FIELD OF INVENTION:

The present invention relates to the field of microorganism assisted phyto-remediation. More particularly the present invention relates to selective removal of elemental copper by accumulation of contaminated elemental copper from the soil by microorganism and removal of accumulated copper from the decayed microorganism by scavenger plants.

BACKGROUND OF THE INVENTION:

Land contaminated by toxic metals threatens both the environment and human health. Generally, the contamination has arisen through human activities, such as the burning of fossil fuels, the disposal of sewage and municipal wastes, or the use of fertilizers and pesticides in agriculture. To return this land to agricultural or amenity use, the toxic metals must be removed or rendered inaccessible. Conventional approaches to the reclamation of contaminated land are expensive. However, the use of plants to remove toxic metals ('Phyto-remediation') affords a low-cost alternative, which is ecologically preferable because it reclaims the original soil permanently.

Phyto-remediation is an emerging technology that uses various plants to degrade, extract, contain, or immobilize contaminants from soil and water. This technology has been receiving attention lately as an innovative, cost-effective alternative to the more established treatment methods used at hazardous waste sites Phytoremediation applications can be classified based on the contaminant namely degradation, extraction, containment, or a combination of these. Phytoremediation applications can also be classified based on the mechanisms involved. Such mechanisms include extraction of contaminants from soil or groundwater; concentration of contaminants in plant tissue; degradation of contaminants by various biotic or abiotic processes; volatilization or transpiration of volatile contaminants from plants to the air;
immobilization of contaminants in the root zone; hydraulic control of contaminated groundwater (plume control); and control of runoff, erosion, and infiltration by vegetative covers etc.

In practice, metal accumulation by plants is restricted by metal bioavailability in the soil. As accumulator plants have access only to metal in the soil solution, which might represent only a small fraction of the metal in the soil, this limits the amount that can be extracted. The use of chemical chelates, which remove metals from their binding sites in the soil, was once proposed as a solution to this problem. However, although the accumulation of metals by plants can be enhanced by the addition of chemical chelates, these compounds can purge the soil of metals too efficiently, resulting in pollution of the water table. An analogous approach was proposed to employ natural soil microorganisms (bacteria and fungi) to increase the bioavailability of metals in contaminated soils. This has two advantages. First, microbial chelates are likely to occur predominantly in the vicinity of the roots (rhizosphere). Second, their persistence in the soil is likely to be shorter than that of chemical chelates. Thus microorganism assisted phyto-remediation to remove toxic pollutants from the soil is in need in the state of art.

PRIOR ART:

Environment International 31(6), 2005, 861-866 discloses effect of copper-tolerant rhizosphere bacteria on mobility of copper in soil and copper accumulation by Elsholtzia splendens. The role of rhizosphere bacteria in facilitating the solubility of copper (Cu) in contaminated soil and Cu accumulation in plant were studied.

Chemosphere 81(9) 2010, 1149-54 discloses bacterial stimulation of copper phytoaccumulation by bioaugmentation with rhizosphere bacteria. This study employed bioaugmentation with copper resistant bacteria to improve phytoremediation. Oatmeal plant (Avena sativa L.) was used for copper phytoextraction. Three copper resistant bacterial isolates from oatmeal rhizosphere (Pseudomonas putida A1; Stenotrophomonas maltophilia A2 and Acinetobacter calcoaceticus A6) were used for the stimulation of copper phytoextraction. Results suggest potential application of bacterial stimulation of phytoaccumulation of copper for biological removal of copper from contaminated areas.

Biotechnology Advances 28(3) 2010 367-374 envisage the use of soil bacteria to facilitate phytoremediation. The bacteria used to facilitate phyto-remediation includes biodegradative bacteria, plant growth-promoting bacteria and bacteria that facilitate phytoremediation by other means. An overview of bacterially assisted phytoremediation is provided here for both organic and metallic contaminants, with the intent of providing some insight into how these bacteria aid phytoremediation so that future field studies might be facilitated.

J. Sci. Res. Chula. Univ., 31(1) 2006 discloses phytoremediation of copper contaminated soil by Brassica juncea (L.) Czern and Bidens alba (L.) DC. var. radiate.

Journal of Trace Elements in Medicine and Biology 18(4) 2005, 355-364 discloses role of soil microbes in the rhizospheres of plants growing on trace metal contaminated soils in phytoremediation. This article reviews recent developments in in situ bioremediation of trace metal contaminated soils, with particular reference to the microbial dynamics in the rhizospheres of plants growing on such soils and their significance in phytoremediation.

Applied Geochemistry 11(1-2) 1996, 163-167 discloses bioavailability of heavy metals and decontamination of soils by plants, the possibilities of the use of hyperaccumulating higher plants for the decontamination of metal polluted soils, i.e. phytoremediation, are explored.

US6923914 discloses a method wherein hydrocarbon utilizing bacteria are used to remediate heavy metal contaminants. The method includes supplying a hydrocarbon substrate to the site and treating the metal contaminant with hydrocarbon-utilizing bacteria in the presence of at least one hydrocarbon. The applications include the remediation of heavy metals, the remediation of arsenic impacted surface water, groundwater and/or soil, the remediation of acid mine drainage, and the treatment of spent metal plating solutions.

US 5968359 discloses a method of cleaning up heavy metal-laden waters and soils, conditions are established in the soils and waters to be treated so that the microorganism present there will form hydrogen sulfide, and optionally apathogenic facultative anaerobes that are tolerant of heavy metals are added to produce hydrogen sulfide, and the hydrogen sulfide formed by microbiological action is allowed to react with the heavy metals to form metal sulfides. Thus it is possible to encapsulate and immobilize heavy metals in the pollution zone thereby preventing the entry of heavy metals into the food chain.

US 5785735 disclose a process for the removal of metal ions from metal containing soil. The process utilizes plants of the family Brassicaceae. In order to accomplish this at least one member of the family Brassicaceae is contacted with the metal-containing soil environment and the metal-containing soil environment is manipulated in a manner sufficient to increase availability of metal within the metal-containing soil environment. These particular plants will absorb metals into their roots making the metals non-leachable from the soils or will absorb the metal into their roots and transfer them to the shoots and/or roots which can be easily harvested The step of manipulation of the soil involves tilling, addition of chelating agents and associated bacteria.

US 5927005 disclose a process of phytoremediation using the plants of the species Larrea tridentata, the creosote bush. The method is particularly directed at the decontamination of soil containing lead, copper, nickel and cadmium. The creosote bushes contain a substantial amount of scavenged metal in their stems and leaves. Collection and removal of the leaves and stems or of the leaves alone will reduce and should ultimately eliminate heavy metals from the contaminated soil. The creosote roots or the roots and stems can be left to continue removing more heavy metals from the soil while growing more leaves. According to the invention creosote plants can be left growing in an area for many years until the area is decontaminated. The plant tissues, either the leaves and , a portion of the stems or just the leaves alone, being periodically removed from the contaminated area before those plant tissues are allowed to become incorporated into the soil, recontaminating the soil.

US 2003/0126632 Al discloses a method for the phytoremediation treatment of a contaminated medium. The invention discloses the use of a plant associated with an endophytic microorganism which is able to improve phytoremediation by the plant and degradation of the metabolites of said element inside the plant. The plant comprises in its vascular and/or root system a genetically modified endophytic microorganism which is able to express proteins allowing the degradation or the accumulation of at least one element selected from the group consisting of organic pollutants, heavy metals, radionuclides or a mixture thereof.

CN 200710047489 relates to a method for remediation of heavy metals contaminated soil by a plant-microorganism joint direction method. First, plants and microorganism bacteria strains with heavy metal resistance are obtained and then the seeds of the resistant plant is soaked and coated in microorganism resistant bacteria solution. Finally, the seeds are planted in the soil contaminated by heavy metal, therefore the soil can be remedied.

The prior arts discuss the removal of toxic metal once the land is completely contaminated with toxic metals. The land becomes a waste land with no further use ,unless the soil is completely removed of all the toxins. During the whole phytoremediation process the land remains a waste land, the whole process is time consuming and tedious. The problem is more severe when the toxic metals accumulate in agricultural fields due to the continuous use of pesticide and insecticides. Once the land is completely contaminated it is very difficult to remove all the toxic metal from it. The process is time consuming and expensive.

So there exits a need to develop a process which checks the accumulation of heavy metals in the starting stage when the concentration of these metals starts increasing above the safety levels in turn protecting the vegetation and soil from toxic effects of heavy metals. The process also protects the land from becoming waste land.

There are many methods discussed in the prior arts but there is a need for a simple, cost effective and time saving process for the removal of heavy metal contamination happening from the continuous use of pesticides/insecticides in the agricultural lands with standing crops.

More particularly, there is a need for a process that removes metal from soil with standing crops ensuring healthy crop growth. There is a need for an efficient process which effectively protects the crops from the traces of heavy metal left by the continuous use of pesticides and /or insecticides at the same time ensures the complete removal of the heavy metal from the agricultural land, so that the agricultural field is clear of contaminants and ready for the cultivation of the same crop again.

OBJECT OF THE INVENTION:

The main object of the present invention is to remove contaminated elemental copper which is a left over unwanted residue arising out of the pesticides, present in the soil containing the crop, combinedly by microorganism assisted phyto-remediation and by metal scavenger plants.

Another object of the present invention is to mask elemental copper from the soil containing the crop during cultivation.

Another object of the present invention is to introduce micro-organism in copper contaminated soil to selectively accumulate elemental copper from the soil thereby making the crop free of copper contamination thereby masking the elemental copper from the soil in the microorganisms.

Yet another object of the present invention is to scavenge copper from the contaminated microorganism in the soil by planting scavenger plants in the soil after harvesting the crop thereby totally removing the copper contamination from the soil in the microorganisms.

SUMMARY OF THE INVENTION:

The present invention relates to removal of elemental copper from the soil. The elemental copper is a left behind as unwanted residue from pesticides used for cultivation of the crop. The leftover elemental copper is removed from the soil by the introduction of micro-organism into the soil. As step one the micro-organisms selectively accumulate elemental copper into themselves from the soil thereby making the soil free of copper contamination and hence the crop is unaffected by copper contamination. After cultivation of crop the soil is left behind with copper accumulated microorganism. But such copper containing micro-organism if left unattended in the soil will decay over a period of time and resurface in the soil as copper contamination. This is overcome by planting scavenger plants as step two in the soil containing the referred micro-organism.

The scavenger plants bio-accumulates copper contamination from the soil and removes all the copper from the soil thereby totally eliminating the copper contamination from the soil.

DETAILED DESCRIPTION OF THE INVENTION:

The present invention discloses a two step process for removing copper metal from the agricultural land the method comprises of accumulating elemental copper from the soil into the microorganisms by phytoremediation, during the growth of the crop, and thereafter accumulating copper from the contaminated microorganisms into scavenging plants by bioaccumulation during the growth of scavenging plants after the harvest of the crop.

In one of the preferred embodiment the present invention shall disclose a process for removing elemental copper contamination from the soil containing the crop by micro¬organism assisted phyto-remediation. The copper present in the soil is a leftover unwanted residue arises out of pesticides used for the cultivation of the crop. If the copper in the soil is left untreated bio-accumulatoin of copper into the crop will takes place thereby leading to toxification of the crop which results in damaging the crop or disrupting its biochemistry.

According to the invention this phenomenon is overcome by rendering the pollutant copper present in the soil in a passive state by introduction of microorganism into the copper contaminated soil. The micro-organisms selectively accumulate elemental copper into themselves from the soil thereby making the soil free of copper contamination and hence the crop is unaffected by copper contamination. After cultivation of crop the soil is left behind with copper accumulated microorganism. But such copper containing micro-organism if left unattended in the soil will decay over a period of time and resurface in the soil as copper contamination. This is overcome by planting scavenger plants in the soil containing the referred micro-organism. The scavenger plants are capable of scavenging copper from the decayed microorganisms in the soil. The scavenger plant bio-accumulate copper contamination from the soil and removes all the copper from the soil by the process in which water and the metals (copper) move through the soil toward the roots of the scavenger plants, where the extraction flow of the plants takes them into the plants' tissue. The water and metals then move up into the stem and leaves, where the water evaporates. The heavy metals remain in the plant. The copper metal is held in organic complex within the vacuoles present in the scavenger plants which prevent the metals from damaging the plant or disrupting its biochemistry. The metal packets find their way into roots, shoots, and leaves, where they are stored over the lifetime of the plant. Thus scavenger plants acts as a more efficient metal scavengers and thereby totally eliminating the copper contamination from the soil.

Heavy metal like copper is phytotoxic either at all concentrations or above certain threshold levels. Toxic metals are biologically magnified through the food chain. They infect the environment by affecting soil properties its fertility, biomass and crop yields and ultimately human health.

In one of the preferred embodiment the invention shall disclose a method for removing copper metal from an agricultural land with standing crop. The invention shall disclose an efficient method of removal of copper from the soil which involves the first step of introducing and promoting growth population of atleast one microorganism in the soil which is capable of accumulating copper into them. These organisms are introduced into the agricultural land with atleast one standing crop. The organisms take up copper from the soil and accumulate into themselves thereby keeping the soil free of copper contamination during the growth period of the crop. The crop is harvested after its growth period clearing the agricultural land.

Once the agricultural land is cleared after the harvest of crop, as a second step metal scavenger plants are introduced to the same field. These plants absorb toxic metals and help to clean up them from soils .These plants are resistant to heavy metals and are capable of accumulating them into their roots and leaves and transporting these soil pollutants to high concentrations. Copper metal scavenger plants are planted and cultivated in the same agricultural field. The copper metal scavenger plants are capable of scavenging copper from the contaminated microorganisms present in the soil. These plants scavenge copper from the decayed contaminated microorganisms and accumulate the copper into them.

The matured contaminated scavenger plants are harvested and removed from the agricultural land. Once the scavenger plants are completely removed from the agricultural field the agricultural soil becomes free of all the contaminated microorganisms and there by free of all copper. The soil is now ready for cultivation of crop The crop can be cultivated again in the clean agricultural soil.

As per the invention the crop is preferably selected from the group consisting of Solanaceae, Amaranthacea and Leguminosae, but may not restricted to the members of their family or combinations thereof.

The selection of microorganism is an important factor in the process of bioremediation. There are other factors like pH, temperature which affect the efficacy of the microorganisms. As per the invention the microorganism is preferably selected from the group consisting of Bacillus sp., Pseudomonas sp., Frateuria sp., and Thiobacillus sp., but may not restricted to the members of their family or combinations thereof.

The purification capacity of heavy metals by plants is affected by several factors, such as the concentration of the heavy metals, plant species, exposure duration, temperature, pH etc. As per the invention the scavenger plant is preferably selected from the group consisting of Tomato and Amaranthus, but may not restricted to the members of their family or combinations thereof.

Examples:
Example 1:

Soil with increased copper content was treated with micro- organisms

Table 1

From Table1 it is observed that Pseudomonas fluorescens showed higher percentage depletion of 52% and 63% in combinations with F.aurantia

Example 2:
To the soil pre-treated with microbes, scavenger plant - tomato was planted and copper uptake by the
plant was analyzed using AAS after 15, 50 and 60 day. After 60 days, 86% increase in copper level was observed when compared to the control plant. Table 2:

Examples
Table 3

Amaranthus ,when used as scavenger plant it showed up to 26% of copper uptake from the soil after 20 days of planting and harvesting.

Thus the micro-organisms selectively accumulate elemental copper into themselves from the contaminated soil thereby making the soil free of copper contamination and hence the crop is unaffected by copper contamination. As stated in example 1 Pseudomonas sp., accumulate copper up to 51% and 62 % or above in combinations.

The copper metal scavenger plants are capable of scavenging copper from the contaminated microorganisms present in the soil. Tomato and Amaranthus plants scavenge copper from the decayed contaminated microorganisms and accumulate the copper into them. Tomato plant can accumulate up to 86% of copper approximately after 60 days of planting whereas Amaranthus can accumulate up to 25% of copper from the contaminated microorganisms present in soil.

The claim is, by planting and cultivating scavenger plants in the agricultural field the agricultural soil becomes free of all the contaminated microorganisms and there by free of all copper. The soil is now ready for cultivation of crop. The crop can be cultivated again in the clean agricultural soil.

It should be indicated that the claim is made for the effective use of these microbes for the purpose of bioremediation of cleansing the soil from toxic metals like copper, but not restricted to the usage of these microbes only and not restricted to this combination of microbes only, but can cover different microbes of similar category and with different combinations there on for effective usage at different stages and levels, as adopted in the protocol developed and deployed in this our studies.

Although the invention has been described above with reference to the disclosed embodiments, those skilled in the art will readily appreciate that the specific experiments detailed are only illustrative of the invention and not restrictive. It should be understood that various modifications can be made without departing from the spirit of the invention.

WE CLAIM:

1. A method for removing copper metal from an agricultural land , the method comprises of:

a. Introducing and promoting growth population of atleast one microorganism in the soil which microorganisms are capable of accumulating copper into themselves from the said agricultural land with atleast one standing crop thereby keeping the soil free of copper contamination during the growth period of the said crop,

b. planting and cultivating copper metal scavenger plants in the same said soil post harvest of the said crop which plants are capable of scavenging the copper from the said decayed contaminated microorganisms and,

c. harvesting the matured contaminated scavenger plants whereby the said agricultural soil is free of all the contaminated microorganisms and ready for cultivating the crop again in clean agricultural soil free of elemental copper.

the said method characterized in the use of the combination of microorganisms and scavenger plants for removing the copper metal from the agricultural land.

2. The method as claimed in claim 1, wherein the crop is preferably selected from the group consisting of Solanaceae, Amaranthacea and Leguminosae or combinations thereof.

3. The method as claimed in claim 1, wherein the microorganism is preferably selected from the group consisting of Bacillus subtilis, Bacillus megaterium, Pseudomonas fluorescens, Thiobacillus thiooxidans, Frateuria aurantia or combinations thereof.

4. The method as claimed in claim 1, wherein the scavenger plant is preferably selected from the group consisting of Tomato, Amaranthus or combinations thereof.