Strategies for enhancing the phytoremediation of cadmium-contaminated agricultural soils by Solanum nigrum L

Field trials contribute practical information towards the development of phytoremediation strategies that cannot be provided by laboratory tests. We conducted field experiments utilizing the Cd hyperaccumulator plant Solanum nigrum L., on farmland contaminated with 1.91 mg kg⁻¹ Cd in the soil. Our study showed that S. nigrum has a relatively high biomass. Planting density had a significant effect on the plant biomass and thus on overall Cd accumulation. For double harvesting, an optimal cutting position influenced the amount of Cd extracted from soils. Double cropping was found to significantly increase the amount of Cd extracted by S. nigrum. Fertilizing had no significant effect on plant biomass or on the Cd remediation of the soil over the short-term period. Our study indicates that S. nigrum can accumulate Cd from soils where the concentrations are relatively low, and thus has application for use in decontamination of slightly to moderately Cd-contaminated soil.     

Analysis and characterization of cultivable heavy metal-resistant bacterial endophytes isolated from Cd-hyperaccumulator Solanum nigrum L. and their potential use for phytoremediation

This study investigates the heavy metal-resistant bacterial endophytes of Cd-hyperaccumulator Solanum nigrum L. grown on a mine tailing by using cultivation-dependent technique. Thirty Cd-tolerant bacterial endophytes were isolated from roots, stems, and leaves of S. nigrum L. and classified by amplified ribosomal DNA-restriction analysis into 18 different types. Phylogenetic analysis based on 16S rDNA sequences showed that these isolates belonged to four groups: Actinobacteria (43%), Proteobacteria (23%), Bacteroidetes (27%) and Firmicutes (7%). All the isolates were then characterized for their plant growth promoting traits as well as their resistances to different heavy metals; and the actual plant growth promotion and colonization ability were also assessed. Four isolates were re-introduced into S. nigrum L. under Cd stress and resulted in Cd phytotoxicity decrease, as dry weights of roots increased from 55% to 143% and dry weights of above-ground from 64% to 100% compared to the uninoculated ones. The total Cd accumulation of inoculated plants increased from 66% to 135% (roots) and from 22% to 64% (above-ground) compared to the uninoculated ones. Our research suggests that bacterial endophytes are a most promising resource and may be the excellent candidates of bio-inoculants for enhancing the phytoremediation efficiency.     

The presence of Bacillus thuringiensis (Bt) protein in earthworms Eisenia fetida has no deleterious effects on their growth and reproduction

Earthworms Eisenia fetida, bred in substances with stover of two genetically-engineered Bacillus thuringiensis (Bt) corns (5422Bt1 (Event Bt11) and 5422CBCL (MON810)) expressing Cry1Ab and their near-isogenic non-Bt corn (5422), were used to investigate the non-target effects of Bt corn on soil-dwelling organisms. Cry1Ab concentrations in substances, casts and guts of E. fetida were also investigated by Enzyme-linked immunosorbent assay (ELISA). More than 90% individuals of E. fetida survived over a period of 30 d, irrespective of whether they received Bt corn or non-Bt corn. Compared to 5422 treatments, significantly higher relative growth rate and more number of new offspring and cocoons of E. fetida were found in 5422Bt1 and 5422CBCL treatments. These results were unlikely to be directly caused by Cry1Ab released from Bt corns but rather by differences in other factors of plants such as plant components (soluble sugar, total organic carbon, total protein and available phosphorus of Bt corns were more than 5422). ELISA results indicated immunoreactive Cry1Ab was detectable in substances, and the casts, guts of E. fetida from Bt corns treatments, of which the highest levels were detected in substances under the corresponding experimental conditions. With the increase of treated time, a strong decline was observed in Cry1Ab from substances and casts of E. fetida, whereas Cry1Ab in guts of E. fetida from 5422Bt1 treatments gradually increased and that from 5422CBCL treatments increased between 14 and 30 d. Therefore, the presence of Cry1Ab in E. fetida had no deleterious effects on their growth and reproduction.     

Isolation and characterization of lead-tolerant Ochrobactrum intermedium and its role in enhancing lead accumulation by Eucalyptus camaldulensis

In this study, the potential of rhizospheric bacteria in promoting the growth and Pb accumulation by the woody plant Eucalyptus camaldulensis under hydroponic conditions was investigated for the first time. Three Pb-tolerant bacteria were isolated from the rhizosphere of E. camaldulensis grown in Pb-contaminated soils in the Bo Ngam Pb mine, Thailand. Based on analysis of partial 16S rRNA gene sequence, the three isolates were identified as Microbacterium paraoxydans BN-2, Ochrobactrum intermedium BN-3, and Bacillus fusiformis BN-4. Among these strains, O. intermedium BN-3 showed the highest tolerance to not only Pb but also Cd and Zn. After growth in the presence of Pb, the membranes of O. intermedium BN-3 cells exhibited an increase in unsaturated fatty acid levels but a decrease in fluidity. In hydroponic studies, inoculation of O. intermedium BN-3 significantly increased the biomass and Pb accumulation by E. camaldulensis compared to the uninoculated control. The results suggested the role of the natural rhizospheric bacteria localized to the root surface of E. camaldulensis in promoting Pb accumulation and plant growth. Our results indicate that O. intermedium BN-3 and other indigenous rhizospheric bacteria have the potential to improve the efficiency of phytoremediation of Pb-contaminated sites.     

The growth response of Alternanthera philoxeroides in a simulated post-combustion emission with ultrahigh [CO2] and acidic pollutants

Although post-combustion emissions from power plants are a major source of air pollution, they contain excess CO₂ that could be used to fertilize commercial greenhouses and stimulate plant growth. We addressed the combined effects of ultrahigh [CO₂] and acidic pollutants in flue gas on the growth of Alternanthera philoxeroides. When acidic pollutants were excluded, the biomass yield of A. philoxeroides saturated near 2000 μmol mol⁻¹ [CO₂] with doubled biomass accumulation relative to the ambient control. The growth enhancement was maintained at 5000 μmol mol⁻¹ [CO₂], but declined when [CO₂] rose above 1%, in association with a strong photosynthetic inhibition. Although acidic components (SO₂ and NO₂) significantly offset the CO₂ enhancement, the aboveground yield increased considerably when the concentration of pollutants was moderate (200 times dilution). Our results indicate that using excess CO₂ from the power plant emissions to optimize growth in commercial green house could be viable.     

Cd-tolerant Suillus luteus: A fungal insurance for pines exposed to Cd

Soil metal pollution can trigger evolutionary adaptation in soil-borne organisms. An in vitro screening test showed cadmium adaptation in populations of Suillus luteus (L.: Fr.) Roussel, an ectomycorrhizal fungus of pine trees. Cadmium stress was subsequently investigated in Scots pine (Pinus sylvestris L.) seedlings inoculated with a Cd-tolerant S. luteus, isolated from a heavy metal contaminated site, and compared to plants inoculated with a Cd-sensitive isolate from a non-polluted area. A dose-response experiment with mycorrhizal pines showed better plant protection by a Cd-adapted fungus: more fungal biomass and a higher nutrient uptake at high Cd exposure. In addition, less Cd was transferred to aboveground plant parts. Because of the key role of the ectomycorrhizal symbiosis for tree fitness, the evolution of Cd tolerance in an ectomycorrhizal partner such as S. luteus can be of major importance for the establishment of pine forests on Cd-contaminated soils.     

Direct and indirect impact of sewage sludge compost spreading on Quercus coccifera monoterpene emissions in a Mediterranean shrubland

Monoterpene emissions of Quercus coccifera L. were repeatedly measured during the two years following the spreading of a sewage sludge compost at rates of 50 Mg ha⁻¹ and 100 Mg ha⁻¹, in a twelve-year-old post-fire Mediterranean shrubland. We also monitored the patterns of change in soil and leaf nutrient content, plant water potential, chlorophyll fluorescence, and plant growth. Compost spreading resulted in weak changes in leaf nutrient content and plant water status, and therefore no significant effect on monoterpene emissions at leaf scale, except during one summer sampling, probably related to advanced leaf maturity with the highest compost rate. However, compost increased plant growth, particularly the leaf biomass. The results suggest that compost spreading in Mediterranean shrublands has no strong short-term effect on Q. coccifera monoterpene emissions at leaf level, but may indirectly increase volatile organic compound fluxes at the stand scale, which may contribute to regional ozone pollution.     

Seabird feathers as monitors of the levels and persistence of heavy metal pollution after the Prestige oil spill

We measured heavy metal concentrations in yellow-legged gulls (n = 196) and European shags (n = 189) in order to assess the temporal pattern of contaminant exposure following the Prestige oil spill in November 2002. We analysed Pb, Cu, Zn, Cr, Ni and V levels in chick feathers sampled at four colonies during seven post-spill years (2003-2009), and compared results with pre-spill levels obtained from feathers of juvenile shag corpses (grown in spring/summer 2002). Following the Prestige wreck, Cu (4.3-10 μg g⁻¹) and Pb concentrations (1.0-1.4 μg g⁻¹) were, respectively, between two and five times higher than pre-spill levels (1.5-3.6 and 0.1-0.4 μg g⁻¹), but returned to previous background concentrations after three years. Our study highlights the suitability of chick feathers of seabirds for assessing the impact of oil spills on heavy metal contamination, and provides the best evidence to date on the persistence of oil pollution after the Prestige incident.     

Effects of sub-lethal glyphosate concentrations on growth and photosynthetic performance of non-target species Bolboschoenus maritimus

Glyphosate use has increased over the last decades for the control of invasive plant species in wetland ecosystems. Although glyphosate has been considered ‘environmentally’ safe, its repeated use could increase the toxicological risk derived from diffuse pollution of surface and groundwater on non-target vegetation. A glasshouse study was designed to determine the effect produced by the addition of different sub-lethal doses of glyphosate herbicides (5–30 mg L⁻¹) to the nutrient solution on the growth and photosynthetic apparatus of Bolboschoenus maritimus. Although B. maritimus plants were able to grow and survive after 20 d of exposure to glyphosate, the presence of this herbicide affected their growth, through a direct interaction with the root system. Particularly, at 30 mg L⁻¹ glyphosate, B. maritimus showed ca. 30% of biomass decrease. The reduction in B. maritimus growth was due to a decrease in net photosynthetic rate (A), which ranged between values ca. 11.5 and 5.5 μmol m⁻² s⁻¹ CO₂ for the control and the highest glyphosate treatment, respectively. The response of A to glyphosate could be largely accounted for by non-stomatal limitations, since stomatal conductance was similar in all glyphosate treatments. Thus, A decrease was prompted by the negative impact of herbicide on photochemical (PSII) apparatus, the reduction in the absorption of essential nutrients, the reduction of photosynthetic pigments and possibly the reduction in Rubisco carboxilation capacity. Moreover, glyphosate excess caused photoinhibitory damage. In conclusion, in this study we have shown that herbicide water pollution could be a source of indirect phytotoxicity for B. maritimus.     

Biodegradation of 4-aminobenzenesulfonate by Ralstonia sp. PBA and Hydrogenophaga sp. PBC isolated from textile wastewater treatment plant

A co-culture consisting of Hydrogenophaga sp. PBC and Ralstonia sp. PBA, isolated from textile wastewater treatment plant could tolerate up to 100 mM 4-aminobenzenesulfonate (4-ABS) and utilize it as sole carbon, nitrogen and sulfur source under aerobic condition. The biodegradation of 4-ABS resulted in the release of nitrogen and sulfur in the form of ammonium and sulfate respectively. Ninety-eight percent removal of chemical oxygen demand attributed to 20 mM of 4-ABS in cell-free supernatant could be achieved after 118 h. Effective biodegradation of 4-ABS occurred at pH ranging from 6 to 8. During batch culture with 4-ABS as sole carbon and nitrogen source, the ratio of strain PBA to PBC was dynamic and a critical concentration of strain PBA has to be reached in order to enable effective biodegradation of 4-ABS. Haldane inhibition model was used to fit the degradation rate at different initial concentrations and the parameters μ_max, K_s and K_i were determined to be 0.13 h⁻¹, 1.3 mM and 42 mM respectively. HPLC analyses revealed traced accumulation of 4-sulfocatechol and at least four unidentified metabolites during biodegradation. This is the first study to report on the characterization of 4-ABS-degrading bacterial consortium that was isolated from textile wastewater treatment plant.     

Indication of airborne pollution by birch and spruce in the vicinity of copper smelter

The aim of the study was determination of air pollution impact of the copper smelter in Bor and its surroundings (Serbia) by assessing the suitability of birch (Betula pendula Roth.) and spruce (Picea abies L.) for the purposes of biomonitoring and comparing it with previously published data from the same study area. The concentrations of Cu, Zn, Pb and Mn in leaves/needles, branches, roots and soil were determined. Sampling was performed during 2009 in two zones with high load of air pollution due to copper mining and smelting activities, and one background zone. Metal accumulation and translocation was evaluated in terms of biological factors. In addition, plant enrichment factor was calculated. According to the results, plant foliage was not enriched through soil, which indicates absorption from the air, with both species acting as excluders of Cu, Pb, Zn and Mn. Leaves were more enriched with all the metals than needles, indicating a better response of birch to airborne pollution than spruce. Cluster analysis showed different level of pollution at the sites, while correlations between Cu and Pb obtained by Principal Component Analysis indicated their anthropogenic origin. Regarding previously published results, beside birch leaves, pine needles (which showed higher level of response to pollution compared to linden leaves) could be applied in air biomonitoring surveys near copper smelters.     

Zinc tolerance and accumulation in the salt-marsh shrub Halimione portulacoides

The halophytic shrub Halimione portulacoides is known to be capable of growth in soils containing extremely high concentrations of Zn. This study evaluated in detail the tolerance and accumulation potential of H. portulacoides under moderate and high external Zn levels. A greenhouse experiment was conducted in order to investigate the effects of a range of Zn concentrations (0-130 mmol L⁻¹) on growth and photosynthetic performance by measuring relative growth rate, total leaf area, specific leaf area, gas exchange, chlorophyll fluorescence parameters and photosynthetic pigment concentrations. We also determined the total zinc, nitrogen, phosphorus, calcium, magnesium, sodium, potassium, iron and copper concentrations in the plant tissues. H. portulacoides demonstrated hypertolerance to Zn stress, since it survived with leaf concentrations of up to 2300 mg Zn kg⁻¹ dry mass, when treated with 130 mmol Zn L⁻¹. Zinc concentrations greater than 70 mmol L⁻¹ in the nutrient solution negatively affected plant growth, in all probability due to the recorded decline in net photosynthesis rate. Our results indicate that the Zn-induced decline in the photosynthetic function of H. portulacoides may be attributed to the adverse effect of the high concentration of the metal on photosynthetic electron transport. Growth parameters were virtually unaffected by leaf tissue concentrations as high as 1500 mg Zn kg⁻¹ dry mass, demonstrating the strong capability of H. portulacoides to protect itself against toxic Zn concentrations. The results of our study indicate that this salt-marsh shrub may represent a valuable tool in the restoration of Zn-polluted areas.     

水质对紫外消毒在两种典型再生水中应用的影响

以大肠杆菌为对象，研究了再生水水质变化对紫外消毒效果和光复活的影响。结果表明，紫外对大肠杆菌有很强的灭活作用，在紫外剂量为4mJ／cm^2时，大肠杆菌的灭活率达到了4．41个对数级。腐殖酸、铁和2种再生水水体中其他溶解性物质会影响光吸收和紫外透射率，但对紫外消毒动力学无影响。在颗粒物浓度为0～200mg／L的范围内，外源高岭土和活性污泥等颗粒物的投加对紫外消毒效果影响较小，而再生水水样W1中原有的2．61／1：g／L的颗粒物则会极大地影响消毒效果，使UV对细菌的灭活出现明显的拖尾现象。腐殖酸会增强紫外损伤大肠杆菌的光复活能力，但2种再生水中细菌的光复活能力相对磷酸盐缓冲溶液（PBS）中减弱，减弱程度在不同水样中有所不同。     

Hydrocarbon degradation, plant colonization and gene expression of alkane degradation genes by endophytic Enterobacter ludwigii strains

The genus Enterobacter comprises a range of beneficial plant-associated bacteria showing plant growth promotion. Enterobacter ludwigii belongs to the Enterobacter cloacae complex and has been reported to include human pathogens but also plant-associated strains with plant beneficial capacities. To assess the role of Enterobacter endophytes in hydrocarbon degradation, plant colonization, abundance and expression of CYP153 genes in different plant compartments, three plant species (Italian ryegrass, birdsfoot trefoil and alfalfa) were grown in sterile soil spiked with 1% diesel and inoculated with three endophytic E. ludwigii strains. Results showed that all strains were capable of hydrocarbon degradation and efficiently colonized the rhizosphere and plant interior. Two strains, ISI10-3 and BRI10-9, showed highest degradation rates of diesel fuel up to 68% and performed best in combination with Italian ryegrass and alfalfa. All strains expressed the CYP153 gene in all plant compartments, indicating an active role in degradation of diesel in association with plants.     

Bioaccumulation of the pharmaceutical 17α-ethinylestradiol in shorthead redhorse suckers (Moxostoma macrolepidotum) from the St. Clair River, Canada

17α-ethynylestradiol (EE2), a synthetic estrogen prescribed as a contraceptive, was measured in Shorthead Redhorse Suckers (ShRHSs) (Moxostoma macrolepidotum) collected near a wastewater treatment plant (WWTP) in the St. Clair River (Ontario, Canada). We detected EE2 in 50% of the fish samples caught near the WWTP (Stag Island), which averaged 1.6 ± 0.6 ng/g (wet weight) in males and 1.43 ± 0.96 ng/g in females. No EE2 was detected in the samples from the reference site (Port Lambton) which was 26 km further downstream of the Stag Island site. Only males from Stag Island had VTG induction, suggesting the Corunna WWTP effluent as a likely source of environmental estrogen. EE2 concentrations were correlated with total body lipid content (R² = 0.512, p < 0.01, n = 10). Lipid normalized EE2 concentrations were correlated with δ¹⁵N (R² = 0.436, p < 0.05, n = 10), suggesting higher EE2 exposures in carnivores. Our data support the hypothesis of EE2 bioaccumulation in wild fish.     

Effects of ammonia from livestock farming on lichen photosynthesis

This study investigated if atmospheric ammonia (NH₃) pollution around a sheep farm influences the photosynthetic performance of the lichens Evernia prunastri and Pseudevernia furfuracea. Thalli of both species were transplanted for up to 30 days in a semi-arid region (Crete, Greece), at sites with concentrations of atmospheric ammonia of ca. 60 μg/m³ (at a sheep farm), ca. 15 μg/m³ (60 m from the sheep farm) and ca. 2 μg/m³ (a remote area 5 km away). Lichen photosynthesis was analysed by the chlorophyll a fluorescence emission to identify targets of ammonia pollution. The results indicated that the photosystem II of the two lichens exposed to NH₃ is susceptible to this pollutant in the gas-phase. The parameter PI_ABS, a global index of photosynthetic performance that combines in a single expression the three functional steps of the photosynthetic activity (light absorption, excitation energy trapping, and conversion of excitation energy to electron transport) was much more sensitive to NH₃ than the F_V/F_M ratio, one of the most commonly used stress indicators.     

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.     

The effect of γ-Fe2O3 nanoparticles on Escherichia coli genome

Extensive production and application of γ-Fe₂O₃ magnetic nanoparticles (MNPs) has increased their potential risk on environment and human health. This report illustrates a genetic impact of γ-Fe₂O₃ magnetic nanoparticles (MNPs) on Escherichia coli (E. coli). After 3000-generation incubation with MNPs addition, obvious genomic variations were revealed by using repetitive extragenic palindromic PCR (rep-PCR) DNA fingerprint technique. The physicochemical interactions between MNPs and bacteria could be responsible for such genomic responses. It was revealed that Fe³⁺ concentration increased in the medium. Transmission electronic microscopy (TEM) and flow cytometry (FCM) analysis consistently demonstrated the occurrences of adsorption and membranes-internalization of MNPs outside and inside cells. Both increased Fe³⁺ ion and the uptake of MNPs facilitated Fe binding with proteins and DNA strands, resulting in enhancing the mutation frequency of E. coli. Our results would be of great help to assessing the potential impact of MNPs on human and environment.     

Influence of anthropogenic stress on fitness and behaviour of a key-species of estuarine ecosystems, the ragworm Nereis diversicolor

Fitness, (biometric measurements, reproduction) and behaviour that are ecologically relevant biomarkers in assessing the quality of estuarine sediments were studied by comparing the responses of the polychaete worm Nereis diversicolor - a key species in estuaries - along a pollution gradient. Intersite differences were shown for all the measured parameters: size-weight relationships, energy reserves as glycogen and lipids, sexual maturation patterns, total number of oocytes per female, total and relative fecundity, burrowing behaviour. The physiological and behavioural status of N. diversicolor was consistently disturbed in the larger, most contaminated estuaries (Loire and Seine, Fr.) compared to reference sites (Bay of Bourgneuf, Goyen estuary, Fr.). Many classes of potentially toxic chemicals present in these estuaries most likely contribute to these impairments but food availability may act as a confounding factor, interfering with the potential impact of contaminants.     

Application of phosphate-solubilizing bacteria for enhancing bioavailability and phytoextraction of cadmium (Cd) from polluted soil

In this study, phosphate-solubilizing bacteria (PSB), Bacillus megaterium, were used to enhance Cd bioavailability and phytoextractability of Cd from contaminated soils. This strain showed a potential for directly solubilizing phosphorous from soils more than 10 folds greater than the control without inoculation. The results of pot experiments revealed that inoculation with B. megaterium significantly increased the extent of Cd accumulation in Brassica juncea and Abutilon theophrasti by two folds relative to the uninoculated control. The maximum Cd concentrations due to inoculation were 1.6 and 1.8 mg Cd g⁻¹ plant for B. juncea and A. theophrasti after 10 wk, respectively. The total biomass of A. theophrasti was not significantly promoted by the inoculation treatment, yet the total biomass of B. juncea increased from 0.087 to 0.448 g. It is also worth to mention that B. juncea predominantly accumulates Cd in its stems (39%) whereas A. theophrasti accumulates it in its leaves (68%) after 10 wk. The change of the Cd speciation indicated that inoculation of B. megaterium as PSB increased the bioavailabilty of Cd and consequently enhanced its uptake by plants. The present study may provide a new insight for improving phytoremediation using PSB in the Cd-contaminated soils.