Rhizoremediation of Lead Using Alternanthera Versicolor

Rhizoremediation of Lead Using Alternanthera Versicolor Shalini S., Priyasha G., Sohini B. and Jabez Osborne W* Abstract: Anthropogenic development has been a major drawback due to the increased discharge of heavy metals into the environment containing the saturated and unsaturated zones of soil. Heavy metals are those which have density more than 5gram per cm cube. Lead causes major health hazards like Plumbism (lead poisoning) at higher concentrations. In the present study the ability of Alternenthera versicolor to phytoremediate toxic lead contamination was assessed at different concentrations of lead. To enhance the uptake capabilities of Alternenthera versicolor, bacterial organism capable of lead degradation was supplemented. The samples were uprooted at an interval of 10, 20 and 30 days. The uptake of lead was assessed by AAS, chlorophyll and anthocyanin content was also checked. Further the effective strain were characterised by 16s rRNA sequencing. Keywords: Rhizoremediation, 16s rRNA, PGPR, AAS, Anthocyanin, Chlorophyll INTRODUCTION The most common heavy metals found 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 water and surface water (Gade,.2000). Heavy metal contamination is a major concern because they cannot be degraded (Lone et al,. 2008). Plant growth promoting rhizobacteria are heterogeneous group of bacteria that can be found in the rhizosphere, and in association with roots. The plant-bacteria synergistic interaction in the rhizosphere is determinants of plant growth and fertility. These involve the ability to produce indole acetic acid which is an important growth regulatory hormone, to produce siderophores which enable the uptake of solubilised mineral phosphate from the soil, HCN production and Ammonia production. Morphological studies involve the root and shoot length. Hence depending upon the results obtained for each concentration, we can determine the maximum concentrat ion at which the bacteria can withstand lead contamination. Thus the present study involves the phytoremediation of lead supply by Alternenthera versicolor followed by the rhizoremedial clean up by the synergistic interaction between the effective rhizobacterial isolate and the plant. MATERIALS AND METHODS Sample Collection For the isolation of lead degrading bacteria, tannery sludge was collected from four locations in Vellore, Tamil Nadu, India. All the samples were collected using sterile polyethylene bags and were preserved immediately (McGrath et al,. 2001). Isolation of lead resistant bacteria from tannery sludge sample Lead resistant bacteria were isolated from tannery sludge using serial dilution technique and 10-4 dilutions onto MSM agar plates supplemented with 150 mg/L lead concentration. The obtained pure colonies were purified on MSM agar plates with 150mg/L lead. Screening Techniques The effective isolates obtained from the tannery sludge sample, were screened for its PGPR traits. Indole Acetic Acid Production For estimation of IAA 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  µl of inocula from overnight culture 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 few drops of Salkowski’s reagent was added to it. Optical density was taken at 530 nm using spectrophotometer. (Hussain et. al,. 2013) Phosphate Solubilisation For the detection of phosphate solubilising capability of the effective isolate, Pikovskaya agar media was inoculated with effective isolate and zone of inhibition was observed after 4 days incubation at RT. (Hussain et. al.  2013) Growth Kinetics Study For growth kinetics study, seed culture of the effective isolate VITPSS1J was prepared 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 was plotted against O.D. and time interval. Preparation of pot cultures 64 pots were prepared each consisting of one control and three test pots (Cosio et al, .2004). 2 kgs of sieved garden soil was used to plant Alternanthera versicolor plantlets (20 cm in length) in each pot. Pots were placed in greenhouse for 10 days 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 adaptation was completed, various concentrations (25mg/L, 50mg/L, 75mg/L and 100 mg/L) of lead was added at regular interval. Fig 1: Pot cultures of Alternanthera versicolor prepared in 2 kg soil with varying lead concentration Treatment of effective strain in pots Seed culture of effective isolate VITPSS1J was prepared in Nutrient broth (incubated at RT for 24 hrs). The bacterial culture was further mass multiplied by inoculating 100ÃŽ ¼l 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 characterization The root and shoot length of the uprooted plants was calculated. The length of root and shoot of the test plants were compared with that of the control plants. Comparison of the root-shoot length was also done among plants uprooted from various concentrations. The colour of the plants was also observed. Anthocyanin Estimation Anthocyanin is a pigment responsible for the red, purple, blue colour of several fruits and vegetables, cereals and grains. Anthocyanin can also be used as a pH indicator. Each plant was studied for the amount of anthocyanin present. The leaves of a plant were taken and ground with 70% ethanol and 7% acetic acid (in the ratio 3:2) in a mortar-pestle and transferred to Falcon tubes. Sterile water was added in all tubes including the controls. The tubes were kept in rotary shaker for 10 minutes and then centrifuged (15 mins, 4 ºC, 4500 rpm). The supernatant was removed and 400  µl of methanol was added in each tubes. The anthocyanin content was measured by taking the UV spectrophotometric reading at 530 nm and 657 nm. The same procedure was followed for all the 12 plants. Chlorophyll Estimation Each plant was also studied for determining the chlorophyll content. The leaves were cut into small pieces and mixed 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. RESULTS Isolation of lead resistant bacteria A total of three morphologically distinct colonies were observed in MSM agar plates supplemented with 150mg/l of lead and named VITPSS1J, VITPSS2J, VITPSS3J Fig 2: Mother culture plates of the isolated bacteria from tannery sludge B. PGPR analysis of isolates Indole 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 positive 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 Kinetics: Growth kinetics was performed for effective isolateVITPSS3J. Stationary phase was achieved at 270 min (Graph1) B. Morphological Characterisation: The 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 turned green. Root and shoot length were measured for Alternanthera versicolor at regular interval of 10, 20 days to check the effect of toxic lead concentration of plant growth (Table1, Table2). Table 2: Root and shoot measurement after 10 days of observation Table 3: Root and Shoot measurement after 20 days of observation Anthocyanin and Chlorophyll concentration Anthocyanin is a pigment responsible for purple, blue colour of several fruits grains and flowers. Chlorophyll is a pigment which provides green colour to the plant. (Table 3, 4) Table 4: Anthocyanin and Chlorophyll estimation after 10 days of observation Table 5: Anthocyanin and Chlorophyll estimation after 20 days of observation Figure 1 Estimation of anthocyanin after 10 days of observation Figure 2 Estimation of chlorophyll b by using spectrophotometer (652nm) Figure 3Fig 5: Estimation of chlorophyll a using spectrophotometer (665 nm) after 10 days of observation Figure 4. Estimation of Anthocyanin after 20 days of observation Figure 5Fig 7: Estimation of chlorophyll b by using spectrophotometer (652 nm) after 10 days of observation Figure 6Fig 8: Estimation of chlorophyll a by using spectrophotometer (665 nm) after 20 days of observation Molecular characterization The 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 (GGTTACCTTGTTTTACGACTT-3†). DNA extracts from cell’s DNA and the 16s Rrna sequence was determined by fluorescent dye terminator method using the sequencing kit. Products were obtained and run on a sequencer. DISCUSSION The 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 proved 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 proved 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 chl orophyll 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. CONCLUSION Alternenthera 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. REFERENCES 1. Arvind Gulati, Pratibha Vyas, Praveen Rahi, Ramesh Chand Kasana (2009) Plant Growth-Promoting and Rhizosphere-Competent Acinetobacter rhizosphaerae Strain BIHB 723 from the Cold Deserts of the Himalayas 58:371–377 2. 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 431-436 3. Solon A. Gordon, Robert P. Weber (1950) Colorimetric Estimation of Indole acetic acid 4. Shirin Heydari1, Parviz Rezvani Moghadam2, Seyyed Mehdi Arab3 Hydrogen Cyanide Production Ability by Pseudomonas Fluorescence Bacteria and their Inhibition Potential on Weed Germination 5. 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 Rhizobium leguminosarum strain IARI 312 6. 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 613-620 7. Simcha Lev-Yadun and Kevin S. Gould (2009) Role of Anthocyanins in Plant Defence 978-0-387-77 8. Irene Kuiper, Ellen L. Lagendijk, Guido V. Bloemberg, and Ben J. J. Lugtenber (2003) Rhizoremediation: A Beneficial Plant-Microbe Interaction1105-01 9. Sushma Bondre, Pallavi Patil, Amaraja Kulkarni, M. M. Pillai (2012) Study on isolation and purification of anthocyanins and its application as pH indicator698-702 10. Sabyasachi Chatterjee*, Anindita Mukherjee, Agniswar Sarkar, Pranab Roy (2012)Bioremediation of lead by lead-resistant microorganisms, isolated from industrial sample290-295 11. 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 Wastewater75:290–296

Causes of Air Pollution Essay

There are several main causes of air pollution, the vast majority of them can be attributed to man. Some natural sources of air pollution include forest fires, dust storms, and volcanic eruptions. Plants such as ragweed contaminate the air with pollen. Decaying leaves and other forms of vegetation release gases that contribute to air pollution and cause haze. (Morgan) Air pollution is the introduction into the atmosphere of chemicals, particulates, and biological matter that cause harm to humans, other living organisms, or cause damage to the natural environment. Stratospheric ozone depletion (contributed to air pollution) has long been recognized as a threat to human health as well as to the Earth’s ecosystems. The Earth is capable of cleaning itself of a certain level of pollution, but man-made pollutant have become too numerous for the Earth’s natural mechanisms to remove. We are seeing the results of this overload in the form of acid rain, smog, and the variety of health problems that can be contributed to our environment. (Godish) One of the main causes of air pollution is manufacturing. This source of pollution spews particulate matter and chemicals into the atmosphere. The exhaust from a factory includes, sulfur oxides, nitrogen oxides, carbon monoxide and dioxide, as well as volatile organic compounds and particulates. There is not an area of the Earth’s atmosphere or an ecosystem that has not been altered by the long term effects of the pollution created by manufacturing. Strides have been made to filter the material coming from manufacturing plants, but it may take the Earth millennia to completely recover from the damage already done. (universe) A surprisingly link may exist between ocean fertility and air pollution over land, according to Georgia Institute of Technology research reported in the Feb. 16 issue of the Journal of Geophysical Research – Atmospheres. The work provides new insight into the role that ocean fertility plays in the complex cycle involving carbon dioxide and other greenhouse gases in global warming. (Cain) Finally, the burning of fossil fuels is a part of everyday life of every human on the planet. We burn fossil fuels in our cars, fossil fuel is burned to extract fossil fuel from the Earth, and fossil fuel is used to process fossil fuel into its individual components. Every step of the way releases sulfur and nitrogen oxides, carbon monoxide, heavy metals and particulates into the air. Each step in the process increases the number of asthma cases in the world, raises a person’s chance of having cancer, and increases the chance that your child will be born with congenital defects. (Turco) References Cain, Fraser. (2005). Air Pollution Linked to Growth of Life in Oceans. Retrieved from http://www.universetoday.com/10263/air-pollution-linked-to-gr Godish, Thad. 1997. Air Quality, 3rd ed. Lewis Publishers, Boca Raton, Fl. Morgan, M. T. (2003). Environmental Health, (3rd ed.). Belmont: CA: Wadsworth Turco, Richard P. 2001. Earth Under Siege: From Air Pollution to Global Change, 2nd ed. Oxford University Press, New York.

A comparison of two media articles Essay

The Daily Express and The Independent write about the same event, England’s 2-0 victory over Greece in one of England’s world cup qualifier matches. It is obvious from looking at the articles that they are set out and written differently, this is because of their target audiences. The Independent is a more formal newspaper in A2 size, aimed at the more educated person. It is generally read by people in the social groups of A, B and C1, this includes people such as doctors, policemen and other such skilled workers. The Daily Express however is a much less formal newspaper, in A3 size, its target audience are people who are generally less educated and belong in the social groups of C1 and C2, people like builders, receptionists and other unskilled workers. The Daily Express’ article dominates the entire back page with very little else but a small sub article and some small adverts, the Daily Express sets it out in this way to attract its readers to the paper and article, it is bold and stands out. The Independent is different in this as the article takes up roughly half of the page, with enough room to have another article on the same page this is because of its much larger page. The Independent does not use big bold headlines or make it to try and attract the reader into the story; instead they set the article out in a more professional form making it look elegant and neat. This is done as The Independents readers do not want to be enticed into reading an article just because it looks attractive and stands out, they will read what is of interest to them and for content of the story. The Daily Express however uses the bold headlines and dominating layout to attract it’s readers into the article and paper. The Daily Express includes two pictures in its article, one for the main article of the match and the other one shows the gossip of the match. The Daily Star does this as their readers like pictures to attract them to the story and also like to read about the gossip as well, in this case the bottles being thrown at David Beckham’s head. The picture for the main article that the Daily Star chooses to show is that of David Beckham celebrating his goal, however they have blocked everything else out of the picture and just have David shown with a bold black outline. The picture is laid out across the page in an untidy fashion making it bolder and more attracting to its reader. This is done as the readers of the paper recognise David as a big celebrity and so want to read what is said about him. The Independent however is very different it has only one picture; it dominates the article but is set out in a square as a neat part of the article. The picture is the same as Daily stars however it is much broader as it shows more than just David Beckham; it is left showing us the stadium and the crowd’s celebrations. The chooses to do this as its readers prefer a much more in depth view as to what has happened when reading their articles, with this they can see the sort of atmosphere there would have been in the Stadium. The Daily Express uses a type of language typical of most tabloid newspapers, but not usually seen in broadsheet newspapers; puns, a play on words. The Daily Express uses pun in the headline of its article which focuses solely on David Beckham. The headline is â€Å"Haircules†, deriving from the ancient Greek God of Hercules, a mighty, strong warrior. The Daily Express has done this as it shows David Beckham as the mighty warrior of the football match. The beginning part â€Å"Her†, has been altered to â€Å"Hair† to apply more to David Beckham and his famous, forever changing hair cut. The Daily Express uses war imagery; the writer uses words, phrases and sentences which compare the football match to a bloody battle or war. The sub header is a great example of this war imagery language with the phrase â€Å"cool under fire†. We would more often than not associate this sort of term with a soldier who had kept his cool whilst being shot at, however in this case it has been changed to describe an aspect of the football match. Also, â€Å"missiles†, â€Å"victory† and â€Å"stoop to conquer† are more war imagery terms which the writer has used to elevate the match and make it seem more exciting. The Independent has not used war imagery as this is a very convincing language, the paper would much rather its readers made up their own minds rather than have them made for them. Sentence Length, sentence structure and sentence variety are another three things which sets these two articles apart. Whereas the Daily Express uses very simple, basic, short sentence structures The Independent uses longer more complex sentence structures which appeal more to its readers. The Daily express does not break down its sentences with punctuation, punctuation is very basic in this article for example, take the opening sentence of each, â€Å"Skipper David Beckham provided the perfect answer to the Greek thugs who targeted him last night with missiles as he curled in the free kick which put the seal on victory in Athens and kept England’s world cup hopes alive†, not a single comma is used to break down that very large sentence. ‘†Bring on the Germans† was the cry from the sunburned, shaven-headed masses in the northern arc of the Spiro Louis Stadium last night and there would not have been a single man in the England dressing room who would disagree’, in this the Independents the sentence is broken down by a quote and comma. Both of the articles input interviews from David Beckham; they are very similar but The Independents comments contain better vocabulary as to appeal more to its readers. The Independent inputs phrases such as â€Å"the importance of maintaining our discipline†, so that the comments appear more intellectual and appeal more to its readers. The Daily Express however writes this comment as â€Å"It was important we kept our discipline†, keeping the phrase more simple, so that it’s less sophisticated readers are not put off by complex language. The independent also contains much more dialogue than the Daily Express, this is because the Independent would like to get across as many view points as possible on the match, so that it’s readers can think for themselves and come to their own decision on the what kind of match the game was. In conclusion both of the articles are effective for their different readers. The Daily Express writes more basically and writes more about gossip and facts to appeal to its lesser educated readers. With bold headlines and the picture of David Beckham the paper is going to sell as it attracts its readers. The Independent is very different, it is much more factual, it’s more educated readers are less interested in the gossip and would rather read the facts. The article contains a lot of dialogue and refrains from using war imagery, presenting different view points enabling the reader to make their own decision on the match.

Is Cranberry Juice Effective for Preventing and Treating...

A 65-year-old Chinese woman, who is currently being treated for a suspected urinary tract infection (UTI), has enquired to whether cranberry juice is effective for preventing and treating UTIs. After querying the RN, they too are unsure how effective the therapeutic intervention is. Thus, there must be an investigation utilising evidence-based practice (EBP) to determine the effectiveness of the therapeutic intervention and provide accurate information to the patient. Furthermore, when delivering the information to the patient, culturally appropriate care must be considered, to ensure clear and coherent communication to the patient. EBP provides health professionals a method to use critically appraised and scientifically proven evidence†¦show more content†¦The question was rephrased as a specific, answerable question using PICO, which refined the case study to its core components; postmenopausal women, cranberry juice and UTIs. The advanced search limits were enabled to craft rather precise research strategies, with Boolean operators and wildcards were utilised in the phrase search; postmenopausal wom*n AND cranberr$ juice AND UTIs (Wolf et al., 2013). The search limits of the Cochrane Library were restricted to Cochrane review and trials, and the publication dates from January 2010 to May 2014. From this refined search of the selected databases, eight papers returned from over 8000 results. Evaluating the eight papers for quality evidence was critical when selecting the two papers to utilise. The evidence hierarchy was considered, aiming to use the highest evidence possible, such as systematic reviews and meta-analyses; and randomized, controlled, double-blind studies, to ensure the most accurate evidence informed the clinical decision for the patient (Bloom, Olinzock, Radjenoic Trice, 2013). When sorting the results, other various factors were also taken into consideration; credibility, reputability, reasonability and support. With these factors taken into account, the two papers selected are highly esteemed in regards to evidence, and are most relevant to the patient (Stichler, 2010). The first paper, selected from the CINAHL database, was the