Molecular Biomonitoring during Rhizoremediation of Oil-Contaminated Soil

Rhizoremediation is the use of microbial populations present in the rhizosphere of plants for environmental cleanup. The idea of this work was that bacteria living in the rhizosphere of a nitrogen-fixing leguminous plant, goat's rue (Galega orientalis), could take part in the degradation of harmful monoaromatic hydrocarbons, such as benzene, toluene and xylene (BTEX), from oil-contaminated soils. In addition to chemical (e.g. pollutant concentration) and physical (e.g. soil structure) information, the knowledge of biological aspects (e.g. bacteria and their catabolic genes) is essential when developing the rhizoremediation into controlled and effective bioremediation practice. Therefore, the need for reliable biomonitoring methods is obvious. The main aims of this thesis were to evaluate the symbiotic G. orientalis - Rhizobium galegae system for rhizoremediation of oil-contaminated soils, to develop molecular methods for biomonitoring, and to apply these methods for studying the microbiology of rhizoremediation. In vitro, Galega plants and rhizobia remained viable in m-toluate concentrations up to 3000 mg/l. Plant growth and nodulation were inhibited in 500 mg/l m-toluate, but were restored when plants were transferred to clean medium. In the greenhouse, Galega showed good growth, nodulation and nitrogen fixation, and developed a strong rhizosphere in soils contaminated with oil or spiked with 2000 mg/l m-toluate. The high aromatic tolerance of R. galegae and the viability of Galega plants in oil-polluted soils proved this legume system to be a promising method for the rhizoremediation of oil-contaminated soils. Molecular biomonitoring methods were designed and/or developed further for bacteria and their degradation genes. A combination of genomic fingerprinting ((GTG)5-PCR), taxonomic ribotyping of 16S rRNA genes and partial 16S rRNA gene sequencing were chosen for molecular grouping of culturable, heterogeneous rhizosphere bacteria. PCR primers specific for the xylE gene were designed for TOL plasmid detection. Amplified enzyme-coding DNA restriction analysis (AEDRA) with AluI was used to profile both TOL plasmids (xylE primers) and, in general, aromatics-degrading plasmids (C230 primers). The sensitivity of the direct monitoring of TOL plasmids in soil was enhanced by nested C23O-xylE-PCR. Rhizosphere bacteria were isolated from the greenhouse and field lysimeter experiments. High genetic diversity was observed among the 50 isolated, m-toluate tolerating rhizosphere bacteria in the form of five major lineages of the Bacteria domain. Gram-positive Rhodococcus, Bacillus and Arthrobacter and gram-negative Pseudomonas were the most abundant genera. The inoculum Pseudomonas putida PaW85/pWW0 was not found in the rhizosphere samples. Even if there were no ecological niches available for the bioaugmentation bacterium itself, its conjugative catabolic plasmid might have had some additional value for other bacterial species and thus, for rhizoremediation. Only 10 to 20% of the isolated, m-toluate tolerating bacterial strains were also able to degrade m-toluate. TOL plasmids were a major group of catabolic plasmids among these bacteria. The ability to degrade m-toluate by using enzymes encoded by a TOL plasmid was detected only in species of the genus Pseudomonas, and the best m-toluate degraders were these Pseudomonas species. Strain-specific differences in degradation abilities were found for P.oryzihabitans and P. migulae: some of these strains harbored a TOL plasmid - a new finding observed in this work, indicating putative horizontal plasmid transfer in the rhizosphere. One P. oryzihabitans strain harbored the pWW0 plasmid that had probably conjugated from the bioaugmentation Pseudomonas. Some P. migulae and P. oryzihabitans strains seemed to harbor both the pWW0- and the pDK1-type TOL plasmid. Alternatively, they might have harbored a TOL plasmid with both the pWW0- and the pDK1-type xylE gene. The breakdown of m-toluate by gram-negative bacteria was not restricted to the TOL pathway. Also some gram-positive Rhodococcus erythropolis and Arthrobacter aurescens strains were able to degrade m-toluate in the absence of a TOL plasmid. Three aspects of the rhizosphere effect of G. orientalis were manifested in oil-contaminated soil in the field: 1) G. orientalis and Pseudomonas bioaugmentation increased the amount of rhizosphere bacteria. G. orientalis especially together with Pseudomonas bioaugmentation increased the numbers of m-toluate utilizing and catechol positive bacteria indicating an increase in degradation potential. 2) Also the bacterial diversity, when measured as the amount of ribotypes, was increased in the Galega rhizosphere with or without Pseudomonas bioaugmentation. However, the diversity of m-toluate utilizing bacteria did not significantly increase. At the community level, by using the 16S rRNA gene PCR-DGGE method, the highest diversity of species was also observed in vegetated soils compared with non-vegetated soils. Diversified communities may best guarantee the overall success in rhizoremediation by offering various genetic machineries for catabolic processes. 3) At the end of the experiment, no TOL plasmid could be detected by direct DNA analysis in soil treated with both G. orientalis and Pseudomonas. The detection limit for TOL plasmids was encountered indicating decreased amount of degradation plasmids and thus, the success of rhizoremediation. The use of G. orientalis for rhizoremediation is unique. In this thesis new information was obtained about the rhizosphere effect of Galega orientalis in BTEX contaminated soils. The molecular biomonitoring methods can be applied for several purposes within environmental biotechnology, such as for evaluating the intrinsic biodegradation potential, monitoring the enhanced bioremediation, and estimating the success of bioremediation. Environmental protection by using nature's own resources and thus, acting according to the principle of sustainable development, would be both economically and environmentally beneficial for society. Keywords: molecular biomonitoring, genetic fingerprinting, soil bacteria, bacterial diversity, TOL plasmid, catabolic genes, horizontal gene transfer, rhizoremediation, rhizosphere effect, Galega orientalis, aerobic biodegradation, petroleum hydrocarbons, BTEX

Diversitet och tillväxtfrämjande egenskaper hos Stenotrophomonas-endofyter isolerade från Calliandra calothyrsus Meisn. rotknölar

The bacterial genus Stenotrophomonas comprises 12 species. They are widely found throughout the environment and particularly S. maltophilia, S. rhizophila and S. pavanii are closely associated with plants. Strains of the most common Stenotrophomonas species, S. maltophilia, promote plant growth and health, degrade natural and man-made pollutants and produce biomolecules of biotechnological and economical value. Many S. maltophilia –strains are also multidrug resistant and can act as opportunistic human pathogens. During an INCO-project (1998-2002) rhizobia were collected from root nodules of the tropical leguminous tree Calliandra calothyrsus Meisn. from several countries in Central America, Africa and New Caledonia. The strains were identified by the N2-group (Helsinki university) and some strains turned out to be members of the genus Stenotrophomonas. Several Stenotrophomonas strains induced white tumor- or nodule-like structures on Calliandra?s roots in plant experiments. The strains could, besides from root nodules, also be isolated from surface sterilized roots and stems. The purpose of my work was to investigate if the Stenotrophomonas strains i) belong to a new Stenotrophomonas species, ii) have the same origin, iii) if there are other differences than colony morphology between phase variations of the same strain, iv) have plant growth-promoting (PGP) activity or other advantageous effects on plants, and v) like rhizobia have ability to induce root nodule formation. The genetic diversity and clustering of the Stenotrophomonas strains were analyzed with AFLP fingerprinting to get indications about their geographical origin. Differences in enzymatic properties and ability to use different carbon and energy sources were tested between the two phases of each strain with commercial API tests for bacterial identification. The ability to infect root hairs and induce root nodule formation was investigated both using plant tests with the host plant Calliandra and PCR amplification of nodA and nodC genes for nodulation. The PGP activity of the strains was tested in vitro mainly with plate methods. The impact on growth, nitrogen content and nodulation in vivo was investigated through greenhouse experiments with the legumes Phaseolus vulgaris and Galega orientalis. Both the genetic and phenotypic diversity among the Stenotrophomonas strains was small, which proposes that they have the same origin. The strains brought about changes on the root hairs of Calliandra and they also increased the amount of root hairs. However, no root nodules were detected. The strains produced IAA, protease and lipase in vitro. They also showed plant a growth-promoting effect on G. orientalis, both alone and together with R. galegae HAMBI 540, and also activated nodulation among efficient rhizobia on P. vulgaris in greenhouse. It requires further research to get a better picture about the mechanisms behind the positive effects. The results in this thesis, however, confirm earlier studies concerning Stenotrophomonas positive impact on plants.