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My name is Devashi Nemdharry and I am currently enrolled in an

undergraduate degree namely Biotechnology and Molecular Biology and

Biomedical Sciences as Minor.

Biological, physical and chemical processes and their applications in a

real life situation have always been and will always be one of my

interests. One of the most serious problems that our planet is experiencing

nowadays. Pollution has severe effect on the climate, biotic and abiotic

components of the environment and can also affect human health. I was

very amazed by the complex mechanism of detoxification that plants

possess and how they can be of a bioremediation to the environment. This

technology includes Phytoremediation ( plants ) and rhizoremediation

(plant & microbe interaction).

In fact, Australia has already implemented this technique for the

remediation of mining affected land (Brett Robinson et al.2007). This

technique has maintained Australia's 'Green-clean' image abroad. Since

many people are not aware of this emerging technology, I decided to

design this webpage so that to pass on knowledge to others.

Key- words Explained:

Phytoremediation- means the use of plants to remove pollutants from the environment.

Rhizofiltration- is the process that plants use to remove toxic metals/ pollutants from the

Environment. It is basically the use of plant roots & microbes to remove heavy metals mainly from

water.

Phytoremediation of Heavy metals from the Environment by the process Rhizofiltration

Author: Devashi Devi Nemdharry, 31624575

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Rapid Industrialization, increased anthropogenic activities, modern agricultural

methods and flawed waste disposal methods have boost up stream pollution by

increasing the concentration of pollutants in water and also Soil. These

pollutants are highly toxic and is harmful to living organisms. The common

being: Lead, Copper, Cadmium, Chromium, Nickel and Zinc (Kumar et

al.1995).For example; Cadmium is one of the most toxic metal. Cadmium

inhibits root and shoot growth, affect nutrients uptake and if it is consumed by

animals and humans, it can cause severe disease ( Belimov et al.2005).

Pollutants are both Ecological and human health hazard and this has lead to the

development of technologies based on ion exchange or chemical or microbial

precipitation. However these technologies have different efficiencies for

different metals and maybe very costly if large volume, low metal concentration

and high clean-up standards are required such as large surface soil

contamination (Susan Eapen et al.2004)

Due to these reasons, other cost effective and reliable technologies have been

developed and implemented. According to Research, plants have been identified

to have the ability to absorb heavy metals and such remediation is called

Phytoremediation1. This remediation is cheap, non destructive to the

environment, and also helps in removing high level of carbon dioxide in the

atmosphere. Plants that have the ability to remove toxic metals and other

pollutants from contaminated soil or water act as biosorbers and they do this via

a process known as Rhizofiltration2.

This whole process is actually controlled by some metal tolerant bacteria known

as rhizosphere bacteria and they have been isolated from the root tips of the

Indian Mustard( Brassica Juncea) plant. Brassica Juncea plant is most efficient

due to its high growth rate ( Belimov et al.2005).

They have bacteria which can hydrolyse the toxic metals and use it as their

energy source for growth. This will be explained later in the second paper.

Background:

Rhizospere bacteria

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A Brief Explanation:

All the processes at root level is known as Rhizofiltration.

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1. Rhizofiltration: the use of plants to remove heavy metals from aqueous streams' Viatcheslav Dushenkov, P.B.A.Nanda Kumar, Harry Motto and Ilya Raskin. 1995. Agbiotech Centre and Department of Environmental Sciences Rutgers University, Cook College, New Brunswick, New Jersey 08903-0231. doi: 10.1021/es00005a015

2. Cadmium- tolerant plant growth- promoting bacteria associated with the roots of Indian Mustard ( Brassica juncea L.Czern ) A.A Belimov, N. Hontzeas, V.I.Safronova, S.V.Demchinskaya, G.Piluzza, S. Bullitta, B.R. Glick.2004.All Russia Research institute for Agricultural Microbiology, Department of Biology, university of waterloo, Canada

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Both paper argue that Phytoremediation/rhizofiltration is an emerging

low cost and ecologically benign technology for the refinement of soils

by using plants to absorb, store and to render contaminants in soil and

water less harmful through physical, chemical and biological processes.

Rhizofiltration is mainly controlled by rhizosphere bacteria found in the

roots of certain terrestrial plants.

Both papers showed that the plant Indian Mustard known as Brassica

Juncea, is one of the most suitable plant for this purpose and hence

shows that phytoremediation is one of the cheapest remedies for

contaminated soil and Environment. The plants carry this through

Rhizofiltration. The positive points about these papers are that, they

show the effect of different Toxic metals on the same plant species but

the second one talks more about the microbes involved. The papers also

show that the Indian Mustard Plant is one of the most effective plants to

use for this purpose due to its high growth rate.

The only difference between the papers is that the second paper focuses more on the heavy metal resistant bacteria that were isolated from the roots of the Indian Mustard.

Executive Summary:

Image taken from Bioremediation , its application to contaminated

sites in India by Dr Prasad et al.2011

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Summary Paper 1:

Summary of Paper 1: Rhizofiltration: the use of plants to remove heavy metals from aqueous streams'

Roots of many hydroonically grown terrestrial plants, e.g., Indian Mustard (Brassica juncea ), Sunflower (Helianthus annuus L.) and other various grasses effectively removed toxic metals such as Cu2+, Cd2+, Cr6+, Ni2+, Pb2+, and Zn2+ from aqueous solutions. This paper demonstrate that hydroponically grown roots of the terrestrial plant Indian Mustard (Brassica juncea) effectively removed heavy metals from the aqueous solution by the process Rhizofiltration. Brassica juncea was chosen for a model in this experiment as it can accumulate large amount of Lead and other heavy metals in Screen that used a number of commercially cultivated plant species. Moreover, several members of the Brassica.j species usually have the ability to absorb large amount of heavy metals in both their roots and shoots. The following heavy metals were used in the experiment: Lead, Cadmium, Copper, Zinc, Chromium, Nickel because they are ubiquitous pollutants present in Industrial waste, agricultural and municipial Waste. The results obtained clearly demonstrate that the efficiency of removal compares favourably with currently employed water treatment technologies and this experiment mostly studies the effect of different lead concentration on the speed and magnitude of Pb removal by the Indian Mustard plants. The results obtained for the absorption of Lead by the plant:

Figure 1: Lead absorption by roots over time. (Kumar et al.1995)

Figure 1 shows the effect of different Pb concentrations on Pb removal from water by the roots of the Indian Mustard plant. Plants were exposed to different concentrations of Lead for 72 hours as shown from the graph. The control contained aerated Pb solution without any roots. Samples were taken from the aliquot and was measured for metal content using atomic absorption spectrophotometry.

It can be concluded that with the

presence of the roots of the Indian

Mustard plant, the level of Pb in

solution decreased greatly compared

to the control ( in which no roots

were present). This therefore suggest

that Indian mustard is an effective

plant for the removal of lead from a

contaminated water or soil.

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Figure 2: Relationship between initial Pb concentration and the time required for B.juncea roots to reduce Pb concentration in the solution by 50% ( Kumar et al.1995)

Figure 2 shows the time taken

for the Pb concentration to be

halved by the B.juncea roots. So

this also suggest that the plant

can quickly remove high level

of pollutants from the soil. This

will therefore not affect the soil

fauna (micro-organism) too

much.

Figure 3 shows the removal of different metals from aqueous solutions by the roots of B.juncea.

Figure 3 shows that Indian

Mustard plant can effectively

remove various toxic metals from

aqueous solution over a period of

8 hours. We can therefore

conclude that these plants are not

specific to metals. One plant

species can remove various heavy

metals at different concentrations.

The results of the experiment shows that the roots of terrestrial plants have an intrinsic ability to absorb and precipitate heavy metals from solution at a relatively high speed. Hence we can conclude that the use of terrestrial roots of plants can to remove heavy metals from aqueous solution can end up in a novel water treatment technology. Rhizofiltration can be used treat surface and underground water, industrial and domestic waste, downwashes from powerlines, storm waters, acid mine drainage, and radionuclide contaminated solutions ( Kumar et al.1995).

Summary paper 1 Continued:

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Summary of Paper 2: Cadmium- tolerant plant growth- promoting bacteria associated with the roots of Indian Mustard ( Brassica juncea L.Czern ) Among heavy metals which are widespread pollutants of the surface soil layer, Cadmium is one of the most toxic metals. In plants, Cd acts as an inhibitor and affect shoot and root growth, affect nutrient uptake and homeostasis. Thus, when humans and animals consume these plants they can suffer from diseases. Cadmium as well as other heavy metals affect the biodiversity and the activity of soil microbial communities ( Belimov et al.2005). Plants suitable for phytoremediation should have a high biomass production with enhanced metal tolerance and metal uptake potential. Most of the metal accumulators belong to the Brassicaceae family ( Belimov et al.2005). It has been found in this study that Rhizofiltration is mainly carried out by the help of Bacteria. The bacteria associated with plant roots may have profound effects on the plant growth and their mode of nutrition through processes such as Nitrogen fixation, production of phytohormones, and siderophores and transformation of nutrient elements. This paper focus more on the isolation and characterization of the Cd- tolerant bacteria associated with the roots of the metal accumulating plant and how they actually render toxic metal to less toxic compounds. The isolated strains included Variovorax paradoxus, Rhodococcus sp, and Flavobacterium sp. Theses isolated bacteria were able to stimulate root elongation in the B.Juncea sp. in the presence or absence of toxic Cd concentration. Some of the strains of the above bacteria produce indoles or siderophores, but none possessed C2H2- reduction activity. All the stains except Flavobacterium sp .strain 5P-3, contained the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase which hydrolyse ACC ( the immediate precursor of plant hormone ethylene) to NH3 and alpha- ketobutyrate. The V. paradoxus sp utilize ACC as a sole source of N or Energy. The isolated bacteria has stimulating effect on the root growth of the Indian mustard plant and also offer promise for the development of plant inoculant systems useful for phytoremediation of polluted soils ( Belimov et al.2005) The strains of bacteria were identify by determination of 16S rRNA gene sequences. This was done by carrying out a Colony PCR. The sequence obtained were then compared with similar databases on the BLAST analysis ( Basic Logical alignment search tool). The results obtained showed the identification and isolation of the bacteria. In total 42 Cd-tolerant bacteria strains were isolated from the rhizoplane of plants grown in contaminated soils, contaminated sewage sludge and mining waste. All of the strains had stimulatory effect, seven strains had no effect and one strain had an inhibitory effect on root elongation and were characterized by the determination of 16S rRNA gene sequencing. Then, appropriate tests were used to compare the biochemical properties of the isolated strains with their closest genetic relatives obtained from the BLAST database as shown in Table 3 in next page:

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Summary Paper 2 cont.

The results obtained showed the identification and isolation of the bacteria. In total 42 Cd-tolerant bacteria strains were isolated from the rhizoplane of plants grown in contaminated soils, contaminated sewage sludge and mining waste. All of the strains had stimulatory effect, seven strains had no effect and one strain had an inhibitory effect on root elongation and were characterized by the determination of 16S rRNA gene sequencing. Then, appropriate tests were used to compare the biochemical properties of the isolated strains with their closest genetic relatives obtained from the BLAST database as shown in Table 3:

(Results as shown in Belimov et al.2005 paper)

Root Length Promotion: The effect of each identified strain on root elongation of B. Juncea VIR -3129 in the absence of Cd is shown in the following table:

It can be concluded from the table that the addition of 8 um of Cd inhibited root elongation of un inoculated seedlings by 30%. However, the maximum root length promoting effect on Cd- treated plants were observed after inoculation with the V. Paradoxus strains as indicated by the arrow above. The Ralstonia sp. on the other hand increased root elongation.

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Characteristics of the Bacteria: The isolated bacteria from table above had a high tolerance to Cd. The most tolerant strain and also those having a stimulatory effect on root elongation being V.paradoxus 5C-2 and 5P-3 whereas least tolerant Cd-tolerant strains being Rhodococcus sp.4N-4, and V.paradoxus 2C-1and 2P-1. The ACC deaminase Assay: The ACC deaminase activity of the cell-free extracts was determined by monitoring the amount of alpha ketobutyrate generated by enzymatic hydrolysis of ACC. The results of the experiment therefore concludes that Cd-tolerant bacteria are present in the root zone of metal of metal accumulating plant Brassia juncea grown in soils contaminated with heavy metals. The isolated strains belonging to the V.paradoxus are of a particular interest. Therefore the study of these bacteria can be of a great importance to Phytoremediation in the near future.

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Comparison of Paper:

Comparison of Both papers: Both papers proved that Phytoremediation is one the most cost effective and non destructive technology to treat contaminated soil and water. Both papers involved the study of the Indian Mustard Plant ( Brassia.J sp) in response to their metal accumulating abilities. According to both papers, the Indian Mustard plant might be slow in accumulating heavy toxic metals but their high growth rate make them very apt for this purpose compared to other metal accumulating plants. The results from Kumar et al.2005 showed that hydroponically grown terrestrial Indian Mustard plant can successfully remove toxic metals from aqueous solution by the process Rhizofiltration. The experiment focus mainly on the capacity of the Indian Mustard plant in absorbing heavy metals and the time they take to remove or reduce the concentration of the heavy metal from an aqueous solution. The experiment was mainly based on Lead but few samples also included other heavy metals such as Copper, Zinc, Cadmium, Nickel and Chromium. Basically, we can say that the experiment technique was mainly based on the rate of disappearance or reduction of a certain Heavy metal from an aqueous solution. The paper Belimov et al. 2005 on the other hand focus more on the bacteria involved in the process Rhizofiltration. Belimov et al.2005 succeeded to prove that the process of Rhizofiltration or metal accummulating process is mainly due to some strains of metal tolerant bacteria. Metal tolerant bacteria were isolated from the roots of the Indian Mustard Plant and analysed. The study was also mostly studying the effect of Cd on different Indian Mustard plant root's growth and their bacteria were then Isolated. The Belimov et al.2005 paper were more into DNA sequencing and the most metal tolerant strain were identified. This strain was then isolated and through genetic engineering can be implanted in other Brassia.J .species to produce more efficient Cd absorbing plant.

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Critical and Personal Comments:

Both papers demonstrated that terrestrial plants such as the Indian Mustard plants have the ability to accumulate metals in their roots and even in the shoot. Therefore they can be used for phytoremediation. The reason I have picked a paper such as Kumar et al. 1995 is because first of all: it is a very good paper in terms it is very clear about what is being done and very easy to understand what actually Phytoremediation and Rhizofiltration is whereas paper such as Belimov et al.2005 is more into the details microbial process involved in rhizofiltration and the different strains of microbe that can best be used as biosorbers were found by firstly isolating the bacteria from the root region of different Brassia. J sp. It is very interesting but I personally think that It is a bit hard for those that do not have a scientific background or too much of genetic background. So I believe the first paper (Kumar et al.1995) is best in terms of understanding the process and make people realise that planting more of metal accumulating tree or constructing wetlands can be a remediation to contaminated soil or water. The second reason is because of the difference in methodology used in both papers. The method/ technology used for Belimov et al.2005 paper were more sophisticated and more reliable. This is because when we compare the year of the research for the respective paper, we can definiely say that back in the year 1995, such cutting edge technology was not available so as to go deep into the rhizofiltration process and isolate the bacteria that is involved in the accumulation of heavy metals. The use of PCR, was useful in identifying the strain of the bacteria and they were successfully cultured in a batch culture are techniques that in 1995 did not exist maybe. One important point in Belimov et al.2005 paper, the researchers have been able to isolate the bacteria that can accumulate more metals and that can stimulate root growth in plants. According to me, I believe we can use this

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References:

A.A. Belimov, N. Hontzeas, V.I. Safronova, S.V.Demchinskaya, G.Piluzza, S.Bullitta, B.R. Glick .2005. Cadmium tolerant plant growth- promoting bacteria associated with the roots of Indian Mustard ( Brassica juncea L.Czern). Soil Biology and Biochemistry 37 (2005) 241- 250. Viatcheslav Dushenkov, P.B.A. Nanda Kumar, Harry Motto and Ilya Raskin. 1995. Rhizofiltration: the use plants to remove Heavy metals from Aqueous streams. Agbiotech centre and Department of Enviromental Sciences, Rutgers University, Cook College, New Brunswick, New Jersey Susane Eapen and S.F.D'Souza.2004. Prospect of genetic engineering of plants for phytoremediation of toxic metals. Biotechnology advances 97-114. India: Nuclear agriculture and Biotechnology Division , Bhabha Atomic research Centre Mumbai.