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Bacteria immobilisation on hydroxyapatite surface for heavy metals removal

Publication . Piccirillo, C.; Pereira, S. I. A.; Marques, A. P. G. C.; Pullar, R. C.; Tobaldi, D. M.; Pintado, M. E.; Castro, P. M. L.

Selected bacterial strains were immobilised on the surface of hydroxyapatite (Ca-10(PO4)(6)(OH)(2) - HAp) of natural origin (fish bones). The capacity of the material, alone and in combination with the bacterial strains to act as heavy metal removers from aqueous streams was assessed. Pseudomonas fluorescens (S3X), Micro bacterium oxydans (EC29) and Cupriavidus sp. (1C2) were chosen based on their resistance to heavy metals and capacity of adsorbing the metals. These systems were tested using solutions of Zn(II), Cd(II) and in solutions containing both metals. A synergistic effect between the strains and HAp, which is effective in removing the target heavy metals on its own, was observed, as the combination of HAp with the bacterial strains led to higher adsorption capacity for both elements. For the solutions containing only one metal the synergistic effect was greater for higher metal concentrations; 1C2 and EC29 were the most effective strains for Zn(II) and Cd(II) respectively, while S3X was less effective. Overall, an almost four-fold increase was observed for the maximum adsorption capacity for Zn(II) when 10 was employed - 0.433 mmol/g in comparison of 0.121 mmol/g for the unmodified HAP. For Cd(II), on the other hand, an almost three-fold increase was registered with EC29 bacterial strain - 0.090 vs 0.036 mmol/g for the unmodified HAp. When the solutions containing both metals were tested, the effect was more marked for lower concentrations.

2013Journal article

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Endophytic culturable bacteria colonizing Lavandula dentata L. plants: Isolation, characterization and evaluation of their plant growth-promoting activities

Publication . Pereira, S.I.A.; Monteiro, Cristina M.; Vega, A. L.; Castro, Paula M. L.

Aromatic plants such as lavender are stirring the attention of many researchers due to their content in bioactive secondary metabolites that can be used in traditional medicine. However, information regarding naturally occurring lavender associated bacterial endophytes (BE) is limited. To the best of our knowledge, this is the first study which aims to assess the phylogenetic diversity of the culturable endophytic bacteria of Lavandula dentata cultivated under organic management and to evaluate their potential as plant growth promoting (PGP) agents. BE were grouped by random amplified polymorphic DNA and identified by 16S ribosomal RNA gene sequencing. Endophytes were further characterized for the ability to produce several PGP substances, like ammonia, siderophores, indol-3-acetic acid, and hydrogen cyanide and for the ability to solubilize phosphate. Plant cell-wall degrading enzymes were also determined. Densities of BE were higher in roots (log 6.39 CFU g−1 fresh weight) than in shoots (log 5.56 CFU g−1 fresh weight). Phylogenetic analysis showed that BE were affiliated to two major groups: -Proteobacteria (50%) and Firmicutes (31.6%) and a small part belonged to - (7.9%) and -Proteobacteria (10.5%), being Pseudomonas and Bacillus the most highly represented genera. Higher bacterial diversity was found in the lavender roots, with endophytes belonging to 6 different genera (Pseudomonas, Variovorax, Rhizobium, Caulobacter, Bacillus and Paenibacillus), than in shoots where only 3 genera (Bacillus, Pseudomonas and Xanthomonas) were found. Overall, BE showed ability to produce extracellular enzymes and multiple PGP traits, suggesting their potential use as efficient bioinoculants in sustainable cultivation of medicinal and aromatic plants.

2016Journal article

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Assessment of plant growth promoting bacterial populations in the rhizosphere of metallophytes from the Kettara mine, Marrakech

Publication . Benidire, L.; Pereira, S. I. A.; Castro, P. M. L.; Boularbah, A.

Soil heavy metal contamination resulting from mining activities constitutes a major environmental problem worldwide. The spread of heavy metals is often facilitated by scarce vegetation cover, so there is an urgent need to improve plant survival and establishment in these metalliferous areas. This study is aimed at the isolation and analysis of the phylogenetic relationship of culturable bacteria from the rhizosphere of metallophyte plants growing in the Kettara mine, in Marrakech, in order to select plant growth-promoting rhizobacteria (PGPR), which could be used in assisted-phytoremediation. Bacterial isolates were grouped by random amplified polymorphic DNA analysis and identified by 16S rRNA gene sequencing. Strains were further characterized for the production of plant growth-promoting (PGP) substances, such as NH3, siderophores, indol-3-acetic acid (IAA), hydrogen cyanide, and extracellular enzymes, for ACC-deaminase activity, their capacity to solubilize phosphate, and for their tolerance to heavy metals and acidic pH. Rhizosphere soils were highly contaminated with Cu and Zn and presented low fertility. Phylogenetic analysis showed that the rhizobacteria were affiliated to three major groups: γ-Proteobacteria (48 %), β-Proteobacteria (17 %), and Bacilli (17 %). The most represented genera were Pseudomonas (38 %), Bacillus (10 %), Streptomyces (10 %), and Tetrathiobacter (10 %). Overall, rhizobacterial strains showed an ability to produce multiple, important PGP traits, which may be helpful when applied as plant growth promoter agents in contaminated soils. PGPR were also able to withstand high levels of metals (up to 2615.2 mg Zn l(-1), 953.29 mg Cu l(-1), and 1124.6 mg Cd l(-1)) and the order of metal toxicity was Cd > Cu > Zn. The rhizobacterial strains isolated in the present study have the potential to be used as efficient bioinoculants in phytoremediation strategies for the recovery of Kettara mine soils.

2016Journal article

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Selection of metal resistant plant growth promoting rhizobacteria for the growth and metal accumulation of energy maize in a mine soil — Effect of the inoculum size

Publication . Moreira, Helena; Pereira, Sofia I. A.; Marques, Ana P. G. C.; Rangel, António O. S. S.; Castro, Paula M. L.

Heavy metals, such as Cd and Zn, are spilled in soils by several anthropogenic sources, including mining activities. Their toxic effects can be minimized using plants especially when paired with plant growth promoting rhizobacteria (PGPR), under phytomanagement strategies. Several factors can contribute to the failure of rhizobacterial inoculation, such as bacteria selection and the inoculum size. In this work five metal resistant PGPR (Ralstonia eutropha 1C2, Chryseobacterium humi ECP37, Pseudomonas fluorescens S3X, Rhizobium radiobacter EC1B and Pseudomonas reactans EDP28) were investigated for their in vitro growth promoting traits and for their ability to induce growth of maize seedlings exposed to Zn and Cd. PGPR inoculum size (10 and 20 mL) and inoculation effectiveness was assessed in energy maize sowed in a mine soil. The results showed that some bacteria only exhibited or enhanced PGP traits when exposed to metals. The bacterial strains ECP37 and EDP28 were the most efficient in improving seedling growth with increasing metal concentrations, followed by S3X. When inoculated in energy maize grown in mine soil, these same strains also outperformed the others by increasing shoot biomass and elongation, metal accumulation, and by decreasing it in roots. The most evident effect of doubling the inoculum size was the increase in Cd accumulation, which was of 17% and 31% in roots and shoots, respectively. Other effects included a slight reduction in shoots' biomass (13%) and a general decrease in P tissue content. The results obtained suggest that PGPR selection prior to inoculation in the target soils should be primarily based in seedling growth promotion under metal exposure. Additionally, the size of the inoculum applied in the soil rhizosphere appears to be important in remediation processes and should be taken into account when planning phytomanagement strategies, especially when the biomass of plants is an important demand.

2016Journal article

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Mine land valorization through energy maize production enhanced by the application of plant growth-promoting rhizobacteria and arbuscular mycorrhizal fungi

Publication . Moreira, Helena; Pereira, Sofia I. A.; Marques, Ana P. G. C.; Rangel, António O. S. S.; Castro, Paula M. L.

The use of heavy metals (HM) contaminated soils to grow energy crops can diminish the negative impact of HM in the environment improving land restoration. The effect of two PGPR (B1—Chryseobacterium humi ECP37T and B2—Pseudomonas reactans EDP28) and an AMF (F—Rhizophagus irregularis) on growth, Cd and Zn accumulation, and nutritional status of energy maize plants grown in a soil collected from an area adjacent to a Portuguese mine was assessed in a greenhouse experiment. Both bacterial strains, especially when co-inoculated with the AMF, acted as plant growth-promoting inoculants, increasing root and shoot biomass as well as shoot elongation. Cadmium was not detected in the maize tissues and a decrease in Zn accumulation was observed for all microbial treatments in aboveground and belowground tissues—with inoculation of maize with AMF and strain B2 leading to maximum reductions in Zn shoot and root accumulation of up to 48 and 43 %, respectively. Although microbial single inoculation generally did not increase N and P levels in maize plants, co-inoculation of the PGPR and the AMF improved substantially P accumulation in roots. The DGGE analysis of the bacterial rhizosphere community showed that the samples inoculated with the AMF clustered apart of those without the AMF and the Shannon-Wiener Index (H′) increased over the course of the experiment when both inoculants were present. This work shows the benefits of combined inoculation of AMF and PGPR for the growth energy maize in metal contaminated soils and their potential for the application in phytomanagement strategies.

2016Journal article

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