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.     

Effect of endophyte-infection on growth parameters and Cd-induced phytotoxicity of Cd-hyperaccumulator Solanum nigrum L

The aim of this work was to evaluate effects of endophytic bacterium inoculation on plant growth and assess the possible mechanism of endophyte in heavy metal phytoremediation. Seeds of Solanum nigrum L. were inoculated with endophyte Serratia nematodiphila LRE07 and were subjected to Cd in the growing medium. Cd produced a significant inhibition on plant growth and a reduction in the content of photosynthetic pigments. The inoculation of endophytic bacterium alleviated the Cd-induced changes, resulting in more biomass production and higher photosynthetic pigments content of leaves compared with non-symbiotic ones. The beneficial effect was more obvious at relatively low Cd concentration (10 μM). Based on the alteration of nutrient uptake and activated oxygen metabolism in infected plants, the possible mechanisms of endophytic bacterium in Cd phytotoxicity reduction can be concluded as uptake enhancement of essential mineral nutrition and improvement in the antioxidative enzymes activities in infected plant.     

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.     

Temporal variations and bioaccumulation of heavy metals in different Suaeda salsa marshes of the Yellow River estuary,China

To understand the temporal variations and bioaccumulation of heavy metals in the coastal marshes, the concentrations of heavy metals (Cr, Ni, Pb, and Cu) in the two Suaeda salsa marshes [middle S. salsa marsh (MM) and low S. salsa marsh (LM)] of the Yellow River estuary were determined from May to November in 2008 by in situ sampling and inductively coupled plasma mass spectrometry (ICP-MS) analysis. Results showed that heavy metal concentrations in S. salsa of MM and LM were generally in the order of Cu?>?Cr?>?Pb?>?Ni, while those in sediments fell in the order of Cr?>?Ni?>?Cu?>?Pb. Heavy metal concentrations of S. salsa in MM and LM were different, and significant differences were observed in stems (F?=?4.797, p?=?0.046) and litters (F?=?6.799, p?=?0.026) for Ni. Litter was the main stock of heavy metals, and the allocations of Cr, Ni, and Pb reached 31.25–51.31, 28.49–42.58, and 29.55–66.79 % (in MM) and 36.73–48.60, 41.70–57.87, and 33.30–60.64 % (in LM), respectively. The ratios of roots/leaves (R/L) and roots/stems (R/S) for Cr and Ni in MM were mostly greater than 1, while those ratios in LM were mostly less than 1, indicating that Cr and Ni in S. salsa at LM had greater mobility compared with those at MM. Moreover, the [accumulation factor, AF]_plant of Cr, Ni, Cu, and Pb in LM, especially [AF]_root and [AF]_stem of Cr and [AF]_litter of Ni, was also higher than that in MM. These indicated that S. salsa grown in LM was more suitable for potential biomonitor or phytoremediation of Cr, Ni, Cu, and Pb if intertidal sediments were seriously contaminated with an increase of pollutant loading (especially heavy metals) in the Yellow River estuary. The use of biomonitor (S. salsa) living and growing in LM could yield valuable information not only on the presence of anthropogenic stressors, but, more importantly, on the adverse influence the stressors are having on the environment.     

Effect of birch (Betula spp.) and associated rhizoidal bacteria on the degradation of soil polyaromatic hydrocarbons, PAH-induced changes in birch proteome and bacterial community

Two birch clones originating from metal-contaminated sites were exposed for 3 months to soils (sand-peat ratio 1:1 or 4:1) spiked with a mixture of polyaromatic hydrocarbons (PAHs; anthracene, fluoranthene, phenanthrene, pyrene). PAH degradation differed between the two birch clones and also by the soil type. The statistically most significant elimination (p ≤ 0.01), i.e. 88% of total PAHs, was observed in the more sandy soil planted with birch, the clearest positive effect being found with Betula pubescens clone on phenanthrene. PAHs and soil composition had rather small effects on birch protein complement. Three proteins with clonal differences were identified: ferritin-like protein, auxin-induced protein and peroxidase. Differences in planted and non-planted soils were detected in bacterial communities by 16S rRNA T-RFLP, and the overall bacterial community structures were diverse. Even though both represent complex systems, trees and rhizoidal microbes in combination can provide interesting possibilities for bioremediation of PAH-polluted soils.     

Clonal differences in survival capacity, copper and zinc accumulation, and correlation with leaf polyamine levels in poplar: A large-scale field trial on heavily polluted soil

Three ex situ collections of poplar clones from natural populations of Populus alba and P. nigra growing in northern Italy were assessed for their genetic dissimilarity (GD) by means of amplified fragment length polymorphism (AFLP). The high GD evidenced within populations was exploited for screening 168 clones in a field trial on heavy metal-polluted soil. After one growth season, clonal differences in plant survival and growth were observed. On the basis of performance, six clones were singled out, and used to evaluate copper and zinc accumulation in different organs. Clonal differences in metal concentrations were most evident for leaves and stems; one clone of P. alba (AL35) had a distinctly higher concentration of both metals in the roots. Leaf polyamine (putrescine, spermidine, spermine) profiles correlated with tissue metal concentrations, depending on the clone, plant organ and metal. In particular, the high metal-accumulating clone AL35 exhibited a dramatically higher concentration of free and conjugated putrescine. Overall, the results indicate that, given the high GD of Populus even within populations, it is possible to identify genotypes best suited for soil clean-up, and useful also for investigating physiological markers associated with high metal accumulation/tolerance     

Microbial diversity,community composition and metabolic potential in hydrocarbon contaminated oily sludge: prospects for in situ bioremediation

Microbial community composition and metabolic potential have been explored in petroleum-hydrocarbon-contaminated sludge of an oil storage facility. Culture-independent clone library-based 16S rRNA gene analyses revealed that the bacterial community within the sludge was dominated by the members of β-Proteobacteria (35 %), followed by Firmicutes (13 %), δ-Proteobacteria (11 %), Bacteroidetes (10 %), Acidobacteria (6 %), α-Proteobacteria (3 %), Lentisphaerae (2 %), Spirochaetes (2 %), and unclassified bacteria (5 %), whereas the archaeal community was composed of Thermoprotei (54 %), Methanocellales (33 %), Methanosarcinales/Methanosaeta (8 %) and Methanoculleus (1 %) members. Methyl coenzyme M reductase A (mcrA) gene (a functional biomarker) analyses also revealed predominance of hydrogenotrophic, methanogenic Archaea (Methanocellales, Methanobacteriales and Methanoculleus members) over acetoclastic methanogens (Methanosarcinales members). In order to explore the cultivable bacterial population, a total of 28 resident strains were identified and characterized in terms of their physiological and metabolic capabilities. Most of these could be taxonomically affiliated to the members of the genera Bacillus, Paenibacillus, Micrococcus, Brachybacterium, Aerococcus, and Zimmermannella, while two strains were identified as Pseudomonas and Pseudoxanthomonas. Metabolic profiling exhibited that majority of these isolates were capable of growing in presence of a variety of petroleum hydrocarbons as sole source of carbon, tolerating different heavy metals at higher concentrations (≥1 mM) and producing biosurfactant during growth. Many strains could grow under a wide range of pH, temperature, or salinity as well as under anaerobic conditions in the presence of different electron acceptors and donors in the growth medium. Correlation between the isolates and their metabolic properties was estimated by the unweighted pair group method with arithmetic mean (UPGMA) analysis. Overall observation indicated the presence of diverse groups of microorganisms including hydrocarbonoclastic, nitrate reducing, sulphate reducing, fermentative, syntrophic, methanogenic and methane-oxidizing bacteria and Archaea within the sludge community, which can be exploited for in situ bioremediation of the oily sludge.     

Characterization of heavy metal-resistant endophytic bacteria from rape (Brassica napus) roots and their potential in promoting the growth and lead accumulation of rape

Two lead (Pb)-resistant endophytic bacteria were isolated from rape roots grown in heavy metal-contaminated soils and characterized. A pot experiment was conducted for investigating the capability of the two isolates to promote the growth and Pb uptake of rape from Pb-amended soil. The two isolates were identified as Pseudomonas fluorescens G10 and Microbacterium sp. G16 based on the 16S rDNA gene sequence analysis. Strains G10 and G16 exhibited different multiple heavy metal and antibiotic resistance characteristics and increased water-soluble Pb in solution and in Pb-added soil. Root elongation assays demonstrated increases in root elongation of inoculated rape seedlings compared to the control plants. Strain G16 produced indole acetic acid, siderophores and 1-aminocyclopropane-1-carboxylate deaminase. Increases in biomass production and total Pb uptake in the bacteria-inoculated plants were obtained compared to the control. The two strains could colonize the root interior and rhizosphere soil of rape after root inoculation.     

Model evaluation of the phytoextraction potential of heavy metal hyperaccumulators and non-hyperaccumulators

Evaluation of the remediation ability of zinc/cadmium in hyper- and non-hyperaccumulator plant species through greenhouse studies is limited. To bridge the gap between greenhouse studies and field applications for phytoextraction, we used published data to examine the partitioning of heavy metals between plants and soil (defined as the bioconcentration factor). We compared the remediation ability of the Zn/Cd hyperaccumulators Thlaspi caerulescens and Arabidopsis halleri and the non-hyperaccumulators Nicotiana tabacum and Brassica juncea using a hierarchical linear model (HLM). A recursive algorithm was then used to evaluate how many harvest cycles were required to clean a contaminated site to meet Taiwan Environmental Protection Agency regulations. Despite the high bioconcentration factor of both hyperaccumulators, metal removal was still limited because of the plants' small biomass. Simulation with N. tabacum and the Cadmium model suggests further study and development of plants with high biomass and improved phytoextraction potential for use in environmental cleanup.     

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.     

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.     

重金属污染土壤中提高植物提取修复功效的探讨

随着对重金属超积累植物研究的加深 ,用植物提取修复技术来改良重金属污染的土壤已逐步进入实用阶段。本文所探讨的提高此技术功效的方法基于两个方面 :提高土壤溶液中重金属的浓度 ,促进植物对重金属的吸收 ;根据已了解的超积累的生理机制可能采取的一些措施     

Long-term Hg pollution-induced structural shifts of bacterial community in the terrestrial isopod (Porcellio scaber) gut

In previous studies we detected lower species richness and lower Hg sensitivity of the bacteria present in egested guts of Porcellio scaber (Crustacea, Isopoda) from chronically Hg polluted than from unpolluted environment. Basis for such results were further investigated by sequencing of 16S rRNA genes of mercury-resistant (Hg^r) isolates and clone libraries. We observed up to 385 times higher numbers of Hg^r bacteria in guts of animals from polluted than from unpolluted environment. The majority of Hg^r strains contained merA genes. Sequencing of 16S rRNA clones from egested guts of animals from Hg-polluted environments showed elevated number of bacteria from Pseudomonas, Listeria and Bacteroidetes relatives groups. In animals from pristine environment number of bacteria from Achromobacter relatives, Alcaligenes, Paracoccus, Ochrobactrum relatives, Rhizobium/Agrobacterium, Bacillus and Microbacterium groups were elevated. Such bacterial community shifts in guts of animals from Hg-polluted environment could significantly contribute to P. scaber Hg tolerance.     

Molecular characterization of microbial communities and quantification of <Emphasis Type="Italic">Mycobacterium immunogenum</Emphasis> in metal removal fluids and their associated biofilms

A number of human health effects have been associated with exposure to metal removal fluids (MRFs). Multiple lines of research suggest that a newly identified organism, Mycobacterium immunogenum (MI), appears to have an etiologic role in hypersensitivity pneumonitis (HP) in case of MRFs exposed workers. However, our knowledge of this organism, other possible causative agents (e.g., Pseudomonads), and the microbial ecology of MRFs in general, is limited. In this study, culture-based methods and small subunit ribosomal RNA gene clone library approach were used to characterize microbial communities in MRF bulk fluid and associated biofilm samples collected from fluid systems in an automobile engine plant. PCR amplification data using universal primers indicate that all samples had bacterial and fungal contaminated. Five among 15 samples formed colonies on the Mycobacteria agar 7H9 suggesting the likely presence of Mycobacteria in these five samples. This observation was confirmed with PCR amplification of 16S rRNA gene fragment using Mycobacteria specific primers. Two additional samples, Biofilm-1 and Biofilm-3, were positive in PCR amplification for Mycobacteria, yet no colonies formed on the 7H9 cultivation agar plates. Real-time PCR was used to quantify the abundance of M. immunogenum in these samples, and the data showed that the copies of M. immunogenum 16S rRNA gene in the samples ranges from 4.33?×?10⁴ copy/ml to 4.61?×?10⁷ copy/ml. Clone library analysis revealed that Paecilomyces sp. and Acremonium sp. and Acremonium-like were dominant fungi in MRF samples. Various bacterial species from the major phylum of proteobacteria were found and Pseudomonas is the dominant bacterial genus in these samples. Mycobacteria (more specifically MI) were found in all biofilm samples, including biofilms collected from inside the MRF systems and from adjacent environmental surfaces, suggesting that biofilms may play an important role in microbial ecology in MRFs. Biofilms may provide a shield or sheltered microenvironment for the growth and/or colonization of Mycobacteria in MRFs.     

Isolation and characterization of Bacillus cereus IST105 from electroplating effluent for detoxification of hexavalent chromium

Purpose

Electroplating industries are the main sources of heavy metals, chromium, nickel, lead, zinc, cadmium and copper. The highest concentrations of chromium (VI) in the effluent cause a direct hazards to human and animals. Therefore, there is a need of an effective and affordable biotechnological solution for removal of chromium from electroplating effluent.

Methods

Bacterial strains were isolated from electroplating effluent to find out higher tolerant isolate against chromate. The isolate was identified by 16S rDNA sequence analysis. Absorbed chromium level of bacterium was determined by inductively coupled plasma-atomic emission spectrometer (ICP-AES), atomic absorption spectrophotometer (AAS), scanning electron microscope (SEM), transmission electron microscope (TEM) and energy dispersive X-ray analysis (EDX). Removal of metals by bacterium from the electroplating effluent eventually led to the detoxification of effluent confirmed by MTT assay. Conformational changes of functional groups of bacterial cell surface were studied through Fourier transform infrared spectroscopy.

Results

The chromate tolerant isolate was identified as Bacillus cereus. Bacterium has potency to remove more than 75% of chromium as measured by ICP-AES and AAS. The study indicated the accumulation of chromium (VI) on bacterial cell surface which was confirmed by the SEM-EDX and TEM analysis. The biosorption of metals from the electroplating effluent eventually led to the detoxification of effluent. The increased survivability of Huh7 cells cultured with treated effluent also confirmed the detoxification as examined by MTT assay.

Conclusion

Isolated strain B. cereus was able to remove and detoxify chromium (VI). It would be an efficient tool of the biotechnological approach in mitigating the heavy metal pollutants.     

Impact of metal stress on the production of secondary metabolites in <Emphasis Type="Italic">Pteris vittata</Emphasis> L. and associated rhizosphere bacterial communities

Plants adapt to metal stress by modifying their metabolism including the production of secondary metabolites in plant tissues. Such changes may impact the diversity and functions of plant associated microbial communities. Our study aimed to evaluate the influence of metals on the secondary metabolism of plants and the indirect impact on rhizosphere bacterial communities. We then compared the secondary metabolites of the hyperaccumulator Pteris vittata L. collected from a contaminated mining site to a non-contaminated site in Vietnam and identified the discriminant metabolites. Our data showed a significant increase in chlorogenic acid derivatives and A-type procyanidin in plant roots at the contaminated site. We hypothesized that the intensive production of these compounds could be part of the antioxidant defense mechanism in response to metals. In parallel, the structure and diversity of bulk soil and rhizosphere communities was studied using high-throughput sequencing. The results showed strong differences in bacterial composition, characterized by the dominance of Proteobacteria and Nitrospira in the contaminated bulk soil, and the enrichment of some potential human pathogens, i.e., Acinetobacter, Mycobacterium, and Cupriavidus in P. vittata’s rhizosphere at the mining site. Overall, metal pollution modified the production of P. vittata secondary metabolites and altered the diversity and structure of bacterial communities. Further investigations are needed to understand whether the plant recruits specific bacteria to adapt to metal stress.     

功能微生物对污染农田土壤中铅锌的溶出实验

用微生物修复重金属污染的土壤是当今的研究热点之一。试图寻找一类可以增加土壤中重金属铅、锌的可溶态的功能微生物,并研究该微生物在不同土壤含水率和不同投菌比条件下对铅、锌溶出作用的影响。最终筛选出符合条件的菌JK3,并发现当土壤含水率为35%,投菌比为0.15 mL/g土(菌液OD=1)时,土壤中铅和锌的可溶态增加量最多。其中,可溶态铅的含量由原来的0.49 mg/kg增加到了5.04 mg/kg,可溶态锌的含量由原来的2.23 mg/kg增加到了22.44mg/kg。该方法为后续用植物提取或化学淋洗等方法将土壤中的重金属彻底去除提供了可能。     

Interactions between plant and rhizosphere microbial communities in a metalliferous soil

In the present work, the relationships between plant consortia, consisting of 1-4 metallicolous pseudometallophytes with different metal-tolerance strategies (Thlaspi caerulescens: hyperaccumulator; Jasione montana: accumulator; Rumex acetosa: indicator; Festuca rubra: excluder), and their rhizosphere microbial communities were studied in a mine soil polluted with high levels of Cd, Pb and Zn. Physiological response and phytoremediation potential of the studied pseudometallophytes were also investigated. The studied metallicolous populations are tolerant to metal pollution and offer potential for the development of phytoextraction and phytostabilization technologies. T. caerulescens appears very tolerant to metal stress and most suitable for metal phytoextraction; the other three species enhance soil functionality. Soil microbial properties had a stronger effect on plant biomass rather than the other way around (35.2% versus 14.9%). An ecological understanding of how contaminants, ecosystem functions and biological communities interact in the long-term is needed for proper management of these fragile metalliferous ecosystems.     

Bioremediation of Cd and carbendazim co-contaminated soil by Cd-hyperaccumulator Sedum alfredii associated with carbendazim-degrading bacterial strains

The objective of this study was to develop a bioremediation strategy for cadmium (Cd) and carbendazim co-contaminated soil using a hyperaccumulator plant (Sedum alfredii) combined with carbendazim-degrading bacterial strains (Bacillus subtilis, Paracoccus sp., Flavobacterium and Pseudomonas sp.). A pot experiment was conducted under greenhouse conditions for 180 days with S. alfredii and/or carbendazim-degrading strains grown in soil artificially polluted with two levels of contaminants (low level, 1 mg kg^?1 Cd and 21 mg kg^?1 carbendazim; high level, 6 mg kg^?1 Cd and 117 mg kg^?1 carbendazim). Cd removal efficiencies were 32.3–35.1 % and 7.8–8.2 % for the low and high contaminant level, respectively. Inoculation with carbendazim-degrading bacterial strains significantly (P?<?0.05) increased Cd removal efficiencies at the low level. The carbendazim removal efficiencies increased by 32.1–42.5 % by the association of S. alfredii with carbendazim-degrading bacterial strains, as compared to control, regardless of contaminant level. Cultivation with S. alfredii and inoculation of carbendazim-degrading bacterial strains increased soil microbial biomass, dehydrogenase activities and microbial diversities by 46.2–121.3 %, 64.2–143.4 %, and 2.4–24.7 %, respectively. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis revealed that S. alfredii stimulated the activities of Flavobacteria and Bradyrhizobiaceae. The association of S. alfredii with carbendazim-degrading bacterial strains enhanced the degradation of carbendazim by changing microbial activity and community structure in the soil. The results demonstrated that association of S. alfredii with carbendazim-degrading bacterial strains is promising for remediation of Cd and carbendazim co-contaminated soil.