Eeva Heinaru

Grouping of phenol hydroxylase and catechol 2,3-dioxygenase genes among phenol- and p-cresol-degrading Pseudomonas species and biotypes

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.

Comparison of API 20NE and Biolog GN identification systems assessed by techniques of multivariate analyses

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 Mantels 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.

Survival and catabolic performance of introduced Pseudomonas strains during phytoremediation and bioaugmentation field experiment.

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.

Limnobacter spp. as newly detected phenol-degraders among Baltic Sea surface water bacteria characterised by comparative analysis of catabolic genes.

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.

Conjugal transfer and mobilization capacity of the completely sequenced naphthalene plasmid pNAH20 from multiplasmid strain Pseudomonas fluorescens PC20.

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.

Evaluation of different phenol hydroxylase-possessing phenol-degrading pseudomonads by kinetic parameters

Phenol-degrading pseudomonads possessing different phenol hydroxylases (PH) were evaluated by the values of apparent half-saturation constant for phenol-oxygenating activity (KS), maximum specific growth rate (μmax), lag-time length (λ), inhibition constant (KI) and growth yield factor (YX/S). Strains of the same PH type showed similar kinetic parameters: single-component PH (sPH) harbouring strains had higher values of KS and lower values of μmax than the strains having multicomponent PH (mPH). However, the values of KI and the dependencies of the lag-time length on initial phenol concentration were strain-specific. The elevated ratio between specific activities of catechol 1,2-dioxygenase (C12O) and muconate cycloisomerase in sPH-strains caused irreversible accumulation of a high amount of exogenous cis,cis-muconate (CCM) which resulted in decreased YX/S values. Co-presence of sPH and mPH genes did not give the strains PC16 and P69 any extra advantage and according to determined kinetic parameters only one PH was active during phenol degradation. At the same time simultaneous functioning of catechol ortho and meta cleavage pathways (strain PC20) resulted in higher μmax and YX/S values. Evaluation of strains showed that the type of PH determined the efficiency of phenol degradation, whereas the tolerance to elevated phenol concentrations was strain-specific.

Occurrence of plasmids in the aromatic degrading bacterioplankton of the baltic sea.

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.

Analysis of river pollution data from low-flow period by means of multivariate techniques: a case study from the oil-shale industry region, northeastern Estonia.

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.

FORMATION OF MICROBIAL COMMUNITIES IN OIL SHALE CHEMICAL INDUSTRY SOLID WASTES DURING PHYTOREMEDIATION AND BIOAUGMENTATION

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.

Biodegradation of dimethylphenols by bacteria with different ring-cleavage pathways of phenolic compounds.

The biodegradation of 3,4, 2,4, 2,3, 2,6 and 3,5-di-methylphenol in combination with phenol andp-cresol by axenic and mixed cultures of bacteria was investigated. The strains, which degrade phenol andp-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-di-methylphenol 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 inPseudomonas mendocina PC1, wheremeta cleavage pathway was functional during the degradation ofp-cresol. In the case of strains PC18 and PC24 ofP. fluorescens, the degradation ofp-cresol occurred via the protocatechuateortho 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.