Leena Suominen

Evaluation of the Galega-Rhizobium galegae system for the bioremediation of oil-contaminated soil.

The bioremediation potential of a nitrogen-fixing leguminous plant, Galega orientalis, and its microsymbiont Rhizobium galegae was evaluated in BTX (benzene, toluene, xylene)-contaminated soils in microcosm and mesocosm scale. To measure the intrinsic tolerance of the organisms to m-toluate, a model compound representing BTX, G. orientalis and R. galegae were cultivated under increasing concentrations of m-toluate alone and in association with Pseudomonas putida pWWO, a bacterial strain able to degrade toluene-derived compounds. The test plants and rhizobia remained viable in m-toluate concentrations as high as 3000 ppm. Plant growth was inhibited in concentrations higher than 500 ppm, but restituted when plants were transferred into m-toluate-free medium. Nodulation was blocked under the influence of m-toluate, but was restored after the plants were transferred into the non-contaminated media. In the mesocosm assay the Galega plants showed good growth, nodulation and nitrogen fixation, and developed a strong rhizosphere in soils contaminated with oil or spiked with 2000 ppm m-toluate. Thus, this legume system has good potential for use on oil-contaminated sites

Description of two biovars in the Rhizobium galegae species: biovar orientalis and biovar officinalis.

Twenty-six Rhizobium galegae strains, representing the center of origin of the host plants Galega orientalis and G. officinalis as well as other geographic regions, were used in a polyphasic analysis of the relationships of R. galegae strains. Phage typing, lipopolysaccharide (LPS) profiling, pulsed field gel electrophoresis (PFGE) profiling and rep-PCR (use of repetitive sequences as PCR primers for genomic fingerprinting) with REP and ERIC primers investigated nonsymbiotic properties, whereas plasmid profiling and hybridisation with a nif gene probe, and with nodB, nodD, nod box and an IS sequence from the symbiotic region as probes, were used to reveal the relationships of symbiotic genes. The results were used in pairwise calculations of distances between the strains, and the distances were visualised as a dendrogram. Indexes of association were compared for all tests pooled, and for chromosomal tests and symbiotic markers separately, to display the input of the different categories of tests on the grouping of the strains. Our study shows that symbiosis related genetic traits in R. galegae divide strains belonging to the species into two groups, which correspond to strains forming an effective symbioses with G. orientalis and G. officinalis respectively. We therefore propose that Rhizobium galegae strains forming an effective symbiosis with Galega orientalis are called R. galegae bv. orientalis and strains forming an effective symbiosis with Galega officinalis are called R. galegae bv. officinalis.

Changes in hydrocarbon groups, soil ecotoxicity and microbiology along horizontal and vertical contamination gradients in an old landfarming field for oil refinery waste

Horizontal and vertical contaminant gradients in an old landfarming field for oil refinery waste were characterised with the aim to assess parallel changes in hydrocarbon groups and general, microbiological and ecotoxicological soil characteristics. In the surface soil polar compounds were the most prevalent fraction of heptane-extractable hydrocarbons, superseding GC-FID-resolvable and high-molar-mass aliphatics and aromatics, but there was no indication of their relatively higher mobility or toxicity. The size of the polar fraction correlated poorly with soil physical, chemical and microbiological properties, which were better explained by the total heptane-extractable and total petroleum hydrocarbons (TPH). Deleterious effects on soil microbiology in situ were observed at surprisingly low TPH concentrations (0.3%). Due to the accumulation of polar and complexed degradation products, TPH seems an insufficient measure to assess the quality and monitor the remediation of soil with weathered hydrocarbon contamination.

Ecological inference on bacterial succession using curve-based community fingerprint data analysis, demonstrated with rhizoremediation experiment

Nucleic acid-based community fingerprinting methods are valuable tools in microbial ecology, as they offer rapid and robust means to compare large series of replicates and references. To avoid the time-consuming and potentially subjective procedures of peak-based examination, we assessed the possibility to apply direct curve-based data analysis on community fingerprints produced with bacterial length heterogeneity PCR (LH-PCR). The dataset comprised 180 profiles from a 21-week rhizoremediation greenhouse experiment with three treatments and 10 sampling times. Curve-based analysis quantified the progressive effect of the plant (Galega orientalis) and the reversible effect of the contaminant (fuel oil) on bacterial succession. The major observed community shifts were assigned to changes in plant biomass and contamination level by canonical correlation analysis. A novel method to extract relative abundance data from the fingerprint curves for Shannon diversity index revealed contamination to reversibly decrease community complexity. By cloning and sequencing the fragment lengths, recognized to change in time in the averaged LH-PCR profiles, we identified Aquabacterium (Betaproteobacteria) as the putative r-strategic fuel oil degrader, and K-strategic Alphaproteobacteria growing in abundance later in succession. Curve-based community fingerprint analysis can be used for rapid data prescreening or as a robust alternative for the more heavily parameterized peak-based analysis.

Activation of the nodA promoter by the nodD genes of Rhizobium galegae induced by synthetic flavonoids or Galega orientalis root exudate

Rhizobial nodD genes produce transcriptional regulators that, together with appropriate inducer compounds, activate the other symbiotic nodulation (nod) genes and initiate the nodule formation process. Two nodD homologues, nodD1 and nodD2, are present in the Rhizobium galegae strain HAMBI 1174. In this work we analysed their ability to induce the nodA promoter with synthetic inducers known to activate nod genes in other rhizobia. According to phylogenetic analysis, the inducer-specific carboxy-terminal part of the R. galegae nodD protein sequence groups together with those of Rhizobium leguminosarum and Sinorhizobium meliloti. However, the respective inducer compounds for their NodD proteins are not highly effective with R. galegae nodD products. The best inducer discovered with R. galegae nodD1 was the root exudate of the host plant of R. galegae, Galega orientalis. HPLC analyses revealed the presence of many divergent flavonoid compounds in the G. orientalis root exudate. The most effective HPLC fractions induced R. galegae nodD1 up to the level obtained by intact G. orientalis root exudate while apigenin and luteolin, which were also present in the root exudate, were only moderate inducers. A UV-Vis diode array spectrum of the most active peak indicated that the main inducer present in the G. orientalis root exudate is an unidentified chalcone-type compound. In the Galega-R. galegae interaction the first recognition between the NodD protein and the flavonoid inducer secreted from the roots of Galega is specific for these organisms, and thus partly responsible of the strict host specificity of this symbiosis.

Bacterial and archaeal communities in long‐term contaminated surface and subsurface soil evaluated through coextracted RNA and DNA

Soil RNA and DNA were coextracted along a contamination gradient at a landfarming field with aged crude oil contamination to investigate pollution-dependent differences in 16S rRNA and rRNA gene pools. Microbial biomass correlated with nucleic acid yields as well as bacterial community change, indicating that the same factors controlled community size and structure. In surface soil, bacterial community evenness, estimated through length heterogeneity PCR (LH-PCR) fingerprinting, appeared higher for RNA-based than for DNA-based communities. The RNA-based community profiles resembled the DNA-based communities of soil with a lower contamination level. Cloning-based identification of bacterial hydrocarbon-degrading taxa in the RNA pool, representing the viable community with high protein synthesis potential, indicated that decontamination processes still continue. Analyses of archaea revealed that only Thaumarchaeota were present in the aerobic samples, whereas more diverse communities were found in the compacted subsurface soil with more crude oil. For subsurface bacteria, hydrocarbon concentration explained neither the community structure nor the difference between RNA-based and DNA-based communities. However, rRNA of bacterial taxa associated with syntrophic and sulphate-reducing alkane degradation was detected. Although the same prokaryotic taxa were identified in DNA and RNA, comparison of the two nucleic acid pools can aid in the assessment of past and future restoration success.

Simplified MPN method for enumeration of soil naphthalene degraders using gaseous substrate

We describe a simplified microplate most-probable-number (MPN) procedure to quantify the bacterial naphthalene degrader population in soil samples. In this method, the sole substrate naphthalene is dosed passively via gaseous phase to liquid medium and the detection of growth is based on the automated measurement of turbidity using an absorbance reader. The performance of the new method was evaluated by comparison with a recently introduced method in which the substrate is dissolved in inert silicone oil and added individually to each well, and the results are scored visually using a respiration indicator dye. Oil-contaminated industrial soil showed slightly but significantly higher MPN estimate with our method than with the reference method. This suggests that gaseous naphthalene was dissolved in an adequate concentration to support the growth of naphthalene degraders without being too toxic. The dosing of substrate via gaseous phase notably reduced the work load and risk of contamination. The result scoring by absorbance measurement was objective and more reliable than measurement with indicator dye, and it also enabled further analysis of cultures. Several bacterial genera were identified by cloning and sequencing of 16S rRNA genes from the MPN wells incubated in the presence of gaseous naphthalene. In addition, the applicability of the simplified MPN method was demonstrated by a significant positive correlation between the level of oil contamination and the number of naphthalene degraders detected in soil.