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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.

The major environmental problem in the northeastern Estonia is the semi-coke mounds in oilshale industry areas, Alternatives to the chemical methods (sorption, ozonation) for removingxenobiotic compounds from leachate are biological methods, like bioaugmentation, where theproperly selected microorganisms are used. Determination of the kinetic constants (maximumspecific growth rates (µmax), lag times (A.), half saturation constants (Kso for oxygenatingactivity and Ksa for growth) and inhibition constants (Ki)) will give us information about therate of pollutant degradation and is the basis for the selection of the most effective bacteria forbioaugmentation. In phenol degradation the initial and rate-limiting enzyme is phenolhydroxylase, encoded by different genes, The aim of this work was to carry out a kineticstudy of the aerobic degradation of phenol using single strains (Pseudomonas mendocinaPC 1, P. fluorescens PC 18, PC20 and PC24) isolated from river water continuously pollutedwith phenolic compounds, The strains PC 1 and PC 18 contain genes for multicomponentphenol hydroxylase, whereas single-component phenol hydroxylase (pheBA operon)characterizes the strains PC20 and PC24, The phenol-oxygenating activity (Kso) was obtainedfrom substrate-dependent oxygen uptake data (oxygen concentrations were measured with aClark-type oxygen electrode) using Michaelis-Mentens model. Specific growth rates µ andlag times). were calculated from absorbance growth curves on phenol concentrations 0,2-10.6mM and the growth kinetic constants (µmax, Ksa, Ki) were estimated using Haldanes, Edwardsand Aiba-Edwards model. The Kso values for phenol were one order of magnitude lower instrains PC 1 and PC 18 than in strains PC20 and PC24.

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).

Although the production of oil shale energy and oil has decreased steadily during the last years with a corresponding decrease in wastes, the semi-coke mounds constitute one of the major adverse environmental challenges in Estonia The processed oil shale (semicoke) contains several organic and inorganic compounds (oil fractions, sulphides, phenolic compounds, polycyclic aromatic hydrocarbons). Laboratory and field experiments were carried out in order to test the effect of phytoremediation and bioaugmentation for remediation of pollutants in semi-coke. Four pilot test plots (50 m2 ) were established at semi-coke depository in July 2001. For bioaugmentation experiment the set of bacteria consisting of three biodegradative strains isolated from nearby area was selected. Several molecular microbiological methods were used to assess and compare the microbial community structure and diversity as well as the presence and diversity of biodegradative genes in collected samples. The dominant bacterial species based on 16S rDNA sequences in semi-coke samples were also identified. These analyses revealed that semi-coke microbial community is characterized by few dominant populations and possesses low diversity. The phytoremediation increased the number of bacteria and diversity of microbial community in semi-coke. Within a one and half year period starting from establishment of test plots, the concentration of phenolic compounds decreased up to 50% and oil products up to three times at plots with vegetation compared to control. Bioaugmentation experiment, performed in summer 2002 increased biodegradation intensity of oil products up to 50% compared to untreated planted controls. The plots, which were supplemented with laboratory-selected bacteria, were characterized by higher microbial activity and showed changes in microbial community structure. Our findings also indicate that plant growth; particularly rooting depth and belowground biomass could be enhanced by adding mixture of selected bacterial strains to semi-coke.

Several years ago, a laboratory-constructed plasmid with a single-component phenol monooxygenase gene (pheBA operon) flanked by two IS elements was released to a phenol-polluted area. During the following years, we found in the test area widely distributed pheBA operon-containing bacteria. The new pheBA+ strains belong predominantly to the Pseudomonas fluorescens group, and they did not arise via selection of the released PHE plasmid. On the contrary, the formation of several different types of PHE plasmids occurred, namely pPHE101 (60,958 bp) from the IncP-9 group, non-transferable plasmid pPHE69 (44,717 bp), mobilizable plasmid pPHE20 (39,609 bp) and the IncP-7 type plasmid pPHE24ΔpheBA (120,754 bp), in which the pheBA operon was translocated from the plasmid to the chromosome. In two cases, PHE plasmid-bearing strains exist in a multi-plasmid state, also containing the non-catabolic plasmids pG20 (133,709 bp) and pG69 (144,433 bp) with backbones sharing 97% DNA identity and with redundant genes for the initiation of replication, repA1and repA2, of which only one was active. Seemingly, several other plasmids and bacterial features besides the pheBA operon were involved in selective distribution of catabolic operons in the natural environment. The comparison of the genetic structure of plasmids and IS elements' functions, as well as resistance to heavy metals of seven completely sequenced plasmids, are discussed.

Phenol- and naphthalene-degrading indigenous Pseudomonas pseudoalcaligenes strain C70 has great potential for the bioremediation of polluted areas. It harbours two chromosomally located catechol meta pathways, one of which is structurally and phylogenetically very similar to the Pseudomonas sp. CF600 dmp operon and the other to the P. stutzeri AN10 nah lower operon. The key enzymes of the catechol meta pathway, catechol 2,3-dioxygenase (C23O) from strain C70, PheB and NahH, have an amino acid identity of 85%. The metabolic and regulatory phenotypes of the wild-type and the mutant strain C70ΔpheB lacking pheB were evaluated. qRT-PCR data showed that in C70, the expression of pheB- and nahH-encoded C23O was induced by phenol and salicylate, respectively. We demonstrate that strain C70 is more effective in the degradation of phenol and salicylate, especially at higher substrate concentrations, when these compounds are present as a mixture; i.e., when both pathways are expressed. Moreover, NahH is able to substitute for the deleted PheB in phenol degradation when salicylate is also present in the growth medium. The appearance of a yellow intermediate 2-hydroxymuconic semialdehyde was followed by the accumulation of catechol in salicylate-containing growth medium, and lower expression levels and specific activities of the C23O of the sal operon were detected. However, the excretion of the toxic intermediate catechol to the growth medium was avoided when the growth medium was supplemented with phenol, seemingly due to the contribution of the second meta pathway encoded by the phe genes.

In the present study we describe functional redundancy of bacterial multicomponent monooxygenases (toluene monooxygenase (TMO) and toluene/xylene monooxygenase (XylAM) of TOL pathway) and cooperative genetic regulation at the expression of the respective catabolic operons by touR and xylR encoded regulatory circuits in five phenol- and toluene-degrading Pseudomonas stutzeri strains. In these strains both toluene degradation pathways (TMO and Xyl) are active and induced by toluene and phenol. The whole genome sequence of the representative strain 2A20 revealed the presence of complete TMO- and Xyl-upper pathway operons together with two sets of lower catechol meta pathway operons, as well as phenol-degrading operon in a single 292,430 bp contig. The much lower GC content and analysis of the predicted ORFs refer to the plasmid origin of the approximately 130 kb region of this contig, containing the xyl, phe and tou genes. The deduced amino acid sequences of the TMO, XylA and the large subunit of phenol monooxygenase (LmPH) show 98–100% identity with the respective gene products of the strain Pseudomonas sp. OX1. In both strains 2A20 and OX1 the meta-cleavage pathways for catechol degradation are coded by two redundant operons (phe and xyl). We show that in the strain 2A20 TouR and XylR are activated by different effector molecules, phenol and toluene, respectively, and they both control transcription of the xyl upper, tou (TMO) and phe catabolic operons. Although the growth parameters of redundant strains did not show advantage at toluene biodegradation, the functional redundancy could provide better flexibility to the bacteria in environmental conditions.

p-Cresol methylhydroxylase (PCMH), a key enzyme responsible for the catabolism of p-cresol via the protocatechuate ortho pathway, was used as a tool to characterize catabolic differences between phenol- and p-cresol-degrading Pseudomonas fluore-scens strains PC18 and PC24. Although both strains catabolize p-cresol using PCMH, different whole-cell kinetic parameters for this compound were revealed. Affinity for the substrate and the specific growth rate were higher in PC18, whereas maximum p-cresol tolerance was higher in PC24. In addition, PCMH of strain PC18 was induced during growth on phenol. In both strains, the pchACXF operon, which encodes p-hydroxybenzaldehyde dehydrogenase and PCMH, was sequenced. Transcriptional regulation of these operons by PchR, a putative sigma(54)-dependent regulator, was shown. Although the promoters of these operons resembled sigma(54)-controlled promoters, they differed from the consensus sequence by having T instead of C at position -12. Complementation assays confirmed that the amino acid sequence differences of the PchR regulators between the two strains studied led to different effector-binding capabilities of these proteins: (1) phenol was a more efficient effector for PchR of PC18 than p-cresol, (2) phenol did not activate the regulator of PC24, and (3) both regulators responded similarly to p-cresol.

The complete 83 042-bp nucleotide sequence of the IncP-9 naphthalene degradation plasmid pNAH20 from Pseudomonas fluorescens PC20 exhibits striking similarity in size and sequence to another naphthalene (NAH) plasmid pDTG1. However, the positions of insertion sequence (IS) elements significantly alter both catabolic and backbone functions provided by the two plasmids. In pDTG1, insertion of a pCAR1 ISPre1-like element disrupts expression of the lower naphthalene operon and this strain utilizes the chromosomal pathway for complete naphthalene degradation. In pNAH20, this operon is intact and functional. The transfer frequency of pNAH20 is 100 times higher than that of pDTG1 probably due to insertion of the pCAR1 ISPre2-like element into the mpfR gene coding for a putative repressor of the mpf operon responsible for mating pilus formation. We also demonstrate in situ plasmid transfer - we isolated a rhizosphere transconjugant strain of pNAH20, P. fluorescens NS8. The plasmid pNS8, a derivative of pNAH20, lacks the ability to self-transfer as a result of an additional insertion event of ISPre2-like element that disrupts the gene coding for VirB2-like major pilus protein MpfA. The characteristics of the strain PC20 and the conjugal transfer/mobilization capacity of pNAH20 (or its backbone) make this strain/plasmid a potentially successful tool for bioremediation applications.

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.

The processed oil shale (semi-coke) contains several organic and inorganic compounds (oil fractions, sulphides, phenolic compounds, polycyclic aromatic hydrocarbons) and is highly toxic. The solid waste deposit is a source of toxic phenolic leachate, which is discharged into nearby bodies of water without any treatment. Field experiments were carried out in order to test the effect of phytoremediation to enhance in-situ microbial biodegradation of pollutants in semi-coke. Four pilot test plots (50 m 2 ) were established at a semi-coke deposit in July 2001. The growth rate of plants was approximately twice higher in the case of soil amendment. The phytoremediation increased the number of biodegradative bacteria and diversity of microbial community in semi-coke. The presence and diversity of multi-component phenol hydroxylase (mPH) genes in the environment were assessed using the microbial DNA directly extracted from semi-coke. The changes in the genotypes of mPHs indicated shifts in the microbial community structure towards the more efficient degradation of pollutants due to plant treatment. Within a four-month period starting from the establishment of test plots in July, the concentration of phenolic compounds decreased three times in average and the concentration of oil products up to ten times, compared to the control plot.

Rivers in the northeastern part of Estonia have been for decades under constant input of phenolic leachate water from semi-coke mounds formed as by-product of oil-shale processing. The dynamics of chemical and microbiological parameters were studied along river system polluted by ash-dump leachate in May-September 1995. The structure of the bacterial community was characterized by general microbial functional groups (heterotrophic bacteria), by biodegradative bacterial groups (phenol, m-toluate, benzoate, salicylate, camphor, naphtalene and heptane degrading bacteria), and by community-level physiological profile (Biolog microplates). The taxonomic diversity of the isolated phenol-utilizing bacterial strains was studied using commercial identification systems (Biolog and API). Multivariate statistical techniques were applied to relate different aspects of bacterial community structure to environmental parameters, and to assess the relationships between different estimates of community stricture. Pollution markedly altered all aspects of the studied microbial diversity. The structure and functioning of the bacterial communities changed significantly along the river and revealed remarkable adaptation to phenolic compounds in environment. Based on the data observed the concentration of volatile phenols decreased as an average 85 % along the river system, and the most significant process involved in self-purification is biodegradation of these compounds.

In the present study we report the complete nucleotide sequence of the toluene catabolic plasmid pD2RT of Pseudomonas migulae strain D2RT isolated from Baltic Sea water. The pD2RT is 129,894 base pairs in size with an average G+C content of 53.75%. A total of 135 open reading frames (ORFs) were predicted to encode proteins, among them genes for catabolism of toluene, plasmid replication, maintenance and conjugative transfer. ORFs encoding proteins with putative functions in stress response, transposition and site-specific recombination were also predicted. Analysis of the organization and nucleotide sequence of pD2RT backbone region revealed high degree of similarity to the draft genome sequence data of the plant-pathogenic pseudomonad Pseudomonas syringae pv. glycinea strain B076, exhibiting relatedness to pPT23A plasmid family. The pD2RT backbone is also closely related to that of pGRT1 of Pseudomonas putida strain DOT-T1E and pBVIE04 of Burkholderia vietnamiensis strain G4, both plasmids are associated with resistance to toluene. The ability of pD2RT to self-transfer by conjugation to P. putida recipient strain PaW340 was experimentally determined. Genetic organization of toluene-degrading (xyl) genes and flanking DNA segments resembles the structure of Tn1721-related class II transposon Tn4656 of TOL plasmid pWW53 of P. putida strain MT53. The complete sequence of the plasmid pD2RT extends the known range of xyl genes carriers, being the first completely sequenced TOL plasmid, which is not related to well-studied IncP plasmid groups. We also verified the functionality of the catabolic route encoded by pD2RT by monitoring the expression of the xylE gene in pD2RT bearing hosts along with bacterial strains containing TOL plasmid of IncP-9 group. The growth kinetics of plasmid-bearing strains was found to be affected by particular TOL plasmid.

A set of phenol-degrading strains of a collection of bacteria isolated from Baltic Sea surface water was screened for the presence of two key catabolic genes coding for phenol hydroxylases and catechol 2,3-dioxygenases. The multicomponent phenol hydroxylase (LmPH) gene was detected in 70 out of 92 strains studied, and 41 strains among these LmPH+ phenol-degraders were found to exhibit catechol 2,3-dioxygenase (C23O) activity. Comparative phylogenetic analyses of LmPH and C23O sequences from 56 representative strains were performed. The studied strains were mostly affiliated to the genera Pseudomonas and Acinetobacter. However, the study also widened the range of phenol-degraders by including the genus Limnobacter. Furthermore, using a next generation sequencing approach, the LmPH genes of Limnobacter strains were found to be the most prevalent ones in the microbial community of the Baltic Sea surface water. Four different Limnobacter strains having almost identical 16S rRNA gene sequences (99%) and similar physiological properties formed separate phylogenetic clusters of LmPH and C23O genes in the respective phylogenetic trees.

Plasmids are mobile genetic elements that provide their hosts with many beneficial traits including in some cases the ability to degrade different aromatic compounds. To fulfill the knowledge gap regarding catabolic plasmids of the Baltic Sea water, a total of 209 biodegrading bacterial strains were isolated and screened for the presence of these mobile genetic elements. We found that both large and small plasmids are common in the cultivable Baltic Sea bacterioplankton and are particularly prevalent among bacterial genera Pseudomonas and Acinetobacter. Out of 61 plasmid-containing strains (29% of all isolates), 34 strains were found to carry large plasmids, which could be associated with the biodegradative capabilities of the host bacterial strains. Focusing on the diversity of IncP-9 plasmids, self-transmissible m-toluate (TOL) and salicylate (SAL) plasmids were detected. Sequencing the repA gene of IncP-9 carrying isolates revealed a high diversity within IncP-9 plasmid family, as well as extended the assumed bacterial host species range of the IncP-9 representatives. This study is the first insight into the genetic pool of the IncP-9 catabolic plasmids in the Baltic Sea bacterioplankton.

p-Cresol methylhydroxylase (PCMH), a key enzyme responsible for the catabolism of p-cresol via the protocatechuate ortho pathway, was used as a tool to characterize catabolic differences between phenol- and p-cresol-degrading Pseudomonas fluore-scens strains PC18 and PC24. Although both strains catabolize p-cresol using PCMH, different whole-cell kinetic parameters for this compound were revealed. Affinity for the substrate and the specific growth rate were higher in PC18, whereas maximum p-cresol tolerance was higher in PC24. In addition, PCMH of strain PC18 was induced during growth on phenol. In both strains, the pchACXF operon, which encodes p-hydroxybenzaldehyde dehydrogenase and PCMH, was sequenced. Transcriptional regulation of these operons by PchR, a putative sigma(54)-dependent regulator, was shown. Although the promoters of these operons resembled sigma(54)-controlled promoters, they differed from the consensus sequence by having T instead of C at position -12. Complementation assays confirmed that the amino acid sequence differences of the PchR regulators between the two strains studied led to different effector-binding capabilities of these proteins: (1) phenol was a more efficient effector for PchR of PC18 than p-cresol, (2) phenol did not activate the regulator of PC24, and (3) both regulators responded similarly to p-cresol.

A long-term field experiment was carried out to estimate the efficiency of bioaugmentation in combination with phytoremediation for oil shale chemical industry solid waste dump area remediation. Soil samples for microbiological and chemical analysis were collected during 3 years after bacterial biomass application. Microbial communities in soil samples were analysed using both culture-based and molecular methods. The survival of the introduced bacterial strains was confirmed by cultivation-based Box-PCR genomic fingerprints and denaturing gradient gel electrophoresis fingerprinting of the 16S rRNA and lmPH genes. The introduced bacterial strains as well as corresponding catabolic genes were recovered several years after biomass application, predominantly from the rhizosphere of birches. Soil samples from bioaugmented plots showed an elevated potential for degradation of phenolic compounds even 40 months after treatment. Based on our results we can conclude that the introduced Pseudomonas strains both survived, and their metabolic traits have persisted at the contaminated site over a long period of time.

Oil‐shale chemical industry creates approximately 600 000 tons of thermally processed oil shale solid wastes (semi‐coke) every year in Estonia. A field phytoremediation and bioaugmentation experiment has been monitored for three years in the solid waste depository area of oil‐shale chemical industry. We found enhanced degradation rates of pollutants in plots with vegetation and added bacterial biomass. The concentration of volatile phenols had decreased almost by 100 %, and the concentration of oil products had decreased approximately 3 times in planted plots compared to the control plots. The degradation rates were the highest in the upper soil layer which has the highest root density. Vegetation also changed the microbial community structure in comparison with the control plots. In addition to the vegetation, properties of the substrate had an essential effect on the microbial community. Mikroorganizmų dirvožemyje kaita vykdant fitoatkūrimo lauko eksperimentą Santrauka Kasmet Estijoje naftos skalūnų chemijos pramonėje susidaro apytiksliai 600 000 t termiškai apdorotų naftos skalūnų kietųjų atliekų (pusiau kokso). Fitoatkūrimo ir biopapildymo lauko eksperimentas buvo vykdomas trejus metus naftos skalūnų chemijos pramonės kietųjų atliekų saugojimo zonoje. Pastebėta, kad padidėjo teršalų degradacijos greitis plotuose, kur yra augalijos, ir pridėta bakterinės biomasės. Lakiųjų fenolių koncentracija sumažėjo beveik 100 %, o naftos produktų koncentracija sumažėjo apytiksliai 3 kartus apsodintuose plotuose, palygti su kontroliniais plotais. Degradacijos greitis buvo didžiausias viršutinimame dirvožemio sluoksnyje, kuriame yra didžiausias šaknų tankis. Augalija taip pat pakeitė mikrobiologinės bendrijos struktūrą, palyginti su kontroliniais plotais. Be augalijos, dar ir substrato savybės turėjo didelės įtakos mikrobiologinei bendrijai. Reikšminiai žodžiai: naftos skalūnai, chemijos pramonės kietosios atliekos, biopapildymas, fitoatkūrimas, mikrobiologinė bendrija. Смена микроорганизмов в почве во время полевого эксперимента с применением фиторемедиации Резюме В химической промышленности по обработке нефтяных сланцев Эстонии ежегодно примерно 600 000 т составляют термически обработанные твердые отходы нефтяных сланцев. Эксперимент по полевой фиторемедиации и биодополнению проводился в течение трех лет на территории хранения твердых отходов химической промышленности по обработке нефтяных сланцев. Установлено, что скорость деградации загрязнителей увеличивается на площадях, где имеется растительность и добавлена бактерицидная биомасса. На засаженных площадях концентрация летучих фенолей уменьшилась почти в два раза, а нефтяных продуктов – приблизительно в 3 раза по сравнению с контрольными площадями. Скорость деградации была наибольшей в верхнем слое почвы, в котором больше всего корней. Растительность также изменила структуру микробиологического сообщества по сравнению с контрольными площадями. Кроме растительности, существенное воздействие на микробиологическое сообщество оказали также свойства субстрата. Ключевые слова: нефтяной сланец, твердые отходы химической промышленности, биодополнение, фиторемедиация, микробиологическое сообщество. First Published Online: 14 Oct 2010

Oil shale thermal processing has resulted in solid waste dump sites containing up to 100 million tons of solid waste. The processed oil shale contains complex mixture of organic and inorganic compounds and is highly toxic. Laboratory and field experiments were carried out in order to test the effect of phytoremediation and bioaugmentation for remediation of pollutants in semi-coke. Microbial community of aged (ca 10 ten years) semi-coke is characterized by few dominant populations and possesses low diversity. Changes in microbial community structure and activity occurred in semi- coke as a result of phytoremediation and bioaugmentation. The phytoremediation increased the number of bacteria and diversity of microbial community in semi-coke as well as microbial biomass. The general trend was the increase of proportion of biodegradable bacterial numbers within microbial community due to the treatment. Highest values for all measured microbiological parameters were found in rhizosphere samples. Within a two and half year period starting from establishment of test plots, the concentration of phenolic compounds decreased up to 100% and oil products more than three times at plots with vegetation compared to control. Bacterial biomass consisting of three bacterial strains was applied to three experimental plots. These three bacterial strains Pseudomonas mendocina PC1, P. fluorescens PC24 and P. fluorescens PC18 degrade phenols via catechol meta, catechol or protocatechuate ortho or via the combination of catechol meta and protocatechuate ortho pathways, respectively. Bioaugmentation increased biodegradation intensity of oil products up to 50% compared to untreated planted controls and enhanced plant growth, but the effect of bioaugmentation on microbial community parameters was short- term. Our results indicate that increased biodegradation activity was due to proliferation of specific microbial groups, changes in taxonomic diversity of bacterial community and catabolic genes.

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 oil-shale industry has created serious pollution problems in northeastern Estonia. Untreated, phenol-rich leachate from semi-coke mounds formed as a by-product of oil-shale processing is discharged into the Baltic Sea via channels and rivers. An exploratory analysis of water chemical and microbiological data sets from the low-flow period was carried out using different multivariate analysis techniques. Principal component analysis allowed us to distinguish different locations in the river system. The riverine microbial community response to water chemical parameters was assessed by co-inertia analysis. Water pH, COD and total nitrogen were negatively related to the number of biodegradative bacteria, while oxygen concentration promoted the abundance of these bacteria. The results demonstrate the utility of multivariate statistical techniques as tools for estimating the magnitude and extent of pollution based on river water chemical and microbiological parameters. An evaluation of river chemical and microbiological data suggests that the ambient natural attenuation mechanisms only partly eliminate pollutants from river water, and that a sufficient reduction of more recalcitrant compounds could be achieved through the reduction of wastewater discharge from the oil-shale chemical industry into the rivers.

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.

A total of 39 phenol- and p-cresol-degraders isolated from the river water continuously polluted with phenolic compounds of oil shale leachate were studied. Species identification by BIOLOG GN analysis revealed 21 strains of Pseudomonas fluorescens (4, 8 and 9 of biotypes A, C and G, respectively), 12 of Pseudomonas mendocina, four of Pseudomonas putida biotype A1, one of Pseudomonas corrugata and one of Acinetobacter genospecies 15. Computer-assisted analysis of rep-PCR fingerprints clustered the strains into groups with good concordance with the BIOLOG GN data. Three main catabolic types of degradation of phenol and p-cresol were revealed. Type I, or meta-meta type (15 strains), was characterized by meta cleavage of catechol by catechol 2,3-dioxygenase (C23O) during the growth on phenol and p-cresol. These strains carried C23O genes which gave PCR products with specific xylE-gene primers. Type II, or ortho-ortho type (13 strains), was characterized by the degradation of phenol through ortho fission of catechol by catechol 1,2-dioxygenase (C12O) and p-cresol via ortho cleavage of protocatechuic acid by protocatechuate 3,4-dioxygenase (PC34O). These strains carried phenol monooxygenase gene which gave PCR products with pheA-gene primers. Type III, or meta-ortho type (11 strains), was characterized by the degradation of phenol by C23O and p-cresol via the protocatechuate ortho pathway by the induction of PC34O and this carried C23O genes which gave PCR products with C23O-gene primers, but not with specific xylE-gene primers. In type III strains phenol also induced the p-cresol protocatechuate pathway, as revealed by the induction of p-cresol methylhydroxylase. These results demonstrate multiplicity of catabolic types of degradation of phenol and p-cresol and the existence of characteristic assemblages of species and specific genotypes among the strains isolated from the polluted river water.

The increasing use of commercial multitest systems for identification of environmental bacteria creates the problem of how to compare the identification results obtained from different systems. The limited use of species designations in such comparisons is caused by low usage of environmental bacteria in the development of commercial identification schemes. Two multivariate statistical methods, the Mantel's test and the co-inertia analysis, were applied to analyze data derived from the Biolog GN and the API 20NE systems of identification for 50 environmental bacterial strains. We found these two methods to be useful for revealing the relationship between the two sets of numerical taxonomic traits. Both of these methods showed that the distances according to the Biolog GN results between the studied strains were related to those derived from the API 20NE results, despite the differences in the test sets of the two systems. In addition, the co-inertia analysis allowed us to visualise the relationships between classifications of strains derived from the two identification systems and, simultaneously, to estimate the contribution of particular tests to the differentiation of bacterial strains.

Horizontal transfer of genes of selective value in an environment 6 years after their introduction into a watershed has been observed. Expression of the gene pheA, which encodes phenol monooxygenase and is linked to the pheBA operon (A. Nurk, L. Kasak, and M. Kivisaar, Gene 102:13-18, 1991), allows pseudomonads to use phenol as a growth substrate. Pseudomonas putida strains carrying this operon on a plasmid were used for bioremediation after an accidental fire in the Estonia oil shale mine in Estonia in 1988. The water samples used for studying the fate of the genes introduced were collected in 1994. The same gene cluster was also detected in Pseudomonas strains isolated from water samples of a nearby watershed which has been continuously polluted with phenols due to oil shale industry leachate. Together with the more frequently existing counterparts of the dmp genes (V. Shingler, J. Powlowski, and U. Marklund, J. Bacteriol. 174:711-724, 1992), the pheA gene was also represented in the phenol-degrading strains. The area where the strains containing the pheA gene were found was restricted to the regular route of phenolic leachate to the Baltic Sea. Nine Pseudomonas strains belonging to four different species (P. corrugata, P. fragi, P. stutzeri, and P. fluorescens biotypes B, C, and F) and harboring horizontally transferred pheBA operons were investigated. The phe genes were clustered in the same manner in these nine phe operons and were connected to the same promoter as in the case of the original pheBA operon. One 10.6-kb plasmid carrying a pheBA gene cluster was sequenced, and the structure of the rearranged pheBA operon was described. This data indicates that introduced genetic material could, if it encodes a beneficial capability, enrich the natural genetic variety for biodegradation.

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.