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 MANUSCRIPT RECEIVED 24 APRIL, 2015; ACCEPTED 2 JULY, 2015 Copyright 2015, Fine Focus all rights reserved FinalJournal.indd 95 9/25/15 11:38 AM 96 • FINE FOCUS, VOL. 1 (2) ABSTRACT 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 sunfower (Helianthus annuus) have been shown to sequester heavy metals in their root systems, thus fltering contaminants such as lead (Pb) from soil, a CORRESPONDING process termed rhizofltration. In the present study, Bacillus megaterium was applied to the root system of AUTHOR sunfowers growing in Pb-contaminated soil and the effciency of rhizofltration was examined. Lead levels *Rebekah J. Ward in the rhizosphere of the Bacillus megaterium and EDTA Georgia Gwinnett College amended plants were almost 100 mg/kg soil higher than 1000 University Center Lane those without treatment, suggesting the amendment Lawrenceville, GA 30043 may have been effective in augmenting lead Email: [email protected] sequestration. In order to further elucidate these lead- sequestering communities, preliminary phylogenetic KEYWORDS assays were conducted on the soil with and without the presence of the plant. Although complete coverage • Bioremediation of the community phylogeny was not possible, there • Sunfowers was evidence indicating that the rhizosphere may have • Rhizofltration induced changes in the composition of the bacterial • Lead community. These studies offer simple methods for • Phylogenetics enhancing bioremediation in agriculture. INTRODUCTION Heavy metals are defned as metals that have Environmental Protection Agency (EPA), a specifc density > 5g/cm3 (17). Elevated lead can be found in the air, soil, water, and concentrations of heavy metals in soil can in homes because of its presence in gasoline, have a devastating effect on human health industrial facilities, paint, ceramics, pipes, and the environment. This is especially true batteries, cosmetics, ammunition, and even for gardens located in urban areas. Human food (30).These make lead a priority heavy health is most adversely affected by certain metal for study in urban environments. heavy metals, namely cadmium, mercury, The problem of heavy metal soil arsenic, and lead (17). Excess Pb is especially contamination can be addressed using toxic to humans because of the effects it a technique known as bioremediation. has on kidneys and the nervous system, Bioremediation is the process of introducing which can cause headaches, weakness, organisms, such as bacteria, into a cramps, anemia, and may lead to mental contaminated environment in order to health disorders (1). According to the U.S. FinalJournal.indd 96 9/25/15 11:38 AM APPLIED AND ENVIRONMENTAL MICROBIOLOGY • 97 remove the pollutants. Phytoremediation an infuence on the effectiveness is a subset of bioremediation that exploits of phytoremediation. Addition of plants in order to clean up contamination. ethylenediaminetetraacetic acid (EDTA) has Certain plants termed hyperaccumulators shown an increased Pb uptake by almost can store the heavy metals in tissues or the 20% in previous studies, and has also been rhizosphere (root system and surrounding shown to facilitate phytoremediation in soil), therefore taking it out of biological plants (13, 20). EDTA is a common and circulation. This process is termed powerful chelating agent that has been rhizofltration. This study was conducted added recently into heavy metal treatment to quicken the remediation process and systems and works especially well with increase its effciency without hampering Pb and copper (4). This demonstrates cost effectiveness. Previous studies (13, 15, that certain chemical additives can help 18, 20) have indicated that adding chemicals sunfowers absorb higher levels of Pb, or biological elements to the soil may help therefore ultimately reducing the cost, plants sequester metals in the tissues. This, amount of land used, and amount of plants in turn, reverses some of the effects of the needed to grow in a particular plot of land metals by lowering the concentration in when used for bioremediation. the surrounding area, allowing more plants Bacteria that reside in rhizospheres of that are less metal-tolerant to grow. After plants can play a role in reducing the a rhizosphere absorbs as much metal as it toxic effects of heavy metals on the plants can, the plant can be uprooted, allowing for (15). These microorganisms can protect removal of contaminants from the soil. This the plant from damage and, in return, can alleviate some of the harmful impacts of beneft from living in the rhizosphere metals on the environment. However, one of systems, therefore creating a mutualistic the problems with phytoremediation is that relationship with the plant. In this study, it is a slow process that produces a low yield Bacillus megaterium was used because this (10). bacterium has been shown to absorb and One plant that has been shown to sequester store Pb intracellularly, therefore making it heavy metals in the rhizosphere is the resistant to elevated levels of Pb (25). This sunfower (Helianthus annuus) (18). Sunfowers species is also a common soil bacteria that is are considered hyperaccumulators and have considered part of plant growth promoting been used for various environmental cleanup rhizobacteria (PGPR), which helps improve projects, (7). Sunfowers also are more tolerant growth by releasing a key auxin (indole-3- to pH variation than many other common acetic acid) to encourage cell proliferation plants, capable of growing in soil ranging (3). Bacterial cultures were added to from pH 5.7 to over 8.0, while optimal soil germinating seedlings to help improve pH for other plants is 6.4 (24). Sunfowers also the health of the plants for rhizofltration produce more roots, shoots, and total biomass and increase the concentration of Pb in than many other common plants. This means the rhizosphere through the intracellular that they can potentially hold larger amounts sequestration used by B. megaterium. The of pollutants and fewer plants are required to purpose of this experiment was to analyze recover the same amount of pollutants, thereby the impact of the addition of bioavailable becoming economically sustainable. phosphate and heavy-metal tolerant bacteria on the Pb concentrations in highly Bioavailability of phosphate also has contaminated soil. FinalJournal.indd 97 9/25/15 11:38 AM 98 • FINE FOCUS, VOL. 1 (2) MATERIALS AND METHODS COLLECTION, SETUP, AND mL of freshly made bacterial culture once, MAINTENANCE and then watered with 40 mL tap water on alternate days. The bacterial culture For this experiment, three fve-gallon was then serially diluted onto LB agar plastic containers of soil were collected plates to determine the original cell count. from two sites in an urban garden in Sunfower height was then measured Atlanta, Georgia, USA. These samples seven times for a total of three months and were then transported to the laboratory at recorded. The measurements were taken Georgia Gwinnett College. The soil was from the base of the stem to the tip of the characterized as being a crumbly, fne soil. tallest leaf. The soil (2.5 grams, manually homogenized) was put into 500 mL conical Falcon tubes SOIL FILTRATION with 7.5 mL distilled H O and stored at 2 Tubes with the most contaminated soil, -80˚C for DNA extraction. according to the analysis results, were A sample was taken from the middle of thawed, and 1 mL 0.1 M EDTA was added, each container prior to planting seedlings, along with 200 µL Tris buffer and distilled homogenized, and sent to the University water to balance the tubes. They were of Georgia Soil and Water Analysis Lab centrifuged at 1000 rcf for 1 min. at 25˚C, for determination of Pb concentration. A then the supernatant was removed and Teddy Bear sunflower (Helianthus annuus) 10 mL of Tris buffer was added. After seed was planted one inch deep in nine thorough mixing with a vortex mixer, 10” x 12” containers with each weighing the tubes were weighed and water was approximately 0.95 kg per container. added to balance them within 0.1 g. This The samples were watered with 40 mL was repeated three times. After the fourth of tap water on alternate days for three centrifugation, the liquid was poured months. After the seeds sprouted, a 40 through muslin to flter out particles and Watt Growlux, wide-spectrum grow light 1 mL 1X Tris Borate EDTA (TBE) was (Grower’s Supply, Dexter, MI) was placed added to the soil, weighed out in tubes, on a timer for eight hours daily. Unplanted and centrifuged one more time at the same soil was maintained as the control settings. The supernatant was transferred experiment. to two microcentrifuge tubes for storage at -80˚C for fve days. Sterile Luria broth (LB, 100 mL) was inoculated with Bacillus megaterium DNA EXTRACTION AND ATCC14581 and placed in a shaker for CLONING 24 h at 37°C. The nine containers of soil The method of Tsai and Olson (29) was were then separated into three different used for DNA extraction in the present categories. Three control containers study with the following modifcations: were watered with 40 mL tap water on Tris EDTA was used in place of Tris HCl, alternate days. Three EDTA containers the tubes were stored in a -80˚C freezer were watered with 80µL EDTA once then instead of in dry ice, and the tubes were 40 mL tap water on alternate days. Three centrifuged at 6000 rcf for 10 min. before EDTA plus B. megaterium containers were starting the protocol. Ten new PCR watered with 80 µL 0.1 M EDTA once, 1 FinalJournal.indd 98 9/25/15 11:38 AM APPLIED AND ENVIRONMENTAL MICROBIOLOGY • 99 samples were made with Cetyltrimethyl elongation, and the positive control used ammonium bromide (CTAB) and 5 g of Bacillus subtilis DNA (48 µL).