The potential of microbial ecological indicators to guide ecosophisticated management of hydrocarbon-contaminated soils

Soil is an unrenewable natural resource under increasing anthropogenic pressure. One of the main threats to soils, compromising their ability to provide us with the goods and ecosystem services we expect, is pollution. Oil hydrocarbons are the most common soil contaminants, and they disturb not just the biota but also the physicochemical properties of soils. Indigenous soil micro-organisms respond rapidly to changes in the soil ecosystem, and are chronically in direct contact with the hydrophobic pollutants on the soil surfaces. Soil microbial variables could thus serve as an intrinsically relevant indicator of soil quality, to be used in the ecological risk assessment of contaminated and remediated soils. Two contrasting studies were designed to investigate soil microbial ecological responses to hydrocarbons, together with parallel changes in soil physicochemical and ecotoxicological properties. The aim was to identify quantitative or qualitative microbiological variables that would be practicable and broadly applicable for the assessment of the quality and restoration of oil-polluted soil. Soil bacteria commonly react on hydrocarbons as a beneficial substrate, which lead to a positive response in the classical microbiological soil quality indicators; negative impacts were accurately reflected only after severe contamination. Hydrocarbon contaminants become less bioavailable due to weathering processes, and their potentially toxic effects decrease faster than the total concentration. Indigenous hydrocarbon degrader bacteria, naturally present in any terrestrial environment, use specific mechanisms to improve access to the hydrocarbon molecules adsorbed on soil surfaces. Thus when contaminants are unavailable even to the specialised degraders, they should pose no hazard to other biota either. Change in the ratio of hydrocarbon degrader numbers to total microbes was detected to predictably indicate pollutant effects and bioavailability. Also bacterial diversity, a qualitative community characteristic, decreased as a response to hydrocarbons. Stabilisation of community evenness, and community structure that reflected clean reference soil, indicated community recovery. If long-term temporal monitoring is difficult and appropriate clean reference soil unavailable, such comparison could possibly be based on DNA-based community analysis, reflecting past+present, and RNA-based community analysis, showing exclusively present conditions. Microbial ecological indicators cannot replace chemical oil analyses, but they are theoretically relevant and operationally practicable additional tools for ecological risk assessment. As such, they can guide ecologically informed and sustainable ecosophisticated management of oil-contaminated lands.

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

Microbial activities in boreal soils: Biodegradation of organic contaminants at low temperature and ammonia oxidation

This thesis deals with the response of biodegradation of selected anthropogenic organic contaminants and natural autochthonous organic matter to low temperature in boreal surface soils. Furthermore, the thesis describes activity, diversity and population size of autotrophic ammonia-oxidizing bacteria (AOB) in a boreal soil used for landfarming of oil-refinery wastes, and presents a new approach, in which the particular AOB were enriched and cultivated in situ from the landfarming soil onto cation exchange membranes. This thesis demonstrates that rhizosphere fraction of natural forest humus soil and agricultural clay loam soil from Helsinki Metropolitan area were capable of degrading of low to moderate concentrations (0.2 50 µg cm-3) of PCP, phenanthrene and 2,4,5-TCP at temperatures realistic to boreal climate (-2.5 to +15 °C). At the low temperatures, the biodegradation of PCP, phenanthrene and 2,4,5-TCP was more effective (Q10-values from 1.6 to 7.6) in the rhizosphere fraction of the forest soil than in the agricultural soil. Q10-values of endogenous soil respiration (carbon dioxide evolution) and selected hydrolytic enzyme activities (acetate-esterase, butyrate-esterase and β-glucosidase) in acid coniferous forest soil were 1.6 to 2.8 at temperatures from -3 to +30 °C. The results indicated that the temperature dependence of decomposition of natural autochthonous soil organic matter in the studied coniferous forest was only moderate. The numbers of AOB in the landfarming (sandy clay loam) soil were determined with quantitative polymerase chain reaction (real-time PCR) and with Most Probable Number (MPN) methods, and potential ammonium oxidation activity was measured with the chlorate inhibition technique. The results indicated presence of large and active AOB populations in the heavily oil-contaminated and urea-fertilised landfarming soil. Assessment of the populations of AOB with denaturing gradient gel electrophoresis (DGGE) profiling and sequence analysis of PCR-amplified 16S rRNA genes showed that Nitrosospira-like AOB in clusters 2 and 3 were predominant in the oily landfarming soil. This observation was supported by fluorescence in situ hybridization (FISH) analysis of the AOB grown on the soil-incubated cation-exchange membranes. The results of this thesis expand the suggested importance of Nitrosospira-like AOB in terrestrial environments to include chronically oil-contaminated soils.

Bioavailability aspects of hydrophobic contaminant degradation in soils

This thesis concentrates on bioavailability of organic soil contaminants in the context of bioremediation of soil contaminated with volatile or non-volatile hydrophobic pollutants. Bioavailability and biodegradation was studied from four viewpoints: (i) Improvement of bioavailability and biodegradation of volatile hydrocarbons in contained bioremediation systems at laboratory - and pilot-scale. (ii) Improvement of bioavailability of non-volatile, hydrophobic compounds in such systems. (iii) Biodegradation of a non-volatile hydrophobic compound in soil organic matter in microcosms. (iiii) Bioavailability of nitrogen in an open, full-scale bioremediation system. It was demonstrated that volatility of organic compounds can be controlled by amending the soil with adsorbents. The sorbed hydrocarbons were shown to be available to soil microbiota. As the result, biodegradation of the volatile hydrocarbons was greatly favored at the expense of volatilization. PAH compounds were shown to be mobilized and their bioavailability improved by a hydrophobic, non-toxic additive, vegetable oil. Bioavailability of the PAHs was recorded as an increased toxicity of the soil. In spite of the increased bioavailability, biodegradation of the PAHs decreased. In microcosms simulating boreal forest organic surface soil, PAH-compound (pyrene) was shown to be removed from soil biologically. Therefore hydrophobicity of the substrate does not necessarily mean low availability and biodegradation in organic soil. Finally, in this thesis it was demonstrated that an unsuitable source of nitrogen or its overdose resulted in wasteful spending of this nutrient and even harmful effects on soil microbes. Such events may inhibit rather than promote the bioremediation process in soil.

Tropical dryland agroforestry on clay soils: : Analysis of systems based on Acacia senegal in the Blue Nile region, Sudan

Acacia senegal, the gum arabic producing tree, is the most important component in traditional dryland agroforestry systems in the Blue Nile region, Sudan. The aim of the present study was to provide new knowledge on the potential use of A. senegal in dryland agroforestry systems on clay soils, as well as information on tree/crop interaction, and on silvicultural and management tools, with consideration on system productivity, nutrient cycling and sustainability. Moreover, the aim was also to clarify the intra-specific variation in the performance of A. senegal and, specifically, the adaptation of trees of different origin to the clay soils of the Blue Nile region. In agroforestry systems established at the beginning of the study, tree and crop growth, water use, gum and crop yields, nutrient cycling and system performance were investigated for a period of four years (1999 to 2002). Trees were grown at 5 x 5 m and 10 x 10 m spacing alone or in mixture with sorghum or sesame; crops were also grown in sole culture. The symbiotic biological N2 fixation by A. senegal was estimated using the 15N natural abundance (δ15N) procedure in eight provenances collected from different environments and soil types of the gum arabic belt and grown in clay soil in the Blue Nile region. Balanites aegyptiaca (a non-legume) was used as a non-N-fixing reference tree species, so as to allow 15N-based estimates of the proportion of the nitrogen in trees derived from the atmosphere. In the planted acacia trees, measurements were made on shoot growth, water-use efficiency (as assessed by the δ13C method) and (starting from the third year) gum production. Carbon isotope ratios were obtained from the leaves and branch wood samples. The agroforestry system design caused no statistically significant variation in water use, but the variation was highly significant between years, and the highest water use occurred in the years with high rainfall. No statistically significant differences were found in sorghum or sesame yields when intercropping and sole crop systems were compared (yield averages were 1.54 and 1.54 ha-1 for sorghum and 0.36 and 0.42 t ha-1 for sesame in the intercropped and mono-crop plots, respectively). Thus, at an early stage of agroforestry system management, A. senegal had no detrimental effect on crop yield, but the pattern of resource capture by trees and crops may change as the system matures. Intercropping resulted in taller trees and larger basal and crown diameters as compared to the development of sole trees. It also resulted in a higher land equivalent ratio. When gum yields were analysed it was found that a significant positive relationship existed between the second gum picking and the total gum yield. The second gum picking seems to be a decisive factor in gum production and could be used as an indicator for the total gum yield in a particular year. In trees, the concentrations of N and P were higher in leaves and roots, whereas the levels of K were higher in stems, branches and roots. Soil organic matter, N, P and K contents were highest in the upper soil stratum. There was some indication that the P content slightly increased in the topsoil as the agroforestry plantations aged. At a stocking of 400 trees ha-1 (5 x 5 m spacing), A. senegal accumulated in the biomass a total of 18, 1.21, 7.8 and 972 kg ha-1of N, P, K and OC, respectively. Trees contributed ca. 217 and 1500 kg ha-1 of K and OC, respectively, to the top 25-cm of soil over the first four years of intercropping. Acacia provenances of clay plain origin showed considerable variation in seed weight. They also had the lowest average seed weight as compared to the sandy soil (western) provenances. At the experimental site in the clay soil region, the clay provenances were distinctly superior to the sand provenances in all traits studied but especially in basal diameter and crown width, thus reflecting their adaptation to the environment. Values of δ13C, indicating water use efficiency, were higher in the sand soil group as compared to the clay one, both in leaves and in branch wood. This suggests that the sand provenances (with an average value of -28.07 ) displayed conservative water use and high drought tolerance. Of the clay provenances, the local one (Bout) displayed a highly negative (-29.31 ) value, which indicates less conservative water use that resulted in high productivity at this particular clay-soil site. Water use thus appeared to correspond to the environmental conditions prevailing at the original locations for these provenances. Results suggest that A. senegal provenances from the clay part of the gum belt are adapted for a faster growth rate and higher biomass and gum productivity as compared to provenances from sand regions. A strong negative relationship was found between the per-tree gum yield and water use efficiency, as indicated by δ13C. The differences in water use and gum production were greater among provenance groups than within them, suggesting that selection among rather than within provenances would result in distinct genetic gain in gum yield. The relative δ15N values ( ) were higher in B. aegyptiaca than in the N2-fixing acacia provenances. The amount of Ndfa increased significantly with age in all provenances, indicating that A. senegal is a potentially efficient nitrogen fixer and has an important role in t agroforestry development. The total above-ground contribution of fixed N to foliage growth in 4-year-old A. senegal trees was highest in the Rahad sand-soil provenance (46.7 kg N ha-1) and lowest in the Mazmoom clay-soil provenance (28.7 kg N ha-1). This study represents the first use of the δ15N method for estimating the N input by A. senegal in the gum belt of Sudan. Key words: Acacia senegal, agroforestry, clay plain, δ13C, δ15N, gum arabic, nutrient cycling, Ndfa, Sorghum bicolor, Sesamum indicum

Temperature sensitivity of soil organic matter decomposition in boreal soils

The temperature sensitivity of decomposition of different soil organic matter (SOM) fractions was studied with laboratory incubations using 13C and 14C isotopes to differentiate between SOM of different age. The quality of SOM and the functionality and composition of microbial communities in soils formed under different climatic conditions were also studied. Transferring of organic layers from a colder to a warmer climate was used to assess how changing climate, litter input and soil biology will affect soil respiration and its temperature sensitivity. Together, these studies gave a consistent picture on how warming climate will affect the decomposition of different SOM fractions in Finnish forest soils: the most labile C was least temperature sensitive, indicating that it is utilized irrespective of temperature. The decomposition of intermediate C, with mean residence times from some years to decades, was found to be highly temperature sensitive. Even older, centennially cycling C was again less temperature sensitive, indicating that different stabilizing mechanisms were limiting its decomposition even at higher temperatures. Because the highly temperature sensitive, decadally cycling C, forms a major part of SOM stock in the organic layers of the studied forest soils, these results mean that these soils could lose more carbon during the coming years and decades than estimated earlier. SOM decomposition in boreal forest soils is likely to increase more in response to climate warming, compared to temperate or tropical soils, also because the Q10 is temperature dependent. In the northern soils the warming will occur at a lower temperature range, where Q10 is higher, and a similar increase in temperature causes a higher relative increase in respiration rates. The Q10 at low temperatures was found to be inversely related to SOM quality. At higher temperatures respiration was increasingly limited by low substrate availability.

Phosphorus retention in forest soils and the functioning of buffer zones used in forestry

Phosphorus (P) retention properties of soils typical for boreal forest, i.e. podzolic soil and peat soils, vary significantly, but the range of this variation has not been sufficiently documented. To assess the usefulness of buffer zones used in forestry in removing P from the discharge by chemical sorption in soil, and to estimate the risk of P leaching after forestry operations, more data is needed on soil P retention properties. P retention properties of soils were studied at clear-cut areas, unharvested buffer zones adjoining the clear-cut and at peatland buffer zone areas. Desorption-sorption isotherms were determined for the humus layer, the mineral soil horizons E, B and C of the Podzol profile and for the surface layer peat (0-15 cm) and the subsurface layer peat (15-30 cm). The efficiency of buffer zones in retaining P was studied at six peatland buffer zone areas by adding P-containing solute in the inflow. A tracer study was conducted at one of the buffer zone areas to determine the allocation of the added P in soil and vegetation. Measured sorption or desorption rather than parameter values of fitted sorption equations described P desorption and sorption behaviour in soil. The highest P retention efficiency was in the B horizon and consequently, if contact occurred or was established between the soluble P in the water and the soil B horizon, the risk of P leaching was low. Humus layer was completely incapable of retaining P after clear-cutting. In the buffer zones, the decrease in P retention properties in the humus layer and the low amount of P sorbed by it indicated that the importance of the layer in the functioning of buffer zones is low. The peatland buffer zone areas were efficient in retaining soluble P from inflow. P sorption properties of the peat soil at the buffer zone areas varied largely but the contribution of P sorption in the peat was particularly important during high flow in spring, when the vegetation was not fully developed. Factors contributing to efficient P retention were large buffer size and low hydrological load whereas high hydrological load combined with the formation of preferential flow paths, especially during early spring or late autumn was disadvantageous. However, small buffer zone areas, too, may be efficient in reducing P load.

Phosphorus in agricultural soils of Finland - characterization of reserves and retention in mineral soil profiles

An overwhelming majority of all the research on soil phosphorus (P) has been carried out with soil samples taken from the surface soils only, and our understanding of the forms and the reactions of P at a soil profile scale is based on few observations. In Finland, the interest in studying the P in complete soil profiles has been particularly small because of the lack of tradition in studying soil genesis, morphology, or classification. In this thesis, the P reserves and the retention of orthophosphate phosphorus (PO4-P) were examined in four cultivated mineral soil profiles in Finland (three Inceptisols and one Spodosol). The soils were classified according to the U.S. Soil Taxonomy and soil samples were taken from the genetic horizons in the profiles. The samples were analyzed for total P concentration, Chang and Jackson P fractions, P sorption properties, concentrations of water-extractable P, and for concentrations of oxalate-extractable Al and Fe. Theoretical P sorption capacities and degrees of P saturation were calculated with the data from the oxalate-extractions and the P fractionations. The studied profiles can be divided into sections with clearly differing P characteristics by their master horizons Ap, B and C. The C (or transitional BC) horizons below an approximate depth of 70 cm were dominated by, assumingly apatitic, H2SO4-soluble P. The concentration of total P in the C horizons ranged from 729 to 810 mg kg-1. In the B horizons between the depths of 30 and 70 cm, a significant part of the primary acid-soluble P has been weathered and transformed to secondary P forms. A mean weathering rate of the primary P in the soils was estimated to vary between 230 and 290 g ha-1 year-1. The degrees of P saturation in the B and C horizons were smaller than 7%, and the solubility of PO4-P was negligible. The P conditions in the Ap horizons differed drastically from those in the subsurface horizons. The high concentrations of total P (689-1870 mg kg-1) in the Ap horizons are most likely attributable to long-term cultivation with positive P balances. A significant proportion of the P in the Ap horizons occurred in the NH4F- and NaOH-extractable forms and as organic P. These three P pools, together with the concentrations of oxalate-extractable Al and Fe, seem to control the dynamics of PO4-P in the soils. The degrees of P saturation in the Ap horizons were greater (8-36%) than in the subsurface horizons. This was also reflected in the sorption experiments: Only the Ap horizons were able to maintain elevated PO4-P concentrations in the solution phase − all the subsoil horizons acted as sinks for PO4-P. Most of the available sorption capacity in the soils is located in the B horizons. The results suggest that this capacity could be utilized in reducing the losses of soluble P from excessively fertilized soils by mixing highly sorptive material from the B horizons with the P-enriched surface soil. The drastic differences in the P characteristics observed between adjoining horizons have to be taken into consideration when conducting soil sampling. Sampling of subsoils has to be made according to the genetic horizons or at small depth increments. Otherwise, contrasting materials are likely to be mixed in the same sample; and the results of such samples are not representative of any material present in the studied profile. Air-drying of soil samples was found to alter the results of the sorption experiments and the water extractions. This indicates that the studies on the most labile P forms in soil should be carried out with moist samples.