A comparative analysis of endophytic bacterial communities associated with hyperaccumulators growing in mine soils

Interactions between endophytic bacterial communities and hyperaccumulators in heavy metal-polluted sites are not fully understood. In this study, the diversity of stem-associated endophytic bacterial communities of two hyperaccumulators (Solanum nigrum L. and Phytolacca acinosa Roxb.) growing in mine soils was investigated using molecular-based methods. The denaturing gradient gel electrophoresis (DGGE) analysis showed that the endophytic bacterial community structures were affected by both the level of heavy metal pollution and the plant species. Heavy metal in contaminated soil determined, to a large extent, the composition of the different endophytic bacterial communities in S. nigrum growing across soil series (five sampling spots, and the concentration of Cd is from 0.2 to 35.5 mg/kg). Detailed analysis of endophytic bacterial populations by cloning of 16S rRNA genes amplified from the stems of the two plants at the same site revealed a different composition. A total of 51 taxa at the genus level that included α-, β-, and γ-Proteobacteria (68.8% of the two libraries clones), Bacteroidetes (9.0% of the two libraries clones), Firmicutes (2.0% of the two libraries clones), Actinobacteria (16.4% of the two libraries clones), and unclassified bacteria (3.8% of the two libraries clones) were found in the two clone libraries. The most abundant genus in S. nigrum was Sphingomonas (23.35%), while Pseudomonas prevailed in P. acinosa (21.40%). These results suggest that both heavy metal pollution and plant species contribute to the shaping of the dynamic endophytic bacterial communities associated with stems of hyperaccumulators.     

Fungicide impacts on microbial communities in soils with contrasting management histories

The impacts of the fungicides azoxystrobin, tebuconazole and chlorothalonil on microbial properties were investigated in soils with identical mineralogical composition, but possessing contrasting microbial populations and organic matter contents arising from different management histories. Degradation of all pesticides was fastest in the high OM/biomass soil, with tebuconazole the most persistent compound, and chlorothalonil the most readily degraded. Pesticide sorption distribution coefficient (K(d)) did not differ significantly between the soils. Chlorothalonil had the highest K(d) (97.3) but K(d) for azoxystrobin and tebuconazole were similar (13.9 and 12.4, respectively). None of the fungicides affected microbial biomass in either soil. However, all fungicides significantly reduced dehydrogenase activity to varying extents in the low OM/biomass soil, but not in the high OM/biomass soil. The mineralization of subsequent applications of herbicides, which represents a narrow niche soil process was generally reduced in both soils by azoxystrobin and chlorothalonil. 16S rRNA-PCR denaturing gradient gel electrophoresis (DGGE) indicated that none of the fungicides affected bacterial community structure. 18S rRNA PCR-DGGE analysis revealed that a small number of eukaryote bands were absent in certain fungicide treatments, with each band being specific to a single fungicide-soil combination. Sequencing indicated these represented protozoa and fungi. Impacts on the specific eukaryote DGGE bands showed no relationship to the extent to which pesticides impacted dehydrogenase or catabolism of herbicides.     

Measurement of symbiotic nitrogen-fixation in leguminous host-plants grown in heavy metal-contaminated soils amended with sewage sludge

Rates of nitrogen fixation by Rhizobium in symbiosis with leguminous host-plants including white clover, broad bean and peas have been established in soils that have been amended experimentally with heavy metal-contaminated sewage sludges. Results from 15N-dilution experiments for the measurement of N2 fixation have shown that adverse heavy metal effects are apparent on symbiotic N2 fixation rates for white clover grown in inter-specific competition with ryegrass under mixed sward conditions, compared to white clover grown in pure sward. Further experiments on broad bean and pea indicated a significant, but minor-inhibitory metal-related effect on the rate of N2 fixation compared to untreated soils and soils amended with a relatively uncontaminated sludge. The implications of the results with respect to sludge utilisation in agriculture are discussed.     

Interactions of mycorrhizal fungi with Pteris vittata (As hyperaccumulator) in As-contaminated soils

A greenhouse trial was conducted to investigate the role of arbuscular mycorrhizas (AM) in aiding arsenic (As) uptake and tolerance by Pteris vittata (As hyperaccumulator) and Cynodon dactylon (a multi-metal root accumulator). Plants inoculated with lived and killed native mycorrhizas isolated from an As mine site were grown in a sterile and slightly acidic soil. The infectious percentage of mycorrhizas (0 mg/kg As: 26.4%, 50 mg/kg As: 30.3%, 100 mg/kg As: 40.6%) and the average biomass of shoots in infected P. vittata increased (0 mg/kg As: 2.45 g/pot, 50 mg/kg As: 2.48 g/pot, 100 mg/kg As: 10.9 g/pot) according to the increase of As levels when compared to control. The indigenous mycorrhizas enhanced As accumulation (0 mg/kg As: 3.70 mg/kg, 50 mg/kg As: 58.3 mg/kg; 100 mg/kg As: 88.1 mg/kg) in the As mine populations of P. vittata and also sustained its growth by aiding P absorption. For C. dactylon, As was mainly accumulated in mycorrhizal roots and translocation to shoots was inhibited.     

Microbial communities in pesticide-contaminated soils in Kyrgyzstan and bioremediation possibilities

In Kyrgyzstan, many former storehouses and dump sites for obsolete pesticides exist. In 2009/2010, an inventory and assessment of these sites including risks of environmental hazard has been conducted by FAO and the World Bank. Monitoring revealed high concentration of pesticides listed as persistent organic pollutants (POPs). The purpose of this research was to study the microbial structural complexes of the pesticide-contaminated soils in these dumping zones, and to search for and select microorganism’s destructors with cytochrome P450 genes for pesticide degradation. Culture-dependent and culture-independent approaches were used to determine the taxonomic composition of these bacterial communities. The universal primer set for the 16S ribosomal RNA (rRNA) gene and the specific primer set P450R were used to amplify the cytochrome P450 hydroxylase gene. In soils from Suzak A and B and soils from Balykchy dumping sites, the bacteria from the Actinobacteria phylum (Micrococcus genus) were dominant. These bacteria made up 32–47% of the indigenous local microflora; bacteria species from the Pseudomonas genus (Gammaproteobacteria phylum) made up 23% in Suzak, 12% in Balykchy soils. Bacillus species from the Firmicutes phylum were found only in Suzak soils. The 16S rRNA analyses and the specific primer set P450R have revealed bacteria with cytochrome genes which are directly involved in the degradation process of organic carbon compounds. Experiments were carried out to help select active degraders from the bacterial populations isolated and used to degrade Aldrin in laboratory. Active bacterial strains from the Pseudomonas fluorescens and Bacillus polymyxa population were selected which demonstrated high rates of degradation activity on Aldrin.     

Inactivation of fungi associated with barley grain by gaseous ozone

The use of gaseous ozone as a fungicide to preserve stored barley was studied. The effects of the following operating parameters on the fungicidal efficacy of ozone were examined: 1) the applied ozone dose, 2) ozonation time, 3) water activity of barley, and 4) temperature of barley. The effect of ozonation on germination of barley was also investigated. The experimental results showed that ozone was very effective in inactivation of fungi associated with the barley regardless of whether the fungi were in the forms of spores or mycelia. However, the mycelia were less resistant to ozone. With 5 minutes of ozonation, 96% of inactivation were achieved for spores as well as for mixtures of spores and small amount of mycelia by applying 0.16 and 0.10 mg of ozone/(g barley) x min, respectively. In addition, for sealed storage silos, inactivation of fungi continued when the ozone-containing gas was held inside the silos following a continuous ozone supply. The experimental results also revealed that increases in water activity and temperature of barley enhanced the fungicidal efficacy of ozone. Results of this study also indicated that the inactivation processes could be controlled by simply monitoring the exit ozone from the reactor instead of performing the time-consuming microbial examination. This finding would make the application of ozone in the preservation of cereal grains easier, simpler, and more practically applicable. The experimental results demonstrated that although ozonation above certain strength may reduce barley germination, inactivation of fungi was achieved with ozonation strengths far below the critical point.     

The influences of fly ash on stabilization for Cd in contaminated soils

Soil contaminated with potentially toxic metals (PTMs) has being a global environmental issue, which needs to be addressed on the priority basis. Fly ash (FA) is a kind of low-cost alkaline materials, which has been widely used in remediation of soil contaminated by PTMs, while the effects of FA on the stability for PTMs in contaminated farmland soil are still not clearly evaluated. In this study, cadmium (Cd) contaminated soil samples, collected from Shaanxi (SX), Hubei (HB), and Zhejiang (ZJ) province of China, were amended with FA addition (0, 1%, 2.5%, 5%, and 10% dose), and 1-year changes of Cd availability in soil samples were focused on. In addition, biological assessment method through pot culture was carried out to evaluate the reuse potential of Cd contaminated soils amended by FA. The result indicated that FA had a notable impact on decreasing the Cd mobility of SX soil (sand type), with 18.2~52.1% reduction in the DTPA extractable solution, followed by HB soil with 5.9~16.7% reduction, but no obvious effect of FA on ZJ soil (clay type) was observed. Furthermore, the results of pot experiment revealed that FA application could increase the biomass of Chinese cabbage. However, the DTPA extractable Cd in soils after planation and the Cd accumulation of plant increased. The results revealed that FA was not a promising soil stabilizer to immobilize HMs in Cd contaminated soil, and careful consideration should be given to Cd contaminated soils with FA restoration especially in their using for farmland productive due to the remaining risk of Cd bioavailability. These results also contributed to provide references for similar soil pollution remediation.

     

Dynamic of inorganic nitrogen and amino sugar to glucosamine addition in forest soils

Environmental Science and Pollution Research - Amino sugars (AS) are routinely used as microbial biomarkers to investigate the dynamics of soil carbon (C) and nitrogen (N) under different...     

Effects of soil attributes and straw accumulation on the sorption of hexazinone and tebuthiuron in tropical soils cultivated with sugarcane

Brazil is the largest sugarcane producer in the world in which hexazinone (3-cyclohexyl-6-dimethylamino-1-methyl-1,3,5-triazine-2,4-dione) and tebuthiuron (1-(5-tert-butyl-1,3,4-thiadiazol-2-yl)-1,3-dimethylurea) are heavily used. Sugarcane harvesting is changing from the manual system with previous straw burning to the mechanized system without straw burning. The lack of burning results in soil organic carbon accumulation mainly in clayey soils, which should affect herbicides availability and fate. Therefore, we evaluated sorption of these herbicides in soil samples with and without straw burning. Both herbicides presented low apparent sorption coefficients (mean K_d,app= 0.6 and 2.4 L kg^?1 for hexazinone and tebuthiuron, respectively), suggesting that they may leach to groundwater. Moreover, their sorption correlated primarily with soil organic carbon (SOC), but iron oxide contents extracted with ammonium oxalate (Fe₂O₃^AOX) also affected it (K_d,app = ?0.228 + 0.0397 SOC + 0.117 Fe₂O₃^AOX for hexazinone and K_d,app = ?1.407 + 0.201 SOC + 0.348 Fe₂O₃^AOX for tebuthiuron). Soil organic carbon accumulation due to straw maintenance in the field positively affected sorption of both herbicides, but its effects were not enough to classify them as “non-leachers.”     

Survival strategies of plants associated with arbuscular mycorrhizal fungi on toxic mine tailings

A field survey of metal concentrations and arbuscular mycorrhizal (AM) components of plants growing on five mining sites was conducted in Chenzhou City, Hunan Province, Southern China and a control site in Hong Kong. Significant differences were observed in the average concentrations of total heavy metals (Pb, Zn, Cu, Cd) and one metalloid (As) in contaminated soils compared with the control site. Gramineae and Compositae were the dominant plant families growing on mine tailings, with Chrysanthemum moritolium (common chrysanthemum), Cynodon dactylon (Bermuda grass), Miscanthus florodulus (Sword grass) and Pteris vittata (Ladder brake fern) commonly found at all sites. AM fungal colonization was detected in most of the plants. Comparing the four common plant species, three components of mycorrhizal colonization (arbuscules, vesicles and coiled hyphae) were found in the roots of C. dactylon and P. vittata growing at Do Shun Long (DSL) mine site. Concentrations of As in fronds were 24-fold higher than in roots of P. vittata with the highest mycorrhizal colonization rate (73%) among all sampling sites. Extensive mycorrhizal colonization (85%) was also recorded in the roots of C. dactylon with As accumulation 57 times higher than in shoots. The four common plants found in metal contaminated sites had developed different strategies for survival in the contaminated sites with the aid of indigenous AM fungi.     

Effect of phosphate addition on the sorption-desorption reaction of selenium in Japanese agricultural soils

Desorption levels of soil-sorbed selenium (Se) were studied by adding phosphate to 22 typical Japanese agricultural soils. Soil-soil solution distribution coefficients of Se (Kd-Se) were measured using a batch process as an index of Se sorption level, adding 75Se as a tracer. After the Kd measurement, extraction of soil-sorbed 75Se with a 0.1 M or 1 M Na2HPO4 solution followed to determine the amount of 75Se desorbed by the phosphate. When the 0.1 M Na2HPO4 solution was used, 18-70% of soil-sorbed Se was extracted (average: 47%). However, when the 1 M Na2HPO4 solution was used, 27-83% of soil-sorbed Se was extracted (average: 57%). The observed 75Se desorption percentage indicated the maximum Se removability by phosphate addition. The desorption percentage of Se with 1 M Na2HPO4 correlated with Kd-Se values, suggesting that the soil sample with higher Kd-Se contained more reactive components for phosphate-sorption than the soil sample with lower Kd-Se. To evaluate the effect of phosphate concentration on the Se sorption, the Kd-Se was measured for two typical soils under different levels of phosphate (0.1-10 mM PO4). The Kd values were decreased by phosphate addition for both soils. The Kd decrease was observed even for just 1 mM PO4. The phosphate addition with 1 mM PO4 is the same level as in P fertilizer applied to paddy fields in Japan. Therefore, it was suggested that Se desorption should occur in Japanese soils due to the phosphate input.     

Dissipation of racemic mecoprop and dichlorprop and their pure R-enantiomers in three calcareous soils with and without peat addition

Two racemic herbicides, mecoprop (R,S-MCPP) and dichlorprop (R,S-DCPP), as well as their enantiopure R-forms, were incubated in three calcareous soils at 15 degrees C and 80% of their field capacity to try to elucidate their behaviour in soil and compare the dissipation rates when racemic and enantiopure compounds are used. Quantitation of pesticides is made by HPLC and the R/S ratio by GC-MS. The inactive S-enantiomer from the racemic forms persists longer than the R-forms in silt and sandy loam soils, but for shorter time in the clay loam soil. The pure R-enantiomers, both for MCPP and DCPP, after incubation in soil, are partially converted into their S-forms. In all cases, the dissipation of racemic and pure enatiomeric forms is lower in the clay loam soil than in the silt and sandy loam soils. The R-forms' peristence, in the three soils, is approximately two times lower when they are incubated alone than when they are incubated as racemic compounds. When peat is added, the persistence of these herbicides in the silt and sandy loam soils increases, while in the clay loam soil it decreases. Besides, in the clay loam soil, the enantiomeric ratio (ER) changes from its S-preferential degradation to a preferential degradation of its R-form, so an increase in the persistence of the inactive S-form occurs.     

Microbial metabolic activities in soils of old-field communities following eleven years of nutrient enrichment

Effects of 11 years (1978-1988) of nutrient enrichment (fertilizer or sludge) on microbial metabolic activity in soil samples collected from contrasting types of old-field communities were studied during September 1989. During the 1989 growing season, subplots were manipulated by tilling and/or liming to evaluate mechanisms of ecosystem recovery or were left undisturbed. Metabolic activities of soil microorganisms were determined by measuring dehydrogenase activity within soil samples collected from these subplots. The amounts of 2,3,5-triphenyltetrazolium formazan formed during incubation by the reduction of 2,3,5-triphenyltetrazolium chloride were used to evaluate dehydrogenase activity. Plots that had received long-term applications of sludge or fertilizer had significantly lower rates of microbial activity (P<0.05) than did control plots. Fertilizer and sludge plots treated with lime had significantly higher microbial metabolic activity (P<0.05) than those not receiving lime. Whereas liming stimulated microbial activity to near control levels, tilling had no significant treatment effect.     

Remediation of metal-contaminated soils with the addition of materials - part II: leaching tests to evaluate the efficiency of materials in the remediation of contaminated soils

The effect of the addition of materials on the leaching pattern of As and metals (Cu, Zn, Ni, Pb, and Cd) in two contaminated soils was investigated. The examined materials included bentonites, silicates and industrial wastes, such as sugar foam, fly ashes and a material originated from the zeolitization of fly ash. Soil + material mixtures were prepared at 10% doses. Changes in the acid neutralization capacity, crystalline phases and contaminant leaching over a wide range of pHs were examined by using pH_stat leaching tests. Sugar foam, the zeolitic material and MX-80 bentonite produced the greatest decrease in the leaching of pollutants due to an increase in the pH and/or the sorption capacity in the resulting mixture. This finding suggests that soil remediation may be a feasible option for the reuse of non-hazardous wastes.     

Structural changes in soil communities after triclopyr application in soils invaded by Acacia dealbata Link

Triclopyr is a commonly used herbicide in the control of woody plants and can exhibit toxic effects to soil microorganisms. However, the impact on soils invaded by plant exotics has not yet been addressed. Here, we present the results of an 18-month field study conducted to evaluate the impact of triclopyr on the structure of fungal and bacterial communities in soils invaded by Acacia dealbata Link, through the use of denature gradient gel electrophoresis. After triclopyr application, analyses of bacterial fingerprints suggested a change in the structure of the soil bacterial community, whereas the structure of the soil fungal community remained unaltered. Bacterial density and F:B ratio values changed across the year but were not altered due to herbicide spraying. On the contrary, fungal diversity was increased in plots sprayed with triclopyr 5 months after the first application. Richness and diversity (H´) of both bacteria and fungi were not modified after triclopyr application.     

2,4-D mineralization in unsaturated and near-saturated surface soils of an undulating, cultivated Canadian prairie landscape

The herbicide 2,4-D [2,4-(dichlorophenoxy) acetic acid] is one of the most widely used pesticides in the Canadian prairies and is frequently detected as a ground and surface water contaminant. The objective of this paper was to determine the magnitude and extent of variation of 2,4-D mineralization in a cultivated undulating prairie landscape. Microcosm incubation experiments, using a 4 x 3 x 2 factorial experimental design (soil moisture, 4 levels: 60, 85, 110, 135% of field capacity; slope position, 3 levels: upper-, mid- and lower-slopes; soil depth, 2 levels: 0-5 and 5-15 cm), were used to assess 2,4-D mineralization. The first-order mineralization rate constant (k(1)) varied from 0.03 to 0.22 day(- 1), while total 2,4-D mineralization varied from 31 to 52%. At near-saturated conditions (110 and 135% of field capacity), the onset of 2,4-D degradation was delayed in soil obtained from the upper- and mid-slopes but not in soils obtained from the lower-slope position. The k(1) and total 2,4-D mineralization was significantly influenced by all three factors and their interactions. The Freundlich sorption coefficient of 2,4-D ranged from 0.83 to 2.46 microg (1-1/n)g(- 1) mL(1/n) and was significantly influenced by variations in soil organic carbon content across slope positions. The infield variability of 2,4-D sorption and mineralization observed across slope positions in this undulating field was comparable in magnitude and extent to the regional variability of 2,4-D sorption and mineralization observed in surface soils across Manitoba. The large variability of 2,4-D mineralization and sorption at different slope positions in this cultivated undulating field suggests that landform segmentation models, which are used to delineate slope positions, are important considerations in pesticide fate studies.     

2,4-D mineralization in unsaturated and near-saturated surface soils of an undulating,cultivated Canadian prairie landscape

The herbicide 2,4-D [2,4-(dichlorophenoxy) acetic acid] is one of the most widely used pesticides in the Canadian prairies and is frequently detected as a ground and surface water contaminant. The objective of this paper was to determine the magnitude and extent of variation of 2,4-D mineralization in a cultivated undulating prairie landscape. Microcosm incubation experiments, using a 4 × 3 × 2 factorial experimental design (soil moisture, 4 levels: 60, 85, 110, 135% of field capacity; slope position, 3 levels: upper-, mid- and lower-slopes; soil depth, 2 levels: 0–5 and 5–15 cm), were used to assess 2,4-D mineralization. The first-order mineralization rate constant (k₁) varied from 0.03 to 0.22 day^{? 1}, while total 2,4-D mineralization varied from 31 to 52%. At near-saturated conditions (110 and 135% of field capacity), the onset of 2,4-D degradation was delayed in soil obtained from the upper- and mid-slopes but not in soils obtained from the lower-slope position. The k₁ and total 2,4-D mineralizationwas significantly influenced by all three factors and their interactions. The Freundlich sorption coefficient of 2,4-D ranged from 0.83 to 2.46 ug ^1–1/ng^{? 1} mL^1/n and was significantly influenced by variations in soil organic carbon content across slope positions. The infield variability of 2,4-D sorption and mineralization observed across slope positions in this undulating field was comparable in magnitude and extent to the regional variability of 2,4-D sorption and mineralization observed in surface soils across Manitoba. The large variability of 2,4-D mineralization and sorption at different slope positions in this cultivated undulating field suggests that landform segmentation models, which are used to delineate slope positions, are important considerations in pesticide fate studies.     

Dynamic changes in functional gene copy numbers and microbial communities during degradation of pyrene in soils

This study investigates the dynamics of pyrene degradation rates, microbial communities, and functional gene copy numbers during the incubation of pyrene-spiked soils. Spiking pyrene to the soil was found to have negligible effects on the bacterial community present. Our results demonstrated that there was a significant difference in nidA gene copy numbers between sampling dates in QZ soil. Mycobacterium 16S rDNA clone libraries showed that more than 90% mycobacteria detected were closely related to fast-growing PAH-degrading Mycobacterium in pyrene-spiked soil, while other sequences related to slow-growing Mycobacterium were only detected in the control soil. It is suggested that nidA gene copy number and fast-growing PAH-degrading Mycobacterium could be used as indicators to predict pyrene contamination and its degradation activity in soils.     

The toxicity and fate of phenolic pollutants in the contaminated soils associated with the oil-shale industry

Phenol, cresols, dimethylphenols and resorcinols are considered major pollutants in the oil-shale semi-coke dump leachates (up to 380 mg phenols/L) that contaminate the surrounding soils and pose a threat to the groundwater in the North-East of Estonia. However, despite high residual concentrations of polyaromatic hydrocarbons (PAHs) and oil products in these soils, the concentration of phenols (especially their water-extractable fraction) was low, not exceeding 0.7 mg/kg dwt. The aim of the current study was to evaluate the role of biodegradation and aging on the decrease of hazard caused by phenolic pollution. The extractability of phenols (phenol, cresols, dimethylphenols and resorcinols) and their biodegradability by the microbial population was studied in the 13 soils sampled from the Estonian oil-shale region, territories of former gas stations, and from presumably non-polluted areas. Phenol, 5-methylresorcinol, p-cresol and resorcinol could be considered easily degradable in the soils as the microbial populations from majority of the soils studied were able to grow on mineral medium supplemented with these phenols as a single source of carbon. 2,3- and 2,4- and 3,4-dimethylphenols could be considered less easily biodegradable.The semi-coke dump leachate polluted soil (containing no dibasic phenols, 43 mg of monobasic phenols, 1348 mg of oil products and 35 mg of PAHs per g dwt) was analyzed chemically (HPLC) and toxicologically (Flash-Assay usingVibrio fischeri) for the leaching of phenols during shaking of soil-water slurries for 24 h. Only 5.8% of the total concentration of phenols was water-extractable, whereas about 50% of the leached amount was biodegraded by the soil microorganisms. Phenol and cresols were biodegraded by 80%, but the concentration of dimethyl-phenols practically did not change. The pollutants (measured as total water-extractable toxicity) were desorbed from the soil particles by the 8^th h of extraction, whereas the toxicity of the aqueous phase continued to increase, probably due to the formation of toxic metabolites. The concentration of water-extractable phenols was too low to explain the toxicity of the extract. Also the impact of PAHs and oil products was excluded. Thus, the relatively low concentration of phenols in the oil-shale region soils is most probably the reflection of both natural attenuation and pollution aging. Therefore, the impact of phenolic compounds to the net bioavailable hazard is probably not so remarkable as it has been considered. The actual pollutants causing the toxicity of the soils from the oil-shale region, however, need to be elucidated.