Title of Invention

"A PROCESS FOR BIOREMEDIATION OF P-NITROPHENOL CONTAMINATED SOIL"

Abstract The present invention relates to a process for bioremediation of agricultural soil, sludge, effluents and other sites contaminated with the nitroaromatic compound p-nitrophenol which is a toxic compound of anthropogenic origin. The preferred method of carrying out the invention comprises immobilizing the cells of a p-nitrophenol degrading bacteria Arthrobacter protophormiae on a carrier material and introducing the immobilized cells into PNP contaminated site. The bacteria are able to degrade PNP in a few days thereby remediating the site. The present invention thus provides an economical and efficient method for biodegradation and elimination of PNP in situ.
Full Text A PROCESS FOR BIOREMEDIATION OF P- NITROPHENOL CONTAMINATED SOIL OF DIFFERENT SITES.
The present invention relates to a process for bioremediation of p-nitrophenol contaminated soil of different sites such as farmlands, dumping sites, sites of accidental spillage and industrial manufacturing sites of organophosphate pesticides/PNP. More particularly, it relates to a process for degrading p-nitrophenol (hereinafter referred to as PNP) with the help of bacteria in order to eliminate this pollutant and clean up PNP-contaminated areas. Background and prior art of the invention:
1
The utility of this invention is to bioremediate land areas contaminated by PNP which has been classified as a priority pollutant by the United States Environmental Protection Agency owing to its high toxicity towards humans, animals and plants, and its removal from the environment is therefore an urgent need. The use of pesticides, particularly those belonging to the organophosphate class of pesticides has risen tremendously over the years. PNP which is formed from hydrolysis of organophosphate pesticides is a stable and toxic compound which remains in the soil. Being highly soluble in water, it dissolves in underground water and spreads to the surrounding areas thereby contaminating them. According to the present invention, bioremediation of PNP will be brought about by contacting the PNP-contaminated areas with cells of a PNP-degrading bacterium. Bioaugmentation i.e. the addition of exogenous microorganisms into the contaminated environment is being increasingly used successfully as a biological remedy for pollution reduction/removal. There are several advantages of bioaugmentation over the traditional physico-chemical methods such as landfilling, excavation, incineration etc. since it allows the destruction of target contaminants in situ and is a safe, environment friendly and cost effective process. Further, the present invention utilizes the pollutant degrading ability of a naturally occurring microorganism and not a genetically modified one.
Nitroaromatic compounds such as nitrophenols exhibit high toxicity/mutagenicity for many living organisms and are some of the most refractory substances present in industrial wastewaters. PNP, an important member of the nitrophenol group is used in the manufacturing of pesticides.
dyes, pigments, pharmaceuticals and engineering polymers and as a fungicide for leather. It is released into the environment during manufacturing and processing, frequently appearing in industrial effluents, and is also released from breakdown of organophosphate pesticides such as parathion, methyl parathion, fluorodifen and nitrofen. It can thus build up in the soil and can enter groundwater reservoirs.
Conventional decontamination methods are not necessarily viable and feasible in terms of cost and operability and are generally not environmentally sound. Accordingly there is need for a cost effective and environmentally acceptable process for remediating soil which is contaminated with PNP. The most efficient and safe method from the viewpoint of ecology and economy is the biological treatment method which utilizes the ability of microorganisms to degrade contaminating compounds. Several bacterial strains have been isolated for their ability to degrade PNP. PNP degrading organisms that have been described in the prior art include Flavobactehum (Raymond DGM & Alexander M 1971. Pestic. Biochem. Physiol. 1: 123-130), Moraxella (Spain JC, Wyss O & Gibson DT 1979. Biochem. Biophys. Res. Comm. 88:634-641), Nocardia (Hanne LF, Kirk LL, Appel SM, Narayan AD & Bains KK 1993. Appl. Environ. Microbiol. 59:3505-3508), Pseudomonas (Nishino SF & Spain JC 1993. Environ. Sci. Technol. 27:489-494), Arthrobacter (Jain RK, Dreisbach JH & Spain JC 1994. Appl. Environ. Microbiol. 60:3030-3032), Bacillus sphaericus (Kadiyala V & Spain JC 1998. Appl. Environ. Microbiol. 64:2479-2784), Sphingomonas (Zablotowicz RM, Leung KT, Alber T, Cassidy MB, Trevors JT, Lee H, Veldhuis L & Hall JC 1999. Can. J. Microbiol. 45:840-848), Ralstonia sp. (Samanta SK, Bhushan B, Chauhan A & Jain RK 2000. Biochem. Biophys. Res. Comm. 269:117-123), Rhodococcus (Heiss G, Trachtmann N, Abe Y, Takeo M & Knackmuss HJ 2003. Appl. Environ. Microbiol. 69:2748-2754). Reference may also be made to publications (Bhatti, Zl, Toda H & Furukawa K 2002. Water Res. 36:1135-1142; Heitkamp MA, Camel V, Reuter TJ & Adams WJ 1990. Appl. Environ. Microbiol. 56:2967-2973) wherein PNP degrading bacteria or biomass cultivated from activated sludge were used for the biological degradation of PNP in water. The drawback in these reports is that although they provide some evidence for the presence of intermediates in the degradation of PNP, or describe the
complete biochemical pathway(s) for degradation of PNP, but they do not provide any method for bioremediating soils contaminated with this compound. There is no information available on practical approaches or means to bioremediate PNP-contaminated soils using these organisms.
The main object of the present invention is to provide a process for bioremediation of p-nitrophenol contaminated soil and other sites such as farmlands, dumping sites, sites of accidental spillage and industrial manufacturing sites of organophosphate pesticides/PNP which obviates the drawbacks as detailed above.
Another object of the present invention is to provide a process which can completely degrade PNP leaving no detectable or environmentally significant amounts of PNP in the soil.
Yet another object of the present invention is to provide a simple process for bioremediating PNP- and organophosphate pesticide-contaminated soil and other sites such as farmlands, dumping sites, sites of accidental spillage and industrial manufacturing sites of organophosphate pesticides/PNP in a cost effective, efficient and easy manner, particularly in the field since it does not require the use of a bioreactor or any other complex equipment or procedure.
Novelty of the present invention with respect to prior art: Novelty of the present invention lies in the fact that no other report in prior art describes a practical method for bioremediation of PNP contaminated soil. Biodegradation is emerging as a promising technology for reclaiming contaminated soils but since there is no report in the prior art describing a process for bioremediating PNP-contaminated soil, there exists a need for an inexpensive and environmentally acceptable method for remediating PNP contaminated sites. A. protophormiae RKJ100 has been reported to degrade PNP efficiently and metabolize it completely. The present invention makes use of the excellent PNP-degrading properties of strain RKJ100 and is carried out by growing this organism in presence of an inexpensive carbon source which is easily available as an industrial waste; immobilizing the cells (after harvesting) on a cheap and easily available carrier material and introducing the immobilized cells into PNP contaminated soil or sludge which results in
depletion of PNP within a few days, thereby demonstrating a novel process for the bioremediation of areas/sites contaminated with PNP.
Accordingly, the present invention provides a process for bioremediation of p-nitrophenol contaminated soil of different sites which comprises:
growing of p-nitrophenol degrading organism Arthrobacter protophormiae RKJ 100 in sugarcane molasses based minimal medium; harvesting the cells by centrifugation and resuspending the culture in a minimal medium comprising;
Immobilizing the said organism suspended in minimal medium by mixing with a carrier material in form of corn cob powder in a ratio of about 6:1 (v/w) to get a homogenous mixing of bacterial cells;
mixing the immobilized Arthrobacter protophormiae as obtained in step (ill) with para-nitrophenol contaminated soil in a ratio ranging 1:10 to 1:1000 to have a inoculum level of soil in the range of 2x10^-2x10°CFUs/g soil. In an embodiment of the present invention the p-nitrophenol degrading organism is Arthrobacter protophormiae RKJ100 and is deposited at IMT Chandigarh and having accession number MTCC 6511. In another embodiment of the present invention the p-nitrophenol degrading organism is Arthrobacter protophormiae RKJ 100 and having the biochemical characteristics of ability to grow on sugarcane molasses and also the ability to utilize para-nitrophenol as the sole source of carbon, nitrogen and energy and is is gram positive .
In yet another embodiment of the present invention the medium used for cultivation for Arthrobacter protophormiae RKJ 100 comprises of sugarcane molasses ranging 1-1 Og, disodium hydrogen phosphate about 2g, potassium dihydrogen phosphate about 1 gmbeing dissolved in distilled water 1L with addition of 1ml of trace element solution and t, ammonium sulphate about 0.4gm, agmnesium sulphate about 0.4gm and the ingredients he said composition of trace element mg/L consisting of 0.1 g AI(0H)3, 0.05g SnCl2.2H20, 0.05g Kl, 0.05g LiCI, 0.8gMnSO4.4H2O, 0.5g H3BO3, O.lg ZnS04, O.lg C0CI2.6H2O, 0.1gNiSO4.6H2O, 0.05g BaCb and 0.05g (NH4)6M07O24.4H2O.
In still another embodiment of the present invention the carrier material used in form of corn cob is highly absorbent, granular, high volume low weight, completely biodegradable and inexpensive and has water holding capacity of about 700%.
In still another embodiment of the present invention the depletion of p-nitrophenol from contaminated soil in situ is in the range of 90-98% within a period ranging 5-7 days
In still another embodiment of the present invention the process is eco-friendly as it uses naturally occurring micoorganism.
DETAILED DESCRIPTION OF THE INVENTION:
The organism Arthrobacter protophormiae strain RKJ100 (hereinafter referred to as RKJ100) which is a gram positive bacterium, reported to have the ability to utilize PNP as the sole source of carbon, nitrogen and energy (Chauhan A, Chakraborti AK and Jain RK. 2000. Biochem. Biophys. Res. Commun. 270:733-740) was used for the purpose of the present invention. The biochemical pathway of PNP degradation by this organism has been found to proceed with the formation of p-benzoquinone and hydroquinone which is further degraded via the p-ketoadipate pathway. In the natural soil environment, growth and degradation activity of bacteria is difficult to predict due to factors such as temperature, moisture content, pH, and other parameters of soil which therefore need to be studied in order to recover the soil from contaminant. Therefore, to test the efficiency of RKJ100 in degrading PNP in soil, field studies were carried out under natural environmental conditions. The organism was grown in minimal medium containing sugarcane molasses as a cheap carbon source and after harvesting the cells, they were immobilized on a carrier material before adding them to PNP-spiked soil. PNP degradation was studied in the field by introducing the immobilized cells of strain RKJ100 directly into agricultural soil contaminated with PNP in small plots (1 m x 1 m x 30 cm). Soil samples from the plots were removed at different time intervals and analyzed for the amount of PNP remaining in the soil.
The following examples are given by way of illustration and therefore should not be construed to limit the scope of the present invention: Example-1
Cells of A. protophormiae strain RKJ100 were grown in minimal medium with
the following composition for one liter: 2g Na2HP04, 1gKH2P04, 0.4g
(NH4)2S04, 0.4g MgS04. 7H20 and 1 mil of trace element solution
containing, per liter, 0.1 g AI(0H)3, 0.05g SnCI2.2H20, 0.05g Kl, 0.05g
LiCI, 0.8gMnSO4.4H2O, 0.5g H3B03, 0.1 g ZnS04, O.lg CoCI2.6H20, O.lg
NiS04.6H20, 0.05g BaCI2 and 0.05g (NH4)6M07O24.4H2O. To grown this
strain cost effectively for mass cultivation, sugarcane molasses were used as
the carbon and energy source. The organism was grown in medium
containing10% molasses which was treated with 0.1% of potassium
hexacyanoferrate.
Example-2
A protophormiae strain RKJ100 was grown in the presence of molasses as
the carbon source as described in Example 1 and after harvesting the cells,
they were resuspended in minimal medium. These cells were then
immobilized on carrier material. The carrier material used for this
purpose was corncob powder which is highly absorbent, granular, completely
biodegradable, easily available and inexpensive. The cells of strain
RKJ100 re-suspended in minimal medium were mixed with dry corncob
powder to get cell suspension:corncob ratio of 6:1 (v/w) which allowed
homogenous mixing of bacterial cells with corncob powder. The cells thus
immobilized on carrier matrix were added to the soil contaminated with PNP.
Example-3
Cells of A. protophormiae strain RKJ100 were grown on minimal medium containing sugarcane molasses as the carbon and energy source. Strain RKJ100 can be grown on different concentrations of molasses ranging from 1% to 10%. Other carbohydrates such as glucose, sucrose etc were also used as the carbon and energy sources. In a preferred mode of the invention, for mass cultivation of this organism, it is grown in medium containing 10% molasses which is treated with 0.1% potassium hexacyanoferrate to precipitate metal ions present in molasses.
Example- 4
Strain RKJ100 was grown on minimal medium containing 10% molasses as the carbon source. After harvesting the cells by centrifugation they were resuspended in minimal medium. The bacterial cells were immobilized by mixing them with carrier material. The water holding capacity of the carrier material being very high (-700%), additional medium was added to allow proper and thorough mixing of cells with the carrier matrix. These cells were then added to soil contaminated/spiked with PNP to get the Inoculum level of 2x10^-2x108 CFUs/g soil. The ratio of soil and carrier material can range from 1:10 to 1:1000. Cells immobilized on carrier material provide a protective niche to the bacteria in contaminated soil protecting them from predation and contaminant toxicity, hence exhibiting more stability and better degradation capability than free cells. The immobilized bacterial cells can be stored for several months at temperatures ranging from 4°C to -20°C providing a ready to use formulation whenever required. Example- 5
Soil was air dried and sieved through 2 mm mesh. To 1 kg soil, 400 ml distilled water containing 70 ppm PNP was added and mixed thoroughly to form a thick slurry which was again air dried and pulverized. This soil is hereinafter referred to as PNP-spiked soil. Microcosms were prepared in glass beakers using 20 g of PNP-spiked soil (pH 7.5) in each beaker. A. protophormiae RKJ100 cells grown in minimal medium containing 10% molasses and induced with 0.2mM PNP were diluted to a cell density of 2x106 CFUs/g soil and added to the microcosms. PNP depletion was also studied in flooded soil for which an additional 30 ml medium was added to the microcosms to create flooded conditions. Un-inoculated soil microcosms served as control. All beakers were incubated for 20 days at 30°C and PNP levels were measured in 1 g soil samples withdrawn at 24 h time intervals upto 7 days and subsequently at 10, 15 and 20 days. Residual PNP from the soil was extracted by taking 1 g soil sample and mixing it with 10 ml of 5% NaOH solution. The soil suspension was centrifuged at 5000 rpmJor 20 min. and the supernatant was collected in a separating funnel. It was extracted with double the volume of ethyl acetate and then the pH of the aqueous phase was adjusted to 2.0 with 5 N HCI and again extracted with twice the volume of
ethyl acetate. Both the extracts were pooled, collected in a round bottom flask, evaporated to dryness under vacuum in a rotavapor and finally dissolved in 1 ml methanol. PNP was quantified by High Performance Liquid Chromatography using Waters 600 model equipped with a Waters 996 photodiode array detector operating at 315 nm. Separation was carried out with a Waters Spherisorb 5µm C8 column and the mobile phase was acetonitrile:water (80:20 v/v) containing 0.1% trifluoroacetic acid, with a flow rate of 1.0 ml/min. Degradation of PNP by strain RKJ100 occurs in the inoculum range of 2x10^-2x109 CFUs/g soil, temperature range of 20-40°C, pH range of 7-9.5 and in both flooded and unflooded conditions; however the most preferred mode of carrying out the invention is using an inoculum level of 2x108 CFUs/g soil at neutral pH and at a temperature of 30°C which resulted in about 98% PNP depletion within 7 days. PNP depletion in flooded soil by the said organism makes it suitable for use in water logged areas, sludge etc.
Example-6
Soil was spiked with 70 ppm PNP in the same manner as described in Example 3. Cells of strain RKJ100 were mixed with carrier material and added to the soil (ratio of soil and carrier material was 1:10) so as to reach a final concentration of 2 x 10° CFUs/g soil. The immobilized cells were further mixed with 3.5 kg of PNP-spiked soil and filled in pots measuring 20 cm in length and having top and bottom diameters of 20 and 12.5 cms respectively. Another set of pots contained PNP-spiked soil alone. To another set of pots, 2 X 10° CFUs/g soil were added to 3.5 kg soil in pots without being immobilized on carrier material (hereinafter referred to as free cells). Moisture levels in the pots were maintained at 40-50% of the water holding capacity of soil during the study. All the pots were incubated at 30°C. Soil samples were collected from each pot after every 24 hours until 7 days and thereafter at 10, 15, 20 and 30 days. PNP was extracted and analyzed as described in Example 3. The results showed that 98% depletion of PNP was achieved within 7 days by immobilized cells of RKJ100 while in case of free cells, PNP depletion occurred in 25 days.
Example- 7
Four plots of 1 m X 1 m and 30 cm deep were prepared and lined by a plastic sheet (30 cm deep) to control runoff. Agricultural soil contaminated with 9 ppm PNP was sieved and added to the plots as follows:
Plot 1: Soil without carrier material or bacterial cells.
Plot 2: Soil mixed with carrier material without bacteria.
Plot 3: Soil mixed with bacteria without the carrier material.
Plot 4: Soil mixed with bacteria immobilized on carrier material. The
carrier material was premixed with bacterial cells to give 106 bacteria/g
soil and each plot contained approximately 200 kg soil. Arthrobacter protophormiae RKJ100 cells (2 x 1011) suspended in 200 ml of minimal medium were mixed with the carrier material, which was thoroughly mixed into 5 kg soil. This inoculated soil was then mixed with the rest of the soil. Water was added to the soil to bring the moisture level to 40-50% of the water holding capacity of soil. This moisture level was maintained in all the plots throughout the period of study by irrigating with water as and when required. Soil samples were collected using a hollow pipe/soil core of ~1 inch diameter from six different random positions, pooled, mixed thoroughly and used for analysis of residual PNP. Sampling was done at the following time points: 0, 3, 7, 14, 21 and 28 days. PNP was extracted and analyzed as described in Example 3. The results showed that PNP was completely depleted by immobilized cells of RKJ100 (plot 4) within 5 days while free cells (plot 3) depleted about 90% PNP in 20 days. In plots 1 and 2, only about 20% PNP was depleted by the end of the study.
The main advantages of the present invention are:
1. The present invention describes a novel method for bioremediation of PNP-contaminated soil. PNP is used extensively in the industry and agriculture, resulting in its accumulation in soil and water resources. The present invention can be used for bioremediation of soil, water-logged areas, sludge and effluents contaminated with PNP.
2. The present invention involves immobilization of the bacterial cells on a carrier material which protects the organism from soil predators and toxic contaminants thereby increasing the stability and degradation capability of the biodegrading bacteria.
3. The present invention uses a microorganism which can be easily grown on sugarcane molasses, a cheap and easily available carbon source. The carrier material used for immobilization of bacterial cells is also easily available and inexpensive. Also it involves the addition of organism directly to the waste site and hence allows destruction of target contaminant in situ, eliminating transportation expenses.
4. The present invention is highly effective in eliminating PNP from contaminated soil, and since PNP is formed from organophosphate pesticides parathion and methyl parathion, this process can be used for remediation and reclamation of areas contaminated with this group of pesticides.
5. Strain RKJ100 used for the bioremediation of PNP contaminated soil as described in the present invention is highly advantageous since it is a naturally occurring organism and not a genetically engineered one, the use of which is still limited and controversial. In addition, it is simple to cultivate and requires only ordinary nutrients to support its growth and to degrade PNP.
6. In addition, the invention is a non-polluting method since it generates no intermediate metabolites and uses harmless bacteria. Thus the present invention will be a safe, useful and efficient means to bioremediate PNP contaminated areas.












We Claim
1. A process for bioremediation of Para Nitrophenol contaminated soil comprising
the steps of:
a. growing cells of Arthrobacter protophormiae, strain RKJ100 on molasses
having concentration in the range of 1% to 10%;
b. harvesting the cells obtained in step(a) by centrifugation;
c. resuspending the harvested cells obtained in step (b) in molasses having
concentration in the range of 1% to 10%;
d. mixing the cells as obtained in step (c) with carrier material to obtain
immobilized cells;
e. adding immobilized cells as obtained in step (d) to microcosm of soil
contaminated/spiked with Para Nitro Phenol (PNP) at a PNP inoculum level
in the range of 2xl05-2xl09 CFUs/g to obtain oculated microcosm;
f. incubating the oculated microcosm as obtained in step (e) at a temperature in
the range of 20-40°C, pH in the range of 7-9.5 for a period in the range of 20-
30 days;
g. measuring residual PNP of the soil at regular intervals of time by analytical
methods.
2. The process as claimed in step (a) of claim 1, wherein mass cultivation of Arthrobacter protophormiae, strain RKJ100 is carried out in medium containing 10%) molasses which is treated with 0.1% potassium hexacyanoferrate to precipitate metal ions present in molasses.
3. The process as claimed in step (d) of claim 1, wherein the carrier material used is highly absorbent, granular, high volume low weight, completely biodegradable and inexpensive such as corncob powder.
4. The process as claimed in step (d) of claim 1, wherein the immobilized cells can be stored for several months at temperatures ranging from 4°C to -20°C providing a ready to use formulation whenever required.
5. The process as claimed in step (e) of claim 1, wherein the spiked soil contains 70ppm Para nitrophenol.
6. The process as claimed in step (e) of claim 1, wherein the ratio of soil: carrier material is in the range of 1:10 to 1:1000.
7. The process as claimed in step (e) of claim 1, wherein moisture level in the microcosms is maintained at 40-50% of the water holding capacity of the soil.
8. A process for bioremediation of PNP contaminated soil substantially as herein described with reference to the examples accompanying this specification.

Documents:

1871-del-2004-Abstract-(08-11-2010).pdf

1871-del-2004-abstract.pdf

1871-del-2004-Claims-(08-11-2010).pdf

1871-del-2004-claims.pdf

1871-del-2004-Correspondence-Others-(08-11-2010).pdf

1871-del-2004-correspondence-others.pdf

1871-del-2004-description (complete).pdf

1871-del-2004-Form-1-(08-11-2010).pdf

1871-del-2004-form-1.pdf

1871-del-2004-form-18.pdf

1871-del-2004-Form-2-(08-11-2010).pdf

1871-del-2004-form-2.pdf

1871-del-2004-form-3.pdf

1871-del-2004-form-5.pdf


Patent Number 245068
Indian Patent Application Number 1871/DEL/2004
PG Journal Number 01/2011
Publication Date 07-Jan-2011
Grant Date 30-Dec-2010
Date of Filing 29-Sep-2004
Name of Patentee COUNCIL OF SCIENTIFIC & INDUSTRIAL RESEARCH
Applicant Address RAFI MARG, NEW DELHI-110001, INDIA.
Inventors:
# Inventor's Name Inventor's Address
1 RAKESH KUMAR JAIN IMT, INDIA.
2 SUMEED LABANA IMT, INDIA.
3 GUNJAN PANDEY IMT, INDIA.
PCT International Classification Number B09C 1/10
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA