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Field and laboratory experiments were carried out in order to estimate the suitability ofphytoremediation and bioaugmentation for oil shale chemical industry solid waste (semicoke) dump area remediation as well as influence of plants and laboratory selecteddegradative bacterial strains on the microbial communities in semi-coke, Field test plots (each50 m2) were established at semi-coke depository in July 200 I and samples for microbiologicaland chemical analysis were collected in October 2002 and 2003, Microbial communities insemi-coke were · analysed using both culture-based and molecular methods, Changes inmicrobial community structure and activity occurred in semi-coke as a result ofphytoremediation and bioaugmentation, Phytoremediation increased the number of oildegrading bacteria and diversity of microbial community in semi-coke as well as microbialbiomass. A comparison of 16S rRNA gene-based DGGE fingerprints of semi-coke samplesusing multivariate analysis showed variation between the bacterial community profiles fromdifferent treatments. Degradation rates of pollutants did not differ significantly between plotswith vegetation except for sod, showing negligible effect of soil amendment typeonbiodegradation activity. Our results indicate that increased biodegradation activity was due toproliferation of specific microbial groups, changes in taxonomic and metabolic diversity ofbacterial community and shifts in the structure of catabolic genes, Based on our findings weconclude that phytoremediation and bioaugmentation could be considered as an alternativemanagement option for remediation of oil shale solid waste.

Successful bioaugmentation requires that bacterial strains introduced into the polluted area must be able to adapt to new environmental conditions and retain high enough catabolic activity. The strains should degrade pollutant present at high concentrations, while having high affinity for the pollutants for their thorough degradation. The transfer of genetic information from introduced donor strain to indigenous bacterial population increases the biodegradation potential. As laboratory-selected strains can be poor survivors and lose catabolic activity in mixed microbial ecosystems, the indigenous biodegradative strains isolated from the river water continuously polluted with phenolic compounds of oil shale leachate may serve as inoculants for bioaugmentation. We have shown that the native phenol- and p-cresol-degrading community could be grouped according to the presence of catabolic genes involved in catabolism of aromatic compounds. The selected representative strains of different catabolic types of degradation of phenol and p-cresol were identified as Pseudomonas mendocina (strain PCl) and P. jluorescens (strains PC! 8, PC24). Catabolic potential of these strains was studied on the basis of phenol hydroxylase, p-cresol methylhydroxylase and catechol 2,3-dioxygenase genes. The occurrence and conjugation of plasmid DNA were revealed in these strains. The ability of the selected strains to degrade several phenolic compounds in natural phenolic wastewater in which the compounds were present in multicomponent mixtures, was investigated through laboratory microcosm studies, To elucidate the extent of interactions among the used bacterial strains single and mixed cultures were set up. The biodegradation activity of strains in microcosms was examined through viable counts, consumption of phenolic compounds and detecting the presence of catabolic genes by hybridization, During the experiments (30 days) the introduced bacteria remained viable even when the substrates were depleted. The mixture of strains was more effective in the decomposition of phenolic compounds from the natural wastewater as compared with the single culture conditions and the metabolic activity and cell density of each strain were co-ordinated within a specific time scale. The behaviour of strains in the phenolic leachate depended on the growth kinetics of the strains (K,,µ).

Denaturing gradient gel electrophoresis of amplified fragments of genes coding for 16S rRNA and for the largest subunit of multicomponent phenol hydroxylase (LmPH) was used to monitor the behaviour and relative abundance of mixed phenol-degrading bacterial populations (Pseudomonas mendocina PC1, P. fluorescens strains PC18, PC20 and PC24) during degradation of phenolic compounds in phenolic leachate- and oil-amended microcosms. The analysis indicated that specific bacterial populations were selected in each microcosm. The naphthalene-degrading strain PC20 was the dominant degrader in oil-amended microcosms and strain PC1 in phenolic leachate microcosms. Strain PC20 was not detectable after cultivation in phenolic leachate microcosms. Mixed bacterial populations in oil-amended microcosms aggregated and formed clumps, whereas the same bacteria had a planktonic mode of growth in phenolic leachate microcosms. Colony hybridisation data with catabolic gene specific probes indicated that, in leachate microcosms, the relative proportions of bacteria having meta (PC1) and ortho (PC24) pathways for degradation of phenol and p-cresol changed alternately. The shifts in the composition of mixed population indicated that different pathways of metabolism of aromatic compounds dominated and that this process is an optimised response to the contaminants present in microcosms.

Phenol- and p-cresol-degrading pseudomonads isolated from phenol-polluted water were analysed by the sequences of a large subunit of multicomponent phenol hydroxylase (LmPH) and catechol 2,3-dioxygenase (C23O), as well as according to the structure of the plasmid-borne pheBA operon encoding catechol 1,2-dioxygenase and single component phenol hydoxylase. Comparison of the carA gene sequences (encodes the small subunit of carbamoylphosphate synthase) between the strains showed species- and biotype-specific phylogenetic grouping. LmPHs and C23Os clustered similarly in P. fluorescens biotype B, whereas in P. mendocina strains strong genetic heterogeneity became evident. P. fluorescens strains from biotypes C and F were shown to possess the pheBA operon, which was also detected in the majority of P. putida biotype B strains which use the ortho pathway for phenol degradation. Six strains forming a separate LmPH cluster were described as the first pseudomonads possessing the Mop type LmPHs. Two strains of this cluster possessed the genes for both single and multicomponent PHs, and two had genetic rearrangements in the pheBA operon leading to the deletion of the pheA gene. Our data suggest that few central routes for the degradation of phenolic compounds may emerge in bacteria as a result of the combination of genetically diverse catabolic genes.

The biodegradation of 3,4, 2,4, 2,3, 2,6 and 3,5-dimethylphenol in combination with phenol and p-cresol by axenic and mixed cultures of bacteria was investigated. The strains, which degrade phenol and p-cresol through different catabolic pathways, were isolated from river water continuously polluted with phenolic compounds of leachate of oil shale semicoke ash heaps. The proper research of degradation of 2,4 and 3,4-dimethylphenol in multinutrient environments was performed. The degradation of phenolic compounds from mixtures indicated a flux of substrates into different catabolic pathways. Catechol 2,3-dioxygenase activity was induced by dimethylphenols in Pseudomonas mendocina PC1, where meta cleavage pathway was functional during the degradation of p-cresol. In the case of strains PC18 and PC24 of P. fluorescens, the degradation of p-cresol occurred via the protocatechuate ortho pathway and the key enzyme of this pathway, p-cresol methylhydroxylase, was also induced by dimethylphenols. 2,4 and 3,4-dimethylphenols were converted into the dead-end products 4-hydroxy-3-methylbenzoic acid and 4-hydroxy-2-methylbenzoic acid. In the degradation of 3,4-dimethylphenol, the transient accumulation of 4-hydroxy-2-methylbenzaldehyde repressed the consumption of phenol from substrate mixtures. A mixed culture of strains with different catabolic types made it possible to overcome the incompatibilities at degradation of studied substrate mixtures.

Accumulation of key catabolic intermediates during degradation of phenol, p-cresol and benzoate was studied in two-substrate batch cultivations by the strains Pseudomonas mendocina PC1, Pseudomonas fluorescens PC18 and P. fluorescens PC24. According to sequence analysis of 16S rRNA genes the strains belonged to different monophyletic clusters of Pseudomonas. The catechol 2,3-dioxygenase (C23O) gene, xylE, of strain PC1 and the phenol monooxygenase gene, pheA, of PC24 were localised on the chromosome, while the C23O gene, xylE, of strain PC18 and the p-cresol methylhydroxylase gene, pchF, of strains PC18 and PC24 were on plasmids. It was shown that, if the substrates were degraded from mixtures using either catechol meta, catechol ortho or catechol ortho and protocatechuate ortho pathways, then both substrates were catabolised simultaneously (nondiauxic growth) without the accumulation of intermediates. Exceptionally, degradation of phenol and benzoate via the catechol ortho pathway caused irreversible accumulation of cis,cis-muconate without detectable effect on simultaneous consumption of substrates. When the substrates were degraded from mixtures through meta and ortho catabolic pathways, the sequential consumption of substrates (diauxic growth) was observed due to the reversible accumulation of the catabolic intermediates p-hydroxybenzoate or catechol. Regulation of parallel catabolic pathways by the accumulation of catabolic intermediates depended on the concentration of growth substrates. At low concentrations simultaneous degradation occurred and the antagonistic effect of p-hydroxybenzoate on the degradation of phenol was diminished. In strain PC18 only the accumulation of p-hydroxybenzoate during growth on a phenol–p-cresol mixture seems to be directly metabolically regulated because phenol also induces the catabolic pathway for p-cresol degradation. Partial sequencing of the pchF genes of strains PC18 and PC24 showed considerable differences.