Rhizoremediation of Lead Using Alternanthera Versicolor

Rhizoremediation of Lead Using genus Alternanthera VersicolorShalini S., Priyasha G., Sohini B. and Jabez Osborne W*Abstr acquit anthropogenic development has been a reflect drawback due to the increased discharge of telling metals into the environment containing the saturated and unsaturated geographical zones of smear. Heavy metals are those which have closeness more than 5gram per cm cube. Lead ca procedures study health hazards like saturnism ( cut poisoning) at higher concentrations. In the present study the ability of Alternenthera versicolor to phytoremediate toxi jakest exact contamination was assessed at different concentrations of ternary. To enhance the economic consumption capabilities of Alternenthera versicolor, bacteriuml organism capable of head up degradation was supplemented. The samples were up descented at an interval of 10, 20 and 30 age. The uptake of star was assessed by AAS, chlorophyl and anthocyanin content was besides checked. Further the in effect(p) strain were characterised by 16s rRNA sequencing.Keywords Rhizoremediation, 16s rRNA, PGPR, AAS, Anthocyanin, ChlorophyllINTRODUCTIONThe around common enceinte metals instal in hazardous sites are Lead(Pb), Copper(Cu), Chromium(Cr),Nickel(Ni) and Zinc(Zn).Ionic and hydroxide forms are the general forms of these heavy metals found in soil, ground piss and surface water (Gade,.2000). Heavy metal contamination is a major concern because they passelnot be degraded (Lone et al,. 2008). Plant growth promoting rhizo bacterium are tangled group of bacteria that can be found in the rhizosphere, and in association with roots. The be-bacteria synergistic interaction in the rhizosphere is determinants of place growth and fertility. These exact the ability to produce indole acetic acid which is an important growth regulatory hormone, to produce siderophores which enable the uptake of solubilised mineral orthophosphate from the soil, HCN production and ammonia production. Morphological studies involve the root and lease length. Hence depending upon the results obtained for apiece concentration, we can determine the maximum concentration at which the bacteria can guard sink contamination. Thus the present study involves the phytoremediation of lead supply by Alternenthera versicolor followed by the rhizoremedial clean up by the synergistic interaction surrounded by the rough-and-ready rhizobacterial seclude and the plant.MATERIALS AND METHODSSample CollectionFor the closing off of lead corrupting bacteria, tannery sludge was compile from four locations in Vellore, Tamil Nadu, India. All the samples were collected development sterile polyethylene bags and were preserved immediately (McGrath et al,. 2001).Isolation of lead resistant bacteria from tannery sludge sampleLead resistant bacteria were insulate from tannery sludge victimisation serial dilution technique and 10-4 dilutions onto MSM agar plates supplemented with one hundred fifty mg /L lead concentration. The obtained pure colonies were purified on MSM agar plates with 150mg/L lead.Screening TechniquesThe rough-and-ready isolates obtained from the tannery sludge sample, were screened for its PGPR traits.Indole Acetic Acid outturnFor bringing close together of indoleacetic acid production, the method described by (Gordon et al,2000). was followed. The isolates were inoculated in Nutrient broth and were incubated in the shaker at 120 rpm for 24hours. 100 l of inocula from overnight civilization of NB was inoculated in MS Media that was supplemented with 5mM L-tryptophan and was incubated in shaker for 48hours. The cultures obtained were centrifuged at 10000 rpm for 10 minutes. 2ml of supernatant was taken and fewer drops of Salkowskis reagent was added to it. Optical density was taken at 530 nm development spectrophotometer. (Hussain et. al,. 2013)Phosphate SolubilisationFor the detection of phosphate solubilising capability of the effective isolate, Pikov skaya agar media was inoculated with effective isolate and zone of inhibition was spy later on 4 geezerhood incubation at RT. (Hussain et. al.2013)Growth Kinetics StudyFor growth kinetics study, seed culture of the effective isolate VITPSS1J was ready in MSM. 2% inoculum of 0.5 OD culture was added in LB broth. O.D readings were taken at every 30 mins till stationary phase was achieved and a plot of land was plotted against O.D. and time interval.Preparation of quite a little cultures64 pots were prepared either(prenominal) consisting of one control and three test pots (Cosio et al, .2004). 2 kgs of sieved garden soil was use to plant Alternanthera versicolor plantlets (20 cm in length) in each pot. Pots were placed in greenhouse for 10 long time for the plantlets to get adapted. The pot culture sets were prepared according to two criteria 1. According to various concentrations of lead being added and 2. According to the number of days of study (10, 20). After the edition was completed, various concentrations (25mg/L, 50mg/L, 75mg/L and 100 mg/L) of lead was added at even interval.Fig 1 Pot cultures of Alternanthera versicolor prepared in 2 kg soil with varying lead concentrationTreatment of effective strain in potsSeed culture of effective isolate VITPSS1J was prepared in Nutrient broth (incubated at RT for 24 hrs). The bacterial culture was further push-down storage multiplied by inoculating 100l of seed culture into 4 Erlenmeyer flasks, each containing 250ml NB. 10 ml of this mass multiplied culture was added to each pot and left for next 10 days (Hussain et al,. 2013).After 10 days, first set of plants were uprooted from the pots (10th day pots of all concentrations).Further studies were carried out on the uprooted plants in order to study the uptake of lead by these plants in association with bacteria in 10 days.Morphological characterizationThe root and institutionalize length of the uprooted plants was calculated. The length of root and sh oot of the test plants were compared with that of the control plants. Comparison of the root-shoot length was also make among plants uprooted from various concentrations. The warp of the plants was also observed.Anthocyanin EstimationAnthocyanin is a pigment obligated for the red, purple, blue colour of several fruits and vegetables, cereals and grains. Anthocyanin can also be utilise as a pH indicator. Each plant was studied for the bar of anthocyanin present. The leaves of a plant were taken and ground with 70% ethanol and 7% acetic acid (in the ratio 32) in a mortar-pestle and transferred to falcon tubes. Sterile water was added in all tubes including the controls. The tubes were kept in lot shaker for 10 minutes and then centrifuged (15 mins, 4C, 4500 rpm). The supernatant was distant and 400 l of methanol was added in each tubes. The anthocyanin content was careful by taking the UV spectrophotometric reading at 530 nm and 657 nm. The equivalent procedure was followed f or all the 12 plants.Chlorophyll EstimationEach plant was also studied for determining the chlorophyll content. The leaves were cut into small pieces and combine with 70% ethanol and ground using mortar-pestle. The mixture was then transferred to falcon tubes and centrifuged at 6000 rpm for 10 mins. Supernatant was used for spectrophotometer assay of chlorophyll at 665nm and 652 nm.RESULTSIsolation of lead resistant bacteriaA gist of three morphologically distinct colonies were observed in MSM agar plates supplementedwith 150mg/l of lead and named VITPSS1J, VITPSS2J, VITPSS3JFig 2 Mother culture plates of the isolated bacteria from tannery sludge B. PGPR analysis of isolatesIndole Acetic Acid Production The ability of obtained isolates for IAA production was assessed. Pink coloration was observed for isolate VITPSS3J. Hence VITPSS3J was used for further studies. Phosphate Solubilisation For the detection of phosphate solubilisation, a clear zone of inhibition is considered as a c oercive conformation for solubilisation. As the isolates were not able to produce any clear zone observed in the Pikovskaya media plate, the isolates were considered negative for phosphate solubilisation.Growth KineticsGrowth kinetics was performed for effective isolateVITPSS3J. Stationary phase was achieved at 270 min (Graph1)B. Morphological CharacterisationThe leaves of Alternanthera versicolor are originally reddish purple in colour. But it was observed that after the supply of lead in the soil the colour of leaves glowering green. Root and shoot length were measured for Alternanthera versicolor at well-ordered interval of 10, 20 days to check the effect of toxic lead concentration of plant growth ( set back1, Table2).Table 2 Root and shoot measurement after 10 days of ceremonyTable 3 Root and Shoot measurement after 20 days of placardAnthocyanin and Chlorophyll concentrationAnthocyanin is a pigment responsible for purple, blue colour of several fruits grains and flowers. Ch lorophyll is a pigment which provides green colour to the plant. (Table 3, 4)Table 4 Anthocyanin and Chlorophyll estimation after 10 days of manifestationTable 5 Anthocyanin and Chlorophyll estimation after 20 days of observationFigure 1 Estimation of anthocyanin after 10 days of observationFigure 2 Estimation of chlorophyll b by using spectrophotometer (652nm)Figure 3Fig 5 Estimation of chlorophyll a using spectrophotometer (665 nm) after 10 days of observationFigure 4. Estimation of Anthocyanin after 20 days of observationFigure 5Fig 7 Estimation of chlorophyll b by using spectrophotometer (652 nm) after 10 days of observationFigure 6Fig 8 Estimation of chlorophyll a by using spectrophotometer (665 nm) after 20 days of observationMolecular characterizationThe obtained isolate was characterized molecularly by 16s rRNA sequencing (Sarita et al,. 2005). Effective bacterial strains were characterized using the universal primers 27F (5-AGAGTTTGATCCTGGCTCAG-3) and 1492R (GGTTACCTTGTTTT ACGACTT-3). DNA extracts from carrells DNA and the 16s Rrna sequence was determined by fluorescent tint terminator method using the sequencing kit. Products were obtained and run on a sequencer. discourseThe present study deals with rhizoremedial treatment of the plant Alternenthera versicolor using bacterial isolates of tannery sludge. The plant has been tested as a hyper accumulator of lead which is a toxic heavy metal. Many plants have been worked upon for phytoremedial and rhizoremedial studies using n Alternenthera versicolor different heavy metals such as isolation of potent PGPR isolate tannery sludge was serially diluted and plated onto MSM agar plates. Carassco et al,.2005 also isolated lead degrading bacteria using Nutrient Agar Medium.Alternenthera versicolor was tested for its ability to be turn out as a potent hyperaccumulator of lead. Hyperaccumulators are plants that have the abilities to take up higher concentration of toxic contaminants or compounds. Some of the hyperaccumulators already be are Agrostis castellana(McCutcheon et al,.2003),Brassica juncea(E.lombi et al,. 2001),Brassica napus(McCutcheon et al,. 2003),Helianthus annuuus(Schmidt et al,.2003). The ability of VITPSS1J to act as a potent PGPR isolate was assessed. VITPSS1J was found to be positive for Indole Acetic Acid production (Hussain et al,.2013), but it showed negative result phosphate solubilizing agent, indicating it has effectiveness as a PGPR organism. Alternenthera versicolor was tested for its ability to phytoremediate toxic lead concentration at 25mg/L, 50mg/L, 75mg/L and 100mg/L concentrations of lead. Earlier reports have suggested the use of Brassica juncea as a potent hyper accumulator for lead. Thus shoot and root length was checked at regular interval and it was found be similar as compared to the control suggesting the effectiveness of Alternenthera versicolor in remediating lead contamination.(Hussain et al,. 2013). Anthocyanin and chlorophyll estimation also suggest the similar result as anthocyanin and chlorophyll content of a plant are a major indicating factor for its toxic uptake studies. Higher level of anthocyanin and chlorophyll at day 10 and 20 suggest that toxic effect of lead has been neutralised by various mechanism taking place in the plant like phytostabalisation and phytovolatalisation (Irene Kuiper et al,.2003).Thus the present study proves that Alternenthera versicolor can be used as a hyperaccumulator of lead at concentration ranging from 25 to 100 mg/L.CONCLUSIONAlternenthera versicolor has hence been proved as a potent hyperaccumulator of lead at concentration ranging from 25 to 100mg/L and VITPSS1J can be used to remediate lead contamination. Hence further study on the synergistic interaction between Alternenthera versicolor and VITPSS1J.V. REFERENCES1. Arvind Gulati, Pratibha Vyas, Praveen Rahi, Ramesh Chand Kasana (2009) Plant Growth-Promoting and Rhizosphere-Competent Acinetobacter rhizosphaerae Strain BIHB 723 f rom the ratty Deserts of the Himalayas 583713772. Althaf Hussain Sk. and Srinivas P (2013) Evaluation of Plant Growth Promoting Traits by Pseudomonas and Azotobacter Isolated From Rhizotic Soils of Two Selected Agroforestry Tree Species of Godavari Belt Region, India 431-4363. Solon A. Gordon, Robert P. weber (1950) Colorimetric Estimation of Indole acetic acid4. Shirin Heydari1, Parviz Rezvani Moghadam2, Seyyed Mehdi Arab3 Hydrogen Cyanide Production Ability by Pseudomonas Fluorescence Bacteria and their Inhibition Potential on Weed Germination5. Brianne L. Clark, Dr. Ranjan Chakraborty, Chair Dr. Bert C. Lampson ,Dr. Lee M. Pike (2004) Characterization of a Catechol-Type Siderophore and the Detection of a Possible Outer Membrane Receptor Protein from genus Rhizobium leguminosarum strain IARI 3126. E. Nicole Bridgersa, Mari S. Chinnb, Van-Den Troungc (2010) Extraction of anthocyanins from industrial purple-fleshed sweetpotatoes and enzymatic hydrolysis of residues for fermentable sugars 613-6207. Simcha Lev-Yadun and Kevin S. Gould (2009) Role of Anthocyanins in Plant Defence 978-0-387-778. Irene Kuiper, Ellen L. Lagendijk, Guido V. Bloemberg, and Ben J. J. Lugtenber (2003) Rhizoremediation A Beneficial Plant-Microbe Interaction1105-019. Sushma Bondre, Pallavi Patil, Amaraja Kulkarni, M. M. Pillai (2012) Study on isolation and purification of anthocyanins and its application as pH indicator698-70210. Sabyasachi Chatterjee*, Anindita Mukherjee, Agniswar Sarkar, Pranab Roy (2012)Bioremediation of lead by lead-resistant microorganisms, isolated from industrial sample290-29511. A. Rehman, S. Ashraf, J. I. Qazi, A. R. Shakoori (2005) Uptake of Lead by a Ciliate, Stylonychia mytilus, Isolated from industrial Effluents Potential Use in Bioremediation of Wastewater75290296