Role of plant growth- promoting bacteria on Phytoextraction process of Cu(II) and Cr(VI) by Helianthus annuus and Zea mays

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(1)1. Role of Plant Growth-Promoting Bacteria on Phytoextraction Process of Cu(II) and Cr(VI) by Helianthus annuus and Zea mays. by. B.Sc. DANIEL FERNANDO ROJAS TAPIAS. Thesis. Presented to the Faculty of Science of the Universidad de los Andes in Partial Fulfillment of the Requirements for the Degree of. Master in Biological Sciences: Microbiology. Universidad de los Andes January, 2011.

(2) 2. Role of Plant Growth-Promoting Bacteria on Phytoextraction Process of Cu(II) and Cr(VI) by Helianthus annuus and Zea mays. Directed by. Director: Jenny Dussán Garzón Co-director: Ruth Bonilla Buitrago. Approved by supervising committee. Supervisor: Maria Ximena Rodríguez. Adriana Bernal.

(3) 3. TABLE OF CONTENTS. Summary................................................................................................................................................. 7 Objectives ............................................................................................................................................... 8 General objective .............................................................................................................................. 8 Specific objectives............................................................................................................................. 8 CHAPTER I: Effect of inoculation with plant growth-promoting bacteria on growth and copper uptake by sunflower on a Cu-contaminated soil 1.1 Abstract ............................................................................................................................................. 9 1.2 Keywords .......................................................................................................................................... 9 1.3 Introduction...................................................................................................................................... 9 1.4 Materials and methods................................................................................................................. 11 1.4.1 Strains and culture media..................................................................................................... 11 1.4.2 Genetic characterization of the PGPB................................................................................ 11 1.4.3 Phosphate solubilization, indolic compound production and siderophores synthesis............................................................................................................................................ 12 1.4.3.1 Phosphate solubilization ................................................................................................. 12 1.4.3.2 Indole quantification........................................................................................................ 12 1.4.3.3 Siderophores ..................................................................................................................... 13 1.4.4 Copper accumulation by bacterial strains......................................................................... 13 1.4.5 Soil analysis ............................................................................................................................ 13 1.4.6 Influence of PGPB and Cu on the growth of plant species and copper uptake. ....... 14 1.4.7 Statistical analysis.................................................................................................................. 14 1.5 Results ............................................................................................................................................. 14 1.5.1 Strains, molecular identification and MIC ....................................................................... 14 1.5.2 Plant growth-promoting (PGP) features of strains.......................................................... 15 1.5.3 Copper accumulation ............................................................................................................ 17 1.5.4 Influence of bacterial inoculation on the growth of Helianthus annuus..................... 18 1.5.5 Effect of PGPB inoculation on copper accumulation...................................................... 21.

(4) 4. 1.6 Discussion ...................................................................................................................................... 21 1.7 Bibliography .................................................................................................................................. 25 CHAPTER II: Improvement of growth and Cu(II) uptake by Zea mays mediated by plantgrowth promoting bacteria (PGPB) inoculation. 2.1 Abstract ........................................................................................................................................... 33 2.2 Keywords ........................................................................................................................................ 33 2.3 Introduction.................................................................................................................................... 33 2.4 Materials and Methods ................................................................................................................ 35 2.4.1 Strains and culture media..................................................................................................... 35 2.4.2 Minimal inhibitory concentration of heavy metals ........................................................ 35 2.4.3 Effect of Cu (II) on bacterial growth................................................................................... 36 2.4.4 PGPB features ......................................................................................................................... 36 2.4.4.1 Phosphate solubilization ................................................................................................. 36 2.4.4.2 Indole quantification........................................................................................................ 36 2.4.4.3 Siderophores ..................................................................................................................... 36 2.4.5 Effects of PGPB on the mobility of ions and copper in soil. ......................................... 37 2.4.6 Soil preparation and analysis .............................................................................................. 37 2.4.7 Influence of PGPB and Cu on the growth of plant species and copper uptake. ....... 38 2.4.8 Analysis of plant material .................................................................................................... 39 2.4.9 Statistical analysis.................................................................................................................. 39 2.5 Results and discussion................................................................................................................. 39 2.5.1 Strains characterization and MIC ....................................................................................... 39 2.5.2 Plant growth promoting features ........................................................................................ 41 2.5.2.1 Indole synthesis ................................................................................................................ 41 2.5.2.2 Phosphate solubilization ................................................................................................... 42 2.5.2.3 Siderophores synthesis.................................................................................................... 43 2.5.3 Effects of PGPB on the mobility of ions and copper in soil. ......................................... 43 2.5.4 Effect of copper on bacterial growth .................................................................................. 44 2.5.5 Influence of PGPB and Cu on the growth of maize ........................................................ 46 2.5.5.1 Effect of PGPB inoculation on plant growth ................................................................ 46.

(5) 5. 2.5.5.2 Photosynthetic pigments content .................................................................................. 51 2.5.6 Effect of PGPB inoculation on copper accumulation...................................................... 52 2.6 Bibliography .................................................................................................................................. 54 CHAPTER III: Effect of inoculation with PGPB strains Pseudomonas putida GN4 and Acinetobacter sp. CC30 on growth and Cr(VI) uptake by Zea mays and Helianthus annuus on Cr(VI) contaminated soil 3.1 Abstract ........................................................................................................................................... 64 3.2 Keywords ........................................................................................................................................ 64 3.3 Introduction.................................................................................................................................... 64 3.4 Materials and Methods ................................................................................................................ 66 3.4.1 Strains and culture media..................................................................................................... 66 3.4.2 Minimal inhibitory concentration of heavy metals ........................................................ 66 3.4.3 Plant growth promoting capabilities.................................................................................. 67 3.4.4 Pathogenicity test ................................................................................................................... 67 3.4.5 Effect of Cr(VI) on bacterial growth................................................................................... 67 3.4.6 Root elongation assay............................................................................................................ 68 3.4.7 Influence of PGPB and Cr on the growth of plant species and copper uptake and soil analysis. ..................................................................................................................................... 68 3.4.8 Plant material analysis .......................................................................................................... 69 3.4.9 Statistical analysis.................................................................................................................. 69 3.5 Results and discussion................................................................................................................. 69 3.5.1 Bacterial characterization...................................................................................................... 69 3.5.2 Root elongation assay............................................................................................................ 72 3.5.3 Effect of Cr(VI) on bacterial growth................................................................................... 72 3.5.4 Effect of PGPB inoculation on maize and sunflower on Cr-contaminated soil ........ 74 3.5.4.1 Growth of maize and sunflower .................................................................................... 74 3.5.4.2 Photosynthetic pigments................................................................................................. 77 3.5.5 Uptake of hexavalent chromium......................................................................................... 79 3.6 Bibliography .................................................................................................................................. 81.

(6) 6. Conclusions .......................................................................................................................................... 85.

(7) 7. Summary As a result of inappropriate deposition of organic residues, toxic anthropogenic compounds have become ubiquitous components in soil and waters. Several alternatives have been developed, however, one of the most important is bioremediation. It is a low-cost, effective and environmentally friendly alternative to remediate contaminated soil. In particular, the phytoremediation of heavy metals has become an important tool to alleviate the impact occasioned by inorganic contamination with arsenic, lead, cadmium, copper or chromium, for example. We decided to evaluate the process of phytoextraction of copper by its implications in agricultural productivity and chromium by its negative effects on both human and animal health. Additionally, selected two vegetable species: maize and sunflower. With regard to maize, it is plant specie of fast-growth and high biomass, characteristics desired in a phytoremediation process. While sunflower has been widely reported and characterized by its qualities to extract several heavy metals from soil. Plant growth-promoting bacteria (PGPB) defined as microorganisms with beneficial effect on plant development, have elucidated to be a relevant strategy to improve phytoremediation process. In this study, we exhibited the role of PGPB on improvement of this biotechnological strategy. We evaluated eight bacteria and characterized them by its capacity as PGPR. Also, identified this molecularly. We demonstrated that bacterial inoculation with bacteria Pseudomonas putida GN4 and Acinetobacter sp. CC30 enhanced plant growth and the content of chlorophyll a, chlorophyll b and carotenoids in both maize and sunflower plants under copper (II) and chromium (VI) contamination. With respect to extraction process, bacterial inoculation exerted an important effect on ions mobility in soil. Furthermore, increased the availability of copper extractable from soil. Several bacterial strategies are though to influence the efficiency of phytoremediation process. Bacterial capacities to synthesize indole or siderophores, solubilize phosphate or mineralize ammonia have evidenced to influence significantly on remediation of heavy metals by plants. Hence, in summary, utilization of PGPR to improve phytoremediation process is an important alternative to reduce cost and increased remediation efficiency. Further, it is a sustainable strategy to preserve the quality of environment..

(8) 8. Objectives General objective To evaluate the plant growth-promoting bacteria role on phytoextraction process of copper (II) and chromium (VI) by Helianthus annuus and Zea mays. Specific objectives 1. Evaluate the heavy metal tolerance of bacteria isolated from heavy metal contaminated soil and to characterize its capacity to promote growth plant. 2. Identify the bacterial role of selected strains on phytoremediation process. 3. Assess the effect of inoculation of plant growth-promoting bacteria on the phytoremediation process of copper (II) and chromium (VI) with Helianthus annuus and Zea mays..

(9) 9. CHAPTER I: Effect of inoculation with plant growth-promoting bacteria on growth and copper uptake by sunflower on a Cu-contaminated soil 1.1 Abstract The effect of plant growth-promoting bacteria (PGPB) inoculation on Helianthus annuus growth and copper uptake was investigated. For this, several bacteria were isolated from highly contaminated soil with heavy metals and hydrocarbons and characterized by its plantgrowth promoting capabilities. From forty-three bacteria recovered, four were selected to further studies: CC22, CC24, CC30 and CC33. The selected strains were characterized based on the 16S rDNA sequencing and identified as Pseudomonas putida, Enterobacter sp., Acinetobacter sp. and Acinetobacter sp., respectively. Strains were able to synthesize indole, solubilize phosphorus and produce siderophores. Additionally, were able to accumulate copper. Acinetobacter sp. CC33 exhibited greatest extent of Cu(II) accumulation with at least 50% more than the rest of bacteria. Acinetobacter sp. CC30 was selected to pot experiments. Its inoculation increased significantly plant growth expressed as root dry weight, shoot dry weight, root length, and shoot length, similarly improved photosynthetic pigments total content on non- and Cu-contaminated soil (p<0.05). Additionally, Cu uptake was improved by CC30 inoculation showing a significantly enhance in root Cu content (p<0.05). Thus, the present observations evidenced that the strains CC30 protected the plant against deleterious effect of copper contamination and improved the phytoextraction process of copper.. 1.2 Keywords Helianthus annuus, plant growth-promoting bacteria (PGPB), copper phytoextraction.. 1.3 Introduction Copper is an essential micronutrient for plants (Sommer, 1931; Arnon and Stout, 1939). However, when it is at high concentrations become toxic (Ouzounidou, 1995). Copper contamination is usually generated by human activities (Flemming and Trevors, 1989). Anthropogenic activities by which Cu enters soils and sediments include smelting, mining, plating, steelworks, refineries, domestic emission, application of fertilizers, sewage sludge, fungicides, etc (Flemming and Trevors, 1989). Excess Cu induces a wide range of biochemical effects and metabolic alterations in plants that are responsible for a strong inhibition of.

(10) 10. growth, sometimes accompanied by anomalous development (Fernandes and Henriques, 1991). The mechanisms through Cu causes toxicity are related with interactions with proteins, enzymes, nucleic acids and metabolites. Since of Cu is a redox-active transition metal its metabolism might result in the generation of Reactive Oxygen Species (ROS). These may lead to unspecific oxidation of proteins and membrane lipids, resulting in an increased concentration of thiobarbituric acid-reacting substances (TBARS), which are products of lipid peroxidation. Thus, can alter the biological activity of enzymes, modulating intracellular signaling, and exerting damaging effects on biological macromolecules, including DNA (Abdelly et al., 2008). In the same way, Cu can interfere with the structure and activity of certain proteins, likely, altering the protein function by binding of the metal to sulfhydryl groups, leading to the inhibition of activity or disruption of the structure (Tanyolac et al., 2007). As a consequence, several strategies have been addressed in order to solve this environmental problem (Vandegrift et al., 1992).. Nevertheless, those are usually expensive and also. contaminants (Glick, 2010). Hence, phytoremediation represents a low-cost and efficient technology with the purpose to remove heavy metal contamination from highly polluted soils. However, phytoremediation efficiency is often limited by the availability of the metal in soil, plant root development, and the level of tolerance of the plant to each particular metal (Cataldo and Wildung, 1978). In consequence, some alternatives have been developed in order to increase the removal of heavy metals from soil. The most common imply the use of chelator agents such as EDTA, which can enhance metal availability (Turgut et al., 2004; Turgut et al., 2005; Evangelou et al., 2007). Although, these may become persistent in the environment and may lead to the contamination of groundwater (Tandy et al., 2006). Soil microorganisms have shown to possess several mechanisms capable of altering metal availability for uptake into roots (Lasat, 2002). Additionally, may facilitate plant growth either directly or indirectly. These first involve several ways including the increasing in nutrients availability or plant growth regulation mediated by synthesis of phytohormones (Glick, 2010). There are several ways to increase nutrient availability. Nitrogen fixation allows increasing the nitrogen content in soil. In the same way, phosphate solubilization enhances phosphorus availability through solubilization of phosphate insoluble salts or mineralization of organic phosphorus compounds (RodrÌguez and Fraga, 1999), and siderophores synthesis, which can sequester iron from the soil and provide it to plant cells which can take up the.

(11) 11. bacterial siderophores-iron complex (Glick, 2010). On the other hand, microbial synthesis of phytohormones such as auxins, cytokinins and gibberellins, can enhance plant growth regulating several plant physiological processes (Costacurta and Vanderleyden, 1995). For instance, Sheng and Xia (2006) found increased growth of both root and shoot and Cd accumulation in canola as a result of soil inoculation with Cd- resistant PGPB. In the same way, Ma et al. (2009) showed that the inoculation with PGPB Achromobacter xylosoxidans AX10 increased plant growth and copper uptake, likely, by synthesis of indole, siderophores or phosphate solubilization. Regarding of indirect mechanisms, these are usually associated to protection against phytopatogenic attacks (Glick, 2010). Several reports have been elucidated the sunflower role to remediate heavy metals (Turgut et al., 2004; Lesage et al., 2005; Nehnevajova et al., 2005; Turgut et al., 2005; Mani et al., 2007). Although, to our knowledge few attempts have been carried out with the purpose to evaluate the PGPB role on sunflower phytoextraction process. Thus, the aims of this work were to characterize the plant growth-promoting capabilities of bacteria isolated from heavy-metal contaminated soil and select one to investigated its role on copper phytoextraction process.. 1.4 Materials and methods 1.4.1 Strains and culture media In our laboratory, 43 bacterial strains were isolated from hydrocarbons and heavy metal contaminated soil in Colombia. Four strains were selected by its capacity to synthesize indole and tolerate copper: CC22, CC24, CC30 and CC33. Bacteria were grown on LB culture media at 28°C and 150 rpm (standard conditions). Minimal inhibitory concentration of copper was tested on LBM media (in g/L: tryptone 5.0, yeast extract 5.0 and NaCl 5.0) at 28°C for 48 h. Stock solution was employed at 80 mM for copper in form of CuSO4·5H2O (249.68 gmol-1) and sterilized by filtration. The growth was verified spectrophotometrically (Genezy UV 10, Thermo Corporation) at 600nm after 48 h at standard condition.. 1.4.2 Genetic characterization of the PGPB The bacterial strains were grown for 16 h in LB broth at 28°C. From each culture were taken 25.0 µL and transferred to 1.5-mL microtubes. These were submerged in boiling water for 15 min. After, centrifuged at 13000 rpm for one minute and 0.9-µL supernatant were used as.

(12) 12. DNA templates. The 25.0-µL volume reactions of PCR amplifications were performed using an iCylcer thermocycler (Bio-Rad). Negative controls were done using ultra-pure water instead of DNA. PCR assays for primer sets targeting the 16S rDNA region included 0.9- µL template DNA, 2U of Taq Polymerase (TucanTaq, Corpogen), 1X PCR buffer, 2.5 mM MgCl2, 0.2 mM dNTPs and 0.3 µM of each primer. PCR protocols for 16S rDNA sequence (35-cycle) were performed as previously reported (Fierer and Jackson, 2006). A 622 bp fragment was expected and confirmed by taking 5.0-µL aliquots of PCR product and loading them to 1% agarose gels. These were stained with ethidium bromide and images were captured with a GelDoc imaging system (BioRad). The primers employed were: 352f (5’ GGT TAC CTT GTT ACG ACT T 3’) and 975r (5’ AGA GTT TGA TCC TGG CTC AG 3’). PCR products were purified using Wizard® SV Gel and PCR Clean-Up System kit (Promega). Purified products were sequenced using the BigDye® Terminator v3.1 Cycle Sequencing Kit using 325f primer and 975r primer. Sequences were submitted to the Ribosomal Database Project II using the BLASTn program.. 1.4.3 Phosphate solubilization, indolic compound production and siderophores synthesis. 1.4.3.1 Phosphate solubilization The bacterial cultures were grown in Pikovskaya medium with tricalcium phosphate at standard conditions for 120 h (Pikovskaya, 1948). The supernatants from culture were centrifuged at 10000 rpm for 10 min. Soluble phosphate in the supernatant was estimated by phosphomolybdate-blue method (Fiske and Subbarow, 1925).. 1.4.3.2 Indole quantification Indolic compounds were estimated using the colorimetric assay based on the Salkowsky reagent (Glickmann and Dessaux, 1995) employing the PC reagent (12 g/L FeCl3 in 7.9 H2SO4). The culture medium employed was K-lactate (Carreno-Lopez et al., 2000), the incubation was carried out for 72 h at 150 rpm in dark. The reaction between PC reagent and culture supernatant was in relation 1:1, and allowed to react for 30 min in dark. Indolic compounds were determined spectrophotometrically at 540 nm. Results are expressed in micrograms of total indolic compounds per mg of protein, using IAA as standard. Protein content was estimated as described (Bradford, 1976)..

(13) 13. 1.4.3.3 Siderophores Strains were screened for the quantitatively production of siderophores (Schwyn and Neilands, 1987). The bacteria were cultured on free-iron MM9 culture media for 72 h. Time after, 1.0-mL aliquot from culture was centrifuged at 10000 rpm for 5.0 min and 0.5-mL supernatant taken and allowed to react with 0.5-mL CAS assay solution. The mix was allowed to stabilize for 6.0 hours and after absorbance was measured at 630 nm. The standard curve was elaborated using 20 – 100 µM deferoxamine mesylate (DFMO). Additionally, the presence of diffusible fluorescent pigments in culture supernatants was estimated measuring supernatant absorbance at 400 nm (Alexander and Zuberer, 1991).. 1.4.4 Copper accumulation by bacterial strains Strains were cultured on LB medium for 24 h. After, the cells were centrifuged at 10000 rpm for 5.0 min and rinsed twice in 0,85% NaCl. The OD600 was adjusted at 0.200, again centrifuged and the supernatant discarded. Immediately, the pellets were resuspended in 1.0 mL of 100 µg mL-1 CuSO4·5H2O and incubated for 16 h. After time, the cultures were newly centrifuged and supernatant analyzed for copper content by atomic absorption spectrophotometry (Perkin Elmer, 2380). Additionally, the total protein content of pellets was estimated (Bradford, 1976) to co-relate it with the copper adsorbed (Lu et al., 2006).. 1.4.5 Soil analysis Soil samples were collected from the Corpoica Garden and representative of Altiplano Cundiboyancence. Once collected, the soil was air-dried at room temperature until eight percent of humidity was reached. When the soil was dry this was sieved at 2.0 mm. The basic soil properties were: organic matter 15.15 ± 0.21%; effective cationic interchange coefficient (ECIC) 8.26 ± 1.81 cmol/kg; pH 5.95 ± 0.07; total phosphorus 13.6 ± 0.70 mg/kg; total sulphur 12.6 ± 0.7 mg/kg; electric conductivity 0.69 ± 0.20 dS/m; total iron 134.5 ± 16.2 mg/kg; total copper 1.85 ± 0.07 mg/kg. Soil was autoclaved three times in three consecutive days and finally allowed to stand for eight days to stabilization..

(14) 14. 1.4.6 Influence of PGPB and Cu on the growth of plant species and copper uptake. Helianthus annuus seeds were surface sterilized with 1.0% NaClO for 10 min and rinsed several times in desionized-sterilized water. The seeds were pre-germinated on sterilized peat for 5 days. After, seedlings were submerged in adequate bacterial suspension three times or in sterilized water for controls and planted. The pots contained 400 g soil intentionally contaminated to reach 200 ppm CuSO4·5H2O and allowed to stabilize for 2 weeks. Plants were grown in a glasshouse at 15-25°C and 16:8 day/night regime. After 21 days, the plants were carefully removed from the pots and the root surface was cleaned several times with distilled water. Growth parameters as shoot and root length, and shoot and dry weight (DW), were determined. In the same way, photosynthetic pigment content was measured (Hiscox and Israelstam, 1979) with the equations proposed by Wellburn (1994). For Cu-uptake analysis the copper content was measured on shoot and root. Briefly, vegetal tissue was oven-dried at 60°C for 48 h and ground separately. After, 200 mg of either shoot or root ground were mixed with 65% HNO3 - 30% H2O2 in relation 1:1 to final volume of 12.0 mL. The mixes were subjected to microwave digestion for 2.0 h at 150°C. After process, the sample was completed to volume 20 mL and copper content measured by Atomic Absorption Spectrometry (Perkin Elmer, 2380).. 1.4.7 Statistical analysis The statistical analysis was carried out employing the SPSS 17.0 software. The analyses were done with 95% confidentiality. HSD Tukey test was used to discriminate between pair groups.. 1.5 Results 1.5.1 Strains, molecular identification and MIC We had isolated 43 strains from highly polluted soil with hydrocarbons and heavy metals. Hence, four were selected by its capacity to synthesize indole and tolerate copper (data not shown). The bacterial strains CC22, CC24, CC30 and CC33 were selected and characterized based in its 16S rDNA sequence. On the basis of comparative analysis of the sequence with already available database showed that the strain CC22 were closed to Pseudomonas putida, CC24 to Enterobacter sp., whereas CC30 and CC33 to Acinetobacter sp. On the other hand, we evaluated the copper tolerance of the isolates. For this, employed LB modified broth with less.

(15) 15. content of tryptone and several copper concentrations. Strains were able to tolerate 1.6 mM Cu(II). However, we found an increasing in the minimal inhibitory concentration when employed LB traditional broth (data not shown). The Cu-tolerances were increased almost 2fold. Additionally, when we employed minimal medium MM9 (Maniatis et al., 1982), the copper tolerance was decreased several times (data not shown).. 1.5.2 Plant growth-promoting (PGP) features of strains. We characterized three PGP features in order to select one strain and evaluate its role on phytoextraction process. Indole synthesis, phosphate solubilization and siderophores production were tested for CC22, CC24, CC30 and CC33. Regarding of indole, its synthesis was evaluated on minimal media supplemented with tryptophan. Among the isolates, Pseudomonas sp. CC22 and Acinetobacter sp. CC30 were the best. These were able to synthesize 59.8 and 61.2 µg indole/mg protein from tryptophan. While Enterobacter sp. CC24 and Acinetobacter sp. CC33 produced 39.3 and 46.5 µg indole/mg protein, respectively (Fig. 1). Further, bacteria were able to solubilize phosphate. Strains CC22 and CC30 solubilized the greatest amount of phosphorus, while CC24 and CC33 exhibited lower rate. Pseudomonas sp. CC22 and Acinetobacter sp. CC30 solubilize between two- and four-fold more phosphates than the other strains (Fig. 2). Additionally, all strains were able to synthesize siderophores in culture media MM9 free of iron. However, not significantly differences were observed among strains. The bacteria tested were able to synthesize more than 100 µM siderophores relatives to DFMO (Fig. 3). Additionally, bacteria produced fluorescent pigments except P. putida CC22..

(16) 16. Fig. 1. Indole synthesis (n=3). Error bars represent standard deviation. The letters denotes HSD Tukey homogeneous groups at 95%..

(17) 17. Fig. 2. Phosphate solubilization (n=3). Error bars represent standard deviation. The letters denotes HSD Tukey homogeneous groups at 95%.. Fig. 3. Production of siderophores (n=3). Error bars represent standard deviation. The letters denotes HSD Tukey homogeneous groups at 95%.. 1.5.3 Copper accumulation Bacteria were examined to copper adsorption (Fig. 4). Acinetobacter sp. CC33 exhibited greater extent of Cu accumulation, between 50-90% more than the others bacteria. Nevertheless, these were able to accumulate 34.2, 40.7 and 39.5 mg Cu(II)/ mg of protein. Several times of adsorption were tested, however not differences were found after 5 min of exposition (data not shown)..

(18) 18. Fig. 4. Copper(II) adsorption by bacterial strains. Error bars represent standard deviation. The letters denotes HSD Tukey homogeneous groups at 95%.. 1.5.4 Influence of bacterial inoculation on the growth of Helianthus annuus We tested the influence of PGPB inoculation on both non- and Cu-contaminated soil in order to assess its role on plant growth. Several parameters were measured including shoot length, root length, shoot weight and root weight. Additionally, total chlorophyll content was estimated. The inoculation of Acinetobacter sp. CC30 had significantly effects on shoot length, root length, shoot DW and root DW on non- and Cu-contaminated soil (p<0.05). In noncontaminated soil, the bacterial inoculation showed to increase root length, shoot DW and root DW by 17.9%, 63.2% and 21.4%, respectively (Fig. 5, 6). While in contaminated soil with copper, the inoculation exhibited an improvement in shoot and root length, root DW and shoot DW by 21.3%, 10.9%, 29.9% and 4.14%, respectively (Fig. 5, 6). These results demonstrated that bacterial inoculation could play an important role on plant development and growth. On the other hand, we investigated the role of bacteria on photosynthetic pigment content in sunflower. This content is often altered by the presence of abiotic stresses such as copper contamination. The results showed that sunflower photosynthetic pigment.

(19) 19. content was improved by the inoculation with Acinetobacter sp. CC30. When the soil was noncontaminated, an increasing of 20.7%, 58.3% and 20.6% was observed for chlorophyll A (chlA), chlorophyll B (chlB) and total carotenoids (x+c) content, respectively. While in Cucontaminated soil the enhancing was of 18.2% and 7.5% for chlA and chlB, respectively, but not increase was exhibited in the x+c amount (Table 1).. Fig. 5. Effect of inoculation with CC30 on shoot and root length. (1) and (3) non-contaminated soil; (2) and (4) Cu-contaminated soil. Each value is the mean of seven replicates. Error bars represent standard deviation. One asterisk (*) denotes a value significantly greater than the corresponding control value (p<0.05)..

(20) 20. Fig. 6. Effect of inoculation with CC30 on shoot and root weight. (1) and (3) non-contaminated soil; (2) and (4) Cu-contaminated soil. Each value is the mean of seven replicates. Error bars represent standard deviation. One asterisk (*) denotes a value significantly greater than the corresponding control value (p<0.05).. Table 1 Photosynthetic pigments in maize and sunflower on soil Cu- and non-contaminated. Sunflower. ChlA. ChlB. x+c. µg mL-1mg FW-1. µg mL-1mg FW-1. µg mL-1mg FW-1. Non-contaminated soil Control. 1,352. (±0,134). 0,336. (±0,033). 0,281. (±0,035). CC30. 1,633. (±0,115). 0,532. (±0,008). 0,339. (±0,016). Cu-contaminated soil Control. 1,573. (±0,117). 0,451. (±0,020). 0,366. (±0,026). CC30. 1,736. (±0,004). 0,485. (±0,028). 0,365. (±0,006). ± shows standard deviation. Chlorophyll A (ChlA), chlorophyll B (ChlB) and total carotenoids (x+c). a. Control corresponds to non-inoculated treatment..

(21) 21. 1.5.5 Effect of PGPB inoculation on copper accumulation The PGP activity in soil is sometimes related with an increasing in ion mobility, furthermore, with an increasing in copper mobility. The results evidenced that bacterial inoculation improved the heavy metal uptake in roots, while non-effect was observed on shoot copper content (Fig. 7). When Acinetobacter sp. CC30 was inoculated the copper uptake in roots was improved from 99.0 ± 1.4 to 139.0 ± 21.0 µg Cu mL-1. These results showed that inoculation with CC30 had a significantly effect on copper accumulation in roots.. Fig. 7. Effect of inoculation with CC30 on copper uptake in both shoot and root. Each value is the mean of three replicates. Error bars represent standard deviation. One asterisk (*) denotes a value significantly greater than the corresponding control value (p<0.05).. 1.6 Discussion Several strategies have been proposed in order to remove heavy metals pollution from environment (Pulford and Watson, 2003). Current, one of the most widely accepted is phytoextraction, the utilization of plants to extract metals from soil (Lasat, 2002; Lesage et al., 2005). With the purpose of improve the process, to soil is often added some chelator.

(22) 22. compounds which increment metal availability but occasioned environment contamination by its high persistence (Lesage et al., 2005). The utilization of microorganisms with certain properties such as metal tolerance and PGP features has elucidated a novel strategy both lowcost and non-contaminant to improve the process (Glick, 2010). Previously, we had isolated several strains from highly polluted soil in order to select one with PGP activity and Cutolerance for phytoextraction process (data not shown), in this study, we reported four that were able to synthesize indole and tolerate copper. In order to establish the extent of copper tolerance, we evaluated bacterial growth in LB modified broth under increasing Cu concentrations. Importantly, MIC was severity affected by the culture media employed. As the culture media was more complex, the extent of tolerance was also increased. This could occur by presence of organic or inorganic chelators in culture media that lead to formation of organometallic complexes and insoluble salts, respectively, both not available to interact with bacteria, increasing MIC (Zevenhuizen et al., 1979). In addition, strains were characterized by its PGP activities. For instance, indole synthesis was evaluated on minimal culture media supplemented with tryptophan. All strains evaluated were able to synthesize it (Fig. 1). Indolic compounds are derived from tryptophan metabolism, several types have been described, however the more important one is the 3indoleacetic acid (IAA) (Costacurta and Vanderleyden, 1995). Generally, these have shown to influence strongly plant growth and development controlling many important physiological processes including cell enlargement and division, tissue differentiation, and responses to light and gravity (Teale et al., 2006b). Nevertheless, the effect is not always positively, it depends of type, amount, availability and sensitivity of the plant tissue to indole presence (Spaepen et al., 2007). Moreover, bacteria were tested to phosphorus solubilization. Pseudomonas sp. CC22 and Acinetobacter sp. CC30 exhibited the best results (Fig. 2). This feature usually is related with an increasing in nutrients availability. Despite, the content of P in soil usually is high this in not available for plant uptake (Rodríguez et al., 2006). In alkaline soils P is precipitated as Mg or Ca salts, while in acidic soils is precipitated with Al and Fe. Bacterial presence has shown to influence P availability because of synthesis of organic acids or certain enzymes that mediate the releasing of P from it (RodrÌguez and Fraga, 1999). Additionally, in Cu-contaminated soils these orthophosphates ions may be precipitated as copper salts. In consequence phosphate solubilization can result in copper releasing increasing its availability to plant uptake. Finally, siderophores synthesis was tested on iron-.

(23) 23. free MM9 culture media with low-phosphorus content in order to avoid interferences (Schwyn and Neilands, 1987). All strains tested were able to produce siderophores (Fig. 3). These molecules are synthesized under iron starvation, related with iron chelation and regulated by the iron cellular content status (Crosa, 1989). Its synthesis plays an important role preventing pathogen infection and iron lacking in plants (Neilands, 1995). The utilization of bacteria that possesses these traits has exhibited to increase plant growth even under heavy metal stress (Ma et al., 2009; Glick, 2010). Rajkumar and Freitas (2008), reported the using of two PGPB strains tolerant to heavy metals able to increase the growth of Ricinus communis in a soil contaminated with nickel, copper and zinc. In the same way, Sheng et al. (2008) expose that inoculation of Brassica napus with Pseudomonas fluorescens G10 and Microbacterium sp. G16 allowed to increase plant growth with consequent effects on phytoremediation process. In this process always is desired plants with high biomass and rapid growth (Glick, 2010). Phytoremediation process could clean up the heavy metal pollution of soil (Cunningham and Ow, 1996). However, there are several factors that define the efficiency of the process. Some usually are related with plant capacity to extract metals from soil, other are involved with soil properties such as metal availability to plant uptake (Cataldo and Wildung, 1978). We used Helianthus annuus in this study due to high capacity to extract metals (Turgut et al., 2004; Lesage et al., 2005; Mani et al., 2007). However, to our knowledge few attempts have been carried out in order to investigate the bacterial role on its growth, development and Cu accumulation. We evaluated the effect of bacterial inoculation on H. annuus growth employing pot experiment on glasshouse conditions. For this, the strain CC30 was selected on basis to its PGP capabilities and employed to the experiment. These traits have shown to influence significantly plant growth (Reed and Glick, 2005; Sheng and Xia, 2006; He et al., 2009). When Acinetobacter sp. CC30 was inoculated an improvement in sunflower growth was observed in both Cu and non-contaminated soil, reflecting its role as plant growth-promoting bacteria (PGPB). Interestingly, CC30 inoculation exhibited to promote plant growth even at adverse conditions (Fig. 5, 6), for instance, copper contamination. Several authors report that plants exposed to high copper levels usually are severity affected in growth and development (Fernandes and Henriques, 1991; Ouzounidou, 1995). Additionally, the prolonged bacterial exposition to Cu in its free ionic form Cu(II) is generally toxic to microbial cells and results in a selective pressure on microorganisms that lead to the appearing of resistant variants possessing copper resistance genetic determinants (Cervantes and Gutierrez-Corona, 1994)..

(24) 24. The PGPB effects on plant growth could increase particularly both root length and weight with direct implications in heavy metal extraction by an enhancing in root surface exploration. Likely, these effects are due to bacterial synthesis of phytohormones such as auxins, gibberellins or cytokinins (Costacurta and Vanderleyden, 1995), or by an increasing in nutrients availability mediated by phosphate solubilization or siderophores synthesis. Although, there are other mechanisms that may be involved in plant growth such as ACC deaminase activity or organic nitrogen mineralization (Glick, 2010). In consequence, Cu resistant bacteria with PGP capabilities constitute an alternative to promote plant growth even under Cu-contamination. On the other hand, bacterial inoculation was able to increase the content of photosynthetic pigments under both soil conditions (Table 1). The highest bacterial increment was observed under not excessive copper exposition in soil (p<0.05). However, in Cu-contaminated soil the effect was also significantly. Heavy metal contamination in soil is often associated with iron deficiency in some plant species (Wallace et al., 1992). Hence, low iron content generally inhibits chlorophyll biosynthesis (Dell'Amico et al., 2008b). However, bacteria were able to synthesize siderophores, likely improving iron nutrition in the plant. On the other hand, we observed that under contamination with copper the chlorophyll content without bacterial inoculation was higher. Drazkiewicz and Baszynski (2005) reported that maize exposed to increasing cadmium concentrations showed an increase in chlA + chlB at 100 µM Cd(II) in young leaves. Likely, the increase in the pigment content per area unit reflects a stronger effect of Cd(II) on the surface size than on the pigments content per leaf segment. Nevertheless, bacterial presence exerted an important effect on chlorophyll content showing an improvement in plant fitness status (Table 1). Finally, we analyzed the bacterial effect on Cu uptake. Microbial presence in soil could exhibit an increasing in Cu availability (Fig. 7). Chen et al. (2005) reported as bacterial inoculation improved the level of water-soluble copper extracted from soil. In this study, a significantly effect on metal uptake mediated by CC30 inoculation was observed (Fig. 7) (p<0.05). In the same way, Jiang et al. (2008) evidenced, as microorganisms able to solubilize heavy metals such as Pb and Cd were able to increase the plant uptake level in soil. Likely an enhancing in Cu availability may be generated by bacterial activity. Usually, the capacity to solubilize phosphates mediated by organic acid production increases ion mobility in soil (Khan, 2005a; Glick, 2010). Additionally, the synthesis of organic chelates may improve the.

(25) 25. extent of copper released from organic matter (OM) and consequently Cu plant uptake (Lasat, 2002). Soils with high OM can decrease several times copper availability due to the strong binding of Cu by OM and other soil colloids. Its mobility could become severely restricted and the fraction of total Cu available for plants uptake usually low (Cataldo and Wildung, 1978). Thus, microbial metabolisms influenced availability of copper in soil, improving the efficiency of the process. Additionally, Acinetobacter sp. CC30 inoculation resulted in a plant growth promotion. Hence, bacterial activity improved the amount and rate of copper extraction, representing a relevant alternative to improve the copper extraction mediated by plants. In this study, we exhibited as Acinetobacter sp. CC30 activity influenced significantly plant growth, fitness and copper uptake. Likely through several mechanisms related with improvement in plant nutrition or with regulation of plant development through synthesis of like-phytohormone compounds. Also, evidenced the enhancing in rate of copper uptake, probably through metal solubilization or chelation. Therefore, the utilization of PGPB bacteria was able to improve phytoextraction process by Helianthus annuus. As a consequence, further research is necessary in order to use this type of bacteria to optimize phytoextraction process. What is more, to design inoculants based in this or another strains to prove its activity in field and order to solve pollution problems with toxic metals. To our knowledge, this is one of the first studies where is evaluated the bacterial role on the phytoextraction process of copper by sunflower. We exhibited the strong influence of bacteria on sunflower efficiency to remove copper from soil.. 1.7 Bibliography Abdelly, C., Öztürk, M., Ashraf, M., Grignon, C., Mediouni, C., Houlné, G., Chabouté, M.-E., Ghorbel, M.H., Jemal, F., 2008. Cadmium and copper genotoxicity in plants. Biosaline Agriculture and High Salinity Tolerance. Birkhäuser Basel, pp. 325-333. Alexander, D.B., Zuberer, D.A., 1991. Use of chrome azurol S reagents to evaluate siderophore production by rhizosphere bacteria. Biology and Fertility of Soils 12, 39-45. Altomare, C., Norvell, W.A., Bjorkman, T., Harman, G.E., 1999. Solubilization of phosphates and micronutrients by the plant-growth-promoting and biocontrol fungus trichoderma harzianum rifai 1295-22. Appl Environ Microbiol 65, 2926-2933..

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(33) 33. CHAPTER II: Improvement of growth and Cu(II) uptake by Zea mays mediated by plant-growth promoting bacteria (PGPB) inoculation. 2.1 Abstract Heavy-metal pollution is becoming one of the most severe environmental issues. Phytoextraction, defined as removal of these metals through plants from soil represents an important alternative to improve soil quality. In this study, we evaluated the bacterial role to improve heavy-metal removal process through plant growth promotion capabilities and mobility of ions. Most the bacteria were able to produce indole, siderophores, and solubilize phosphate. Acinetobacter sp. CC30 and P. putida GN4 were selected to further studies. Its inoculation and co-inoculation showed to improve maize growth and copper uptake, likely due to synthesis of indole or mobilization of nutrients in soil. Similarly, photosynthetic pigments contents were enhanced by bacterial inoculation. It was possible to evidence a positive effect of microbial inoculation on phytoextraction process. Co-inoculation exhibited the more important effect on copper extraction with root copper content of 214 ppm. In effect, the utilization of this strategy could represent a promising alternative to improve the removal of heavy metals in soil. Having a direct effect on environmental quality and health of living beings.. 2.2 Keywords Phytoextraction,. plant. growth-promoting. bacteria. (PGPB),. copper. mobility. and. photosynthetic pigments.. 2.3 Introduction Heavy metal pollution of both soil and water is becoming one of the most severe environment hazards and has negative impact on human health and agriculture. Some of them are micronutrient and in consequence are indispensable for plant growth and development (Gupta et al., 2008b). However, when they are at high concentrations could become toxic (Bibi and Hussain, 2005; Drazkiewicz and Baszynski, 2005). Copper is one of these essential elements for plant development. This is involved at both conformational and functional level of certain proteins (Pilon et al., 2006). Furthermore, most of metalloenzymes containing Cu are involved in the catalysis of redox reaction in which O2 is the electron.

(34) 34. acceptor. Also, play an important role providing reducing equivalents to photosystem I (PSI) (Fernandes and Henriques, 1991). Nevertheless, when this is found at high levels in water and soil ecosystems represent a danger for the live beings especially to plants. Furthermore, its deleterious effects usually arise at tissue levels only slightly higher that its optimal level (Fernandes and Henriques, 1991). Copper contamination is occasioned by several human activities. For instance, pesticides, fertilizers, the use of sludge or municipal compost, mining, car exhaust and smelting industries (Ma et al., 2009). Furthermore, neither copper nor any heavy metals are susceptible to degradation hence persist in the environment. As a consequence of heavy metal contamination some strategies have been developed to decontaminate environments. Some of them involve the use of chemical or physical process that can result expensive and destructive to soil (Pulford and Watson, 2003; Glick, 2010). Phytoremediation has shown to be an effective and low-cost alternative. This involve the use of plants to adsorb, extract, accumulate and detoxify contaminated soils through physical, chemical and biological process (Glick, 2010). Currently, there are several reports about metal accumulating plants that are used to remove toxic metals from the soil (Komarek et al.; Chen et al., 2005; Purakayastha et al., 2008; Murakami and Ae, 2009). Zea mays an important world crop, has shown be suitable to removal of heavy metal because of its high biomass and rapid growth (Meers et al., 2005; Garcia-Rosales and Colin-Cruz, 2010). However, the success of the phytoremediation process depends of several factors related with soil and plant properties such as metal concentration availability and solubility, soil pH, compactness or microbial activity, etc (Cataldo and Wildung, 1978; Glick, 2010). As an alternative some chelator agents such as EDTA or EDDS are added to soil in order to increase heavy metal availability (Wang et al., 2007; Gupta et al., 2008a). However, it used to be expensive and at worst highly polluting. Hence, the use of microorganisms becomes an important alternative that would allow improving heavy metal availability and plant growth nutrition. Certain microorganisms with heavy-metal resistance have the ability to promote plant growth through of several mechanisms such as indole synthesis, siderophores production, ammonification, phosphate solubilization or ACC deaminase activity (Glick, 2010). These are involved in direct or indirect activity on plant. Syntheses of indole and ACC deaminase are related with changes in plant development process, whereas, siderophores, ammonification and phosphate solubilization are related with the increasing of nutrient availability. Additionally, these last may be involved in copper solubilization because of Cu.

(35) 35. have strong affinity with soil components decreasing availability for plant uptake (Cataldo and Wildung, 1978). It has been previously demonstrated that PGPB can enhance biomass production and tolerance of the plant to heavy metals pollution (Rajkumar et al., 2006a; Kumar et al., 2008; Rajkumar and Freitas, 2008). Therefore, microbial activity can be considered a fundamental component of the phytoremediation technology (Glick, 2010). The goals of this work were to select two prominent strains with plant growth promoting potential and studying the role of its inoculation and co-inoculation on phytoremediation process. Furthermore, to analyze its effect on growth, photosynthetic pigments production and plant copper uptake by Zea mays on copper contaminated soil.. 2.4 Materials and Methods 2.4.1 Strains and culture media. Strains Pseudomonas sp. C61 and C907, Pseudomonas putida CC22 and GN4, Acinetobacter sp. CC30 and CC33, and Enterobacter sp. Pb56 and CC24, were provided by Centro de Investigaciones Microbiológicas, Universidad de los Andes, Colombia. Morphological and biochemical description are listed in table 2. The strains were previously isolated from strongly contaminated soil with heavy metals. The strains were grown in LB modified (LBM) broth (in g/L: tryptone 5.0, yeast extract 5.0 and NaCl 5.0) at standard conditions: 28°C and 150 rpm, unless otherwise indicated. For biochemical description bioMérieux API20NE Kit was employed.. 2.4.2 Minimal inhibitory concentration of heavy metals The tolerance level to Cu(II) and Cr(VI) were evaluated on LBM broth culture media. For this, increasing level of metals ranking between 0 – 4.0 mM of Cu(II) and Cr(VI) were studied. Stock solutions were employed at 80 mM for copper in form of CuSO4·5H2O (249.68 gmol-1) and chromium as K2Cr2O7 (294.185 gmol-1). The stock solutions were sterilized by filtration. The growth was verified spectrophotometrically (Genezy UV 10, Thermo Corporation) at 600nm after 48 h at standard condition..

(36) 36. 2.4.3 Effect of Cu (II) on bacterial growth. Culture flask (250 mL) containing 50 mL of LBM broth supplemented to reach 0.0, 0.4 and 0.8 mM CuSO4·5H2O were employed in order to evaluate the growth of Acinetobacter sp. CC30 and P. putida GN4 under increasing copper concentrations. The flasks were inoculated with over night culture adjusted to OD600= 0.500. As control were employed non-inoculated flasks supplemented or not with adequate copper concentrations. The bacterial growth was monitored for 30 h by measuring the optical density at 600 nm (Ma et al., 2009). Growth velocities were determined (Rojas-Tapias et al., 2009).. 2.4.4 PGPB features 2.4.4.1 Phosphate solubilization Quantitative phosphomolybdate method was employed to determine soluble phosphate. In brief, each strains was grown on Pikovskaya broth with 5.0 g/L Ca3(PO4)2 and incubated for 120 h at 150 rpm (Pikovskaya, 1948). A 1.0mL-aliquot from each flask, including controls, was centrifuged at 10000 rpm and 500-µL supernatant employed for analysis according to (Fiske and Subbarow, 1925).. 2.4.4.2 Indole quantification Indolic compounds were estimated using the colorimetric assay described by (Glickmann and Dessaux, 1995) employing K-lactate culture media with tryptophan at 100 ppm (CarrenoLopez et al., 2000). Incubation was carried out for 72 h at 150 rpm in dark. The reagent used was the PC (12 g/L FeCl3 in 7.9 H2SO4), which is based on the Salkowsky reagent. The reaction between PC reagent and culture supernatant was executed in relation 1:1 and allowed. to. react. for. 30. min. in. dark.. Indolic. compounds. were. examined. spectrophotometrically at 540 nm. Results are expressed in micrograms of total indolic compounds per mg of protein, using IAA as standard. Protein content of cultures was measured according to Bradford (1976).. 2.4.4.3 Siderophores Strains were screened for the quantitatively production of siderophores as described Schwyn and Neilands (1987). The test was carried out employing the CAS assay solution. In.

(37) 37. brief, one colony from each strain was cultured on free-iron MM9 culture media for 48 h. After, the cultures were centrifuged and resuspended in double-desionized water (DDW), adjusted at OD600= 0.200 and employed to inoculate flasks with 15.0 mL of same culture media. The glass flasks were previously washed with 6.0 M HCl and rinsed with DDW several times. The cultures were agitated by 72 h at 28°C. Time after, 1.0-mL aliquot from culture was centrifuged at 10000 rpm for 5.0 min and 0.5-mL supernatant taken and allowed to react with 0,5-mL CAS assay solution. The mix was allowed to stabilize for 6.0 hours and after absorbance was measured at 630 nm. The standard curve was elaborated using 20 – 100 µM deferoxamine mesylate (DFMO). Blank solution consisted of 1,5 mM solution of DFMO. As reference solution was employed non-inoculated culture media.. 2.4.5 Effects of PGPB on the mobility of ions and copper in soil. Batch studies on effect of bacterial presence on ions and copper mobility in soil were investigated employing 50-mL centrifuge tubes. The characteristics of the soil employed are listed in the table 1. One gram of soil was intentionally contaminated to reach 0.8mM Cu(II) and added to each centrifuge tubes. On the other hand, bacterial cultures of strains were grown for 24 h on LB medium. The pellets were rinsed twice in DDW and adjusted to OD600= 1.000. One milliliter of adequate bacterial suspensions was added to the corresponding tube. The soil was incubated at 28°C in dark and agitated at 150 rpm for one week. After this, 10 mL DDW were added to each tube and them centrifuged at 7000 rpm for 10 min. The supernatants were used to measure conductivity and copper content by AAS (Perkin Elmer 2380). The control was established inoculating DDW at the same volume instead of bacterial suspension. The tubes were weighted at begin and final to compensate water evaporation (Rajkumar and Freitas, 2008).. 2.4.6 Soil preparation and analysis The employed soil was provided by Laboratorio de Microbiología de Suelos, Corpoica, Colombia. It is representative of Altiplano Cundiboyancence. Once collected, the soil was airdried at room temperature until 8% of humidity was reached. Immediately, this was sieved at 2.0 mm, autoclaved three times in three consecutive days and finally allowed to stand for 8 days to stabilization. The basic soil properties were analyzed and listed in table 1..

(38) 38. Table 1 Characteristics of soil used for pot experiments. Parameter. Value. pH. 5.95 ± 0.07. Organic matter. 15,15 ± 0.21%. Phosphorus (ppm). 13.6 ± 0.70. Sulphur (ppm). 12.6 ± 0.70. Interchangeable cations (cmol/kg) Calcium. 4.72 ± 0.69. Magnesium. 1.11 ± 0.35. Potassium. 1.95 ± 0.75. Sodium. 0.39 ± 0.15. Effective cationic interchange capacity. 8.26 ± 1.81. Electric conductivity (dS/m). 0.69 ± 0.2. Minor elements (ppm) Iron. 134,.5 ± 16.2. Copper. 1.85 ± 0.07. Manganese. 17.30 ± 0.01. Zinc. 3.45 ± 0.9. 2.4.7 Influence of PGPB and Cu on the growth of plant species and copper uptake. One complete factorial statistical design was employed to evaluate the effect of single inoculation and co-inoculation on plant growth and copper uptake by maize. The soil was contaminated intentionally with CuSO4·5H2O to achieve soil copper concentration of 0.8 mM after sterilization. Soil was allowed to stabilize for 2 weeks. The pots contained 400 g soil. On the other hand, Zea mays var. ICA-508 seeds were surface sterilized with 1.0% sodium hypochlorite for 10 min and rinsed several 5-times in desionized-sterilized water. The seeds were pre-germinated on sterilized peat for 5 days. To inoculate the different treatments the seedlings were submerged in adequate bacterial suspension three times and planted. The seedlings submerged in sterilized-water were employed as control. The seedlings submerged in water were used as control. Plants were grown in a glasshouse at 15-25°C and 16:8 day/night regime. After 21 days the plants were carefully removed from the pots and the root surface was cleaned several times with distilled water. Growth parameters as shoot length, root length, shoot dry weight and root dry weight were measured. Additionally,.