Assessment of bacterial communities and characterization of lead-resistant bacteria in the rhizosphere soils of metal-tolerant Chenopodium ambrosioides grown on lead-zinc mine tailings

Bacterial communities in the rhizosphere soils of metal tolerant and accumulating Chenopodium ambrosioides grown in highly and moderately lead-zinc mine tailings contaminated-soils as well as the adjacent soils with low metal contamination were characterized by using cultivation-independent and cultivation techniques. A total of 69, 73, and 83 bacterial operational taxonomic units (OTUs) having 84.8-100% similarity with the closest match in the database were detected among high, moderate, and low-contamination soil clone libraries, respectively. These OTUs had a Shannon diversity index value in the range of 4.06-4.30. There were 9, 10, and 14 bacterial genera specific to high, moderate, and low metal-contaminated soil clone libraries, respectively. Phylogenetic analysis showed that the Pb-resistant isolates belonged to 8 genera. Pseudomonas and Arthrobacter were predominant among the isolates. Most of the isolates (82-86%) produced indole acetic acid and siderophores. More strains from the highly metal-contaminated soil produced 1-aminocyclopropane-1-carboxylate deaminase than the strains from the moderately and lowly metal-contaminated soils. In experiments involving canola grown in quartz sand containing 200 mg kg⁻¹ of Pb, inoculation with the isolated Paenibacillusjamilae HTb8 and Pseudomonas sp. GTa5 was found to significantly increase the above-ground tissues dry weight (ranging from 19% to 36%) and Pb uptake (ranging from 30% to 40%) compared to the uninoculated control. These results show that C. ambrosioides harbor different metal-resistant bacterial communities in their rhizosphere soils and the isolates expressing plant growth promoting traits may be exploited for improving the phytoextraction efficiency of Pb-polluted environment.     

Diversity and community structure of culturable arsenic-resistant bacteria across a soil arsenic gradient at an abandoned tungsten-tin mining area

We studied the bacterial diversity at a single location (the Terrubias mine; Salamanca province, Spain) with a gradient of soil As contamination to test if increasing levels of As would (1) change the preponderant groups of arsenic-resistant bacteria and (2) increase the tolerance thresholds to arsenite [As(III)] and arsenate [As(V)] of such bacteria. We studied the genetic and taxonomic diversity of culturable arsenic-resistant bacteria by PCR fingerprinting techniques and 16S rRNA gene sequencing. Then, the tolerance thresholds to As(III) and As(V) were determined for representative strains and mathematically analyzed to determine relationships between tolerances to As(III) and As(V), as well as these tolerances with the soil contamination level. The diversity of the bacterial community was, as expected, inversely related to the soil As content. The overall preponderant arsenic-resistant bacteria were Firmicutes (mainly Bacillus spp.) followed by γ-Proteobacteria (mainly Pseudomonas spp.), with increasing relative frequencies of the former as the soil arsenic concentration increased. Moreover, a strain of the species Rahnella aquatilis (γ-Proteobacteria class) exhibited strong endurance to arsenic, being described for the first time in literature such a phenotype within this bacterial species. Tolerances of the isolates to As(III) and As(V) were correlated but not with their origin (soil contamination level). Most of the strains (64%) showed relatively low tolerances to As(III) and As(V), but the second most numerous group of isolates (19%) showed increased tolerance to As(III) rather than to As(V), even though the As(V) anion is the prevalent arsenic species in soil solution at this location. To our knowledge, this is the first study to report a shift towards preponderance of Gram-positive bacteria (Firmicutes) related to high concentrations of soil arsenic. It was also shown that, under aerobic conditions, strains with relatively enhanced tolerance to As(III) predominated over the most As(V)-tolerant ones.     

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.     

Naturally-assisted metal phytoextraction by Brassica carinata: Role ofroot exudates

Due to relatively high chelant dosages and potential environmental risks it is necessary to explore different approaches in the remediation of metal-contaminated soils. The present study focussed on the removal of metals (As, Cd, Cu, Pb and Zn) from a multiple metal-contaminated soil by growing Brassica carinata plants in succession to spontaneous metallicolous populations of Pinus pinaster, Plantago lanceolata and Silene paradoxa. The results showed that the growth of the metallicolous populations increased the extractable metal levels in the soil, which resulted in a higher accumulation of metals in the above-ground parts of B. carinata. Root exudates of the three metallicolous species were analysed to elucidate their possible role in the enhanced metal availability. The presence of metals stimulated the exudation of organic and phenolic acids as well as flavonoids. It was suggested that root exudates played an important role in solubilising metals in soil and in favouring their uptake by roots.     

Assessment of the applicability of a “toolbox” designed for microbially assisted phytoremediation: the case study at Ingurtosu mining site (Italy)

The paper describes the fieldwork at the Italian test site of the abandoned mine of sphalerite and galena in Ingurtosu (Sardinia), with the aim to assess the applicability of a “toolbox” to establish the optimized techniques for remediation of soils contaminated by mining activities. A preliminary characterization—including (hydro)geochemistry, heavy metal concentration and their mobility in soil, bioprospecting for microbiology and botany—provided a data set for the development of a toolbox to deliver a microbially assisted phytoremediation process. Euphorbia pithyusa was selected as an endemic pioneer plant to be associated with a bacterial consortium, established with ten selected native strains, including metal-tolerant bacteria and producers of plant growth factors. The toolbox was firstly assessed in a greenhouse pot experiment. A positive effect of bacterial inoculum on E. pithyusa germination and total plant survival was observed. E. pithyusa showed to be a well-performing metallophyte species, and only inoculated soil retained a microbial activity with a high functional diversity, expanding metabolic affinity also towards root exudates. These results supported the decision to proceed with a field trial, investigating different treatments used singly or in combination: bioaugmentation with bacterial consortia, mycorrhizal fungi and a commercial mineral amendment. Microbial activity in soil, plant physiological parameters and heavy metal content in plants and in soil were monitored. Five months after the beginning, an early assessment of the toolbox under field conditions was carried out. Despite the cold season (October–March), results suggested the following: (1) the field setup as well as the experimental design proved to be effective; (2) plant survival was satisfactory; (3) soil quality was increased and bioaugmentation improved microbial activity, expanding the metabolic competences towards plant interaction (root exudates); and (4) multivariate analysis supported the data provided that the proposed toolbox can be established and the field trial can be carried forward.     

Bioremediation assessment of diesel–biodiesel-contaminated soil using an alternative bioaugmentation strategy

This study investigated the effectiveness of successive bioaugmentation, conventional bioaugmentation, and biostimulation of biodegradation of B10 in soil. In addition, the structure of the soil microbial community was assessed by polymerase chain reaction-denaturing gradient gel electrophoresis. The consortium was inoculated on the initial and the 11th day of incubation for successive bioaugmentation and only on the initial day for bioaugmentation and conventional bioaugmentation. The experiment was conducted for 32 days. The microbial consortium was identified based on sequencing of 16S rRNA gene and consisted as Pseudomonas aeruginosa, Achromobacter xylosoxidans, and Ochrobactrum intermedium. Nutrient introduction (biostimulation) promoted a positive effect on microbial populations. The results indicate that the edaphic community structure and dynamics were different according to the treatments employed. CO₂ evolution demonstrated no significant difference in soil microbial activity between biostimulation and bioaugmentation treatments. The total petroleum hydrocarbon (TPH) analysis indicated a biodegradation level of 35.7 and 32.2 % for the biostimulation and successive bioaugmentation treatments, respectively. Successive bioaugmentation displayed positive effects on biodegradation, with a substantial reduction in TPH levels.     

Soil microbial systems respond differentially to tetracycline,sulfamonomethoxine, and ciprofloxacin entering soil under pot experimental conditions alone and in combination

This study investigated soil microbial responses to the application of tetracycline (TC), sulfamonomethoxine (SMM), and ciprofloxacin (CIP) alone and in combination in a soil culture pot experiment conducted at Hangzhou, China. Multiple approaches were applied for a better and complete depiction. Among the three antibiotics, SMM has a lowest dissipation and shows a most dramatic inhibition on microbial community and metabolism diversity. The combined application (AM) of SMM, CIP, and TC improved the dissipation of each antibiotic; similarly, SMM- and CIP-resistant bacteria showed larger populations in the AM than all single applications. Soils accumulated a large content of NO₃–N at day 20 after multi-antibiotics perturbation. All antibiotics stimulated soil basal respirations and inhibited soil metabolism diversity, whereas the interruption exerted by SMM and AM lasted for a longer time. Six nitrogen-cycling genes including chiA, amoA, nifH, nirK, nirS, and narG were quantified and found to decrease owing to both single- and multi-antibiotics perturbation. Overall, AM was most interruptive for soils, followed by SMM perturbation, while other antibiotics could be less interruptive. These results provide systematic insights into how soil microbial systems would shift under each single- or multi-antibiotics perturbation.     

Impact of the novel neonicotinoid insecticide Paichongding on bacterial communities in yellow loam and Huangshi soils

Insecticides are widely sprayed in modern agriculture for ensuring the crop yield, which could also lead to contamination and insecticide residue in soils. Paichongding (IPP) is a novel neonicotinoid insecticide and was developed recently in China. Soil bacterial community, diversity, and community composition vary widely depending on environmental factors. As for now, little is known about bacterial species thriving, bacterial community diversity, and structure in IPP-spraying soils. In present study, IPP degradation in yellow loam and Huangshi soils was investigated, and bacterial communities and diversity were examined in soil without IPP spray and with IPP spray through pyrosequencing of 16S ribosomal RNA (rRNA) gene amplicons. The degradation ratio of IPP at 60 days after treatment (DAT) reached 51.22 and 34.01 % in yellow loam and Huangshi soil, respectively. A higher richness of operational taxonomic units (OTUs) was found in yellow loam soil (867 OTUs) and Huangshi soil (762 OTUs) without IPP spray while OUTs were relatively low in IPP-spraying soils. The community composition also differed both in phyla and genus level between these two environmental conditions. Proteobacteria, Firmicutes, Planctomycetes, Chloroflexi, Armatimonadetes, and Chlorobi were stimulated to increase after IPP application, while IPP inhibited the phyla of Bacteroidetes, Actinobacteria, and Acidobacteria.     

Community structure of arbuscular mycorrhizal fungi associated to Veronica rechingeri at the Anguran zinc and lead mining region

Root colonization and diversity of arbuscular mycorrhizal fungi (AMF) were analyzed in Veronica rechingeri growing in heavy metal (HM) and non-polluted soils of the Anguran Zn and Pb mining region (Iran). Three species could be separated morphologically, while phylogenetic analyses after PCR amplification of the ITS region followed by RFLP and sequencing revealed seven different AMF sequence types all within the genus Glomus. Rarefaction analysis confirmed exhaustive molecular characterization of the AMF diversity present within root samples. Increasing heavy metal contamination between the sites studied was accompanied by a decrease in AMF spore numbers, mycorrhizal colonization parameters and the number of AMF sequence types colonizing the roots. Some AMF sequence types were only found at sites with the highest and lowest soil HM contents, respectively.     

Effects of petroleum contamination on soil microbial numbers, metabolic activity and urease activity

The influence of petroleum contamination on soil microbial activities was investigated in 13 soil samples from sites around an injection water well (Iw-1, 2, 3, 4) (total petroleum hydrocarbons (TPH): 7.5-78 mg kg⁻¹), an oil production well (Op-1, 2, 3, 4, 5) (TPH: 149-1110 mg kg⁻¹), and an oil spill accident well (Os-1, 2, 3, 4) (TPH: 4500-34 600 mg kg⁻¹). The growth rate constant (μ) of glucose stimulated organisms, determined by microcalorimetry, was higher in Iw soil samples than in Op and Os samples. Total cultivable bacteria and fungi and urease activity also decreased with increasing concentration of TPH. Total heat produced demonstrated that TPH at concentrations less than about 1 g kg⁻¹ soil stimulated anaerobic respiration. A positive correlation between TPH and soil organic matter (OM) and stimulation of fungi-bacteria-urease at low TPH doses suggested that TPH is bound to soil OM and slowly metabolized in Iw soils during OM consumption. These methods can be used to evaluate the potential of polluted soils to carry out self-bioremediation by metabolizing TPH.     

The rhizosphere and PAH amendment mediate impacts on functional and structural bacterial diversity in sandy peat soil

To reveal the degradation capacity of bacteria in PAH polluted soil and rhizosphere we combined bacterial extradiol ring-cleavage dioxygenase and 16S rRNA analysis in Betula pubescens rhizoremediation. Characterisation of the functional bacterial community by RFLP revealed novel environmental dioxygenases, and their putative hosts were studied by 16S rRNA amplification. Plant rhizosphere and PAH amendment effects were detected by the RFLP/T-RFLP analysis. Functional species richness increased in the birch rhizosphere and PAH amendment impacted the compositional diversity of the dioxygenases and the structural 16S rRNA community. A shift from an Acidobacteria and Verrucomicrobia dominated to an Alpha- and Betaproteobacteria dominated community structure was detected in polluted soil. Clone sequence analysis indicated catabolic significance of Burkholderia in PAH polluted soil. These results advance our understanding of rhizoremediation and unveil the extent of uncharacterized functional bacteria to benefit bioremediation by facilitating the development of the molecular tool box to monitor bacterial populations in biodegradation.     

Bioaugmentation of polyethylene succinate-contaminated soil with Pseudomonas sp. AKS2 results in increased microbial activity and better polymer degradation

Pseudomonas sp. AKS2 isolated from soil degrades polyethylene succinate (PES) efficiently in the laboratory. However, this organism may not be able to degrade PES with similar efficiency in a natural habitat. Since in situ remediation is preferred for the effective removal of recalcitrant materials like plastic, in the current study, bioaugmentation potential of this organism was investigated. To investigate the potential of the AKS2 strain to bioaugment the PES-contaminated soil, a microcosm-based study was carried out wherein naturally attenuated, biostimulated, and AKS2-inoculated (bioaugmented) soil samples were examined for their ability to degrade PES. The results showed better degradation of PES by bioaugmented soil than other microcosms. Consistent with it, a higher number of PES-degrading organisms were found in the bioaugmented microcosm. The bioaugmented microcosm also exhibited a higher level of average well color development in BiOLOG ECO plate assay than the other two. The corresponding Shannon–Weaver index and Gini coefficient revealed a higher soil microbial diversity of bioaugmented microcosm than the others. This was further supported by community-level physiological profile of three different microcosms wherein we have observed better utilization of different carbon sources by bioaugmented microcosms. Collectively, these results demonstrate that bioaugmentation of PES-contaminated soil with AKS2 not only enhances polymer degradation but also increases microbial diversity. Bioaugmentation of soil with AKS2 enhances PES degradation without causing damage to soil ecology. Thus, Pseudomonas sp. AKS2 has the potential to be implemented as a useful tool for in situ bioremediation of PES.     

Evaluation of bioaugmentation and biostimulation on arsenic remediation in soil through biovolatilization

Arsenic (As) removal through microbially driven biovolatilization can be explored as a potential method for As bioremediation. However, its effectiveness needs to be improved. Biostimulation with organic matter amendment and bioaugmentation with the inoculation of genetic engineered bacteria could be potential strategies for As removal and site remediation. Here, the experiments were conducted to evaluate the impacts of rice straw and biochar amendment, inoculation of genetic engineered Pseudomonas putida KT2440 (GE P. putida) with high As volatilization activity, on microbial mediated As volatilization and removal from three different arseniferous soils. In general, the addition of rice straw (5%) significantly enhanced As methylation and volatilization in comparison with corresponding non-amended soils. Biochar amendments and inoculation of the GE P. putida increased As methylation and volatilization, respectively, but less than that of rice straw addition. The effectiveness of As volatilizations are quite different in the various paddy soils. The combined amendments of rice straw and GE P. putida exhibited the highest As removal efficiency (483.2 μg/kg/year) in Dayu soil, with 1.2% volatilization of the total As annually. The highest water-soluble As concentration (0.73 mg/kg) in this soil could be responsible for highest As volatilization besides the rice straw and bacteria in this soil.     

Modelling phytoremediation by the hyperaccumulating fern, Pteris vittata, of soils historically contaminated with arsenic

Pteris vittata plants were grown on twenty-one UK soils contaminated with arsenic (As) from a wide range of natural and anthropogenic sources. Arsenic concentration was measured in fern fronds, soil and soil pore water collected with Rhizon samplers. Isotopically exchangeable soil arsenate was determined by equilibration with ⁷³As^V. Removal of As from the 21 soils by three sequential crops of P. vittata ranged between 0.1 and 13% of total soil As. Ferns grown on a soil subjected to long-term sewage sludge application showed reduced uptake of As because of high available phosphate concentrations. A combined solubility-uptake model was parameterised to enable prediction of phytoremediation success from estimates of soil As, ‘As-lability’ and soil pH. The model was used to demonstrate the remediation potential of P. vittata under different soil conditions and with contrasting assumptions regarding re-supply of the labile As pool from unavailable forms.     

Bioremediation of oil-contaminated soil using Candida catenulata and food waste

Even though petroleum-degrading microorganisms are widely distributed in soil and water, they may not be present in sufficient numbers to achieve contaminant remediation. In such cases, it may be useful to inoculate the polluted area with highly effective petroleum-degrading microbial strains to augment the exiting ones. In order to identify a microbial strain for bioaugmentation of oil-contaminated soil, we isolated a microbial strain with high emulsification and petroleum hydrocarbon degradation efficiency of diesel fuel in culture. The efficacy of the isolated microbial strain, identified as Candida catenulata CM1, was further evaluated during composting of a mixture containing 23% food waste and 77% diesel-contaminated soil including 2% (w/w) diesel. After 13 days of composting, 84% of the initial petroleum hydrocarbon was degraded in composting mixes containing a powdered form of CM1 (CM1-solid), compared with 48% of removal ratio in control reactor without inoculum. This finding suggests that CM1 is a viable microbial strain for bioremediation of oil-contaminated soil with food waste through composting processes.     

Plants' use of different nitrogen forms in response to crude oil contamination

In this study, we investigated Phragmites australis’ use of different forms of nitrogen (N) and associated soil N transformations in response to petroleum contamination. ¹⁵N tracer studies indicated that the total amount of inorganic and organic N assimilated by P. australis was low in petroleum-contaminated soil, while the rates of inorganic and organic N uptake on a per-unit-biomass basis were higher in petroleum-contaminated soil than those in un-contaminated soil. The percentage of organic N in total plant-assimilated N increased with petroleum concentration. In addition, high gross N immobilization and nitrification rates relative to gross N mineralization rate might reduce inorganic-N availability to the plants. Therefore, the enhanced rate of N uptake and increased importance of organic N in plant N assimilation might be of great significance to plants growing in petroleum-contaminated soils. Our results suggest that plants might regulate N capture under petroleum contamination.     

Influence of flooding and metal immobilising soil amendments on availability of metals for willows and earthworms in calcareous dredged sediment-derived soils

Soil amendments previously shown to be effective in reducing metal bioavailability and/or mobility in calcareous metal-polluted soils were tested on a calcareous dredged sediment-derived soil with 26 mg Cd/kg dry soil, 2200 mg Cr/kg dry soil, 220 mg Pb/kg dry soil, and 3000 mg Zn/kg dry soil. The amendments were 5% modified aluminosilicate (AS), 10% w/w lignin, 1% w/w diammonium phosphate (DAP, (NH₄)₂HPO₄), 1% w/w MnO, and 5% w/w CaSO₄. In an additional treatment, the contaminated soil was submerged. Endpoints were metal uptake in Salix cinerea and Lumbricus terrestris, and effect on oxidation-reduction potential (ORP) in submerged soils. Results illustrated that the selected soil amendments were not effective in reducing ecological risk to vegetation or soil inhabiting invertebrates, as metal uptake in willows and earthworms did not significantly decrease following their application. Flooding the polluted soil resulted in metal uptake in S. cinerea comparable with concentrations for an uncontaminated soil.     

Aporrectodea caliginosa, a suitable earthworm species for field based genotoxicity assessment?

There is a growing interest for the application of biomakers to field-collected earthworms. Therefore we have evaluated the usability of native populations of endogeic, widely distributed earthworm Aporrectodea caliginosa in the assessment of soil genotoxicity using the Comet assay. Validation of the Comet assay on earthworm coelomocytes has been established using commercially available Eisenia fetida exposed to copper, cadmium, and pentachlorophenol, along with A. caliginosa exposed to copper in a filter paper contact test. Neutral red retention time (NRRT) assay was conducted on copper exposed and field-collected earthworms. Significant DNA and lysosomal damage was measured using Comet and NRRT assays in native populations of A. caliginosa sampled from the polluted soils in the urban area in comparison to the earthworms from the reference site. The results of this study confirm the employment of A. caliginosa as a suitable species for the in situ soil toxicity and genotoxicity field surveys.     

Comparison of solid-phase bioassays and ecoscores to evaluate the toxicity of contaminated soils

Five bioassays (inhibition of lettuce germination and growth, earthworm mortality, inhibition of springtail population growth, avoidance by springtails) were compared, using four coke factory soils contaminated by PAHs and trace elements, before and after biotreatment. For each bioassay, several endpoints were combined in an ‘ecoscore’, a measure of test sensitivity. Ecoscores pooled over all tested bioassays revealed that most organisms were highly sensitive to the concentration of 3-ring PAHs. When four soils were combined, behavioural tests using the springtail Folsomia candida showed higher ecoscores, i.e. they were most sensitive to soil contamination. However, despite overall higher sensitivity of behavioural tests, which could be used for cheap and rapid assessment of soil toxicity, especially at low levels of contamination, some test endpoints were more sensitive than others, and this may differ from a soil to another, pointing to the need for a battery of bioassays when more itemized results are expected.     

Suitability of different salt marsh plants for petroleum hydrocarbons remediation

The suitability of the salt-marsh species Halimione portulacoides, Scirpus maritimus, Juncus maritimus and an association of the last two for remediation of petroleum hydrocarbons (PHC) in soil was investigated. An outdoor laboratory experiment (microcosm-scale) was carried out using contaminated soil collected in a refinery, as a complement of another study carried out in the refinery environment (mesocosm-scale). Soil samples with old contamination (mainly crude oil) and with a mixture of the old and recent (turbine oil) contamination were tested. Studies in both micro- and mesocosm-scale provided results coherent in substance. The presence of S. maritimus caused removal of old contamination which was refractory to natural attenuation (after 7 months of exposure, efficiency was 13% when only old contamination was present and 40% when the soil also contained recent contamination). H. portulacoides (only included in the microcosm-scale study) revealed also potentiality for PHC remediation, although with less efficiency than S. maritimus. Degradation of recent contamination was also faster in the presence of plants (after 7 months: 100% in the presence of S. maritimus vs. 63% in its absence). As these species are common in salt marsh areas in Atlantic coast of Europe, it is probable they will be also useful for recovering coast sediments. In contrast, J. maritimus and association did not reveal capability to remove PHC from soil, the presence of J. maritimus inhibiting the capability of S. maritimus.