RHIZOFILTRATION OF LEAD CONTAMINATED SOIL BY HELIANTHUS ANNUUS AMENDED WITH BACILLUS MEGATERIUM AND EDTA KAITLIN M. PEARCE, ALEXANDRA KURTZ, AND REBEKAH J. WARD* GEORGIA GWINNETT COLLEGE 1000 UNIVERSITY CENTER LANE LAWRENCEVILLE, GA, USA Copyright 2015, Fine Focus all rights reserved MANUSCRIPT RECEIVED 24 APRIL, 2015; ACCEPTED 2 JULY, 2015 FinalJournal.indd 95 9/25/15 11:38 AM Heavy metal contamination causes numerous adverse effects to public health and the environment. Sources of heavy metal contamination are widespread, especially in urban environments. Certain plants such as sunflower (Helianthus annuus) have been shown to sequester heavy metals in their root systems, thus filtering contaminants such as lead (Pb) from soil, a process termed rhizofiltration. In the present study, Bacillus megaterium was applied to the root system of sunflowers growing in Pb-contaminated soil and the efficiency of rhizofiltration was examined. Lead levels in the rhizosphere of the Bacillus megaterium and EDTA amended plants were almost 100 mg/kg soil higher than those without treatment, suggesting the amendment may have been effective in augmenting lead sequestration. In order to further elucidate these lead- sequestering communities, preliminary phylogenetic assays were conducted on the soil with and without the presence of the plant. Although complete coverage of the community phylogeny was not possible, there was evidence indicating that the rhizosphere may have induced changes in the composition of the bacterial community. These studies offer simple methods for enhancing bioremediation in agriculture. ABSTRACT Heavy metals are defined as metals that have a specific density > 5g/cm3 (17). Elevated concentrations of heavy metals in soil can have a devastating effect on human health and the environment. This is especially true for gardens located in urban areas. Human health is most adversely affected by certain heavy metals, namely cadmium, mercury, arsenic, and lead (17). Excess Pb is especially toxic to humans because of the effects it has on kidneys and the nervous system, which can cause headaches, weakness, cramps, anemia, and may lead to mental health disorders (1). According to the U.S. Environmental Protection Agency (EPA), lead can be found in the air, soil, water, and in homes because of its presence in gasoline, industrial facilities, paint, ceramics, pipes, batteries, cosmetics, ammunition, and even food (30).These make lead a priority heavy metal for study in urban environments. The problem of heavy metal soil contamination can be addressed using a technique known as bioremediation. Bioremediation is the process of introducing organisms, such as bacteria, into a contaminated environment in order to INTRODUCTION CORRESPONDING AUTHOR *Rebekah J. Ward Georgia Gwinnett College 1000 University Center Lane Lawrenceville, GA 30043 Email: rward1@ggc.edu KEYWORDS • Bioremediation • Sunflowers • Rhizofiltration • Lead • Phylogenetics 96 • FINE FOCUS, VOL. 1 (2) FinalJournal.indd 96 9/25/15 11:38 AM APPLIED AND ENVIRONMENTAL MICROBIOLOGY • 97 remove the pollutants. Phytoremediation is a subset of bioremediation that exploits plants in order to clean up contamination. Certain plants termed hyperaccumulators can store the heavy metals in tissues or the rhizosphere (root system and surrounding soil), therefore taking it out of biological circulation. This process is termed rhizofiltration. This study was conducted to quicken the remediation process and increase its efficiency without hampering cost effectiveness. Previous studies (13, 15, 18, 20) have indicated that adding chemicals or biological elements to the soil may help plants sequester metals in the tissues. This, in turn, reverses some of the effects of the metals by lowering the concentration in the surrounding area, allowing more plants that are less metal-tolerant to grow. After a rhizosphere absorbs as much metal as it can, the plant can be uprooted, allowing for removal of contaminants from the soil. This can alleviate some of the harmful impacts of metals on the environment. However, one of the problems with phytoremediation is that it is a slow process that produces a low yield (10). One plant that has been shown to sequester heavy metals in the rhizosphere is the sunflower (Helianthus annuus) (18). Sunflowers are considered hyperaccumulators and have been used for various environmental cleanup projects, (7). Sunflowers also are more tolerant to pH variation than many other common plants, capable of growing in soil ranging from pH 5.7 to over 8.0, while optimal soil pH for other plants is 6.4 (24). Sunflowers also produce more roots, shoots, and total biomass than many other common plants. This means that they can potentially hold larger amounts of pollutants and fewer plants are required to recover the same amount of pollutants, thereby becoming economically sustainable. Bioavailability of phosphate also has an influence on the effectiveness of phytoremediation. Addition of ethylenediaminetetraacetic acid (EDTA) has shown an increased Pb uptake by almost 20% in previous studies, and has also been shown to facilitate phytoremediation in plants (13, 20). EDTA is a common and powerful chelating agent that has been added recently into heavy metal treatment systems and works especially well with Pb and copper (4). This demonstrates that certain chemical additives can help sunflowers absorb higher levels of Pb, therefore ultimately reducing the cost, amount of land used, and amount of plants needed to grow in a particular plot of land when used for bioremediation. Bacteria that reside in rhizospheres of plants can play a role in reducing the toxic effects of heavy metals on the plants (15). These microorganisms can protect the plant from damage and, in return, benefit from living in the rhizosphere systems, therefore creating a mutualistic relationship with the plant. In this study, Bacillus megaterium was used because this bacterium has been shown to absorb and store Pb intracellularly, therefore making it resistant to elevated levels of Pb (25). This species is also a common soil bacteria that is considered part of plant growth promoting rhizobacteria (PGPR), which helps improve growth by releasing a key auxin (indole-3- acetic acid) to encourage cell proliferation (3). Bacterial cultures were added to germinating seedlings to help improve the health of the plants for rhizofiltration and increase the concentration of Pb in the rhizosphere through the intracellular sequestration used by B. megaterium. The purpose of this experiment was to analyze the impact of the addition of bioavailable phosphate and heavy-metal tolerant bacteria on the Pb concentrations in highly contaminated soil. FinalJournal.indd 97 9/25/15 11:38 AM 98 • FINE FOCUS, VOL. 1 (2) MATERIALS AND METHODS COLLECTION, SETUP, AND MAINTENANCE For this experiment, three five-gallon plastic containers of soil were collected from two sites in an urban garden in Atlanta, Georgia, USA. These samples were then transported to the laboratory at Georgia Gwinnett College. The soil was characterized as being a crumbly, fine soil. The soil (2.5 grams, manually homogenized) was put into 500 mL conical Falcon tubes with 7.5 mL distilled H2O and stored at -80˚C for DNA extraction. A sample was taken from the middle of each container prior to planting seedlings, homogenized, and sent to the University of Georgia Soil and Water Analysis Lab for determination of Pb concentration. A Teddy Bear sunflower (Helianthus annuus) seed was planted one inch deep in nine 10” x 12” containers with each weighing approximately 0.95 kg per container. The samples were watered with 40 mL of tap water on alternate days for three months. After the seeds sprouted, a 40 Watt Growlux, wide-spectrum grow light (Grower’s Supply, Dexter, MI) was placed on a timer for eight hours daily. Unplanted soil was maintained as the control experiment. Sterile Luria broth (LB, 100 mL) was inoculated with Bacillus megaterium ATCC14581 and placed in a shaker for 24 h at 37°C. The nine containers of soil were then separated into three different categories. Three control containers were watered with 40 mL tap water on alternate days. Three EDTA containers were watered with 80µL EDTA once then 40 mL tap water on alternate days. Three EDTA plus B. megaterium containers were watered with 80 µL 0.1 M EDTA once, 1 mL of freshly made bacterial culture once, and then watered with 40 mL tap water on alternate days. The bacterial culture was then serially diluted onto LB agar plates to determine the original cell count. Sunflower height was then measured seven times for a total of three months and recorded. The measurements were taken from the base of the stem to the tip of the tallest leaf. SOIL FILTRATION Tubes with the most contaminated soil, according to the analysis results, were thawed, and 1 mL 0.1 M EDTA was added, along with 200 µL Tris buffer and distilled water to balance the tubes. They were centrifuged at 1000 rcf for 1 min. at 25˚C, then the supernatant was removed and 10 mL of Tris buffer was added. After thorough mixing with a vortex mixer, the tubes were weighed and water was added to balance them within 0.1 g. This was repeated three times. After the fourth centrifugation, the liquid was poured through muslin to filter out particles and 1 mL 1X Tris Borate EDTA (TBE) was added to the soil, weighed out in tubes, and centrifuged one more time at the same settings. The supernatant was transferred to two microcentrifuge tubes for storage at -80˚C for five days. DNA EXTRACTION AND CLONING The method of Tsai and Olson (29) was used for DNA extraction in the present study with the following modifications: Tris EDTA was used in place of Tris HCl, the tubes were stored in a -80˚C freezer instead of in dry ice, and the tubes were centrifuged at 6000 rcf for 10 min. before starting the protocol. Ten new PCR FinalJournal.indd 98 9/25/15 11:38 AM APPLIED AND ENVIRONMENTAL MICROBIOLOGY • 99 samples were made with Cetyltrimethyl ammonium bromide (CTAB) and 5 g of soil was mixed with 3 mL distilled water and 5 mg/mL lysozyme. The mixture was placed in a shaker for 2 h. Four samples were used to proceed to the transformation and cloning steps. Ammonium acetate (5 mg/mL) was used instead of magnesium acetate. The phenol chloroform extraction was a 1:1 ratio of the working solution to phenol: chloroform: isoamyl alcohol (Sigma, St. Louis, MO). The mixture was centrifuged at maximum speed (13,000 rcf) for 10 min. The top layer was then removed using a pipette and 100 µL of ammonium acetate was subsequently added. Of this volume, 350 µL were transferred into four test tubes. An ethanol precipitation was carried out, after which a Tris-EDTA (TE) suspension and DNA samples were stored at -20°C. For the Polymerase Chain Reaction (PCR) amplification, the primers used were 8F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492R (5’-GGTTACCTTGTTACGACTT-3’) (Integrated DNA Technologies, Coralville, IA). PCR samples were prepared as follows: 1 µL of each primer (Integrated DNA Technologies, Coralville, IA), 2 µL of DNA, 46 µL distilled water (48 µL for the controls), and 50 µL of Master Mix (New England Biosystems, Ipswich, MA). Twenty PCR cycles were used as follows: 94˚C for 30 seconds as a hot start and denaturation, 53˚ C for 30 seconds for annealing, 68˚C for 1 minute for elongation, and 68˚C for 5 minutes for final extension. The samples were analyzed using Nanodrop 2000 by Thermo Scientific and Polymerase Chain Reaction was again conducted as the above paragraph described with the following modifications: 50˚C for 30 seconds for annealing, 72˚C for 45 seconds for elongation, and the positive control used Bacillus subtilis DNA (48 µL). Upon maturation of the remaining plants, each was removed at the base of the stem and 700 g of soil was collected from the middle of the container and manually homogenized. One gram was taken from the homogenized soil and put into a test tube with 5 mL distilled water. They were each mixed and stored at -80˚C for 30 min. Lysozyme (75 mg) was then added to each tube after being thawed in a bead bath at 65˚C for 3 min., after which the tubes were stored at 37˚C for 24 h. Gene cloning of PCR products was done through a Tri-N-Octylphosphine Oxide (TOPO) cloning reaction with standard procedures. A series of minipreps were done from a QIAprep spin miniprep kit (from Qiagen, Germantown, MD) using manufacturer’s instructions. PCR and agarose gel electrophoresis were completed (29). A Nanodrop analysis was then conducted for all 95 control samples. After testing, the data was analyzed using FinchTV, Seaview, BLAST, ClustalW, and Geneious and the samples were transferred to the University of Georgia Genomics Center to be sequenced. SEQUENCING AND PHYLOGENETICS Sequences were manually analyzed for quality using FinchTV. Following BLAST searches, the most similar sequences were selected and aligned via Seaview. ClustalW was used to make a phylogenetic tree including all of the samples. Geneious was used to make an outgroup phylogenetic tree using Methanococcus voltae as the outgroup. Two known organisms (16S genes of Bacillus subtilis and Escherichia coli) were also added to the tree. FinalJournal.indd 99 9/25/15 11:38 AM 100 • FINE FOCUS, VOL. 1 (2) RESULTS Some of the original plants did not survive, leaving only control samples and amended samples. The original cell count was determined to be 2.21 x 107 cfu/mL. The initial Pb concentration was 542 mg/kg (Table 1). The final concentrations of Pb for the “control” plants was 531.2 ppm, while the final concentrations for the “non-vegetated” and “B. megaterium plus EDTA” samples were 567.0 mg/kg and 613.7 mg/kg, respectively. The phylogenetic tree data for all samples from the non-vegetated and control treatment indicate that the samples were dominated by a Gram-negative rhizosphere-associated phylotype (Fig. 1). Figs. 2 and 3 show the phylogenetic trees for the control or non-vegetated samples, analyzed separately. The two known bacterial operational taxonomic units (OTUs) were included in each tree for comparison purposes. Table 2 includes five different classes of bacteria in the four clades in the total phylogenetic tree. Fig. 4 is a representation of the percent breakdown of each class that was discovered in the non-vegetated and control samples. These data reiterate the difference in Proteobacteria between the control and non-vegetated samples. DISCUSSION The control plant did not appear to effectively sequester Pb compared to the non-vegetated soil (Table 1). Explanations for this could include that earlier data for rhizofiltration used a different subspecies, or natural variability that could diminish with a larger sample size. However, the amended rhizosphere shows sequestration outside the range of this hypothetical variation. The final concentrations of the control and non-vegetated samples are within a 30 mg/ kg range, so that variation is unlikely to be a determinant of Pb sequestration, seen in the B. megaterium plus EDTA samples (Table 1). However, the Bacillus megaterium plus EDTA sample had sequestered almost 75 mg/kg more than the initial concentration, or more than a 13% increase in mg/kg, as compared to the control (-1.9%) and non-vegetated (4.6%) samples. This suggests that Bacillus megaterium and EDTA may have helped the sunflower sequester more Pb as compared to the other samples. The EDTA-alone samples were not viable, likely due to too high of a concentration of EDTA, and therefore Pb sequestration cannot be evaluated. For future experiments, the concentration of EDTA may be varied and optimized for the best results. The non-vegetated and control sequences were used because studies have shown that non-amended sunflowers can carry out rhizofiltration and by using these sequences, the variety in the bacterial community could be analyzed (1, 2, 18). The phylogenetic tree (Fig. 1) is split into four clades with five distinct classes of bacteria. The first contains Alphaproteobacteria, which were only found in the control samples and had four different OTUs (Fig. 2). The Alphaproteobacteria also were the main bacterial class found in the control soil (Table 2). This could be because Alphaproteobacteria are the common inhabitants of the rhizosphere and therefore absent in soil without plants. FinalJournal.indd 100 9/25/15 11:38 AM APPLIED AND ENVIRONMENTAL MICROBIOLOGY • 101 Phylogentic Tree for All Samples PlasmidC34 0.0048 Hyphomicrobium.facile 0.00474 Clostridium.aminovalericum 0.03498 Bacillus.subtilis 0.00445 Bacillus.thuringiensis 0.00069 Bacillus.toyonensis 0.00061 Pseudomonas.panacis 0.00157 Pseudomonas.fluorescens 0.00342 Bauldia.litoralis 0.03946 PlasmidC67 0.16065 Sphingomonas.jaspsi 0.00976 PlasmidC63 0.01801 Pandoraea.sputorum 0.03728 PlasmidC51 0.0272 Achromobacter.insuavis 0.00195 Acinetobacter.calcoaceticus 0.00178 PlasmidC59 0.00407 PlasmidN38 0.01286 Escherichia.coli 0.07451 PlasmidC28 0.01132 Granulicella.aggregans 0.07567 PlasmidC20 0.06357 PlasmidC68 0.01394 PlasmidN15 0.01509 Pseudomonas.putida 0.00341 PlasmidN36 0.01056 Bacillus.atrophaeus 0.00048 PlasmidN40 0.01058 PlasmidC64 0.00614 PlasmidN8 0.00664 Aciditerrimonas.ferrireducens 0.04288 PlasmidC40 0.03624 Telmatocola.sphagniphila 0.07971 PlasmidN33 0.04637 Crocinitomix.catalasitica 0.06677 PlasmidN35 0.08944 PlasmidC44 0.00137 Rhizobium.tropici 0.00052 Figure 1. Phylogenetic Tree for all Non-vegetated (N) and Control (C) Samples. FinalJournal.indd 101 9/25/15 11:38 AM 102 • FINE FOCUS, VOL. 1 (2) This class of bacteria is important because they are a large and diverse group often symbiotically associated with plants. There are also species that can fix nitrogen and can be found in legumes and other plants. Legumes are hypogenous and these bacteria have adapted to create various classes of relationships with the root system, which supports the argument that they are an expected component of the rhizosphere. One OTU was found in the control samples that may correspond to bacteria such as Rhizobium tropici, which has been shown to fix nitrogen and be associated with legumes (22). This suggests that the rhizosphere may have recruited nitrogen-fixing bacteria to soil without the help of surrounding plants. In the second clade, two classes are found: Betaproteobacteria and Gammaproteobacteria. Two OTUs which affiliate with Betaproteobacteria are only found in the control samples (Fig. 2). Betaproteobacteria share the same relative community composition in control soils with both Firmicutes and Acidobacteria (18%, Fig. 4). This class of bacteria consists of aerobic or facultative bacteria; they can be found in waste water and other environments and some can fix nitrogen like Alphaproteobacteria. One OTU found Control Phylogenetic Tree Methanococcus_voltae Clostridium.aminovalericum Aciditerrimonas.ferrireducens Granulicella.aggregans Hyphomicrobium.facile Sphingomonas.jaspsi Rhizobium.tropici Bauldia.litoralis Achromobacter.insuavis Pseudomonas.panacis Pandoraea.sputorum Escherichia.coli Bacillus.subtillis Bacillus.toyonensis PlasmidC64 PlasmidC28 PlasmidC68 PlasmidC34 PlasmidC44 PlasmidC67 PlasmidC63 PlasmidC20 PlasmidC40 PlasmidC59 PlasmidC51 0.05Figure 2. Outgroup Phylogenetic Tree for Control Samples. FinalJournal.indd 102 9/25/15 11:38 AM APPLIED AND ENVIRONMENTAL MICROBIOLOGY • 103 in these samples was related to Pandoraea sputorum. The genus Pandoraea is important to environmental health because of its known use of bio-catalytic activities, such as biodegradation (14). It is also closely related to species belonging to the Ralstonia lineage, which encompasses many heavy metal-resistant Betaproteobacteria (12). The OTUs found in these samples correlate with other samples that are found in highly polluted environments, which suggest an environmental selection for heavy metal resistance (28). Gammaproteobacteria are a class of bacteria that contain environmentally important species, some of which are human pathogens. Gammaproteobacteria were found in both the control and non-vegetated samples, with a more plentiful amount found in the soil without plants (Table 2). For the control soil, this class is the least abundant (Fig. 4). However, in the non-vegetated soil, this class was the most abundant of all the other classes. This, along with previously stated data, suggests differences between the composition between the non-vegetated and control soil communities. It also suggests that the addition of the rhizosphere enriched certain types of bacteria suggesting that certain species may be integral to the observed augmented Pb sequestration (18, 26). A total of three different OTUs of Pseudomonas were detected in the samples and Pseudomonas panacis was detected in both the control and non-vegetated soil. P. panacis is root-associated and has been identified in root lesions of various plants (23). Pseudomonas spp. are known for aerobic growth and association with plants. Pseudomonas putida is an environmentally important species because it has been shown effective in improving the chemical and physical properties of polluted soil, bioremediating substances such as crude oil and naphthalene (11,21). Pseudomonas fluorescens is also an important species that inhabits the rhizosphere and is effective in bioremediation (31). This is a highly metal-resistant species that can tolerate millimolar concentrations of selected metals (5). The possibility of an enrichment of organisms with known association to polluted environments suggests that they are adapted to it and can help plants tolerate such environments, thereby supporting the previous studies of the possible use of Initial and Final Lead Concentrations for Samples Soil Type Initial Pb Concentration Final Pb Concentration Total Percent Change C NV 542.0 531.2 -1.992% 542.0 567.0 4.613% 542.0 613.7 13.229%BE Table 1. Initial and Final Lead Concentrations of Soil for Controls (C), Non-vegetated (NV), and Bacillus megaterium plus EDTA (BE) samples. FinalJournal.indd 103 9/25/15 11:38 AM 104 • FINE FOCUS, VOL. 1 (2) bacteria in bioremediation. The third clade from Fig. 1 consists of a class of bacteria known as Firmicutes. Many Firmicutes produce endospores, have a low G+C content, and are known for carbohydrate degradation. If stress is sensed in soil, Firmicutes produce endospores, which can help them survive and resist desiccation. They also are abundant in root exudates with sugars and organic acids, specifically the genera Bacillus and Clostridium (27). Bacillus and Clostridium were both detected in this experiment in both types of soil at 50% (Table 2). Clostridium, particularly Clostridium acetobutylicum (a close relative to the obtained OTU related to Clostridium aminovalericum), is environmentally important, as previous studies have indicated its usefulness for degradation of large biological molecules, toxic organic molecules, and metals (8). This species has also been used in the bioremediation of soil and toxic sludge by chemically reducing and solubilizing the amount of radionuclides and toxic heavy metals (such as uranium (U), iron (Fe), magnesium (Mg), and (Pb)) (6). This species may help in remediation of soils, particularly those in urban locations, because of its potential use around industrial sites, buildings, and waste disposal sites. Three different OTUs of Bacillus were also discovered in the samples. Bacillus may be a normal part of these ecosystems, suggesting that amendment with B. megaterium would not cause serious disruption of the bacterial community. This effect may be due to the ability of Bacillus spp. to survive and flourish in more hostile environments. One OTU that was found, associated with Bacillus atrophaeus, is a non-pathogenic, aerobic spore-forming Non-vegetated Phylogenetic Tree Methanococcus_voltae Bacillus.atrophaeus Bacillus.thuringiensis Pseudomonas.panacis PlasmidN15 Crocinitomix.catalasitica Pseudomonas.putida PlasmidN38 Acinetobacter.calcoaceticus PlasmidN33 Bacillus.subtilis Telmatocola.sphagniphila PlasmidN40 PlasmidN36 Pseudomonas.fluorescens PlasmidN12 PlasmidN8 PlasmidN35 Escherichia.coli 0.04Figure 3. Outgroup Phylogenetic Tree for Non-vegetated Samples. FinalJournal.indd 104 9/25/15 11:38 AM APPLIED AND ENVIRONMENTAL MICROBIOLOGY • 105 Table 2. Tables comparing percentage and species of bacteria found in each class according to the phylogenetic tree. Comparison of Classes Found in Control and Non-vegetated Samples Control Soil Percent of Bacteria in Class 100% 100% 25% 50% 50% Number of Different Bacterial Species in Class 4 2 1 2 2 Class Alphaproteobacteria Betaproteobacteria Gammaproteobacteria Firmicutes Acidobacteria Non-vegetated Soil Percent of Bacteria in Class 0% 0% 75% 50% 50% Number of Different Bacterial Species in Class 0 0 3 2 2 Class Alphaproteobacteria Betaproteobacteria Gammaproteobacteria Firmicutes Acidobacteria type of bacteria related to B. subtilis (9) (Fig. 3). Bacillus thuringiensis, known as the source for the entomopathogenic crystalline endotoxin for insect control, was found in the non-vegetative treatments. The last clade consists of Acidobacteria, a newer phylum comprising a diverse range of bacteria, especially soil-inhabiting species. Acidobacteria include species known to be Pb-tolerant and also sometimes acidophilic. These, like the Firmicutes, were found in equal proportions in the control and non- vegetated samples (Table 2). One OTU found, Aciditerrimonas ferrireducens, has been reported to be iron-reducing, which could have impacts on other heavy metals, such as Pb (16). Phytoremediation, including rhizofiltration, is a vital area of inquiry because it is more cost effective and has fewer negative impacts on public health and the environment (19). These preliminary data suggest that there is a biologically helpful as well as economically viable method for increasing FinalJournal.indd 105 9/25/15 11:38 AM 106 • FINE FOCUS, VOL. 1 (2) the efficiency of phytoextraction of Pb from soil. For example, the flower could be cut and sold to sustain the removal of the Pb- contaminated rhizosphere. It also could be used to address the problems of so-called ‘food deserts’ by eventually detoxifying soil in order to grow fresh produce in urban gardens. This investigation into the rhizosphere community without amendment suggests that Acidobacteria and Firmicutes may be a common component of Pb- contaminated soils but that the presence of the rhizosphere may have shifted the relative abundance of the Proteobacteria away from Gammaproteobacteria and towards Alphaproteobacteria and Betaproteobacteria. This may help elucidate the mechanism through which rhizofiltration occurs. These data aim to contribute to the ongoing process of understanding and improving on methods for removing hazardous pollutants from the environment. For future studies, a larger sample size could be used, as well as a lesser concentration of EDTA in order to see if the sunflowers remain alive for a longer period of time. Percentage Breakdown of Classes in Both Soils Control Soil Non-vegetated Soil 37% 18% 42% 29% 29% 9% 18% 18% Alphaproteobacteria Betaproteobacteria Gammaproteobacteria Firmicutes Acidobacteria Gammaproteobacteria Firmicutes Acidobacteria Figure 4. Pie charts representing the class diversity in the control soil and non-vegetated soil. FinalJournal.indd 106 9/25/15 11:38 AM APPLIED AND ENVIRONMENTAL MICROBIOLOGY • 107 We would like to thank Monica Ponce for providing us with the soil that was used in this experiment. REFERENCES ACKNOWLEDGEMENTS 1. Akhtar, M. S., Chali, B., & Azam, T. 2013. Bioremediation of Arsenic and Lead by Plants and Microbes from Contaminated Soil. Res. Plant Sci. 1:68-73. 2. Camargo, F. A., Okeke, B. C., Bento, F. M. & Frankenberger, W.T. 2003. In-vitro reduction of hexavalent chromium by a cell-free extract of Bacillus sp. ES 29 stimulated by Cu2+. Appl. Microbiol. Biotechnol. 62:569-573. 3. Celleto, V. R., Oliveira A. J. B., Gonçalves, J. E., Watanabe, C. S. F., Matioli, G., & Gonçalves, R. A. C. 2012. Biosynthesis of Indole-3-Acetic Acid by New Klebsiella oxytoca free and immobilized cells on inorganic matrices. Scien. World J. 4. Dipu, S., Kumar, A. A., & Thanga, S. G. 2012. Effect of chelating agents in phytoremediation of heavy metals. Remed. J. 22:133-146. 5. El-Shanshoury, A. E. R., Elsilk, S. E., & Ateya, P. S. 2013. Uptake of some heavy metals by metal resistant Enterobacter sp. isolate from Egypt. Afric. J. Microbiol. Res. 7:2875-2884. 6. Francis, A. J. 1998. Bioremediation of uranium contaminated soils and wastes. International conference and workshop: Uranium-mining and hydrogeology. Freiberg in Sachsen (Germany) 340-346. 7. Fulekar, M. H. 2010. Environmental Biotechnology. Boca Raton, FL: Taylor & Francis Group. 8. Gao, W. & Francis, A.J. 2013. Fermentation and Hydrogen Metabolism Affect Uranium Reduction by Clostridia. ISRN Biotechnol. 2013:1-11. 9. Gibbons, H. S., Broomall, S. M., McNew, L. A., Daligault, H., Chapman, C., Bruce, D., Karavis, M., Krepps, M., McGregor, P. A., Hong, C., Park, K. H., Akmal, A., Feldman, A., Lin, J. S., Chang, W. E., Higgs, B. W., Demirev, P., Lindquist, J., Liem, A., Fochler, E., Read, T. D., Tapia, R., Johnson, S., Bishop-Lilly, K. A., Detter, C., Han, C., Sozhamannan, S., Rosenzweig, C. N., & Skowronski, E. W. 2011. Genomic signatures of strain selection and enhancement in Bacillus atrophaeus var. globigii, a historical biowarfare simulant. PLoS One 6:e17836. 10. Gleba, D., Borisjuk, N. V., Borisjuk, L. G., Kneer, R., Poulev, A., Skarzhinskaya, M., Dushenkov, S., Logendra, S., Gleba, Y. Y., Raskin, I. 1999. Use of plant roots for phytoremediation and molecular farming. Proc. Nat’l Acad. Sci. 96:5973-5977. 11. Gomes, N. C., Kosheleva, I. A., Abraham, W. R., & Smalla, K. 2005. Effects of the inoculant strain Pseudomonas putida KT2442 (pNF142) and of naphthalene contamination on the soil bacterial community. FEMS Microbiol. Ecol. 54:21-33. 12. Goris, J., De Vos, P., Coenye, T., Hoste, B., Janssens, D., Brim, H., Diels, L., Mergeay, M., Kersters, K., & Vandamme, P. 2001. Classification of metal-resistant bacteria from industrial biotopes as Ralstonia campinensis sp. nov., Ralstonia metallidurans sp. nov. and Ralstonia basilensis Steinle et al. 1998 emend. Int. J. Sys. Evol. Microbiol. 51:1773-82. 13. Gupta, D. K., Srivastava, A., & Singh, V. P. 2008. EDTA enhances lead uptake and facilitates phytoremediation by vetiver grass. J. Environ. Biol. 29:903-6. 14. Han-Jen, R. E., Wai-Fong, Y., & Kok-Gan, C. 2013. Pandoraea sp. RB-44, A Novel Quorum Sensing Soil Bacterium. Sensors (Basel) 13:14121–14132. 15. Hayat, R., Ali, S., Amara, U., Khalid, R., & Ahmed, I. 2010. Soil beneficial bacteria and their role in plant growth promotion: a review. Ann. Microbiol. 60:579–598. 16. Itoh, T., Yamanoi, K., Kudo, T., Ohkuma, M., & Takashina, T. 2011. Aciditerrimonas ferrireducens gen. nov., sp. nov., an iron-reducing thermoacidophilic actinobacterium isolated from a solfataric field. Int. J. Sys. Evol. Microbiol. 61:1281-1285. 17. Järup, L. 2003. Hazards of heavy metal contamination. Brit. Med. Bull. 68:167-182. 18. Leeds, M. & Yang, M. 2010. Rhizofiltration using sunflower (Helianthus annuus L.) and bean (Phaseolus vulgaris L. var. vulgaris) to remediate uranium contaminated groundwater. J. Hazard. Mat. 173:589–596. 19. Lone, M. I., He, Z., Stoffella, P. J., & Yang, X. 2008. Phytoremediation of heavy metal polluted soils and water: Progresses and perspectives. J. Zhejiang University SCIENCE B. 9:210–220. FinalJournal.indd 107 9/25/15 11:38 AM 108 • FINE FOCUS, VOL. 1 (2) 20. Lopez, M. L., Peralta-Videa, J. R., Parsons, J. G., Benitez, T., & Gardea-Torresdey, J. L. 2007. Gibberellic Acid, Kinetin, and the Mixture Indole–3-Acetic Acid–Kinetin Assisted with EDTA- Induced Lead Hyperaccumulation in Alfalfa Plants. Environ. Sci. Technol. 41:8165–8170. 21. Nwachukwu, S. C., James, P., & Gurney, T. R. 2001. Inorganic nutrient utilization by “adapted” Pseudomonas putida strain used in the bioremediation of agricultural soil polluted with crude petroleum. J. Environ. Biol. 22:153-62. 22. Ormeño-Orrillo, E., Menna, P., Almeida, L. G. P., Ollero, F. J., Nicolás, M.F., Rodrigues, E. P., Nakatani, A. S., Batista, J. S. S., Chueire, L. M. O., Souza, R. C., Vasconcelos, A. T. R., Megías, M., Hungria, M., & Martínez-Romero, E. 2012. Genomic basis of broad host range and environmental adaptability of Rhizobium tropici CIAT 899 and Rhizobium sp. PRF 81 which are used in inoculants for common bean (Phaseolus vulgaris L.). BMC Genomics 13:735. 23. Park, Y. D., Lee, H. B., Yi, H., Kim, Y., Bae, K. S., Choi, J. E., Jung, H. S., & Chun, J. 2005. Pseudomonas panacis sp. nov., isolated from the surface of rusty roots of Korean ginseng. Int. J. Sys. Evol. Microbiol. 55:1721-1724. 24. Rahman, M. M., Azirun, S. M., & Boyce, A. N. 2013. Enhanced Accumulation of Copper and Lead in Amaranth (Amaranthus paniculatus), Indian Mustard (Brassica juncea) and Sunflower (Helianthus annuus). PLoS ONE 8:e62941. 25. Roane, T. M. 1999. Bioaugmentation with Metal- Resistant Microorganisms in the Remediation of Metal and Organic Contaminated Soil (Doctoral dissertation). University of Arizona, Tucson. 26. Salt, D. E., Blaylock M., Kumar, N. P. B. A., Dushenkov, V., Ensley, B. D., Chet, I. & Raskin, I. 1995. Phytoremediation: A novel Strategy for the Removal of Toxic Metals from the Environment Using Plants. Biotechnology 13:468-474. 27. Shi, S., Richardson, A. E., O’Callaghan, M., DeAngelis, K. M., Jones, E. E., Stewart, A., Firestone, M. K., & Condron, L. M. 2011. Effects of selected root exudate components on soil bacterial communities. FEMS Microbiol. Ecol. 77:600-610. 28. Silver, S., & Phung, L. T. 1996. Bacterial heavy metal resistance: new surprises. Ann. Rev. Microbiol. 50:753-789. 29. Tsai, Y. L. & Olson, B. H. 1991. Rapid method for direct extraction of DNA from soil and sediment. Appl. Environ. Microbiol. 57:1070–1074. 30. United States Environmental Protection Agency. 2014. Learn about Lead. Retrieved From the Environmental Protection Agency website: http:// www2.epa.gov/lead/learn-about-lead 31. Varivarn, K., Champa, L. A., Silby, M. W., & Robleto, E. A. 2013. Colonization strategies of Pseudomonas fluorescens Pf0-1: activation of soil-specific genes important for diverse and specific environments. BMC Microbiology 13:92. FinalJournal.indd 108 9/25/15 11:38 AM