Multiangular Spectrometry and Optical Properties of Debris Covered Surfaces

Due to the recent development in CCD technology aerial photography is now slowly changing from film to digital cameras. This new aspect in remote sensing allows and requires also new automated analysis methods. Basic research on reflectance properties of natural targets is needed so that computerized processes could be fully utilized. For this reason an instrument was developed at Finnish Geodetic Institute for measurement of multiangular reflectance of small remote sensing targets e.g. forest understorey or asphalt. Finnish Geodetic Institute Field Goniospectrometer (FiGIFiGo) is a portable device that is operated by 1 or 2 persons. It can be reassembled to a new location in 15 minutes and after that a target's multiangular reflectance can be measured in 10 - 30 minutes (with one illumination angle). FiGIFiGo has effective spectral range approximately from 400 nm to 2000 nm. The measurements can be made either outside with sunlight or in laboratory with 1000 W QTH light source. In this thesis FiGIFiGo is introduced and the theoretical basis of such reflectance measurements are discussed. A new method is introduced for extraction of subcomponent proportions from reflectance of a mixture sample, e.g. for retrieving proportion of lingonberry's reflectance in observation of lingonberry-lichen sample. This method was tested by conducting a series of measurements on reflectance properties of artificial samples. The component separation method yielded sound results and brought up interesting aspects in targets' reflectances. The method and the results still need to be verified with further studies, but the preliminary results imply that this method could be a valuable tool in analysis of such mixture samples.

Geological factors affecting on after-use of Finnish cut-over peatlands : with implications on the carbon accumulation

In Finland, peat harvesting sites are utilized down almost to the mineral soil. In this situation the properties of mineral subsoil are likely to have considerable influence on the suitability for the various after-use forms. The aims of this study were to recognize the chemical and physical properties of mineral subsoils possibly limiting the after-use of cut-over peatlands, to define a minimum practice for mineral subsoil studies and to describe the role of different geological areas. The future percentages of the different after-use forms were predicted, which made it possible to predict also carbon accumulation in this future situation. Mineral subsoils of 54 different peat production areas were studied. Their general features and grain size distribution was analysed. Other general items studied were pH, electrical conductivity, organic matter, water soluble nutrients (P, NO3-N, NH4-N, S and Fe) and exchangeable nutrients (Ca, Mg and K). In some cases also other elements were analysed. In an additional case study carbon accumulation effectiveness before the intervention was evaluated on three sites in Oulu area (representing sites typically considered for peat production). Areas with relatively sulphur rich mineral subsoil and pool-forming areas with very fine and compact mineral subsoil together covered approximately 1/5 of all areas. These areas were unsuitable for commercial use. They were recommended for example for mire regeneration. Another approximate 1/5 of the areas included very coarse or very fine sediments. Commercial use of these areas would demand special techniques - like using the remaining peat layer for compensating properties missing from the mineral subsoil. One after-use form was seldom suitable for one whole released peat production area. Three typical distribution patterns (models) of different mineral subsoils within individual peatlands were found. 57 % of studied cut-over peatlands were well suited for forestry. In a conservative calculation 26% of the areas were clearly suitable for agriculture, horticulture or energy crop production. If till without large boulders was included, the percentage of areas suitable to field crop production would be 42 %. 9-14 % of all areas were well suitable for mire regeneration or bird sanctuaries, but all areas were considered possible for mire regeneration with correct techniques. Also another 11 % was recommended for mire regeneration to avoid disturbing the mineral subsoil, so total 20-25 % of the areas would be used for rewetting. High sulphur concentrations and acidity were typical to the areas below the highest shoreline of the ancient Litorina sea and Lake Ladoga Bothnian Bay zone. Also differences related to nutrition were detected. In coarse sediments natural nutrient concentration was clearly higher in Lake Ladoga Bothnian Bay zone and in the areas of Svecokarelian schists and gneisses, than in Granitoid area of central Finland and in Archaean gneiss areas. Based on this study the recommended minimum analysis for after-use planning was for pH, sulphur content and fine material (<0.06 mm) percentage. Nutrition capacity could be analysed using the natural concentrations of calcium, magnesium and potassium. Carbon accumulation scenarios were developed based on the land-use predictions. These scenarios were calculated for areas in peat production and the areas released from peat production (59300 ha + 15 671 ha). Carbon accumulation of the scenarios varied between 0.074 and 0.152 million t C a-1. In the three peatlands considered for peat production the long term carbon accumulation rates varied between 13 and 24 g C m-2 a-1. The natural annual carbon accumulation had been decreasing towards the time of possible intervention.

Below-ground processes in meadow soil under elevated ozone and carbon dioxide : - Greenhouse gas fluxes, N cycling and microbial communities

This thesis focuses on how elevated CO2 and/or O3 affect the below-ground processes in semi-natural vegetation, with an emphasis on greenhouse gases, N cycling and microbial communities. Meadow mesocosms mimicking lowland hay meadows in Jokioinen, SW Finland, were enclosed in open-top chambers and exposed to ambient and elevated levels of O3 (40-50 ppb) and/or CO2 (+100 ppm) for three consecutive growing season, while chamberless plots were used as chamber controls. Chemical and microbiological analyses as well as laboratory incubations of the mesocosm soils under different treatments were used to study the effects of O3 and/or CO2. Artificially constructed mesocosms were also compared with natural meadows with regards to GHG fluxes and soil characteristics. In addition to research conducted at the ecosystem level (i.e. the mesocosm study), soil microbial communities were also examined in a pot experiment with monocultures of individual species. By comparing mesocosms with similar natural plant assemblage, it was possible to demonstrate that artificial mesocosms simulated natural habitats, even though some differences were found in the CH4 oxidation rate, soil mineral N, and total C and N concentrations in the soil. After three growing seasons of fumigations, the fluxes of N2O, CH4, and CO2 were decreased in the NF+O3 treatment, and the soil NH4+-N and mineral N concentrations were lower in the NF+O3 treatment than in the NF control treatment. The mesocosm soil microbial communities were affected negatively by the NF+O3 treatment, as the total, bacterial, actinobacterial, and fungal PLFA biomasses as well as the fungal:bacterial biomass ratio decreased under elevated O3. In the pot survey, O3 decreased the total, bacterial, actinobacterial, and mycorrhizal PLFA biomasses in the bulk soil and affected the microbial community structure in the rhizosphere of L. pratensis, whereas the bulk soil and rhizosphere of the other monoculture, A. capillaris, remained unaffected by O3. Elevated CO2 caused only minor and insignificant changes in the GHG fluxes, N cycling, and the microbial community structure. In the present study, the below-ground processes were modified after three years of moderate O3 enhancement. A tentative conclusion is that a decrease in N availability may have feedback effects on plant growth and competition and affect the N cycling of the whole meadow ecosystem. Ecosystem level changes occur slowly, and multiplication of the responses might be expected in the long run.

Effects of recreational use and fragmentation on the understorey vegetation and soil microbial communities of urban forests in southern Finland

The impacts of fragmentation and recreational use on the hemiboreal urban forest understorey vegetation and the microbial community of the humus layer (the phospholipid fatty acid (PLFA) pattern, microbial biomass and microbial activity, measured as basal respiration) were examined in the greater Helsinki area, southern Finland. Trampling tolerance of 1) herb-rich OMT, 2) mesic MT, and 3) sub-xeric VT forests (in decreasing order of fertility) was studied by comparing relative understorey vegetation cover (urban/untrampled reference ratio) of the three forest types. The trampling tolerance of forest vegetation increased with the productivity of the site (sub-xeric < mesic < herb-rich). Wear of understorey vegetation correlated positively with the number of residents (i.e., recreational pressure) around the forest patch. An increase of 15000 residents within a radius of 1 km around a forest patch was associated with ca. 30% decrease in the relative understorey vegetation cover. The cover of dwarf shrub Vaccinium myrtillus in particular decreased with increasing levels of wear. The cover of mosses in urban forests was less than half of that in untrampled reference areas. Cover of tree saplings, mainly Sorbus aucuparia, and some resilient herbs was higher than in the reference areas. In small urban forest fragments, broad-leaved trees, grasses and herbs were more abundant and mosses were scarcer than in larger urban forest areas. Thus, due to trampling and edge effects, resilient herb and grass species are replacing sensitive dwarf shrubs, mosses and lichens in urban forests. Differences in the soil microbial community structure were found between paths and untrampled areas and the effects of paths extended more than one meter from the paths. Paths supported approximately 25-30% higher microbial biomass with a transition zone of at least 1 m from the path edge. However, microbial activity per unit of biomass was lower on paths than in untrampled areas. Furthermore, microbial biomass and activity were 30-45% lower at the first 20 m into the forest fragments, due to low moisture content of humus near the edge. The decreased microbial activity detected at forest edges and paths implies decreased litter decomposition rates, and thus, a change in nutrient cycling. Changes in the decomposition and nutrient supply may in turn affect the diversity and function of plant communities in urban forests. Keywords: boreal forest vegetation, edge effects, phospholipid fatty acids, trampling, urban woodlands, wear

Microbial communities in Pb contaminated boreal forest soil

Lead contamination in the environment is of particular concern, as it is a known toxin. Until recently, however, much less attention has been given to the local contamination caused by activities at shooting ranges compared to large-scale industrial contamination. In Finland, more than 500 tons of Pb is produced each year for shotgun ammunition. The contaminant threatens various organisms, ground water and the health of human populations. However, the forest at shooting ranges usually shows no visible sign of stress compared to nearby clean environments. The aboveground biota normally reflects the belowground ecosystem. Thus, the soil microbial communities appear to bear strong resistance to contamination, despite the influence of lead. The studies forming this thesis investigated a shooting range site at Hälvälä in Southern Finland, which is heavily contaminated by lead pellets. Previously it was experimentally shown that the growth of grasses and degradation of litter are retarded. Measurements of acute toxicity of the contaminated soil or soil extracts gave conflicting results, as enchytraeid worms used as toxicity reporters were strongly affected, while reporter bacteria showed no or very minor decreases in viability. Measurements using sensitive inducible luminescent reporter bacteria suggested that the bioavailability of lead in the soil is indeed low, and this notion was supported by the very low water extractability of the lead. Nevertheless, the frequency of lead-resistant cultivable bacteria was elevated based on the isolation of cultivable strains. The bacterial and fungal diversity in heavily lead contaminated shooting sectors were compared with those of pristine sections of the shooting range area. The bacterial 16S rRNA gene and fungal ITS rRNA gene were amplified, cloned and sequenced using total DNA extracted from the soil humus layer as the template. Altogether, 917 sequenced bacterial clones and 649 sequenced fungal clones revealed a high soil microbial diversity. No effect of lead contamination was found on bacterial richness or diversity, while fungal richness and diversity significantly differed between lead contaminated and clean control areas. However, even in the case of fungi, genera that were deemed sensitive were not totally absent from the contaminated area: only their relative frequency was significantly reduced. Some operational taxonomic units (OTUs) assigned to Basidiomycota were clearly affected, and were much rarer in the lead contaminated areas. The studies of this thesis surveyed EcM sporocarps, analyzed morphotyped EcM root tips by direct sequencing, and 454-pyrosequenced fungal communities in in-growth bags. A total of 32 EcM fungi that formed conspicuous sporocarps, 27 EcM fungal OTUs from 294 root tips, and 116 EcM fungal OTUs from a total of 8 194 ITS2 454 sequences were recorded. The ordination analyses by non-parametric multidimensional scaling (NMS) indicated that Pb enrichment induced a shift in the EcM community composition. This was visible as indicative trends in the sporocarp and root tip datasets, but explicitly clear in the communities observed in the in-growth bags. The compositional shift in the EcM community was mainly attributable to an increase in the frequencies of OTUs assigned to the genus Thelephora, and to a decrease in the OTUs assigned to Pseudotomentella, Suillus and Tylospora in Pb-contaminated areas when compared to the control. The enrichment of Thelephora in contaminated areas was also observed when examining the total fungal communities in soil using DNA cloning and sequencing technology. While the compositional shifts are clear, their functional consequences for the dominant trees or soil ecosystem remain undetermined. The results indicate that at the Hälvälä shooting range, lead influences the fungal communities but not the bacterial communities. The forest ecosystem shows apparent functional redundancy, since no significant effects were seen on forest trees. Recently, by means of 454 pyrosequencing , the amount of sequences in a single analysis run can be up to one million. It has been applied in microbial ecology studies to characterize microbial communities. The handling of sequence data with traditional programs is becoming difficult and exceedingly time consuming, and novel tools are needed to handle the vast amounts of data being generated. The field of microbial ecology has recently benefited from the availability of a number of tools for describing and comparing microbial communities using robust statistical methods. However, although these programs provide methods for rapid calculation, it has become necessary to make them more amenable to larger datasets and numbers of samples from pyrosequencing. As part of this thesis, a new program was developed, MuSSA (Multi-Sample Sequence Analyser), to handle sequence data from novel high-throughput sequencing approaches in microbial community analyses. The greatest advantage of the program is that large volumes of sequence data can be manipulated, and general OTU series with a frequency value can be calculated among a large number of samples.

Plasmids and aromatic degradation in Sphingomonas for bioremediation : Aromatic ring cleavage genes in soil and rhizosphere

Microbial degradation pathways play a key role in the detoxification and the mineralization of polyaromatic hydrocarbons (PAHs), which are widespread pollutants in soil and constituents of petroleum hydrocarbons. In microbiology the aromatic degradation pathways are traditionally studied from single bacterial strains with capacity to degrade certain pollutant. In soil the degradation of aromatics is performed by a diverse community of micro-organisms. The aim of this thesis was to study biodegradation on different levels starting from a versatile aromatic degrader Sphingobium sp. HV3 and its megaplasmid, extending to revelation of diversity of key catabolic enzymes in the environment and finally studying birch rhizoremediation in PAH-polluted soil. To understand biodegradation of aromatics on bacterial species level, the aromatic degradation capacity of Sphingobium sp. HV3 and the role of the plasmid pSKY4, was studied. Toluene, m-xylene, biphenyl, fluorene, phenanthrene were detected as carbon and energy sources of the HV3 strain. Tn5 transposon mutagenesis linked the degradation capacity of toluene, m-xylene, biphenyl and naphthalene to the pSKY4 plasmid and qPCR expression analysis showed that plasmid extradiol dioxygenases genes (bphC and xylE) are inducted by phenanthrene, m-xylene and biphenyl whereas the 2,4-dichlorophenoxyacetic acid herbicide induced the chlorocatechol 1,2-dioxygenase gene (tfdC) from the ortho-pathway. A method to study upper meta-pathway extradiol dioxygenase gene diversity in soil was developed. The extradiol dioxygenases catalyse cleavage of the aromatic ring between a hydroxylated carbon and an adjacent non-hydroxylated carbon (meta-cleavage). A high diversity of extradiol dioxygenases were detected from polluted soils. The detected extradiol dioxygenases showed sequence similarity to known catabolic genes of Alpha-, Beta-, and Gammaproteobacteria. Five groups of extradiol dioxygenases contained sequences with no close homologues in the database, representing novel genes. In rhizoremediation experiment with birch (Betula pendula) treatment specific changes of extradiol dioxygenase communities were shown. PAH pollution changed the bulk soil extradiol dioxygenase community structure and birch rhizosphere contained a more diverse extradiol dioxygenase community than the bulk soil showing a rhizosphere effect. The degradation of pyrene in soil was enhanced with birch seedlings compared to soil without birch. The complete 280,923 kb nucleotide sequence of pSKY4 plasmid was determined. The open reading frames of pSKY4 were divided into putative conjugative transfer, aromatic degradation, replication/maintaining and transposition/integration function-encoding proteins. Aromatic degradation orfs shared high similarity to corresponding genes in pNL1, a plasmid from the deep subsurface strain Novosphingobium aromaticivorans F199. The plasmid backbones were considerably more divergent with lower similarity, which suggests that the aromatic pathway has functioned as a plasmid independent mobile genetic element. The functional diversity of microbial communities in soil is still largely unknown. Several novel clusters of extradiol dioxygenases representing catabolic bacteria, whose function, biodegradation pathways and phylogenetic position is not known were amplified with single primer pair from polluted soils. These extradiol dioxygenase communities were shown to change upon PAH pollution, which indicates that their hosts function in PAH biodegradation in soil. Although the degradation pathways of specific bacterial species are substantially better depicted than pathways in situ, the evolution of degradation pathways for the xenobiotic compounds is largely unknown. The pSKY4 plasmid contains aromatic degradation genes in putative mobile genetic element causing flexibility/instability to the pathway. The localisation of the aromatic biodegradation pathway in mobile genetic elements suggests that gene transfer and rearrangements are a competetive advantage for Sphingomonas bacteria in the environment.

Carbon balance and component CO2 fluxes in boreal Scots pine stands

The forest vegetation takes up atmospheric carbon dioxide (CO2) in photosynthesis. Part of the fixed carbon is released back into the atmosphere during plant respiration but a substantial part is stored as plant biomass, especially in the stems of trees. Carbon also accumulates in the soil as litter and via the roots. CO2 is released into the atmosphere from these carbon stocks in the decomposition of dead biomass. Carbon balance of a forest stand is the difference between the CO2 uptake and CO2 efflux. This study quantifies and analyses the dynamics of carbon balance and component CO2 fluxes in four Southern Finnish Scots pine stands that covered the typical economic rotation time of 80 years. The study was based on direct flux measurements with chambers and eddy covariance (EC), and modelling of component CO2 fluxes. The net CO2 exchange of the stand was partitioned into component fluxes: photosynthesis of trees and ground vegetation, respiration of tree foliage and stems, and CO2 efflux from the soil. The relationships between the component fluxes and the environmental factors (light, temperature, atmospheric CO2, air humidity and soil moisture) were studied with mathematical modelling. The annual CO2 balance varied from a source of about 400 g C/m2 at a recently clearcut site to net CO2 uptake of 200 300 g C/m2 in a middle-aged (40-year-old) and a mature (75-year-old) stand. A 12-year-old sapling site was at the turning point from source to a sink of CO2. In the middle-aged stand, photosynthetic production was dominated by trees. Under closed pine canopies, ground vegetation accounted for 10 20% of stand photosynthesis whereas at the open sites the proportion and also the absolute photosynthesis of ground vegetation was much higher. The aboveground respiration was dominated by tree foliage which accounted for one third of the ecosystem respiration. Rate of wood respiration was in the order of 10% of total ecosystem respiration. CO2 efflux from the soil dominated the ecosystem respiratory fluxes in all phases of stand development. Instantaneous and delayed responses to the environmental driving factors could predict well within-year variability in photosynthetic production: In the short term and during the growing season photosynthesis follows primarily light while the seasonal variation is more strongly connected to temperature. The temperature relationship of the annual cycle of photosynthesis was found to be almost equal in the southern boreal zone and at the timberline in the northern boreal zone. The respiratory fluxes showed instantaneous and seasonal temperature relationships but they could also be connected to photosynthesis at an annual timescale.

Hydrogen soil deposition and atmospheric variations in the boreal zone

This research has been prompted by an interest in the atmospheric processes of hydrogen. The sources and sinks of hydrogen are important to know, particularly if hydrogen becomes more common as a replacement for fossil fuel in combustion. Hydrogen deposition velocities (vd) were estimated by applying chamber measurements, a radon tracer method and a two-dimensional model. These three approaches were compared with each other to discover the factors affecting the soil uptake rate. A static-closed chamber technique was introduced to determine the hydrogen deposition velocity values in an urban park in Helsinki, and at a rural site at Loppi. A three-day chamber campaign to carry out soil uptake estimation was held at a remote site at Pallas in 2007 and 2008. The atmospheric mixing ratio of molecular hydrogen has also been measured by a continuous method in Helsinki in 2007 - 2008 and at Pallas from 2006 onwards. The mean vd values measured in the chamber experiments in Helsinki and Loppi were between 0.0 and 0.7 mm s-1. The ranges of the results with the radon tracer method and the two-dimensional model were 0.13 - 0.93 mm s-1 and 0.12 - 0.61 mm s-1, respectively, in Helsinki. The vd values in the three-day campaign at Pallas were 0.06 - 0.52 mm s-1 (chamber) and 0.18 - 0.52 mm s-1 (radon tracer method and two-dimensional model). At Kumpula, the radon tracer method and the chamber measurements produced higher vd values than the two-dimensional model. The results of all three methods were close to each other between November and April, except for the chamber results from January to March, while the soil was frozen. The hydrogen deposition velocity values of all three methods were compared with one-week cumulative rain sums. Precipitation increases the soil moisture, which decreases the soil uptake rate. The measurements made in snow seasons showed that a thick snow layer also hindered gas diffusion, lowering the vd values. The H2 vd values were compared to the snow depth. A decaying exponential fit was obtained as a result. During a prolonged drought in summer 2006, soil moisture values were lower than in other summer months between 2005 and 2008. Such conditions were prevailing in summer 2006 when high chamber vd values were measured. The mixing ratio of molecular hydrogen has a seasonal variation. The lowest atmospheric mixing ratios were found in the late autumn when high deposition velocity values were still being measured. The carbon monoxide (CO) mixing ratio was also measured. Hydrogen and carbon monoxide are highly correlated in an urban environment, due to the emissions originating from traffic. After correction for the soil deposition of H2, the slope was 0.49±0.07 ppb (H2) / ppb (CO). Using the corrected hydrogen-to-carbon-monoxide ratio, the total hydrogen load emitted by Helsinki traffic in 2007 was 261 t (H2) a-1. Hydrogen, methane and carbon monoxide are connected with each other through the atmospheric methane oxidation process, in which formaldehyde is produced as an important intermediate. The photochemical degradation of formaldehyde produces hydrogen and carbon monoxide as end products. Examination of back-trajectories revealed long-range transportation of carbon monoxide and methane. The trajectories can be grouped by applying cluster and source analysis methods. Thus natural and anthropogenic emission sources can be separated by analyzing trajectory clusters.

Two ex situ fungal technologies to treat contaminated soil

Wood-degrading fungi are able to degrade a large range of recalcitrant pollutants which resemble the lignin biopolymer. This ability is attributed to the production of lignin-modifying enzymes, which are extracellular and non-specific. Despite the potential of fungi in bioremediation, there is still an understanding gap in terms of the technology. In this thesis, the feasibility of two ex situ fungal bioremediation methods to treat contaminated soil was evaluated. Treatment of polycyclic aromatic hydrocarbons (PAHs)-contaminated marsh soil was studied in a stirred slurry-phase reactor. Due to the salt content in marsh soil, fungi were screened for their halotolerance, and the white-rot fungi Lentinus tigrinus, Irpex lacteus and Bjerkandera adusta were selected for further studies. These fungi degraded 40 - 60% of a PAH mixture (phenanthrene, fluoranthene, pyrene and chrysene) in a slurry-phase reactor (100 ml) during 30 days of incubation. Thereafter, B. adusta was selected to scale-up and optimize the process in a 5 L reactor. Maximum degradation of dibenzothiophene (93%), fluoranthene (82%), pyrene (81%) and chrysene (83%) was achieved with the free mycelium inoculum of the highest initial biomass (2.2 g/l). In autoclaved soil, MnP was the most important enzyme involved in PAH degradation. In non-sterile soil, endogenous soil microbes together with B. adusta also degraded the PAHs extensively, suggesting a synergic action between soil microbes and the fungus. A fungal solid-phase cultivation method to pretreat contaminated sawmill soil with high organic matter content was developed to enhance the effectiveness of the subsequent soil combustion. In a preliminary screening of 146 fungal strains, 28 out of 52 fungi, which extensively colonized non-sterile contaminated soil, were litter-decomposing fungi. The 18 strains further selected were characterized by their production of lignin-modifying and hydrolytic enzymes, of which MnP and endo-1,4-β-glucanase were the main enzymes during cultivation on Scots pine (Pinus sylvestris) bark. Of the six fungi selected for further tests, Gymnopilus luteofolius, Phanerochaete velutina, and Stropharia rugosoannulata were the most active soil organic matter degraders. The results showed that a six-month pretreatment of sawmill soil would result in a 3.5 - 9.5% loss of organic matter, depending on the fungus applied. The pretreatment process was scaled-up for a 0.56 m3 reactor, in which perforated plastic tubes filled with S. rugosoannulata growing on pine bark were introduced into the soil. The fungal pretreatment resulted in a soil mass loss of 30.5 kg, which represents 10% of the original soil mass (308 kg). Despite the fact that Scots pine bark contains several antimicrobial compounds, it was a suitable substrate for fungal growth and promoter of the production of oxidative enzymes, as well as an excellent and cheap natural carrier of fungal mycelium. This thesis successfully developed two novel fungal ex situ bioremediation technologies and introduce new insights for their further full-scale application. Ex situ slurry-phase fungal reactors might be applied in cases when the soil has a high water content or when the contaminant bioavailability is low; for example, in wastewater treatment plants to remove pharmaceutical residues. Fungal solid-phase bioremediation is a promising remediation technology to ex situ or in situ treat contaminated soil.