Hybridization Analysis of Microbial DNA from Fuel Oil–Contaminated and Noncontaminated Soil

Abstract The enrichment of several genes (xylE, nahAcd, todC1C2BA, tmoABCDE, alkB) that encode enzymes responsible for key steps in the degradation of hydrocarbons, and one gene specific to rRNA group I of the genus Pseudomonas, was studied in DNA extracted from a fuel oil–contaminated field site, and in laboratory microcosms (with the exception of alkB). Toluene, ethylbenzene, xylene, and naphthalene concentrations were related to the extent of hybridization of the genes in the field studies. Significant differences were observed in the extent of hybridization of some of the genes between contaminated and noncontaminated samples. In the microcosm studies, gasoline at rates ranging from 0.5 mg to 125 mg gasoline/g of soil as applied to soils, and the changes in hybridization intensity of these genes monitored with time. The lower threshold of gene enrichment of these genes in response to gasoline addition was below 0.5 mg/g soil. Small increases were observed at the 0.5-mg exposure level, but hybridization intensity quickly decreased to levels below detection 6–8 days after addition of the gasoline. A dose-response effect was observed from treatments with gasoline concentrations ranging from 0.5 to 35 mg/g soil. Inhibition by toxic components in gasoline was observed at 75 and 125 mg/g soil levels. Hybridization of the Pseudomonas group 1 probe to field DNA was not significantly enriched in the contaminated field site, although these sequences were enriched in the microcosm studies. Among the genes tested, xylE was the most sensitive indicator of low levels of fuel oil contamination. Received: 23 July 1996; Accepted 9 October 1996     

Aerobic Biodegradation of 2,4,6-Trinitrotoluene (TNT) by Bacillus cereus İsolated from Contaminated Soil

In this study, biological degradation of 2,4,6-trinitrotoluene (TNT) which is very highly toxic environmentally and an explosive in nitroaromatic character was researched in minimal medium by Bacillus cereus isolated from North Atlantic Treaty Organization (NATO) TNT-contaminated soils. In contrast to most previous studies, the capability of this bacteria to transform in liquid medium containing TNT was investigated. During degradation, treatment of TNT was followed by high-performance liquid chromatography (HPLC) and achievement of degradation was calculated as percentage. At an initial concentration of 50 and 75 mg L^?1, TNT was degraded respectively 68 % and 77 % in 96 h. It transformed into 2,4-dinitrotoluene and 4-aminodinitrotoluene derivates, which could be detected as intermediate metabolites by using thin-layer chromatography and gas chromatography–mass spectrometry analyses. Release of nitrite and nitrate ions were searched by spectrophotometric analyses. Depending upon Meisenheimer complex, while nitrite production was observed, nitrate was detected in none of the cultures. Results of our study propose which environmental pollutant can be removed by using microorganisms that are indigenous to the contaminated site.     

Bioremediation of Crude Oil–Contaminated Soil By Immobilized Bacteria on an Agroindustrial Waste—Sunflower Seed Husks

This study reports the immobilization and performance of a hydrocarbon-degrading Rhodococcus sp. strain (designated as QBTo) on sunflower seed husks (SH) for the bioremediation of soils polluted with crude oil. The SH performance as inoculants carrier was compared with peat, which is a vegetal material traditionally used in carrier-based inoculants production. The stability of the immobilized culture under storage conditions was assessed by viability at different times when stored at 25°C and 10°C. The catabolic activity of immobilized and free QTBo cells introduced into sandy loam soil, freshly contaminated with crude oil, was studied in microcosms. A higher number of viable QTBo cells were recovered from the inoculants formulated with SH (QTBo-SH) after prolonged storage at 10°C and 25°C. The microcosms amended with QTBo-SH inoculants showed a removal of about 66% of total petroleum hydrocarbons (TPH), whereas in those inoculated with QTBo-peat inoculants, the decrease was of about 47%. In the control microcosms (noninoculated) and liquid culture–amended soils, the TPH removal was about 28%. SH is a waste of edible oil industry, nontoxic, and biodegradable and has demonstrated to confer to the immobilized cultures greater potential to survive not only during storage but also in the soil environment, improving bioremediation process.     

Pyrene Biodegradation by Bacillus spp. Isolated from Coal Tar–Contaminated Soil

Aerobic, mesophilic bacteria from coal tar–contaminated soil were analyzed for pyrene utilization capacity and identified by 16S ribosomal DNA sequencing as members of three genera: Bacillus spp., Pseudomonas sp., and Rhodococcus sp. The soil contained nine different hazardous polyaromatic hydrocarbons (PAHs): benzo[g, h, i]perylene, dibenzo[a, h]anthracene, indeno[1,2,3-c,d]pyrene, pyrene, acenaphthylene, fluorene, phenanthrene, benzo[k]fluoranthene, and benzo[b]fluoranthene. Bacillus spp. (PK-6) MTCC 1005 showed 56.4% utilization of pyrene (C₁₆H₁₀) (50 μg ml^?1) in 4 days, with growth associated biosurfactant activity and resulted in the formation of five new intermediates: phenanthrene (C₁₄H₁₀), 9,10-diphenylphenanthrene (C₂₆H₁₈), 9-methoxyphenanthrene (C₁₅H₁₂O), 5,6,7,8-tetrahydro-1-naphthoic acid (C₁₁H₁₂O₂), and 1,6,7-trimethylnaphthalene (C₁₃H₁₄). The results suggested that Bacillus spp. could be found suitable for practical field application for effective in situ PAH bioremediation.     

Can Physiological Endpoints Improve the Sensitivity of Assays with Plants in the Risk Assessment of Contaminated Soils?

Site-specific risk assessment of contaminated areas indicates prior areas for intervention, and provides helpful information for risk managers. This study was conducted in the Ervedosa mine area (Bragança, Portugal), where both underground and open pit exploration of tin and arsenic minerals were performed for about one century (1857 – 1969). We aimed at obtaining ecotoxicological information with terrestrial and aquatic plant species to integrate in the risk assessment of this mine area. Further we also intended to evaluate if the assessment of other parameters, in standard assays with terrestrial plants, can improve the identification of phytotoxic soils. For this purpose, soil samples were collected on 16 sampling sites distributed along four transects, defined within the mine area, and in one reference site. General soil physical and chemical parameters, total and extractable metal contents were analyzed. Assays were performed for soil elutriates and for the whole soil matrix following standard guidelines for growth inhibition assay with Lemna minor and emergence and seedling growth assay with Zea mays. At the end of the Z. mays assay, relative water content, membrane permeability, leaf area, content of photosynthetic pigments (chlorophylls and carotenoids), malondialdehyde levels, proline content, and chlorophyll fluorescence (F_v/F_m and Φ_PSII) parameters were evaluated. In general, the soils near the exploration area revealed high levels of Al, Mn, Fe and Cu. Almost all the soils from transepts C, D and F presented total concentrations of arsenic well above soils screening benchmark values available. Elutriates of several soils from sampling sites near the exploration and ore treatment areas were toxic to L. minor, suggesting that the retention function of these soils was seriously compromised. In Z. mays assay, plant performance parameters (other than those recommended by standard protocols), allowed the identification of more phytotoxic soils. The results suggest that these parameters could improve the sensitivity of the standard assays.     

Do Current Standards of Practice in Canada Measure What is Relevant to Human Exposure at Contaminated Sites? I: A Discussion of Soil Particle Size and Contaminant Partitioning in Soil

Human health risk estimates for sites with contaminated soils are often based on the assumption that the bulk concentration of substances in outdoor soil samples is a reasonable predictor of exposures via incidental soil ingestion, soil particle inhalation, and dermal absorption. Most underlying conceptual models are grossly simplistic, however, when considered in light of (i) biases in the distribution of contaminants across soil particle sizes, (ii) the size range of particles in soils and dusts that is environmentally available, and (iii) factors that influence desorption from particles and uptake into humans. The available studies indicate that contaminant distribution across soil particle size fractions varies widely between different soil types and contaminant delivery mechanisms, and it cannot be assumed that higher masses of contaminants per unit mass of soil are correlated with smaller particles sizes. Soil data gathered in support of detailed human health risk assessments, therefore, should allow for the examination of distribution across particle sizes of contaminants of concern, and consider those size fractions most critical to human exposure. Soil evaluations for health risk assessments of metals/metalloids should also consider mineralogical characterization.     

Soil Drench Treatment with ?-Aminobutyric Acid Increases Drought Tolerance of Potato

The non-protein amino acid β-aminobutyric acid (BABA) is known to be a priming agent for a more efficient activation of cellular defence responses and a potent inducer of resistance against biotic and abiotic stresses in plants. Nevertheless, most of the studies on priming have been carried out in Arabidopsis. In potato, the effect of BABA was demonstrated only on biotic stress tolerance. We investigated the effect of BABA on the drought tolerance of potato and found that soil drenched with BABA at a final concentration of 0.3 mM improves the drought tolerance of potato. Water loss from the leaves of the primed plants is attenuated and the yield is increased compared to the unprimed drought-stressed plants. The metabolite composition of the tubers of the BABA-treated plants is less affected by drought than the tuber composition of the non-treated plants. Nitric oxide and ROS (reactive oxygen species) production is increased in the BABA-treated roots but not in the leaves. In the leaves of the BABA-treated plants, the expression of the drought-inducible gene StDS2 is delayed, but the expression of ETR1, encoding an ethylene receptor, is maintained for a longer period under the drought conditions than in the leaves of the non-treated, drought-stressed control plants. This result suggests that the ethylene-inducible gene expression remains suppressed in primed plants leading to a longer leaf life and increased tuber yield compared to the non-treated, drought-stressed plants. The priming effect of BABA in potato, however, is transient and reverts to an unprimed state within a few weeks.     

Dielectric Properties Based Detection of Heavy Metal Contaminated Soil in the Frequency Range from 10 MHz to 1 GHz

Physical parameters based electromagnetic methods are promising technologies to detect contaminated sites. In these methods, the dielectric property is a key parameter. In this paper, we studied the dielectric characteristics of heavy metal contaminated soil. The chromium contaminated soil was made into samples, and the open-ended coaxial line was adopted as the measurement method. Experiments were conducted in the frequency band between 10 MHz and 1 GHz. The results showed that the complex permittivity, including the real part and the imaginary part, changes as the ionic content changes. Especially, at low frequencies (<50 MHz), the complex permittivity increases significantly with the increase of ionic content. In addition, it also could be seen that the water content of the soil also affects the complex permittivity. We proposed to adopt the drying method or the Time Domain Reflection method to determine the water content. The dielectric parameters are most affected by the ionic content after knowing the water content. Therefore, it is feasible to detect heavy metal contaminated sites based on dielectric properties.     

Bioremediation of Cadmium-Contaminated Soil through Cultivation of Maize Inoculated with Plant Growth–Promoting Rhizobacteria

A pot experiment was conducted to study the effect of single and co-inoculation of Bacillus mycoides and Micrococcus roseus strains, indigenous to heavy metal (HM)–contaminated soils, on the growth and essential-nutrient and Cd uptake of maize in a soil polluted with 100 and 200 mg Cd kg^?1. Increasing Cd levels significantly decreased shoot and root dry weights, and shoot P, Fe, Zn, and Mn uptake. All bacterial treatments significantly increased biomass and shoot nutrient uptake of plant compared with control in the soil polluted with Cd. Inoculation of plants with B. mycoides and consortium of two bacteria significantly increased, whereas M. roseus significantly decreased, shoot and root Cd uptake, and Cd transfer and translocation factors compared with control in Cd-polluted conditions. The results showed that B. mycoides and consortium of two bacteria had an effective role in phytoextraction and M. roseus was the most effective treatment in phytostabilization of Cd.     

Predicting Soil Salinity with Vis–NIR Spectra after Removing the Effects of Soil Moisture Using External Parameter Orthogonalization

Robust models for predicting soil salinity that use visible and near-infrared (vis–NIR) reflectance spectroscopy are needed to better quantify soil salinity in agricultural fields. Currently available models are not sufficiently robust for variable soil moisture contents. Thus, we used external parameter orthogonalization (EPO), which effectively projects spectra onto the subspace orthogonal to unwanted variation, to remove the variations caused by an external factor, e.g., the influences of soil moisture on spectral reflectance. In this study, 570 spectra between 380 and 2400 nm were obtained from soils with various soil moisture contents and salt concentrations in the laboratory; 3 soil types × 10 salt concentrations × 19 soil moisture levels were used. To examine the effectiveness of EPO, we compared the partial least squares regression (PLSR) results established from spectra with and without EPO correction. The EPO method effectively removed the effects of moisture, and the accuracy and robustness of the soil salt contents (SSCs) prediction model, which was built using the EPO-corrected spectra under various soil moisture conditions, were significantly improved relative to the spectra without EPO correction. This study contributes to the removal of soil moisture effects from soil salinity estimations when using vis–NIR reflectance spectroscopy and can assist others in quantifying soil salinity in the future.     

Stabilization of Soil Organic Matter: Association with Minerals or Chemical Recalcitrance?

Soil organic matter (OM) can be stabilized against decomposition by association with minerals, by its inherent recalcitrance and by occlusion in aggregates. However, the relative contribution of these factors to OM stabilization is yet unknown. We analyzed pool size and isotopic composition (¹⁴C, ¹³C) of mineral-protected and recalcitrant OM in 12 subsurface horizons from 10 acidic forest soils. The results were related to properties of the mineral phase and to OM composition as revealed by CPMAS ¹³C-NMR and CuO oxidation. Stable OM was defined as that material which survived treatment of soils with 6 wt% sodium hypochlorite (NaOCl). Mineral-protected OM was extracted by subsequent dissolution of minerals by 10% hydrofluoric acid (HF). Organic matter resistant against NaOCl and insoluble in HF was considered as recalcitrant OM. Hypochlorite removed primarily ¹⁴C-modern OM. Of the stable organic carbon (OC), amounting to 2.4–20.6 g kg⁻¹ soil, mineral dissolution released on average 73%. Poorly crystalline Fe and Al phases (Fe_o, Al_o) and crystalline Fe oxides (Fe_d−o) explained 86% of the variability of mineral-protected OC. Atomic C_p/(Fe+Al)_p ratios of 1.3–6.5 suggest that a portion of stable OM was associated with polymeric Fe and Al species. Recalcitrant OC (0.4–6.5 g kg⁻¹ soil) contributed on average 27% to stable OC and the amount was not correlated with any mineralogical property. Recalcitrant OC had lower Δ¹⁴C and δ¹³C values than mineral-protected OC and was mainly composed of aliphatic (56%) and O-alkyl (13%) C moieties. Lignin phenols were only present in small amounts in either mineral-protected or recalcitrant OM (mean 4.3 and 0.2 g kg⁻¹ OC). The results confirm that stabilization of OM by interaction with poorly crystalline minerals and polymeric metal species is the most important mechanism for preservation of OM in these acid subsoil horizons.     

Accumulation of Hydrocarbons by Maize (Zea mays L.) in Remediation of Soils Contaminated with Crude Oil

This study has investigated the use of screened maize for remediation of soil contaminated with crude oil. Pots experiment was carried out for 60 days by transplanting maize seedlings into spiked soils. The results showed that certain amount of crude oil in soil (≤2 147 mg·kg^?1) could enhance the production of shoot biomass of maize. Higher concentration (6 373 mg·kg^?1) did not significantly inhibit the growth of plant maize (including shoot and root). Analysis of plant shoot by GC-MS showed that low molecular weight polycyclic aromatic hydrocarbons (PAHs) were detected in maize tissues, but PAHs concentration in the plant did not increase with higher concentration of crude oil in soil. The reduction of total petroleum hydrocarbon in planted soil was up to 52.21–72.84%, while that of the corresponding controls was only 25.85–34.22% in two months. In addition, data from physiological and biochemical indexes demonstrated a favorable adaptability of maize to crude oil pollution stress. This study suggested that the use of maize (Zea mays L.) was a good choice for remediation of soil contaminated with petroleum within a certain range of concentrations.     

Remediation and Safe Production of cd Contaminated Soil Via Multiple Cropping Hyperaccumulator Solanum nigrum L. and Low Accumulation Chinese Cabbage

Multiple crop experiment of hyperaccumulator Solanum nigrum L. with low accumulation Chinese cabbage Fenyuanxin 3 were conducted in a cadmium (Cd) contaminated vegetable field. In the first round, the average removal rate of S. nigrum to Cd was about 10% without assisted phytoextraction reagent addition for the top soil (0–20 cm) with Cd concentration at 0.53–0.97 mg kg^?1 after its grew 90 days. As for assisted phytoextraction reagent added plots, efficiency of Cd remediation might reach at 20%. However, in the second round, Cd concentration in Chinese cabbage was edible, even in the plots with assisted phytoextraction reagent added. Thus, multiple cropping hyperaccumulator with low accumulation crop could normally remediate contaminated soil and produce crop (obtain economic benefit) in one year, which may be one practical pathway of phytoremediating heavy metal contaminated soil in the future.     

Microevolution of Nodule Bacteria upon Generation of Mutants with Altered Survival in the Plant–Soil System

Simulation of cyclic processes in the plant–soil system was used to analyze the effects of factors responsible for the population dynamics of rhizobia on generation of mutants with changedex planta viability. Rhizobial evolution in a system of ecological niches (soil, rhizosphere, nodules) was described with recurrent equations. Computer experiments were carried out with parameters determining the mutation pressure, selection, and amplitude of the population wave arising in soil on the release of bacteria from nodules and the rhizosphere. Analysis of the model showed that (1) mutants with enhanced ex planta viability do not completely replace the parental strain and (2) mutants with impaired ex planta viability may be fixed in the population. The maintenance of genotypes subject to elimination from the soil and rhizosphere by Darwinian selection was associated with frequency-dependent selection (FDS), which is effective in competition for nodulation. The FDS index was proposed to characterize FDS pressure and was shown to determine the population polymorphism for adaptive traits. An increase in population wave amplitude proved to increase the fixation level (the proportion in the limiting state of the system) of mutants with enhanced viability and to decrease it in mutants with low viability. The results obtained with the model agreed with the data that, in edaphic stress, rhizobial populations remain highly polymorphic, which is associated with the maintenance of sensitive strains. The simulation procedure may be employed in estimating the genetic consequences of introduction of modified rhizobial strains in the environment.     

Do Current Standards of Practice in Canada Measure What is Relevant to Human Exposure at Contaminated Sites? II: Oral Bioaccessibility of Contaminants in Soil

Human health risk estimates for sites with contaminated soils are often based on the assumption that the outdoor soil sieved to < 250 μm is a reasonable surrogate for predicting exposures via incidental soil ingestion. In vitro bioaccessibility tests are also increasingly used to “improve” ingestion exposure predictions of contaminants at different sites. However, when considered in terms of factors that influence desorption of contaminants from particles and uptake into humans, available studies indicate that current “standards of practice” with respect to assays of oral bioaccessibility have a number of significant shortcomings, at least in Canada. These shortcomings are discussed and various factors that influence the assessment of bioaccessibility of contaminants in soils are examined. We finish with proposing some minimum data submission requirements to support the application and relevance of bioaccessibility assays at contaminated sites.     

Azo Dye Decolorization under Microaerophilic Conditions by a Bacterial Mixture Isolated from Anthropogenic Dye‐Contaminated Soil

Soil samples isolated from dye-contaminated sites were exploited for isolation of dye decolorizing microorganisms. A novel bacterial mixture, RkNb1, was selected based on its efficiency, showing maximum and faster decolorization of textile dyes. Seven bacterial strains were isolated and identified from the bacterial mixture as Ochrobactrum intermedium (HM480365), Ochrobactrum intermedium strain M16-10-4 (HM030758), Enterococcus faecalis (HM480367), Arthrobacter crystallopoietes (HM480368), Kocuria flavus (HM480369), Bacillus beijingensis (HM480370), and Citrobacter freundii (HM480371) by 16S rRNA gene sequence analysis. This bacterial mixture showed 98.17% decolorization of Reactive Violet 5 (400 mg L^?1) within 8 h. The culture exhibited good decolorization ability at pH 8 and at a temperature of 37°C. Malt extract and peptone was found to enhance the decolorization rate of Reactive Violet 5. Plackett-Burman experimental design was used for elucidation of medium components affecting Reactive Violet 5 decolorization. Dye degradation products obtained during the course of decolorization were analyzed by high-performance thin-layer chromatography (HPTLC), Fourier transform infrared (FTIR), and nuclear magnetic resonance (NMR). The potential of this bacterial mixture to decolorize Reactive Violet 5 dye from manufacturing industry effluent is to be carried out using appropriate bioreactors.     

Biogenic Catalysis of Soil Formation on Mars?

The high iron abundance and the weak ferric iron spectral features of martian surface material are consistent with nanophase (nm-sized) iron oxide minerals as a major source of iron in the bright region soil on Mars. Nanophase iron oxide minerals, such as ferrihydrite and schwertmannite, and nanophase forms of hematite and goethite are formed by both biotic and abiotic processes on Earth. The presence of these minerals on Mars does not indicate biological activity on Mars, but it does raise the possibility. This work includes speculation regarding the possibility of biogenic soils on Mars based on previous observations and analyses. A remote sensing goal of upcoming missions should be to determine if nanophase iron oxide minerals, clay silicates and carbonates are present in the martian surface material. These minerals are important indicators for exobiology and their presence on Mars would invoke a need for further investigation and sample return from these sites.     

Tracking Functional Guilds: “Dehalococcoides” spp. in European River Basins Contaminated with Hexachlorobenzene

Hexachlorobenzene (HCB) has been widely used in chemical manufacturing processes and as a pesticide. Due to its resistance to biological degradation, HCB has mainly accumulated in freshwater bodies and agricultural soils. “Dehalococcoides” spp., anaerobic dechlorinating bacteria that are capable of degrading HCB, were previously isolated from river sediments. Yet there is limited knowledge about the abundance, diversity, and activity of this genus in the environment. This study focused on the molecular analysis of the composition and abundance of active Dehalococcoides spp. in HCB-contaminated European river basins. 16S rRNA-based real-time quantitative PCR and denaturing gradient gel electrophoresis in combination with multivariate statistics were applied. Moreover, a functional gene array was used to determine reductive dehalogenase (rdh) gene diversity. Spatial and temporal fluctuations were observed not only in the abundance of Dehalococcoides spp. but also in the composition of the populations and rdh gene diversity. Multivariate statistics revealed that Dehalococcoides sp. abundance is primarily affected by spatial differences, whereas species composition is under the influence of several environmental parameters, such as seasonal changes, total organic carbon and/or nitrogen content, and HCB contamination. This study provides new insight into the natural occurrence and dynamics of active Dehalococcoides spp. in HCB-contaminated river basins.Halogenated organic compounds are among the most widespread environmental pollutants. Although these compounds were previously believed to be only anthropogenic, a large number of them, including aliphatic, aromatic, and heterocyclic derivatives, are introduced into the environment via biogenic and geogenic sources (9, 21). Hexachlorobenzene (HCB) is believed to be persistent in the environment (22) due to its chemical stability and its resistance to biodegradation. HCB is a hydrophobic and bioaccumulative compound and is listed in the EC Directive (15) as a “priority hazardous substance.” At the peak production of HCB in the early 1980s, thousands of tons were produced to be used as fungicides, wood preservatives, and porosity control agents or in the manufacturing of dyes. The use of HCB is no longer allowed in most countries because of its toxicity and carcinogenicity toward fish and mammals. Nevertheless, it is still being released into the environment as a by-product of various chemical processes, as a result of incomplete combustion, or from old landfills (4, 6, 7). HCB contamination has been reported in different environments. Compared to rivers in sparsely populated regions, lakes, and the sea (32, 42), significantly larger amounts of HCB could be found in river water in agricultural areas and in densely populated or highly industrialized areas. HCB concentrations were shown to positively correlate with organic matter content of sediments and soils, and European soils were observed to have the highest HCB concentrations globally (38). Several authors reported on the fate and behavior of HCB in the environment on regional or global scales. Nevertheless, our knowledge of microbial degradation of this compound in natural environments remains limited. It has been shown that HCB from air and water bodies can be removed via physical processes like volatilization and photolysis (6, 43). Adsorption also plays an important role in the removal of HCB from aquatic environments but in turn results in deposition in sediments. In these light-limited environments, biodegradation offers great potential for transforming this persistent organic pollutant (7, 29). The only known pathway for microbial dehalogenation of HCB is reductive dechlorination under anaerobic conditions, which results in formation of less chlorinated benzenes (1).The reductively dechlorinating bacteria isolated up to now belong to the Deltaproteobacteria and Epsilonproteobacteria (Geobacter, Sulfurospirillum, Desulfuromonas, and Desulfomonile), the Firmicutes (Desulfitobacterium and Dehalobacter), or the Chloroflexi (“Dehalococcoides” and related groups) (51). So far, however, Dehalococcoides is the only bacterial genus whose members are known to transform HCB. Several Dehalococcoides strains that could grow with a broad variety of chlorinated aliphatic and aromatic compounds, including chlorinated benzenes and phenols, biphenyls, chloroethenes, and dioxins, were isolated. Nevertheless, until now only two strains, Dehalococcoides sp. strain CBDB1 (3) and “Dehalococcoides ethenogenes” 195 (17), which can transform HCB to tri- and dichlorobenzenes and use the energy conserved in the process for growth, could be isolated. Besides HCB, Dehalococcoides sp. strain CBDB1 can also reductively dechlorinate chlorinated dioxins (11) and chlorophenols (2), whereas Dehalococcoides ethenogenes 195 can dechlorinate various chlorinated ethenes, 1,2-dichloroethane, and vinyl chloride (37).Until now, microbial community analyses of Dehalococcoides spp. largely focused on chlorinated ethene-contaminated aquifers or soils. The presence of Dehalococcoides spp. in uncontaminated and contaminated (with tetrachloroethene [PCE], trichloroethene [TCE], or vinyl chloride) sites from North America, Europe, and Japan was reported elsewhere (24, 26, 30, 34, 60). Furthermore, quantitative analyses targeting the Dehalococcoides 16S rRNA gene in chlorinated ethene bioremediation sites showed that 8.6 × 10³ to 2.5 × 10⁶ copies/g aquifer material (33) and 1.9 × 10² to 1.1 × 10⁷ copies/g soil (50) could be detected depending on the type of treatment applied. Although reductive dechlorination by Dehalococcoides spp. is an energy-yielding process, microcosm studies conducted under controlled environmental conditions showed that growth of the organisms is relatively slow (28). Moreover, the presence of other halorespiring species may result in competition for chlorinated compounds or electron donors. This may adversely affect the success of the reductive dechlorination of HCB in natural environments. Hence, monitoring of the indigenous dechlorinating species is needed to understand their diversity and activity in contaminated sites.The aim of this study was to assess the diversity of active Dehalococcoides spp. in HCB-polluted river basins and to reveal the links between species composition and abundance with changing environmental parameters, using 16S rRNA and reductive dehalogenase-encoding gene-targeted molecular analyses, in combination with multivariate statistics. River sediment, floodplain, and agricultural soil samples were collected from two European rivers, the Ebro (Spain) and the Elbe (Germany), between 2004 and 2006. This study provides new insights on the natural occurrence and dynamics of reductively dechlorinating bacteria, generating important knowledge toward understanding and predicting microbial HCB transformation.     

Uptake and Distribution of Soil Applied Zinc by Citrus Trees—Addressing Fertilizer Use Efficiency with 68Zn Labeling

The zinc (Zn) supply increases the fruit yield of Citrus trees that are grown, especially in the highly weathered soils of the tropics due to the inherently low nutrient availability in the soil solution. Leaf sprays containing micronutrients are commonly applied to orchards, even though the nutrient supply via soil could be of practical value. This study aimed to evaluate the effect of Zn fertilizers that are applied to the soil surface on absorption and partitioning of the nutrient by citrus trees. A greenhouse experiment was conducted with one-year-old sweet orange trees. The plants were grown in soils with different textures (18.1 or 64.4% clay) that received 1.8 g Zn per plant, in the form of either ZnO or ZnSO₄ enriched with the stable isotope ⁶⁸Zn. Zinc fertilization increased the availability of the nutrient in the soil and the content in the orange trees. Greater responses were obtained when ZnSO₄ was applied to the sandy loam soil due to its lower specific metal adsorption compared to that of the clay soil. The trunk and branches accumulated the most fertilizer-derived Zn (Zn_dff) and thus represent the major reserve organ for this nutrient in the plant. The trees recovered up to 4% of the applied Zn_dff. Despite this relative low recovery, the Zn requirement of the trees was met with the selected treatment based on the total leaf nutrient content and increased Cu/Zn-SOD activity in the leaves. We conclude that the efficiency of Zn fertilizers depends on the fertilizer source and the soil texture, which must be taken into account by guidelines for fruit crop fertilization via soil, in substitution or complementation of traditional foliar sprays.