Assessing toxicity of metal contaminated soil from glassworks sites with a battery of biotests.

The present study addresses toxicological properties of metal contaminated soils, using glassworks sites in south-eastern Sweden as study objects. Soil from five selected glassworks sites as well as from nearby reference areas were analysed for total and water-soluble metal concentrations and general geochemical parameters. A battery of biotests was then applied to assess the toxicity of the glassworks soil environments: a test of phytotoxicity with garden cress (Lepidium sativum); the BioTox™ test for toxicity to bacteria using Vibrio fischeri; and analyses of abundancies and biomass of nematodes and enchytraeids. The glassworks- and reference areas were comparable with respect to pH and the content of organic matter and nutrients (C, N, P), but total metal concentrations (Pb, As, Ba, Cd and Zn) were significantly higher at the former sites. Higher metal concentrations in the water-soluble fraction were also observed, even though these concentrations were low compared to the total ones. Nevertheless, toxicity of the glassworks soils was not detected by the two ex situ tests; inhibition of light emission by V. fischeri could not be seen, nor was an effect seen on the growth of L. sativum. A decrease in enchytraeid and nematode abundance and biomass was, however, observed for the landfill soils as compared to reference soils, implying in situ toxicity to soil-inhabiting organisms. The confirmation of in situ bioavailability and negative effects motivates additional studies of the risk posed to humans of the glassworks villages.

Biogas production and methanogenic archaeal community in mesophilic and thermophilic anaerobic co-digestion processes.

Over 258 Mt of solid waste are generated annually in Europe, a large fraction of which is biowaste. Sewage sludge is another major waste fraction. In this study, biowaste and sewage sludge were co-digested in an anaerobic digestion reactor (30% and 70% of total wet weight, respectively). The purpose was to investigate the biogas production and methanogenic archaeal community composition in the anaerobic digestion reactor under meso- (35-37 °C) and thermophilic (55-57 °C) processes and an increasing organic loading rate (OLR, 1-10 kg VS m(-3) d(-1)), and also to find a feasible compromise between waste treatment capacity and biogas production without causing process instability. In summary, more biogas was produced with all OLRs by the thermophilic process. Both processes showed a limited diversity of the methanogenic archaeal community which was dominated by Methanobacteriales and Methanosarcinales (e.g. Methanosarcina) in both meso- and thermophilic processes. Methanothermobacter was detected as an additional dominant genus in the thermophilic process. In addition to operating temperatures, the OLRs, the acetate concentration, and the presence of key substrates like propionate also affected the methanogenic archaeal community composition. A bacterial cell count 6.25 times higher than archaeal cell count was observed throughout the thermophilic process, while the cell count ratio varied between 0.2 and 8.5 in the mesophilic process. This suggests that the thermophilic process is more stable, but also that the relative abundance between bacteria and archaea can vary without seriously affecting biogas production.

Effect of temperature on the reductive dechlorination of 1,2,3,4-tetrachlorodibenzofuran in anaerobic PCDD/F-contaminated sediments.

The effect of temperature on the reductive dechlorination in sediments of the PCDD/F-contaminated Kymijoki River, Finland was assessed with 1,2,3,4-tetrachlorodibenzofuran (1,2,3,4-TeCDF) at various temperatures and with co-amendment of 2,3,4,6-tetrachlorophenol (2,3,4,6-TeCP) in laboratory microcosms. The dechlorination rate of 1,2,3,4-TeCDF increased with incubation temperature, with TeCDF half-lives of 2.1 y at 21°C, 3.9 y at 15°C, and 19.0 y at 4°C. Co-amendment with 2,3,4,6-TeCP reduced the TeCDF half-life to 1.8 y at 21°C. 1,2,3,4-TeCDF was dechlorinated mainly in the lateral position to 1,3,4-TrCDF and then to 1,3-DiCDF over 29 months, but incubation temperature affected the relative molar ratios of the dechlorination products. The abundance of the Dehalococcoides-like Chloroflexi community did not substantially change in microcosms over 24 months incubation at the different temperatures. The dechlorination activity of 1,2,3,4-TeCDF was significantly limited at lower temperatures, which should be considered in predicting the environmental fate of aged PCDD/Fs in sediments of the Kymijoki River.

Biogasification of biowaste and sewage sludge--measurement of biogas quality.

Biogas quality, the presence of some trace components (siloxanes, sulfur compounds, volatile organic compounds, VOCs) in biogas, is in a decisive role when determining the biogas utilization and the purification requirements and equipments. In the present work, the effects of process changes related to reactor loading variations on the concentrations of selected trace compounds in biogas were studied. Source separated biowaste and sewage sludge were co-digested in a mesophilic pilot reactor (200 L) for four months during which the organic load was stepwise increased. The results showed that the process worked steadily up to the load of 8 kgVS m(-3)d(-1). Also the community composition of methanogenic archae stayed largely unaffected by the load increase, and was at all stages typical for a mesophilic biogasification process. Gaseous concentrations of siloxanes, hydrogen sulfide and most VOCs remained at a constant low level, showing no sensitivity to variations in the load and related process changes. However, the total siloxane concentration in the biogas was dependent on feed quality, and the detected concentrations require removal prior to use in turbines or fuel cells. Otherwise, after the removal of siloxanes, the biogas studied in this work is well applicable in various electricity production options, like in gas engines, turbines, microturbines and fuel cells.

Determination of fungal succession during municipal solid waste composting using a cloning-based analysis.

AIMS: The microbiota at industrial full-scale composting plants has earlier been fragmentarily studied with molecular methods. Here, fungal communities from different stages of a full-scale and a pilot-scale composting reactors were studied before and after wood ash amendment. METHODS AND RESULT: The portion of fungal biomass, determined using phospholipid fatty acid analysis, varied between 6.3% and 38.5% in different composting phases. The fungal internal transcribed spacer (ITS) area was cloned and sequenced from 19 samples representing different stages of the composting processes. Altogether 2986 sequenced clones were grouped into 166 phylotypes from which 35% had a close match in the sequence databases. The fungal communities of the samples were related with the measured environmental variables in order to identify phylotypes typical of certain composting conditions. The fungal phylotypes could be grouped into those that dominated the mesophilic low pH initial phases (sequences similar to genera Candida, Pichia and Dipodascaceae) and those found mostly or exclusively in the thermophilic phase (sequences clustering to Thermomyces, Candida and Rhizomucor), but a few were also present throughout the whole process. CONCLUSIONS: The community composition was found to vary between suboptimally and optimally operating processes. In addition, there were differences in fungal communities between processes of industrial and pilot scale. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study reveal the fungal diversity with molecular methods in industrial composting process. This is also one of the first studies conducted with samples from an industrial biowaste composting process.

Microbially treated peat-cellulose fabric as a biodegradable oil-collection cloth.

The aim of this work was to study the use of a fully biodegradable peat-cellulose fabric as a first aid in collecting and removing spilled oil. The fabric itself was made from entirely biodegradable natural components. Another aspect investigated was whether drying microbial suspension--specifically enriched for the degradation of oil hydrocarbons while maintaining a high survival rate and rapid initial growth--to the fabric would improve the degradation of absorbed oil along with the fabric. The results show that the oil absorption capacity of the biodegradable fabric was comparable to commercial products, and that the oil absorbed to the fabric degraded readily when incubated at various conditions. The microbial inoculum enhanced the degradation rate to some degree in sand, but in garden soil no significant difference existed. It was concluded that an oily fabric can be disposed of by biodegradation, e.g., by composting, but that a microbial inoculum is not essential for this purpose.

Use of a by-product of peat excavation, cotton grass fibre, as a sorbent for oil-spills.

The sorbents used to collect oil in case of oil-spills are mostly synthetic, which limits the possibilities of their disposal. We studied the absorption capacities and rates of cotton grass fibre, a by-product of peat excavation, and cotton grass mats for several oil types and compared them with a synthetic, commercially available oil sorbent. We found cotton grass fibre to have superior absorption properties: Cotton grass sorbent absorbed oil approximately two to three times as much, and two to three times as fast as the synthetic one. Cotton grass fibre absorbed no measurable amount of water in the conditions used in the tests making it ideal for absorbing oil from the surface of water. In removing diesel oil from the surface of water, the efficiency was over 99% up to an absorbing factor of 20 times its own weight. The biodegradable cotton grass fibre proved to be an effective oil sorbent with low raw-material costs.

Characterization of the hrp pathogenicity cluster of Erwinia carotovora subsp. carotovora: high basal level expression in a mutant is associated with reduced virulence.

Extracellularly targeted proteins are crucial for virulence of gram-negative phytopathogenic bacteria. Erwinia carotovora subsp. carotovora employs the so-called type II (GSP) pathway to secrete a number of pectinases and cellulases, which cause the typical tissue maceration symptoms of soft-rot disease. The type III (hrp) pathway is the major virulence determinant in the genera Pseudomonas, Ralstonia and Xanthomonas, and in non-macerating species of Erwinia. The hrp cluster was recently partially characterized from E. carotovora sp. carotovora, and shown to affect virulence during early stages of infection. Here we have isolated and characterized 15 hrp genes comprising the remaining part of the cluster. The genes hrpL, hrpXY and hrpS were deduced to be transcribed as separate units, whereas the 11 remaining genes from hrpJ to hrcU form a single large operon. The hrpX gene, which codes for the sensory kinase of the two-component regulatory locus hrpXY was insertionally inactivated by placing a transposon (entranceposon) in the gene. The resulting mutant bacterium expresses the hrp genes at high basal level even in a non-inducing medium. This relative overexpression was shown to be due to the hrpX::entranceposon insertion causing enhanced transcription of the downstream hrpY gene. The hrpX(-)-hrpYC mutant bacterium exhibited a slower growth rate and the appearance of disease symptoms in infected Arabidopsis plants was delayed, as compared to the wild-type strain. The need for hrp gene expression for virulence has been documented in both non-macerating plant pathogens and in soft-rotting Erwinia sp. but this is the first demonstration that high basal-level expression of hrp -regulated genes may actually have a negative impact on disease progress in a susceptible host plant.

Assessing sediment toxicity and arsenite concentration with bacterial and traditional methods.

Three sediment samples LP (pool where logs are stored), LF (brook through landfill area), KN (Kaskesniemi) which is in Lake Pyhäselkä downstream from the mill, were taken from an old sawmill area and one from the unpolluted Lake Höytiäinen. The arsenite concentration was measured by GFAAS and two arsenite biosensing bacterial strains Pseudomonas fluorescens OS8 (pTPT31) and Escherichia coli MC1061 (pTOO31). The toxicity of sediment and pore water samples was determined by using luminescent bacteria (Flash test) and, further, whole sediment toxicity was measured using 10 days growth test and 50 days emergency test with midges (Chironomus riparius). With the flash test a lowered EC50 value was found only in sediment LF (EC50=0.17 v/v%). The Flash test indicated that all sediment samples taken from the sawmill area were highly toxic to bacteria, whereas growth and the emergence of chironomids showed no effects in other samples than LF. The midges tolerate well the contaminated environment. In contrast, bioavailability of arsenite of sediment samples KN and LF was quite high determined using the biosensor-strains in a direct contact assay. The bioavailable fraction of sediment LP was 6-10% out of the total arsenite concentration obtained with GFAAS (0.46-0.77 microg g-1 dw). The results show that the choice of analysis method grossly affects the outcome without any of the method giving an incorrect result. Different methods measure different parameters of a toxic sample and can thus be used to complement each other.

mRNA stability and the secretion signal of HrpA, a pilin secreted by the type III system in Pseudomonas syringae.

Gram-negative bacteria that are pathogenic for animals or plants utilise a specialised Type III secretion system to inject effector proteins into their eukaryotic target cells. The basis for selection of the proteins to be translocated via type III systems is still enigmatic. No clearly defined consensus amino acid sequence that could serve as a specific secretion signal has been identified, and the hypothesis that an mRNA secondary structure acts as the signal has several shortcomings. We have localised a secretion signal that is sufficient to ensure the secretion of the pilin HrpA, a substrate and an indispensable extracellular component of the type III secretion machinery of Pseudomonas syringae pv. tomato DC3000, to the first 15 codons. Transcription of hrpA starts at a single site 42 bp upstream of the first codon. Gene swapping experiments revealed that altering the continuity of the 5' non-translated leader with the region including the secretion signal radically decreased accumulation of the hrpA transcript. These results indicate that an mRNA secondary structure, possibly formed in this region, is important for efficient expression of the gene. The proposed secondary structure is not, however, indispensable for the secretion of HrpA and it does not couple secretion and translation.

Type III secretion contributes to the pathogenesis of the soft-rot pathogen Erwinia carotovora: partial characterization of the hrp gene cluster.

The virulence of soft-rot Erwinia species is dependent mainly upon secreted enzymes such as pectinases, pectin lyases, and proteases that cause maceration of plant tissue. Some soft-rot Erwinia spp. also harbor genes homologous to the hypersensitive reaction and pathogenesis (hrp) gene cluster, encoding components of the type III secretion system. The hrp genes are essential virulence determinants for numerous nonmacerating gram-negative plant pathogens but their role in the virulence of soft-rot Erwinia spp. is not clear. We isolated and characterized 11 hrp genes of Erwinia carotovora subsp. carotovora. Three putative sigmaL-dependent Hrp box promoter sequences were found. The genes were expressed when the bacteria were grown in Hrp-inducing medium. The operon structure of the hrp genes was determined by mRNA hybridization, and the results were in accordance with the location of the Hrp boxes. An E. carotovora strain with mutated hrcC, an essential hrp gene, was constructed. The hrcC- strain was able to multiply and cause disease in Arabidopsis, but the population kinetics were altered so that growth was delayed during the early stages of infection.

Immunocytochemical localization of HrpA and HrpZ supports a role for the Hrp pilus in the transfer of effector proteins from Pseudomonas syringae pv. tomato across the host plant cell wall.

The Hrp pilus, composed of HrpA subunits, is an essential component of the type III secretion system in Pseudomonas syringae. We used electron microscopy (EM) and immunocytochemistry to examine production of the pilus in vitro from P. syringae pv. tomato strain DC3000 grown under hrp-inducing conditions on EM grids. Pili, when labeled with antibodies to HrpA, developed rapidly in a nonpolar manner shortly after the detection of the hrpA transcript and extended up to 5 microm into surrounding media. Structures at the base of the pilus were clearly differentiated from the basal bodies of flagella. The HrpZ protein, also secreted via the type III system, was found by immunogold labeling to be associated with the pilus in vitro. Accumulation and secretion of HrpA and HrpZ were also examined quantitatively after the inoculation of wild-type DC3000 and hrpA and hrpZ mutants into leaves of Arabidopsis thaliana. The functional pilus crossed the plant cell wall to generate tracks of immunogold labeling for HrpA and HrpZ. Mutants that produced HrpA but did not assemble pili were nonpathogenic, did not secrete HrpA protein, and were compromised for the accumulation of HrpZ. A model is proposed in which the rapidly elongating Hrp pilus acts as a moving conveyor, facilitating transfer of effector proteins from bacteria to the plant cytoplasm across the formidable barrier of the plant cell wall.

Role of the DmpR-mediated regulatory circuit in bacterial biodegradation properties in methylphenol-amended soils.

Pathway substrates and some structural analogues directly activate the regulatory protein DmpR to promote transcription of the dmp operon genes encoding the (methyl)phenol degradative pathway of Pseudomonas sp. strain CF600. While a wide range of phenols can activate DmpR, the location and nature of substituents on the basic phenolic ring can limit the level of activation and thus utilization of some compounds as assessed by growth on plates. Here we address the role of the aromatic effector response of DmpR in determining degradative properties in two soil matrices that provide different nutritional conditions. Using the wild-type system and an isogenic counterpart containing a DmpR mutant with enhanced ability to respond to para-substituted phenols, we demonstrate (i) that the enhanced in vitro biodegradative capacity of the regulator mutant strain is manifested in the two different soil types and (ii) that exposure of the wild-type strain to 4-methylphenol-contaminated soil led to rapid selection of a subpopulation exhibiting enhanced capacities to degrade the compound. Genetic and functional analyses of 10 of these derivatives demonstrated that all harbored a single mutation in the sensory domain of DmpR that mediated the phenotype in each case. These findings establish a dominating role for the aromatic effector response of DmpR in determining degradation properties. Moreover, the results indicate that the ability to rapidly adapt regulator properties to different profiles of polluting compounds may underlie the evolutionary success of DmpR-like regulators in controlling aromatic catabolic pathways.

Construction and Use of Broad Host Range Mercury and Arsenite Sensor Plasmids in the Soil Bacterium Pseudomonas fluorescens OS8.

We have generated new sensors for the specific detection and studies of bioavailability of metals by engineering Pseudomonas fluorescens with reporter gene systems. One broad host range mercury (pTPT11) and two arsenite (pTPT21 and pTPT31) sensor plasmids that express metal presence by luminescence phenotype were constructed and transferred into Escherichia coli DH5a and Pseudomonas fluorescens OS8. The maximal induction was reached after 2 h of incubation in metal solutions at room temperature (22 degrees C). In optimized conditions the half maximal velocity of reaction was achieved at acidic pH using a d-luciferin substrate concentration that was nearly sixfold lower for P. fluorescens OS8 than for E. coli DH5a. When using a luciferin concentration (150 mM) that was optimal for E. coli the luminescence declined rapidly in the case of Pseudomonas, for which the substrate level 25 mM gave a stable reading between about 20 min and 3 h. The ability of the strain OS8 to quantitatively detect specific heavy metals in spiked soil and soil extracts is as good, or even better in being a real-time reporter system, than that of a traditional chemical analysis. The Pseudomonas strain used is an isolate from pine rhizosphere in oil and heavy metal contaminated soil. It is also a good humus soil colonizer and is therefore a good candidate for measuring soil heavy metal bioavailability.

Colonization strategies and conjugal gene transfer of inoculated Pseudomonas syringae on the leaf surface.

Survival, colonization and activity of Pseudomonas syringae bacteria inoculated onto the leaf surface of the common bean (Phaseolus vulgaris) was studied. Inoculated Ps. syringae cells shortened by half their size in 100% humidity and by an average of one fifth in 40-60% humidity. The respiring portion of the population, measured by the formation of 5-cyano-2,3-ditolyl tetrazolium chloride (CTC)-formazan crystals, decreased more in 40-60% humidity than in 100% humidity. In scanning electron micrographs, the bacterial cells on leaf surfaces were seen embedded in a mucoid matrix. Intraspecies conjugation of plasmid RP1 also occurred in 40-60% humidity conditions. The portion of transconjugants temporally rose higher than the same portion in 100% humidity conditions. Therefore, although only a small proportion of the inoculated cells remained active on the leaf surface in 40-60% humidity, a relatively high rate of conjugation was still seen. Gene spreading was thus efficient on the leaf surface also when conditions did not allow bacterial population growth.

Members of the amylovora group of Erwinia are cellulolytic and possess genes homologous to the type II secretion pathway.

A cellulase-producing clone was isolated from a genomic library of the Erwinia rhapontici (Millard) Burkholder strain NCPPB2989. The corresponding gene, named celA, encodes an endoglucanase (EC 3.2.1.4) with the extremely low pH optimum of 3.4 and a temperature optimum between 40 and 50 degrees C. A single ORF of 999 nt was found to be responsible for the Cel activity. The corresponding protein, named CelA, showed 67% identity to the endoglucanase Y of E. chrysanthemi and 51.5% identity to the endoglucanase of Cellulomonas uda, and thus belongs to the glycosyl hydrolase family 8. The celA gene, or its homologue, was found to be present in all E. rhapontici isolates analysed, in E. chrysanthemi, and in E. amylovora. The presence of plant cell wall-degrading enzymes in the amylovora group of Erwinia spp. had not previously been established. Furthermore, the DNA of both E. rhapontici and E. amylovora was found to exhibit homology to genes encoding the type II (GSP) secretion pathway, which is known to be responsible for extracellular targeting of cellulases and pectinases in Erwinia spp. that cause soft rotting, such as E. carotovora and E. chrysanthemi. Secretion of the CelA protein by E. rhapontici could not be verified. However, the CelA protein itself was found to include the information necessary for heterologous secretion by E. chrysanthemi.

Effect of Cd-containing wood ash on the microflora of coniferous forest humus.

The use of wood ash in forestry has been questioned because the cadmium (Cd) concentration of ash, which varies between 1 and 20 mg kg(-1) ash, exceeds the level allowed for fertilizers (3 mg kg(-1)) used in agriculture. To investigate the combined and separated effects of Cd and ash on the forest humus microflora, pumice or wood ash, spiked with a water-soluble (CdCl(2)) or -insoluble (CdO) form of Cd at three levels (0, 400 and 1000 mg kg(-1)), were applied at a fertilization level of 5000 kg ha(-1) in a laboratory microcosm study. The trial consisted of 60 microcosms (five replications per treatment), which were incubated in darkness at +20 degrees C and a constant relative air humidity of 60%. After two months the humus in the microcosms was sampled. Analyses of CO(2) evolution to measure the overall microbial activity and of phospholipid fatty acid (PLFA) pattern to measure microbial community structure were performed. The substrate-use patterns of Biolog EcoPlates were analyzed as a measure of bacterial functionality. Finally the bacterial (3)H-thymidine incorporation in the presence of different concentrations of Cd and the number of colony forming units (cfu) of bacteria on nutrient agar in the presence of 0, 5 and 20 mg Cd l(-1) agar were applied to measure Cd tolerance. The use of pumice (pH of humus under the pumice 4.0) did not induce any changes in the above variables compared to two untreated microcosms (humus pH 3.9). Pumice was therefore used to distribute the Cd evenly over the humus surface in order to estimate the possible effect of Cd without ash (pH of humus under the ash 7.0). The application of ash increased the microbial activity, changed the PLFA and substrate-use patterns and increased cfu compared to the humus under pumice. The form and level of Cd in the ash had no further effect on this result. In the humus under pumice the level, but not the form of Cd decreased the microbial activity and changed the PLFA pattern compared to the unspiked pumice. None of the treatments induced bacterial tolerance to Cd. Ash thus protected the humus microflora from the harmful effects of Cd.

Effect of inoculation of a TOL plasmid containing mycorrhizosphere bacterium on development of Scots pine seedlings, their mycorrhizosphere and the microbial flora in m-toluate-amended soil.

The purpose of this study was to evaluate the influence of introduced bacteria containing a contaminant degrading plasmid on the growth and survival of pine seedlings and mycorrhizosphere microbial flora in contaminated soil. The Pseudomonas fluorescens strain OS81, originally isolated from fungal hyphae in contaminated soil, was supplied with the TOL plasmid pWW0::Km (to generate OS81(pWW0::Km)) and inoculated in humus-soil microcosms with and without pine seedlings mycorrhized with Suillus bovinus. After 3 months of regular treatment with m-toluate (mTA) solutions, the introduced catabolic plasmid was found to be disseminated in the indigenous bacterial population of both mycorrhizosphere and soil uncolonized by the fungus. Transconjugants were represented by bacteria of the genera Pseudomonas and Burkholderia and their number correlated positively with the concentration of mTA applied. Indigenous mTA degrading bacteria with low similarity to Burkholderia species were also enriched in microcosms. They were mostly associated with mycorrhizal soil or fungal structures and virtually absent in microcosms without pines. The total number of Tol(+) bacteria was higher in mycorrhizospheric soil compared with bulk soil. Inoculation with P. fluorescens OS81(pWW0::Km) had a positive effect on the development of roots and fungus in contaminated soil. Both inoculation with the P. fluorescens OS81(pWW0::Km) and mTA contamination as well as the presence of mycorrhized pine roots and fungal hyphae had an effect on the microbial community structure of soil as measured by carbon source oxidation patterns. However, the impact of mTA on the microbial community was more prominent. The study indicates that an effect on plant and fungal development can be obtained by manipulating the mycorrhizosphere. Both introduction of the bacterium carrying the degradative plasmid and the plasmid itself are likely to have a positive effect not only on the organisms involved, but also on bioremediation of contaminated soil, a factor that was not directly monitored here.

Means to improve the effect of in situ bioremediation of contaminated soil: an overview of novel approaches.

Different aspects of bacterial degradation of organic contaminants in soil, and how to improve the efficiency and reproducibility is discussed in this review. Although bioremediation in principle includes the use of any type of organism in improving the condition of a contaminated site, most commonly bacteria are the degraders and other organisms, such as soil animals or plant roots, play a role in dissemination of bacteria and, indirectly, plasmids between bacteria, and in providing nutrients and co-substrates for the bacteria active in the degradation process. There are a number of different procedures that have been tested more-or-less successfully in attempts to improve reliability, cost efficiency and speed of bioremediation. The methods range from minimal intervention, such as mere monitoring of intrinsic bioremediation, through in situ introduction of nutrients and/or bacterial inocula or improvement of physico-chemical conditions, all the way to excavation followed by on site or ex situ composting in its different varieties. In the past the rule has been that more intervention (leading to higher costs) has been more reliable, but novel ideas are continuously tried out, both as a means to come up with new truly functional applications and also as a line of studies in basic soil microbial ecology. Both approaches generate valuable information needed when predicting outcome of remediation activities, evaluating environmental risks, deciding on cleaning-up approaches, etc. The emphasis of this review is to discuss some of the novel methods for which the value has not been clearly shown, but that in our view merit continued studies and efforts to make them work, separately or in combination.

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