properties and microbial community evolution was observed. The second stage showed a good balance between floc-forming and filamentous bacteria, with good settling properties and a highly dynamic community in the SBR. In the third stage, an increase in the filamentous bacteria, which became predominant in the system was observed. Again, a good correlation between settling properties and floc size distribution was obtained and a new dominant species was observed in the DGGE patterns, which can be assumed to be a filamentous organism.

Assessment of the estrogenic activity of flue gases from burning processes by means of the yeast based human estrogen receptor (hER) bioassay.

Combustion processes are known to produce organic micro-pollutants in the flue gas at concentrations ranging over several orders of magnitude. Some organic micro-pollutants are suspected of being pseudo-estrogens and as such they can affect the public health. In this study, the possible application of the yeast based human estrogen receptor (hER) bioassay to screen flue gas streams for the presence of estrogenic active micro-pollutants was explored. Specifically, the protocol was modified to allow the detection and quantification of the potential estrogenic active non-polar organic micro-pollutants contained in the flue gas matrix. The modified assay was calibrated using a model estrogenic compound (17-alpha-ethinylestradiol (EE2)) dissolved in methylene chloride at concentrations ranging from 3 ng l(-1) to 3000 ng l(-1). The effective concentration to elucidate a 50% response (EC50) was 87 ng l(-1) of equivalent dissolved in methylene chloride. Samples of methylene chloride used to trap non-polar micro-pollutants in flue gas from combustion of pine wood were found to clearly register estrogenic activity by the bioassay under certain conditions. The combustion tests were performed with pinewood alone and with pine wood in the presence of both Copper-naphthenate and copper(II)chloride at 600 degrees C and 1000 degrees C. These conditions must be considered as experimental rather than practical. Overall, the results suggest that, by means of this modified assay, it is possible and warranted to screen systematically for estrogens in flue gas combustion processes.

Bioaugmentation of soils by increasing microbial richness: missing links.

It is generally assumed that increased microbial diversity corresponds to increased catabolic potential and, hence, to better removal of metabolites and pollutants. Yet, microbial diversity, more specifically richness of species in environmental samples and sites, is difficult to assess. It is proposed to interpret this diversity more in the framework of Pareto's law, i.e. 20% of the species govern 80% of the energy flux of the ecosystem. Ecological studies should attempt to delineate the main energy fluxes and that group of species playing quantitative key roles in the system. Consequently, bioaugmentation should aim at the rearrangement of the group of organisms dominantly involved in the overall energy flux, so that specific catabolic traits necessary for the clean up of pollutants are part of that active group. For soil ecosystems, the capacity of plant roots as creators of physical and chemical discontinuity should be used more strategically to bring about such rearrangements. Overall, this paper identifies a number of ecological concepts, such as the Pareto law, the Gompertz model and plant community-induced microbial competence, which may, given careful underpinning, open new perspectives for microbial ecology and biodegradation.

Microbial sulfate reduction with acetate: process performance and composition of the bacterial communities in the reactor at different salinity levels.

Microbial sulfate reduction with acetate as carbon source and electron donor was investigated at salinity levels between 0.53 and 1.48%. The experiment was carried out in a 2.3-1 upflow anaerobic sludge blanket reactor inoculated with granular methanogenic sludge. A pH of 8.3, a temperature of 32 +/- 1 degrees C and a chemical oxygen demand (COD)/SO4(2-)-S ratio of 2 were maintained in the reactor throughout the experiment. Sulfate reduction and the composition of the dominant bacterial communities in the reactor were monitored. The results showed that a maximal conversion rate for SO4(2-)-S of 14 g l(-1) day(-1) and a conversion efficiency of more than 90% were obtained at a salinity level of 1.26-1.39%. A further increase in the salinity level led to reactor instability. Denaturant gradient gel electrophoresis of 16S rDNA fragments amplified by PCR from total bacterial DNA extracted from the inoculum and reactor sludge showed that salinity level had an impact on the composition of the bacterial communities in the reactor. However, no clear relationship was found between reactor performance and the composition of the dominant bacterial communities in the reactor.

Genetic diversity among 3-chloroaniline- and aniline-degrading strains of the Comamonadaceae.

We examined the diversity of the plasmids and of the gene tdnQ, involved in the oxidative deamination of aniline, in five bacterial strains that are able to metabolize both aniline and 3-chloroaniline (3-CA). Three strains have been described and identified previously, i.e., Comamonas testosteroni I2 and Delftia acidovorans CA28 and BN3.1. Strains LME1 and B8c were isolated in this study from linuron-treated soil and from a wastewater treatment plant, respectively, and were both identified as D. acidovorans. Both Delftia and Comamonas belong to the family Comamonadaceae. All five strains possess a large plasmid of ca. 100 kb, but the plasmids from only four strains could be transferred to a recipient strain by selection on aniline or 3-CA as a sole source of carbon and/or nitrogen. Plasmid transfer experiments and Southern hybridization revealed that the plasmid of strain I2 was responsible for total aniline but not 3-CA degradation, while the plasmids of strains LME1 and B8c were responsible only for the oxidative deamination of aniline. Several transconjugant clones that had received the plasmid from strain CA28 showed different degradative capacities: all transconjugants could use aniline as a nitrogen source, while only some of the transconjugants could deaminate 3-CA. For all four plasmids, the IS1071 insertion sequence of Tn5271 was found to be located on a 1.4-kb restriction fragment, which also hybridized with the tdnQ probe. This result suggests the involvement of this insertion sequence element in the dissemination of aniline degradation genes in the environment. By use of specific primers for the tdnQ gene from Pseudomonas putida UCC22, the diversity of the PCR-amplified fragments in the five strains was examined by denaturing gradient gel electrophoresis (DGGE). With DGGE, three different clusters of the tdnQ fragment could be distinguished. Sequencing data showed that the tdnQ sequences of I2, LME1, B8c, and CA28 were very closely related, while the tdnQ sequences of BN3.1 and P. putida UCC22 were only about 83% identical to the other sequences. Northern hybridization revealed that the tdnQ gene is transcribed only in the presence of aniline and not when only 3-CA is present.

Do conventionally and biologically cultivated soils differ in bacterial diversity and community structure?

The effects of a long-term herbicide treatment on the microbial community of an agricultural soil were investigated. Therefor, molecular techniques were used to evaluate the structure and diversity of the soil microbial community. Eubacterial and group-specific primers for methanotrophs type I and II were used to amplify 16S rRNA gene fragments from total soil DNA. These fragments were subsequently separated by denaturing gradient gel electrophoresis (DGGE). The structure of the methanotrophic community was affected by the herbicides as the patterns obtained from a herbicide treated soil (conventional) clustered separately from the control soil (biological). It seems that group-specific PCR followed by DGGE is a very powerful and sensitive technique to differentiate fields, which have received a herbicide treatment from those who did not. The diversity of the methanotrophic community was quantified by calculating the Shannon-Weaver index of biodiversity. The type I methanotrophs showed a significant decreased biodiversity due to the herbicide treatment but the diversity of the methanotrophs type II was slightly higher in the herbicide treated soil.

Molecular fingerprinting of bacterial populations in groundwater and bottled mineral water.

Monitoring the hygienic quality of drinking waters by determining the concentration of fecal indicators with traditional plate count techniques suffers from important drawbacks. In this work, the potential of PCR-DGGE (polymerase chain reaction - denaturing gradient gel electrophoresis) analysis of 16S rDNA genes to fingerprint the bacterial populations of mineral water and groundwater was investigated. A rapid and simple pretreatment to concentrate and release bacterial DNA prior to PCR was explored. This pretreatment was successful for commercially bottled mineral water. For groundwater, an additional resuscitation step was required to obtain a PCR signal. It was clear that the groundwater under scrutiny contained a more diverse bacterial community than the mineral water. A comparison was made between four kinds of mineral waters and one sample of groundwater using the developed procedures. For each kind of water, bacterial populations cultured on R2A plates were also subjected to PCR-DGGE. Comparison of the fingerprints of the plated samples and the original samples suggested the presence of viable but nonculturable bacteria in the waters. The obtained cluster dendrogram indicated that each kind of water was characterized by a specific molecular fingerprint. The sensitivity of the whole of the procedure was between 10(4) and 10(5) cfu ml(-1) as determined using a pure culture of Escherichia coli. The described PCR-DGGE method can constitute the basis of a new and interesting strategy to monitor in a relatively rapid way (less than 24 h) the bacterial quality of waters such as mineral water, groundwater and certain types of reclaimed water.

Enrichment and molecular characterization of a bacterial culture that degrades methoxy-methyl urea herbicides and their aniline derivatives.

Soil treated with linuron for more than 10 years showed high biodegradation activity towards methoxy-methyl urea herbicides. Untreated control soil samples taken from the same location did not express any linuron degradation activity, even after 40 days of incubation. Hence, the occurrence in the field of a microbiota having the capacity to degrade a specific herbicide was related to the long-term treatment of the soil. The enrichment culture isolated from treated soil showed specific degradation activity towards methoxy-methyl urea herbicides, such as linuron and metobromuron, while dimethyl urea herbicides, such as diuron, chlorotoluron, and isoproturon, were not transformed. The putative metabolic intermediates of linuron and metobromuron, the aniline derivatives 3, 4-dichloroaniline and 4-bromoaniline, were also degraded. The temperature of incubation drastically affected degradation of the aniline derivatives. Whereas linuron was transformed at 28 and 37 degrees C, 3,4-dichloroaniline was transformed only at 28 degrees C. Monitoring the enrichment process by reverse transcription-PCR and denaturing gradient gel electrophoresis (DGGE) showed that a mixture of bacterial species under adequate physiological conditions was required to completely transform linuron. This research indicates that for biodegradation of linuron, several years of adaptation have led to selection of a bacterial consortium capable of completely transforming linuron. Moreover, several of the putative species appear to be difficult to culture since they were detectable by DGGE but were not culturable on agar plates.

Comparison of the spatial homogeneity of physico-chemical parameters and bacterial 16S rRNA genes in sediment samples from a dumping site for dredging sludge.

The homogeneity of the microbial community structure of a sediment landfill was examined by a culture-independent method and compared with physico-chemical parameters, i.e. organic matter, CaCO3 content, pH, and texture. Total genomic DNA was extracted from samples derived from different places and depths. After amplification with two different primer sets of partial bacterial 16S rRNA genes, the products were separated by denaturing gradient gel electrophoresis (DGGE). The DGGE fingerprints of different sediment samples taken in regular patterns at the same depth were similar, which indicates a spatial homogeneity in the numerically dominant bacterial populations in a landfill over 10,000 m2 in size. In a vertical column of approx. 10 m, only some differences in a few bands of the bacterial community structure were observed between samples taken from different depths. This DNA homogeneity coincided with a similar homogeneity of the physico-chemical parameters in the landfill at this site. Nevertheless, the DGGE technique revealed small differences in less prominent bacteria and was capable of separating the upper and lower samples of one column into two clusters. It therefore seems more sensitive than the physico-chemical approach for characterising the homogeneity of an environmental habitat.

Effect of dissemination of 2,4-dichlorophenoxyacetic acid (2,4-D) degradation plasmids on 2,4-D degradation and on bacterial community structure in two different soil horizons.

Transfer of the 2,4-dichlorophenoxyacetic acid (2,4-D) degradation plasmids pEMT1 and pJP4 from an introduced donor strain, Pseudomonas putida UWC3, to the indigenous bacteria of two different horizons (A horizon, depth of 0 to 30 cm; B horizon, depth of 30 to 60 cm) of a 2,4-D-contaminated soil was investigated as a means of bioaugmentation. When the soil was amended with nutrients, plasmid transfer and enhanced degradation of 2,4-D were observed. These findings were most striking in the B horizon, where the indigenous bacteria were unable to degrade any of the 2,4-D (100 mg/kg of soil) during at least 22 days but where inoculation with either of the two plasmid donors resulted in complete 2,4-D degradation within 14 days. In contrast, in soils not amended with nutrients, inoculation of donors in the A horizon and subsequent formation of transconjugants (10(5) CFU/g of soil) could not increase the 2,4-D degradation rate compared to that of the noninoculated soil. However, donor inoculation in the nonamended B-horizon soil resulted in complete degradation of 2,4-D within 19 days, while no degradation at all was observed in noninoculated soil during 89 days. With plasmid pEMT1, this enhanced degradation seemed to be due only to transconjugants (10(5) CFU/g of soil), since the donor was already undetectable when degradation started. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes showed that inoculation of the donors was followed by a shift in the microbial community structure of the nonamended B-horizon soils. The new 16S rRNA gene fragments in the DGGE profile corresponded with the 16S rRNA genes of 2,4-D-degrading transconjugant colonies isolated on agar plates. This result indicates that the observed change in the community was due to proliferation of transconjugants formed in soil. Overall, this work clearly demonstrates that bioaugmentation can constitute an effective strategy for cleanup of soils which are poor in nutrients and microbial activity, such as those of the B horizon.

Bioaugmentation of activated sludge by an indigenous 3-chloroaniline-degrading Comamonas testosteroni strain, I2gfp.

A strain identified as Comamonas testosteroni I2 was isolated from activated sludge and found to be able to mineralize 3-chloroaniline (3-CA). During the mineralization, a yellow intermediate accumulated temporarily, due to the distal meta-cleavage of chlorocatechol. This strain was tested for its ability to clean wastewater containing 3-CA upon inoculation into activated sludge. To monitor its survival, the strain was chromosomally marked with the gfp gene and designated I2gfp. After inoculation into a lab-scale semicontinuous activated-sludge (SCAS) system, the inoculated strain maintained itself in the sludge for at least 45 days and was present in the sludge flocs. After an initial adaptation period of 6 days, complete degradation of 3-CA was obtained during 2 weeks, while no degradation at all occurred in the noninoculated control reactor. Upon further operation of the SCAS system, only 50% 3-CA removal was observed. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes revealed a dynamic change in the microbial community structure of the activated sludge. The DGGE patterns of the noninoculated and the inoculated reactors evolved after 7 days to different clusters, which suggests an effect of strain inoculation on the microbial community structure. The results indicate that bioaugmentation, even with a strain originating from that ecosystem and able to effectively grow on a selective substrate, is not permanent and will probably require regular resupplementation.