CA2313110A1 - Microbiological process and microbial products for the bioremediation of soils or waters - Google Patents

Abstract

This invention concerns the biological remediation of soils or waters contaminated with agricultural, industrial or military chemicals. In particular, it pertains to a microbiological process based on the use of novel, non-genetically engineered, naturally-occurring, non-pathogenic, pollutant-degrading or pollutant-transforming rhizobial strains suitable for the bioremediation or biorestoration of soils or waters contaminated with compounds as simple as fertilizer-nitrate and as complex as substituted aromatic hydrocarbons. The products obtained by the process described can also be used as biofertilizers or bioherbicides for the improvement of the quality of agricultural and recreational sites.

Description

TITLE OF THE INVENTION
Microbiological Process and Microbial Products for the Bioremediation of Soils or Waters FIELD OF THE INVENTION
The present invention relates to the bioremediation of soils or waters contaminated with agricultural, industrial and military chemicals. More specifically, the invention comprises a novel process for decontaminating soil and water ecosystems based on the use of microorganisms from the Rhizobia genera of bacteria.
BACKGROUND OF THE INVENTION
Chemicals play a vital role in our everyday life. One of the consequences of increased industrial and agricultural activity in the latter half of the 20tn century has been a widespread contamination of soil and water ecosystems by a number of chemical products. The primary sources of environmental contaminants are household waste and waste generated by the food, textile, lumber, petroleum, chemical, military and transportation industries (Bollag and Bollag, 1995). The contaminants are often persistent, toxic, carcinogenic, biocumulative and hormone-mimicking. The chemical industry alone is estimated to generate over 5 million tons of waste and more than half of these chemicals are released into the ecosystem.
Agrochemicals have been used almost continuously for more than 100 years in certain agricultural soils, especially in the South. Recent environmental regulations require that contaminated soils either be replaced with clean uncontaminated soil or cleansed of the contaminating entities. This is not only important in protecting soil ecosystems but also in protecting groundwater resources. Several methods of soil decontamination have been developed, some of which have been described in patents and elsewhere.
The magnitude of the contemporary problem of soil and ground water contamination cannot be overlooked, nor can the importance of developing environmentally-sensible techniques for the degradation of environmental pollutants into innocuous materials be over emphasized. The isolation of microorganisms that have the ability to degrade specific chemical contaminants, that are able to survive and catabolize under stressful and continuously changing environmental conditions, and that are ecologically, toxicologically and ecotoxicologically safe, is one important strategy in finding and developing cost-effective and environmentally-sound methods for hazardous chemical use and cleanup.
Many different methods have been proposed for converting pollutants or contaminants in soil and water environments into innocuous substances.
Among these are incineration, chemical transformation and microbiological degradation. Advantageously, microbial degradation of environmental toxins does not result in the production of large amounts of hazardous waste or byproducts (such as fumes produced through incineration, or waste water produced through soil washing), since it does not involve the use of chemical reagents which might themselves be toxic. Microbial degradation has therefore become a preferred method of treating contaminated soils and waters.
Bioremediation may be defined as the use of biological processes, organisms or products for the degradation, detoxification, reduction, or stabilization of hazardous materials or compounds in the environment. It frequently involves the use of microorganisms. The biodegradation of contaminants is the result of the complete mineralization or biotransformation of hazardous compounds into non-toxic or less toxic compounds.
Bioremediation may be effected under aerobic or anaerobic conditions.
The major requirements for an effective bioremediation are: (1 ) a biodegradable or biotransformable substrate (organic or inorganic); (2) an appropriate and active microbial community (consortium); (3) bioavailability of the polluting substrate to the microorganisms; and (4) creation of optimal conditions for microbial metabolism. Occasionally, bioremediation involves biostimulation with nutrients, specific substrates or additives that improve the physical or chemical environment of the working microorganism. It may also require bioaugmentation of the microbial community if the site does not have an appropriate indigenous biodegrading population. Thus, as practiced, bioremediation may involve either one or both of the two following treatment methods:
(1 ) biostimulation (i.e., acceleration of the reproduction and metabolism of indigenous microorganisms capable of biodegradation by amendment of nutrients, etc.); and (2) bioaugmentation (i.e., application or implantation of exogenous or indigenous bacterial strains which have adapted to the environment of interest or which have been genetically manipulated).
Microbial degradation of toxic materials is usually based upon the discovery, development or engineering of a particular microorganism that will metabolize the toxic or unwanted material and convert into to innocuous metabolic products. In the case of organic compounds, the conversion is usually to salts, carbon dioxide and water. Finding microorganisms which can efficiently and safely convert toxic wastes into innocuous metabolic products is a highly complex procedure that involves many strategic and arduous steps, and consequently usually requires a significant expenditure of labour, material and time.

To date, most efforts have focused on finding microorganisms indigenous to and isolated from contaminated soils or waters. One approach involves obtaining a soil or water sample and enriching the sample for a mixture of microorganisms, or purifying a microorganism with the ability to degrade one or more toxic compounds from a complex population. Several studies using mixtures of bacteria obtained by enrichment of a soil sample have shown that such isolates or mixtures of isolates may have the ability to degrade petroleum, PCBs, atrazine, DBTh, PCP, TNT, and other chloro or nitro aromatic hydrocarbons (HCs).
Examples of the application of various microorganisms may be found in the following patents: U.S. Pat. #5,780,290 (for crude oil degradation using mixed bacterial culture, Pseudomonas sp. and Alcaligenes sp., and insoluble nitrogenous fertilizers), and U.S. Pat. #5,508,194 (for PAHs using Achromobacfer and Mycobacterium sp. and nutrients).
Other studies have used cultures of purified strains which have been isolated from soil or water. One such approach is described in U.S. Pat.
#4,493, 895, and the product in U.S. Pat. # 4,477,570 (for halogenated aromatic hydrocarbons using Pseudomonas sp.). Other examples are described in the following patents: U.S. Pat. #4,843,009 (for PCBs using Pseudomonas putida), U.S. Pat. #4,477,570 (for chloro- or nitro- toluenes, 2,4-D, 2,4,5-T, using Pseudomonas sp.), U.S. Pat. #5,429,949 and U.S.
Pat. #5,508,193 (for s-triazines). U.S. Pat #4,664,805, describes the application of either indigenous or nonindigenous microorganisms that metabolize the contaminant but cannot grow on it, in combination with a non-toxic analog of the contaminant serving as a substrate for growth of both the indigenous and non-indigenous microorganisms (to enhance the bioremediation of halogenated organic compounds). Another approach, described in U.S. Pat. #4,511,657, involves a process for treating chemical waste landfill leachates with activated sludge containing undefined mixtures of bacteria capable of metabolizing noxious organics present in the leachates. Yet another approach presented in U.S. Pat. #5,653,675 is based on the application of antiprotozoa chemicals to protect pollutant-degrading bacteria from being preyed upon by other microorganisms, thereby enhancing their intrinsic biodegradative capabilities. U.S. Pat.

5 #5,334,312 describes the use of sodium, potassium or ammonium bicarbonates, in solid or liquid form and in the presence of at least one biodegradative microorganism, to control the pH of the soil or groundwater contaminated with hydrocarbons (diesel fuel or gasoline). This is one form of biostimulation to enhance the degradation process.
Several patents use combined biostimulation and bioaugmentation approaches. Here, nutrients, carbonates or plant materials are simply sprayed or introducted via pipes into the soil (i.e., see U.S. Pat.
#5,525,139 where plant material is used for insecticide degradation; U.S.
Pat. #5,609,667 where cellulose and ammonium sulfate are selected for hydrocarbon remediation; U.S. Pat. #5,501,973 where seaweed and carbamate are utilized for hydrocarbon degradation). In other examples, a combination of physical, chemical and/or biological methods or materials is used for such purposes as those described in U.S. Pat. #5,080,782 and U.S. Pat. #5,753,122 for hydrocarbons and halogenated solvents using thermal and biological treatments.
All of the approaches described above involve the use of microorganisms that are not genetically manipulated. U.S. Pat. #4,535,061 describes a plasmid-assisted breeding procedure for generating pure and mixed cultures, and U.S. Pat. #5,079,166 describes the use of genetically engineered microorganisms (strains of Pseudomonas) obtained by transferring plasmids from other microorganisms that degrade halogenated aromatic organic compounds in order to obtain microorganisms capable of dissimilating environmentally-persistent chemical compounds, such as TCE.

Presently, most microbial strains used for such environmental purposes belong to the following groups: Pseudomonas, Achromobacter, Agrobacterium (U.S. Pat. #5,429,949), Arthrobacter, Aspergillus, Azotobacter, Bacillus, Cunninghamella, Fusarium, Mucor, Nocardia, Penicillium, Phenerochete, Proteus, Rhizoctonia, Rhizopus, Saccharomyces, Sclerotium, Streptomyces, Trichoderma, Verficillium, Xanthomonas, Mycobacterium, Methanocomonas, Desulfovibrio, Micrococcus, Actinomyces, Candida, Aerobacfer, Methylococcus, Streptomyces, etc. (several of which are referenced in U.S. Pat.
#5,334,312 and U.S. Pat. #5,100,455), but not Rhizobium (Ahmad et al., 1997). Despite the efficiency of the processes themselves, the short-term and long-term ecological, ecopathological and ecotoxicological effects resulting from the introduction of these bacteria remain questionable, as certain species belong to groups of well-known opportunistic pathogens.
In summary, bioremediation is the use of biological organisms to degrade contaminants. To be possible, bioremediation requires organisms that have the ability to degrade the contaminants in question. To be safe, the organisms should not be pathogenic to humans, plants, animals or the environment. To be effective, a bioremediation technology should be able to deliver these organisms to all contaminated parts of the soil matrix as well as to provide favourable growth conditions for the organisms. Thus, the first phase concerns the discovery or creation of nonpathogenic organisms with the ability to degrade the target contaminant(s). The second phase concerns the development of a delivery and maintenance system for the organisms.
In light of the above, there is a need for biotechnologies that will make bioremediation a safe and sustainable practice.

SUMMARY OF THE INVENTION
The present invention relates to the discovery, isolation and screening of novel non-genetically engineered, naturally-occurring, non-pathogenic, pollutant-degrading or pollutant-transforming bacterial strains from the genus Rhizobium, and a method for biologically remediating, degrading or cleansing harmful or unwanted industrial, military or agricultural chemicals in soil and water environments. More particularly, it pertains to a microbiological process and microbial products suitable for the bioremediation of soils or waters contaminated with simple substituted aromatic hydrocarbons (such as fertilizer nitrates) to complex substituted aromatic hydrocarbons (such as chloro- and/ or nitro- aromatics) used in herbicides or explosives (such as atrazine, TNT, PCBs, etc.).
Experiments have revealed the potential of Rhizobium sp. as an ecologically and agronomically important group of bacteria for the destruction, reduction and/or stabilization of organic contaminants in soils.
Alternatively, rhizobia have proven useful as biofertilizers (i.e., promoters of plant growth) or bioherbicides (i.e., agents effective in controlling the growth of weeds or unwanted herbs) to improve the quality of agricultural and recreational sites. Advantageously, these bacteria have long been considered to be environmentally safe, since they fix atmospheric nitrogen without causing adverse ecological, ecopathological and ecotoxicological effects and no species in this genus is known to be pathogenic or virulent to humans, animals or plants. For this reason, the use of rhizobia in the bioremediation and in the aforementioned agricultural applications is of great interest.
More specifically, in accordance with the present invention, there is provided a microbiological process and microbial products for the bioremediation of soils or waters. The microbiological process of the invention comprises the following steps: 1 ) collecting a soil or water sample from a contaminated site; 2) isolating native rhizobial species by trapping them with alfalfa, bean, clover, pea, soybean, chickpea, lentil, lupin and other leguminous plant seedlings; 3) screening for their ability to tolerate and to destroy, reduce or stabilize organic contaminants in the sample soil or water, or alternatively, testing for their ex plants nitrogen fixation, phosphate solubilization and iron chelating capability and herbicidal activity.
Plant inoculation may be preceded by a period of adaptation on the relevant contaminant. Alternatively, preselected and prescreened rhizobial strains from a library can also be used. These rhizobial strains, selected by proprietary techniques, used/ applied/ described in this patent, can then be used to treat specific contaminants or weeds either as individual cultures, mixed cultures, or in combination with certain plants (such as alfalfa, clover, pea, soybean or beans), other biodegrading bacterial strains (i.e., bacterial species belonging to a genus other than Rhizobium and having the ability to transform or metabolize harmful or unwanted chemicals), plant growth-promoting rhizobacteria, cosubstrates or plant products that enhance the degradation activity (such as flavonoids, oils or terpenes), chemical nutrients that are required for growth, inducers that bring about the degradation pathways, environmental conditioners, or modifiers that positively affect the degradation activity. The present invention provides a microbial system for treating a selected volume of contaminated soil or water.
In one embodiment, the present invention provides a method for the microbial degradation or transformation of atrazine, TNT, fertlizer-nitrate and PCBs. The rhizobial strains were isolated by plant nodulation and enriched or made to adapt on the substrate prior to application. The method used for the isolation and selection of rhizobial strains has been described previously (Damaj and Ahmad, 1996; Ahmad, et al., 1997) and is herein incorporated by reference.

As described in Examples 3, 5 and 6, a water table management system (U.S. Pat. # 6,027,284) was used to deliver and maintain rhizobial inocula in soil columns. In addition, some of the isolated strains exhibited phytotoxic (herbicidal), or phosphate-mobilizing and iron-chelating (fertilizing) capabilities and hence could be used for agricultural and recreational sites.
Other objects, advantages and features of the present invention will become apparent upon reading the following non-restrictive description of preferred embodiments thereof, with reference to the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
In the appendix drawings:
Figure 1. Shows the distribution pattern of bacterial strains representing the relationship between hydrophobicity and adhesion (characteristics important for bioremediation). The rhizobia are clustered within the circle.
1 and 2 represent C. testosferoni B-356 and E. coli, respectively.
Figure 2. Represents the implantation and survival of R. meliloti strain A-025 at different depths of sterile soil columns 14 days after inoculation.
(mean of three replicates and the SD).
Figure 3. Shows the total change in concentration of Aroclor 1242 congeners after 388 days, as compared with the initial concentration in the presence of CaC03 in sterile soil microcosms augmented with strains a) A - 025, b) Zb57, and c) A-029 (average of duplicates).
Figure 4. Represents the change in concentration of Aroclor 1242 congeners with strain A-025 in the presence of CaC03, as compared with the abiotic control after a) 39, b) 73, c) 165 and d) 388 days (average of duplicates).
Figure 5. Indicates the bacterial populations during the experimental 5 period of 388 days, a) with CaC03 and b) without CaC03 in sterile soil microcosms amended with Aroclor 1242. Strain A-025 was reinoculated on the 123~d day in b) (average of duplicates and SD).
Figure 6. Illustrates the concentration change of PCB congeners in soil 10 columns augmented with strain A-025 as compared with the passive control after a) 20, b) 44, c) 282 and d) 345 days (mean of three replicates).
Figure 7. Reveals the population of R. melilofi strain A - 025 at different depths of soil columns during the experimental period of 377 days (mean of three replicates and SD).
Figure 8. Indicates the total PCB concentration at different depths of soil columns augmented with strain A-025 after 377 days (average of three replicates).
Figure 9. Reveals the concentration of atrazine leached in the drain water of sterile soil columns bioaugmented with R. meliloti A-025 after 80 days of anaerobic and 70 days of aerobic conditions.
Figure 10. Shows the concentration of nitrate leached in the drain water of sterile soil columns bioaugmented with R. meliloti strain A-025 under anaerobic conditions. NA and N represent treatments with nitrate in the simple presence of atrazine and nitrate, respectively.

Figure 11. Indicates the mineralization of ~4C-atrazine by different rhizobial isolates as measured by total dpm in trapped '4CO2 after 72 hours.
Figure 12. The harvested corn yield. Results represent average and SD of triplicates. Treatments are as indicated in Example 11.
Figure 13. Total weight of weeds in the sub-plots and full plots for different treatments after 90 days. The results represent average of triplicates.
Treatment 9 did not have full-plots.
DETAILED DESCRIPTION OF THE INVENTION
To demonstrate the use of rhizobia as a potential microorganism for the degradation of fertilizer-nitrate, aromatic, chloroaromatic or nitroaromatic contaminants, the presence and isolation of a variety of strains of Rhizobium was demonstrated using plant nodulation tests on leguminous plants in soils contaminated with aromatic hydrocarbons, chloroaromatic hydrocarbons, heavy metals, TNT, PCP, and agricultural soils treated with atrazine (Tables 1 & 2). Table 3 shows the ability of different Rhizobial isolates to tolerate atrazine.
R. meliloti strain A-025 (ATCC Accession No. PTA-1457) was selected from an isolated bacterial collection based on membrane characteristics relevant to its transport and distribution through soil matrices (hydrophobicity and adhesion - see Figure 1 ), and its potential to degrade PCBs (Ahmad, et al., 1997). This strain was implanted in sterile columns 200 mm in diameter x 1000 mm in length, which were packed with a sandy loam and which included surface and subsurface irrigation. Results showed that a high number of the bacterial cells distributed in the soil were able to survive at 60, 300, 500, and 700 mm of depth after two weeks of application or implantation (Figure 2). This survival capacity makes strain A-025 particularly suitable for bioremediation technology.
A long-term experiment (i.e., 388 days) with sterile soil microcosms amended with Aroclor 1242 and augmented with R. meliloti strains A-025, Zb57 and A-029 showed depletions of PCBs as compared with initial concentrations (Figure 3). Figure 4 shows the temporal pattern of transformation of PCBs by strain A-025 during the experimental period as compared with an abiotic control. Figure 5 shows the transport and survival of implanted strains in the presence of CaC03. In a different set-up, the change in PCB concentration was followed in columns packed with PCB-contaminated soil and bioaugmented with R. meliloti strain A-025 (Figure 6). The results indicated that the bioaugmented strain survived at all depths for a period of 377 days (Figure 7). The results also indicated that the lowest total PCB concentration was observed at a depth of 590 mm, as compared with depths of 140mm and 340mm (Figure 8).
Bioaugmentation of soil columns with R. meliloti, strain A-025 decreased the concentration of atrazine by 31 % during anaerobic and aerobic cycles (Figure 9). Under anaerobic conditions, this strain reduced the concentration of nitrate in the absence and presence of atrazine in the drain water by as much as 87% and 78%, respectively (Figure 10). Data on the growth characteristics of different species of rhizobia, namely strains B5 of Rhizobium leguminosarum bv. phaseoli (ATCC Accession No. PTA-1458), T10 of Rhizobium leguminosarum by trifolii (ATCC
Accession No. PTA-1459), M8 of Rhizobium meliloti (ATCC No. 700899), P1 of Rhizobium leguminosarum bv. viceae (ATCC No. 700900) and P2 of Rhizobium leguminosarum bv. viceae (ATCC No. 700901 ), all of which were isolated from farm soils treated with atrazine, are presented in Tables 4 and 5. Figure 11 shows the mineralization of radio-labelled atrazine in liquid cultures of these isolates.

Rhizobial strains B5, M8 and T10 exhibited the ability to transform TNT in liquid cultures (Table 6).
Thus, overall results of this work indicate that Rhizobium is an organism that can be used to control environmental pollutants such as PCBs, TNT, atrazine and nitrate.

Table 1: The Origin and History of Soils used for Isolation of rhizobia.
Soil Site Soil Contamination Sample History characteristics (ppm) Heterogeneous m~dure of S-I sl~~ of diesel and g~, sand, wood, PAHs, 5000-85000, bunker rocks, fluids, over 15 oil and grease years g~ss, bricks, pH
(6.50) oorrtamination S-I I na Heterogeneous m~urepAHs of gravel, sand, day, pH (7.88) ManuFac~uring of boat motors and marine equip., Homogenous m~ur~e PCBs > 730, range PCB of 0-S-I I spillage from brokenwood, sandy day, 1400 Arodor 1242, I hydraulic grain see lines on rnachir~es<13 mm, pH (7.75) 1254, o~ and in 1960's grease and 1970's ~N Contaminated with horrx~ger~ous m~6ureRange 600-700 diesel of tiotal over 20 years sand and silt diesel residues Heterogerrous moctiurePCBs 110 , heavy of S-V na gravel, wood, rods, day and sift Wood treatrnt factorynd PCP

S-VI Wood treatrr~nt nd PCP + creosote I factory S-VIII ~minated with TM' Hs m~ure of ~- >~ ~ ppm day and sand Lysimete- used for S-X an atraane o moger~ous mo~ure of H

ding e~erimerrt, ~ , Mlor 3 years ~

old Contaminated with Wood treatr~r>ent nd PCP +
factory Cr~sote S-XI Com field with atraane~ Atrazine applied:

application in 1997 1 2 kg ha Corn field with ~ Atraane applied:1 atraane 2 application in 1998 kg/ha na-not available, nd-not determined Table 2. Tolerance of Different Rhizobial Species to Various Contaminants as Judged by the Presence of Rhizobial Species Trapped on Different Leguminous Plants 5 A) Nodulation of Beans (associated sp.: Rhizobium leguminosarum bv. Phaseol~~
Total Soil type Total Number and fresh matter Leaf color contaminant dry matter of nodules (g) (g) SI PAH 20.60 2.80 133 : Fix+~-Yellow SII PAH 12.54 2.22 0 : Nod- Yellow SIII PCB 12.00 1.62 13 : Fix+~- Green-yellowish SIV Diesel 13.63 1.87 2 : Fix Green-yellowish SV PCB 8.04 1.06 20 : Fix+~- Green-yellowish SVI PCP 8.36 1.18 167: Fix Yellowish SVII Creosote2 225 0: Nod- Green-puny +PCP . . looking SVIII TNT 11.41 2.40 0: Nod- Green Nod- : no nodules 10 Fix+ : pinkish nodules riches in leghemoglobine Fix : white nodules Fix+~-: mixture of pink and white nodules Soils: SI : PAH oil & grease (sandy) ; SII : PAH (sandy) ; SIII : PCB >_ 730 ppm + oil & grease (sandy clay) ; SIV : Diesel (sandy); SV : PCB +
15 heavy metals (clay); SVI: PCP ; SVII: Creosote + PCP and SVIII: TNT (20 000 ppm).

B) Nodulation of Peas (associated sp.: Rhizobium leguminosarum bv.
Viceae) Total Total Number Soil type fresh matterdry matter of nodules deaf color (g) (g) SI 10.57 1.64 169: Fix+ Green SII 4.08 0.49 4: Fix Yellow SIII 6.43 0.81 18: Fix+ Green SIV 3.49 0.42 0: Nod- Greenish-yellow SV 7.95 1.02 30: Fix+ Green SVI 12.36 1.93 78: Fix+ Green-healthy SVII 2 0 0: Nod- Yellow-. . puny looking SVIII 5.19 0.90 0: Nod- Yellowish-green C) Nodulation of Soybean (associated sp.: Bradyrhizobium japonicum) Total Total Number Soil type fresh matterdry matter of nodules deaf color (9) (9) SI 5.38 0.79 Nod- Green SII 0.54 0.04 Nod- Green SI I I 3.29 0.46 Nod- Green SIV 3.76 0.44 Nod- Green SV 4.07 0.62 Nod- Green SVI 1.47 0.19 Nod' Yellow-puny looking SVII 0 0 Nod- Dead plants D) Nodulation of Clover(associated sp.: Rhizobium trifolii~
Total Total Number Soil type fresh matterdry matter of nodules deaf color (9) (9) 15 : Fix Yellow and SI 0.65 (8 plants)0.025 first leaves dead SII 0.52 (8 plants)0.021 0 : Nod-SIII 1.11 (6 plants)0 33 : Fix+ Green:

, healthy plants SIV 0.21 (3 plant)0.018 0 : Nod' Green/necrotic SV 3.45 (6 plants)0.17 126 : Fix+ symptoms on leaflet edge SVI 10.32 1.36 278: Fix+ Green-healthy (10 plants) SVII 0.136 0 0: Nod' Yellow-(7 plants) . puny looking SVIII 0.55 (9 plants)0.053 0: Nod- Green-puny looking E) Nodulation of Alfalfa (associated sp.: Rhizobium melilot~~
Total Total Number Soil type fresh matterdry matter Leaf color of nodules (g) (g) SI 020 FiXOplants) yellow 0 :

(10 plants) .

SII 0'79 017 0 : Nod- Yellow (10 plants) .

SIII 4.22 (8 plants)0.183 37 : Fix+ Green/healthy SIV 0.61 (9 plants)0.013 1 : Fix Yellow SV 0.60 0 0 : Nod- Yellow (10 plants) .

SVI 127 15: Fix Yellow (10 plants) .

SVII 0'38 0 0: Nod- Green- puny (10 plants) . looking SVIII 0.60 (5 plants)0.061 0: Nod-F) Controls for Nodulation Test Total Totaldry Number Plant fresh mattermatter/plant Leaf color of nodules (9) (g) Pea 3.76 0.58 0 Yellow Bean 13.01 2.00 0 Yellow Soybean 3.42 0.70 0 Yellow Alfalfa 0.03 0.008 0 Yellow ~ Clover 0.089 0.02 0 Yellow Table 3: Tolerance of Rhizobial Species to Atrazine (Survival and Competition during Enrichment with Atrazine) Number of Nodules:

Soil: Soil: Inoculated Plants Associated RhizobialSXI SXII with the culture from the 6t"
transfer on MM

Inoculated Species atrazine:
tolerant Rhizobia # Nodules# NodulesSoll:Xl Soll:Xll (#Nodules) (#Nodules) Alfalfa Rhizobium meliloti 22 17 2 N D

Beans:

Rhizobium cv Romano leguminosarum bv. ND ND 20 ~ 18 phaseoli or etli Rhizobium cv Red Kidneyleguminosarum bv. ND ND 19 50 phaseoli or etli cv White Rhizobium Kidney leguminosarum bv. ND ND 21 8 phaseoli or etli Rhizobium cv Pole Beanleguminosarum bv. 5 90 0 0 phaseoli or etli Rhizobium Clover leguminosarum bv. 24 21 9 ND

trifolii Chickpea Rhizobium ciceri 0 0 0 0 ou mediterraneum Fava bean Rhizobium 0 0 0 0 leguminosarum Rhizobium Pea leguminosarum bv. 20 17 0 0 viceae Soybean Bradyrhizobium 0 1 0 0 japonicum Table 4: Growth of Different Rhizobial Isolates on Atrazine (1000 ppm: 0.1%) Minimal Media of Different Compositions Strains +Glucose +KN03 +Gluc+KN03 - C and +Glutamate - N

g5 +++ + ++ + ++

M g +++ ++ +++ + +

p1 +++ N p +++ + +++

p2 +++ N p +++ + +++

T10 +++ + +++ + ++

Table 5: Growth of Different rhizobial Isolates on Cyanurique Acid (500 ppm: 0.05%) Minimal Media of Different Compositions Strains +Glucose +KN03 - C and - N

g5 +++ +/- +/-M g +++

p 1 +++ +/- +/-p2 +++ +/- +/-T 10 +++ + +/-Table 6: Biotransformation of TNT with M8, an Atrazine-Degrading Strain of R.meliloti Isolated from Atrazine-Contaminated Agricultural Soil (initial concentration of TNT: 52 ppm) TNT and metabolites by HPLC/UV1 day 2 days 3 days 4 days TNT 40.07 6.78 0 0 2-ADNT 0.13 2.42 2.62 3.12 4-ADNT 0.23 7.01 8.16 10.06 2-HADNT 0.83 41.86 50.29 14.14 ~4-HADNT -. I 0.81 35.02 54.05- -$.34~
~ ~ ~

~ TNT: Trinitrotoluene ~ 2-ADNT: 2-aminodinitrotoluene (2-NH2-4-6-di-N02-toluene) ~ 4-ADNT: 4-aminodinitrotoluene (4-NH2-2-6-di-N02-toluene) ~ 2-HADNT:2-hydroxylamino-di-N02-toluene ~ 4-HADNT:4-hydroxylamino-di-N02-toluene Table 7: Number of weed plants in the sub-plot of different treatments 90 days after microbial inoculation. The results represent the average number of plants in the three replicates. Treatment means with the same letters for each weed type are not significantly different at a=0.05 based on protected LSD test.
Plant Total numbers treatmentFoxtailBarnyardCrab RedrootLambquarters MonocotDicot Velvetleaf 1 2.67cb0.67a 1.67a1.67a 1.OOa O.OOa 5.OOa 2.67a 2 4.33cb0.33a 0.67a0.67a 3.67a 0.67a 5.33a 5.OOa 3 1.67c 0.33a 2.67a0.67a 0.67a O.OOa 4.67a 1.33a 4 3.67cb0.33a 2.67a2.67a 0.67a 0.33a 6.67a 3.67a 5 3.OOcbO.OOa 2.67a1.33a 1.67a 0.33a 5.67a 3.33a 6 7.33ab1.OOa 3.33a1.67a 0.33a 0.67a 11.67a 2.67a 7 2.33c O.OOa 2.33a1.OOa 3.33a O.OOa 4.67a 4.33a 8 4.OOcbO.OOa 3.OOa2.33a 3.OOa 1.33a 7.OOa 6.67a 9 11.33aO.OOa 1.33a2.OOa 3.OOa 0.67a 12.67a 5.67a EXAMPLES
The following examples are given merely as illustrations of the present invention and are not intended to limit its scope.
EXAMPLE 1: Isolation of Rhizobium sp.

A) Isolation of Rhizobial Strains from Contaminated Soils Seeds of alfalfa, clover, soybean, Romano beans, red and white kidney beans, pole beans, Fava beans, chickpea, lentil, lupin and pea were obtained from the seed farm of the Macdonald Campus of McGill University (Ste. Anne de Bellevue, Quebec, Canada) and commercial centers in Montreal and were surface-sterilized by soaking in 95% ethanol for 10 seconds. This was followed by a 5-minute treatment with 2.8%
NaOCI (household bleach) and thorough rinsing with sterile distilled water.
The seeds were then soaked for 5 hours at room temperature or for 18 hours at 4°C in sterile water for germination. Contaminated soils were used in their original state to sow the seeds, or in suspension form for seed inoculation, or were enriched with degradative microbial populations before inoculation of germinated seedlings. Seeds (2-10) were then placed either on agar plates or slants, or in a sterilized sand-vermiculite-perlite mixture (1:1:1 ) in plastic plant pots (Somasegaran and Hoben, 1994) or in a sterile test tube assembly system (Bromfield et al., 1994;
Ahmad et al. 1997). All media contained nitrogen-free plant nutrient solution (Somasegaran and Hoben, 1994), and were held for germination in a plant growth environmental chamber (Model no. 125L CPM3000, Conviron Controlled Environ. Ltd., Winnipeg, Manitoba, Canada) at a constant temperature of 23°C and a 16 h day' light setting (1000W metal halide lamps that produced 250 Einstein rri 2 sec' at plant height).
Uninoculated controls were included to verify that the seeds used were rhizobium-free. Observations were recorded after 6-8 weeks for nodule formation and for up to 3-4 months for establishing the efficacy of nodules for N2-fixation as judged by the continued healthy growth of plants with nodules as compared with the deteriorating or dead plants resulting from uninoculated nodule-free controls.
B) Isolation of Rhizobium from root nodules A few of the nodules developed with the inoculum from each of the contaminated soils (Table 1 ) were individually harvested, surface sterilized with ethanol for a few seconds followed by a 5-10 minute treatment in 3%
(v/v) of household bleach, and rinsed at least ten times with sterile distilled 5 water. The nodules were then crushed in 100~.L of TYc (Ahmad et al., 1997) in an Eppendorf tube using flamed glass rods. The nodule suspension containing bacteroids was plated on TYc plates. The plates were incubated at 29°C for a few days until colonies were visible. The colonies were purified, characterized for their morphology, retested for 10 their ability to develop effective nodules on respective plants, designated with a number and stored at -70°C in TYc with 20% glycerol.
Conclusions 15 Different species of Rhizobium were present in soils contaminated with atrazine, PCBs, PAHs, diesel, TNT, PCP, creosote and heavy metals (Tables 1 and 2).
EXAMPLE 2: Assay for Hydrophobicity and Adhesion of Bacteria The hydrophobicity of bacteria was determined with a modified version of the method described by Huysman and Verstraete (1993). The rhizobial cells were grown overnight on TYc (Bromfield et al. 1994) or YEMP
(Vincent, 1970; Damaj and Ahmad, 1996) and the non-rhizobial strains, E.
coli JM105 and C. testosteroni B-356, were grown on Luria-Bertani (LB) broth (Sambrook et al. 1989; Damaj and Ahmad, 1996). The bacterial cultures were then centrifuged, washed twice in PBS (each time vortexed for 30 seconds) and resuspended to an optical density at 600 nm (ODsoo) of approximately 1Ø Five mL of the washed cell suspension and 0.5 mL
of octane were added to a test tube and mixed with a vortex mixer for 60 seconds. After 10 minutes of equilibration, the aqueous phase was transferred with a pipette to a cuvette and the OD was measured at 600 nm. The following equation was used to calculate the hydrophobicity of the bacterial strains: [ODi - (Ave.ODf - Ave. ODc)] / ODi x 100, where ODi, ODf and ODc are the ODsoo of the initial, final, and control bacterial cultures, respectively. The control tubes contained only octane and PBS.
All tests were done in triplicate.
The adhesion of bacteria was determined with a modified version of the method described by Huysman and Verstraete (1993). Overnight cell cultures were grown as described for the hydrophobicity assay and centrifuged, washed twice in physiological saline (150 mM NaCI) and resuspended to an ODsoo of approximately 0.7. Ten mL of the washed bacterial suspension was added to 1.0 g of sterilized garden soil (78%
sand, 3% silt, 19% clay, 3.59% organic matter, pH 6.17) in a test tube. The mixture was vortexed for 60 seconds and allowed to settle for 15 minutes.
One mL of the aqueous layer from the top was collected and the ODsoo was measured. A set of tubes containing 10 mL of NaCI and 1.0 g of soil was used as a control for the adjustment of the ODs obtained. All tests were done in triplicate. The following equation was used to calculate the adhesion of bacterial cells to the soil: [ODi - (Ave.ODf - Ave. ODc)] / ODi x 100 where ODi, ODf, and ODc are the ODsoo of the initial, final, and control bacterial cultures, respectively.

Conclusions The behaviour of rhizobial cells in water and soil is influenced by the surface characteristics of the bacteria, which are relevant to the transport and distribution of bacteria during soil bioaugmentation operations. Both hydrophobicity and adhesion characteristics were influenced by the composition of the growth media used (Figure 1 ). All rhizobial strains tested showed characteristics different from those of E. coli or C.
festosteroni.
EXAMPLE 3: Implantation and Survival of Rhizobium meliloti in Sterile Soil Columns R. meliloti strain A-025 (Ahmad et al., 1997) was subcultured in 6 L of fresh TYc medium incubated with shaking for two days at 29°C. A sample of the cultures (approximately 2.3x10$ cell mL-') was plated on TYct agar, a selective medium for strain A-025, to determine initial population counts.
Bacterial cells were then harvested by centrifugation (Sorvall Instruments, Dupont model RCSC) for 5 minutes at 8000 rpm, washed with sterile 0.9%
saline, resuspended in 400 mL of sterilized deionized water (resulting in about 5.8x10$ cell mL-') and mixed thoroughly by vortexing before application at depths of 60mm, 300mm, 500m and 700mm in stainless steel columns packed with sterile soil (1000 mm long and 200 mm in diameter, fitted with sampling ports on the sides). All columns were fitted with a delivery pipe and a subirrigation port at the bottom to supply water and bacterial cells (U.S. Pat. # 6,027,284). The prepared bacterial suspension was added to the columns either through surface irrigation or subirrigation, at a flow rate of 2.4x104 mL hour' (time of inoculation = 1 minute). After 140 hours (about 6 days), continuous saturated flow to all columns was stopped and the columns were kept saturated for two more days before being drained in order to allow the bacterial cells to adhere and stabilize.

Sampling and Microbial Analysis Before inoculation of the soil columns with bacteria, soil-water samples were taken from each column to determine the initial microbial composition and status. Samples of 10 mL were taken from each sampling port at intervals of 14, 28, 56, and 84 hours after inoculation to determine bacterial transport by percolating, ascending and circulating water flows.
The time for one pore volume of water to be replaced in the columns was approximately 14 hours. The samples were kept at 4°C in sterilized glass tubes until the next day, at which point they were plated for microbial analysis and count. Eight days after inoculation, the columns were drained and 10 g soil samples were taken for analysis of bacterial distribution in the soil columns. Bacteria were extracted from soil particles by vortexing 2 g of soil in 10 mL of sterile 0.9% saline solution and serial dilutions were plated on TYct agar plates for counts of viable cells. Six days after inoculation, the columns were drained and soil samples were taken from four different depths to determine the distribution of implanted bacteria in the soil. In addition to using TYct as a selective growth media, the identity of the transported bacteria, R. meliioti, was also confirmed by DNA
hybridization tests (results not shown).
Conclusions R. meliloti strain A-025, when implanted, demostrated the ability to transport and survive over a period of 14 days in a pristine sterile sandy loam under both aerobic and anaerobic conditions and at different depths (Figure 2).
EXAMPLE 4: PCB Biotransformation by Rhizobium meliloti in Sterile Soil Microcosms R. meliloti strains A-025, A-029, and Zb57 were inoculated from precultures into 2 L of TYc and incubated at 29°C in a controlled environment incubator shaker (Psycrotherm, New Brunswick Scientific) for 24 hours. A sample of the culture (about 2.3x10$ cell mL-') was plated on TYct (Kinkle et al., 1994) agar plates for population count. The cells were centrifuged, washed with 0.9% saline solution, resuspended in 100 mL of sterilized deionized water (about 2.3x10" cell mL-') and mixed by vortexing before inoculation into sterile soil jars (about 3.8x10" cell g-' of soil; 600 g of soil per jar). Prior to bacterial inoculation, the soil was artificially contaminated with 100 ppm of Aroclor 1242, with or without CaC03 (6 g per jar). The microcosms were incubated for 388 days at room temperature with loosely screwed tops and were mixed with a spatula at sampling times. The microcosm with strain A-025 in the absence of CaC03 was reinoculated 123 days after the first inoculation, with about 7.1 x109 cell g-' of soil. The second inoculum was added in 50 mL of sterilized deionized water, enough to saturate the soil and produce a 5 mm pond on top of the soil to create anaerobic conditions. Soil samples of 20 g were taken periodically from each microcosm while mixing the soil in the jars (smaller samples from several places in the microcosm were pooled). The experimental period was 388 days after inoculation, except for the microcosm with strain A-025 which underwent 123 days of aerobic conditions (after the 1St inoculation) followed by 265 days of anaerobic conditions (after the 2"d inoculation). Therefore, the conditions at 123 days were considered to be the initial conditions when analyzing the results of the anaerobic period. For all other microcosms, the total loss of PCBs was compared befinreen the initial and 388t" day (Figure 3). However, to eliminate the loss of PCB congeners due to adhesion to soil particles, the pattern of biotransformation during the 388 days was compared to an abiotic control for the strain A-025 microcosm (Figure 4).

The extraction and analysis of PCB was done following the protocol of Barriault and Sylvestre (1993). Two 3-g soil samples from a 20 g soil were mixed in 125 mL flasks with 6 mL of sterilized deionized water and Triton X-100 (30 NL) to enhance desorption of Aroclor from soil particles.
5 Anhydrous sodium sulfate (6 g) was added to prevent the formation of a stable emulsion. Hexane (15 mL) was then added, and the flasks were shaken on a rotary platform for 20 minutes. The hexane fraction was collected on ammonium sulfate and the aqueous phase was extracted two more times. The pooled organic fractions were then evaporated to a final 10 volume of 3 mL, equal to the weight of a soil sample in grams (Barriault and Sylvestre, 1993). The samples were analyzed with a gas chromatograph (Hewlett Packard 5890, Series II) equipped with an electron capture detector (GC/ECD) and a HP-5 (phenyl methyl silicone) capillary column measuring 25 m by 0.32 mm. Helium was used as a 15 carrier gas at a flow rate of 1 mL min-'. The initial temperature (60°C) was held for 2 minutes, then raised at a rate of 5°C min-' to a final temperature of 290°C which was maintained for 15 minutes. Injector and detector temperatures were 275°C and 350°C, respectively. A late eluting peak (51, IUPAC congener no. 110) of Aroclor 1242 resistant to biodegradation was 20 used as an internal standard. The ratio between this peak and other congener peaks at different times of the experiment were compared to determine the changes in the concentration of each congener (Figures 3 and 4) (Barriault and Sylvestre, 1993; Damaj and Ahmad, 1996). These ratios were then compared either to the peak ratios obtained from the 25 abiotic control (Figure 4) or to the ratios at the initial time (Figure 3).
The assignment of congeners to peaks was based on previously published results (Damaj and Ahmad, 1996; Barriault and Sylvestre, 1993;
Ballschmiter and Zell, 1980; Larson, et al., 1992; Erickson, 1997).
30 For microbial population counts, the bacterial cells were extracted from the soil particles by vortexing 2 g of soil in 10 mL of sterilized 0.9% saline solution. Serial dilutions (up to 105) were plated on TYct agar plate medium selective for R. meliloti (Kinkle et al. 1994) and incubated at 29°C
for viable population counts (Figure 5).
Conclusions R. meliloti strains A-025, A-029 and Zb57 showed the ability to transform the more chlorinated congeners to lower chlorinated congeners present in Aroclor 1242, under both aerobic and anaerobic conditions, and the biotransformation appeared to be more rapid in the presence of CaC03 than without it. Furthermore, the survival of the bioaugmented bacterial cells was greater in the presence of CaC03.
EXAMPLE 5: PCB Biotransformation by Rhizobium meliloti in a Weathered Contaminated Soil R. meliloti strain A-025, was precultured in TYc and used to inoculate 1 L
of Tyc incubated at 29°C in a controlled environment incubator shaker (Psycrotherm, New Brunswick Scientific) for 24 hours. The bacterial cultures were plated on TYct agar plates to determine the population count. The bacterial cells were then harvested by centrifugation and the collected cells, 5 g, were washed with 100 mL of 0.9% saline solution and resuspended in 300 mL (7.7x10$ cell mL-~) of sterilized water. The cells were mixed by vortexing the inocula before inoculation of the soil columns.
Soil columns were reinoculated the next day for the 2"d time with 3.971 g of cells. The columns were subirrigated and a water table management (U.S. Pat. # 6,027,284) was applied to create aerobic and anaerobic conditions, as needed.
Soil samples were taken from each column at 140mm, 340mm, and 590mm depths before inoculation of the soil columns with strain A-025 to represent the initial conditions. Soil samples (20g) and water samples (20mL) were collected after each wet and dry cycle and periodically analyzed for their PCB concentrations, pH, and viable population count.
For PCB extraction and analysis, three grams of the 20g soil samples taken from the columns were mixed in a 125mL flask with 6mL of sterilized deionized water and Triton X-100 (30NL) to enhance desorption of Aroclor from soil particles (Barriault and Sylvestre, 1993). Anhydrous sodium sulfate (6g) was added to prevent the formation of stable emulsions (Bedard et al., 1986). Hexane (15mL) was then added and the flasks were shaken on a rotary platform for 20 minutes before collecting the hexane fraction on ammonium sulfate. The aqueous phase was extracted two more times. The pooled organic phases were passed through Florisil~
(5g) column filters and concentrated to 3mL under nitrogen gas. Ten NL of 2,3,4,5,6,2',3',4',5',6'-chlorobiphenyl (333 ppb) were added as an internal standard. Samples were diluted before their injections. The ratio of each peak against the internal standard was determined and these ratios were compared to those of the passive control (Figure 6). The assignment of congeners to peaks was based on previously published references (Ballschmiter and Zell, 1980; Larsen et al., 1992; Erickson, 1997).
The samples were analyzed with a gas chromatograph (Hewlett-Packard 5890, Series II) equipped with an electron capture detector (GC/ECD) and a HP-5 (crosslinked 5% Ph Me silicone) capillary column (25m x 0.2mm x 0.33pm film thickness). Helium was used as a carrier gas at a flow rate of 1 mLmin-'. The initial temperature (60°C) was held for 2 minutes, then raised at a rate of 5°C min'' to a final temperature of 290°C
which was held for 15 minutes. Injector and detector temperatures were 275°C and 350°C, respectively.
For microbial analysis, the bacteria were extracted from the soil of the columns with bioaugmented R. melilofi strain A-025 by vortexing 2 g of soil in 10 mL of sterilized 0.9% saline. Serial dilutions were plated on TYct agar plates for counts of viable populations of strain A-025 (Figure 7). The colonies formed from samples taken on the 377t" day were tested for nodule formation on alfalfa roots using the procedure described in Example 1.
Conclusions The results revealed a higher transformation of PCBs in bioaugmented columns as compared with the passive control, and the patterns were different at different depths (Figure 8). Furthermore, Rhizobium meliloti strain A-025 showed the ability to survive in the presence of PCBs for more than one year at different depths of soil columns.
EXAMPLE 6 : Atrazine and Nitrate Bio~ltration and Bioremediation by Rhizobium meliloti R. meliloti strain A-025 was subcultured in 6 L of fresh Tyc and incubated at 29°C in a controlled environment incubator shaker (Psycrotherm, New Brunswick Scientific) for 24 hours. A sample of the culture was plated on TYct agar plates for microbial population counts. The bacterial cells (approximately 3.3x108 cell mL-') were then collected by centrifugation, washed with 0.9% saline solution, resuspended in 200mL of sterilized deionized water and mixed by vortexing the inoculum before application to sterile soil columns. The columns, 458 mm long and 139 mm in diameter, were fitted with a sampling port on the side at a 298 mm depth and a delivery pipe at the bottom to supply water and bacterial inoculum (U.S.
Pat. # 6,027.284). All soil columns first received 300mL of tap water from the bottom (subirrigation), followed by inoculation with 200mL of cell suspensions for columns assigned to bacterial inoculation (i.e., treatments 1 and 2.). The columns were then saturated from the bottom by adding 2400m1 and 2600mL of water to the treatment and the abiotic control columns, respectively.

Eight days after bacterial augmentation of the soil columns, 300 ~L of atrazine (1000 ppm stock solution, 90% active), and 300 mg of calcium nitrate 4-hydrate, representing approximately 180 Kg hectare-' of fertilizer, were uniformly applied to the soil surface. During the first 44 days after application of atrazine and nitrate, 20 mm (450 mL) of water was applied as rain simulations, on the 9t" day and every 7 days thereafter to all columns for a total application of 120 mm of rainfall. In order to observe the effect of an unusually heavy rainfall, on the 80th day after chemical inoculation the equivalent of 60 mm of rainfall (1,350 mL) was applied. On the 8T" day the columns were drained, and three more rain simulations of 40 mm were applied on the 1041", 112t" and 150t" day.
Water samples were collected at the bottom of the columns after every water application (simulated rainfall) for analysis of leached atrazine, nitrate, and microbial counts. Soil samples (20 g) were collected through sampling ports on the side of the column before each water application during the unsaturated period to analyze atrazine and nitrate residues (Figures 9 and 10). Nitrate was measured by the Soil Testing Laboratory of the Natural Resources Science Department of the Macdonald Campus of McGill University, using a Quikchem Automated Ion Analyser. Atrazine analysis was performed as described by Liaghat et al. (1996), Liaghat and Prasher (1996), and Masse et al. (1994). Water samples were extracted by mixing 200mL of the sample with 50mL of methylene chloride in a separatory funnel. The mixture was hand-shaken for 5 minutes and the organic layer was collected. The process was repeated three times and the extracts were pooled and evaporated to dryness. Residues were dissolved in 10mL of hexane and analyzed by gas chromatography (GC).
Soil samples were extracted by shaking 10 g of soil in 100 mL of methanol for 60 minutes and filtered under suction. The filtrate was then evaporated to dryness in a rotary evaporator at 35°C. Residues were dissolved in 10mL of hexane and analyzed with a GC (Figure 9). The GC was a VarianT"", model 3400, equipped with a TSD detector, an autosampler, and an integrator. The column was a 0.53mm i.d. fused silica MegaboreT"" DB-5. The detector and injector were kept at 290°C and 190°C, respectively.
The column was maintained at 150°C for 10 minutes and then the temperature was raised to 180°C at a rate of 2.5°C min-~~ The helium 5 carrier gas flow was 15mL min-'.
For bacterial population counts, serial dilutions of the drain water (up to 10 4 fold) were spread on TYct agar plates (restrictive media for R. meliloti~
and incubated at 29°C. The plates were incubated for a period of 15 days 10 and the number of colonies determined periodically. The plates from the control treatment showed no growth of bacterial colonies.
Conclusions 15 In conclusion, bioaugmentation of soil amended with atrazine and nitrate with strain A-025 reduced the concentration of atrazine by more than 30%
in the drainage water. The level of nitrate-N was also significantly reduced in the drainage water during anaerobic conditions.
20 EXAMPLE 7 : Tolerance and Survival of Different Species of Rhizobia during Enrichment with Atrazine Native populations of various rhizobial species were isolated directly from agricultural soils (S-XI and S-XII) from farms with atrazine application 25 (Table 1 ) and after 6 cycles of enrichment on atrazine minimal medium, using a standard modulation test on ten varieties of leguminous plant (Example 1; Somasegaran and Hoben, 1994). The results, presented in Table 3 suggest that certain species of rhizobia, such as those modulating chickpea and Fava beans, were absent in the two soils exposed to 30 atrazine. Furthermore, among the species initially present in the soil, some (such as those modulating pole bean, pea and soybean), appeared to be sensitive to enrichment with atrazine, while some others (such as those modulating alfalfa, Romano beans, red and white kidney beans and clover) seemed tolerant. Some of these tolerant species (strains B5, M8, P1, P2, T10) were purified for further study and use.
EXAMPLE 8 : Growth of Different Species of Rhizobia on Atrazine and Cyanuric Acid Five purified strains of rhizobia isolated from farm soils exposed to atrazine (Example 7), namely strains B5, M8, P1, P2, T10, were tested for their ability to use atrazine (0.1 % or 1000 ppm) as a carbon and/or nitrogen source. The test was performed using agar plates with minimal media of different compositions, and the growth of these microorganisms was recorded (Table 4). Since all known metabolic pathways for atrazine lead to the production of cyanuric acid which is then mineralized through other pathways, growth of the selected strains on cyanuric acid minimal media (0.05% or 500 ppm) having different compositions was tested as well (Table 5). All strains seemed to grow best when atrazine and cyanuric acid were provided as a nitrogen source, and glucose as a carbon and energy source.
EXAMPLE 9: Mineralization of '4C-Atrazine by Different Species of Rhizobia Five rhizobial strains isolated from soils with application of atrazine, namely strains B5, M8, P1, P2, and T10, were tested for their ability to mineralize '4C-atrazine ( 0.01 % or 100 ppm, with 50,000 dpm of labelled atrazine) as a N source, essentially following the protocol described by Struthers et al. (1998). The test was performed in serum bottles with assay mixtures inoculated with cells precultured in TYc with atrazine (0.02 %:
200 ppm) to an ODsoo between 0.6 - 0.8, incubated at 29°C with shaking for 72 hours. The total '4C02 trapped in NaOH solution was determined (Figure 11 ). All strains tested showed some level of mineralization, and the performance of strain B5 was better than all other strains tested.

EXAMPLE 10 : Biotransformation of TNT by Rhizobium meliloti Strain M8, isolated from atrazine soil, was tested for its ability to transform TNT (trinitro toluene, an explosive compound) in the presence of glucose (0.5%) and glutamate (0.025%). The test was performed in serum bottles with assay mixtures inoculated with cells precultured in minimal media with TNT (40 ppm) and glucose (0.5%) to an ODsoo of 0.7, incubated at 29C°
under static conditions, essentially following the protocol of Hawari, et al.
(1998). The TNT and metabolites were followed by HPLC. The results showed transformation and depletion of TNT (Table 6). No TNT
transformation was observed in either the chemical or biological control.
EXAMPLE 11: Use of Rhizobial cultures for crop enhancement (Biofertilizer) The fertilizing potential of 3 species of Rhizobia (A025: Rhizobium meliloti # PTA-1457; B5: Rhizobium leguminosarum bv. phaseoli # PTA-1458;
and P1: Rhizobium leguminosarum bv. viceae # ATTC 700900) and native consortium bacteria on corn yield were determined in a corn field study (Figure 12).
The field experiment was performed in a fine sandy loam soil with a pH of 6.8 and 3% organic matter. There was no history of fertilizer and herbicide applications of the site in the preceding two years. The experimental field was divided into twenty four 2 m2 plots. The following eight treatments were performed (in triplicate): 1) 4g of A025 (Rhizobium melilofi~ + corn, 2) 4g of B5 (R. leguminosarum bv. phaseoh~ + corn, 3) 4g of P1 (R.
leguminosarum bv. viceae) + corn, 4) 4g of indigenous consortium of soil microorganisms + corn, 5) 4g mixture of A025, B5, and P1 + corn, 6) 8g mixture of A025, B5, and P1 + corn, 7) 8g of indigenous consortium +
corn, and 8) Control + corn.
('_nnclmeinne The corn seed germination was highest (72% higher than treatment 8, control) in treatments 3 and 6. Treatment 6 also showed 52% higher number of flowering plants than the control, whereas, treatment 2 showed 86% more corncobs than the control. The highest yield in corn (40%
higher than the control) was observed to be in treatment 6 which was augmented with 8 g of Rhizobial mixture (Figure 12).
EXAMPLE 12: Use of Rhizobial cultures as bioherbicides The allelopathic potential of 3 species of Rhizobia (A025: Rhizobium meliloti # PTA-1457; B5: Rhizobium leguminosarum bv. phaseoli # PTA-1458; and P1: Rhizobium leguminosarum bv. viceae # ATTC 700900) and native consortium bacteria on six economically important weeds in vitro and in corn field was studied. More specifically, four bacterial cultures at different concentrations were used to bioaugment field plots to determine their allelopathic effect on six different weed species: Digitaria sanguinalis (crabgrass), Echinochloa crusgalli (barnyard grass), Setaria glauca (yellow foxtail grass), Amaranthus retroflexus (redroot pigweed), Chenopodium album (lambsquarters), and Abutilon theophrastii (velvetleaf). The experimental design was like that described in Example 11. Plots used in treatment 9, control plots without corn plants, were 0.25 m2 in size and were situated in between the plots with corn plants.
Conclusions The results of the field study showed different variability between the treatments for the parameters measured. In the sub-plots, treatments 3 and 7 showed 63% and treatment 3 showed 77% less monocot and dicot weeds as compared to treatment 9, whereas, 33% and 80% less as compared to treatment 8, respectively (Table 7). The lowest weight in monocot weeds, 93% and 66% lower than in treatment 9 and 8, respectively, was obtained in treatment 5. The lowest weight in dicot weeds, 94% and 89% lower than in treatment 9 and 8, respectively, was obtained in treatment 6 (Figure 13).
Deposit Information Six rhizobial strains were deposited with the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, VA 20110. Of the six strains, three were deposited with the Patent Depository and three with the General Bacteriology Collection, as indicated more particularly below.
Rhizobial Strains Deposited with the Patent Depository (Effective date : March 22, 2000) Strain Designation Rhizobium meliloti, A-025 PTA-1457 Rhizobium leguminosarum bv. phaseoli, B5 PTA-1458 Rhizobium leguminosarum bv. trifolii, T10 PTA-1459 Rhizobial Strains Deposited with the General Bacteriology Collection (Effective date : January 7, 2000) Strain Designation Rhizobium meliloti, M8 ATCC 700899 Rhizobium leguminosarum bv. viceae, P1 ATCC 700900 Rhizobium leguminosarum bv, viceae, P2 ATCC 700901 The patent deposits will be maintained in the depository for a period of 30 years, or 5 years after the last request, or for the effective life of the patent, whichever is longer, and will be replaced as necessary during that period.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the invention, as limited only by the scope of the appended claims.

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Claims (28)

44

1. A method for isolating a bacterium from the genus Rhizobium having the ability to bioremediate, degrade or cleanse harmful or unwanted industrial, military or agricultural chemicals in a soil or water environment, comprising the steps of:
1) collecting a soil or water sample from a contaminated environment;
2) isolating native rhizobial species by trapping them with leguminous plant seedlings; and 3) screening said native rhizobial species for their ability to tolerate and to destroy, reduce or stabilize contaminants in said soil or water sample.

2. A method as defined by claim 1, wherein said bean seedlings are selected from the group consisting of alfalfa, bean, clover, pea, soybean, chickpea, lentil, lupin and other leguminous plants.

3. A method as defined in claim 1, wherein said contaminants are organic or inorganic.

4. A method as defined in claim 3, wherein said organic contaminants are PCBs, TNT or atrazine.

5. A method as defined in claim 3, wherein said inorganic contaminants are nitrate.

6. A method for isolating a bacterium from the genus Rhizobium having the ability to act as a biofertilizer in a soil environment, comprising the steps of:
1) collecting a soil or water sample from a contaminated environment;
2) isolating native rhizobial species by trapping them with leguminous plant seedlings; and 3) testing said native rhizobial species for their ex planta nitrogen fixation ability, phosphate solubilization and iron chelating capability.

7. A method for isolating a bacterium from the genus Rhizobium having the ability to act as a bioherbicide in a soil environment, comprising the steps of:
1) collecting a soil or water sample from a contaminated environment;
2) isolating native rhizobial species by trapping them with leguminous plant seedlings; and 3) testing said native rhizobial species for their herbicidal activity.

8. A bacterium from the genus Rhizobium isolated by the method of any one of claims 1 to 7.

9. Use of the bacterium of claim 8 in a process for bioremediating, degrading or cleansing harmful or unwanted industrial, military or agricultural chemicals in a soil or water environment.

10. The use of claim 9 wherein said harmful or unwanted industrial, military or agricultural chemicals are PCBs, TNT, atrazine or nitrate.

11. Use of the bacterium of claim 8 as a biofertilizer or bioherbicide to improve the quality of agricultural or recreational sites.

12. A process for bioremediating, degrading or cleansing harmful or unwanted industrial, military or agricultural chemicals in a soil or water environment comprising the use of a culture of a bacterial species belonging to the genus Rhizobium selected for its ability to transform or metabolize said chemicals.

13. A process for bioremediating, degrading or cleansing harmful or unwanted industrial, military or agricultural chemicals in a soil or water environment comprising the use of a culture of a bacterial species belonging to the genus Rhizobium preselected and prescreened from a library for its ability to transform or metabolize said chemicals.

14. A process for bioremediating, degrading or cleansing harmful or unwanted industrial, military or agricultural chemicals in a soil or water environment comprising the use of a culture of a bacterial species belonging to the genus Rhizobium adapted for its ability to transform or metabolize said chemicals through several cycles of growth in the presence of said chemicals.

15. A process as defined in any one of claims 12 to 14, further comprising the use of one or more components selected from the group consisting of the following: bacterial species belonging to a genus other than Rhizobium and having the ability to transform or metabolize said chemicals; chemical nutrients that are required for growth; inducers that bring about the degradation pathways;
cosubstrates or plant products that enhance the degradation activity; environmental conditioners; and modifiers that positively affect the degradation activity.

16. The process of claim 15, wherein said cosubstrates or plant products are terpenes, oils or flavonoids.

17. A method of treating a soil or water environment contaminated with harmful or unwanted industrial, military or agricultural chemicals consisting of planting a suitable host leguminous plant crop in said soil or water environment in order to speed up the bioremediation, degrading or cleansing of said chemicals by the implanted rhizobial strains.

18. A Rhizobium meliloti strain designated A-025 and having ATCC
Accession No. PTA-1457.

19. A Rhizobium leguminosarum bv. phaseoli strain designated B5 and having ATCC Accession No. PTA-1458.

20. A Rhizobium leguminosarum bv. trifolii strain designated T10 and having ATCC Accession No. PTA-1459.

21. A Rhizobium meliloti strain designated M8 and having ATCC No.
700899.

22. A Rhizobium leguminosarum bv. viceae strain designated P1 and having ATCC No. 700900.

23. A Rhizobium leguminosarum bv. viceae strain designated P2 and having ATCC No. 700901.

24. A process as defined in any one of claims 12 to 16, wherein said bacterial species is selected from the group consisting of: A
Rhizobium meliloti strain designated A-025 and having ATCC
Accession No. PTA-1457; A Rhizobium leguminosarum bv.
phaseoli strain designated B5 and having ATCC Accession No.
PTA-1458; A Rhizobium leguminosarum bv. trifolii strain designated T10 and having ATCC Accession No. PTA-1459; A Rhizobium meliloti strain designated M8 and having ATCC No. 700899; A
Rhizobium leguminosarum bv. viceae strain designated P1 and having ATCC No. 700900; and A Rhizobium leguminosarum bv.
viceae strain designated P2 and having ATCC No. 700901.

25. The process of claim 24, further comprising the use of one or more additional cultures of bacterial species belonging to the genus Rhizobium.

26. The process of claim 25, wherein said bacterial species species is selected from the group consisting of: A Rhizobium meliloti strain designated A-025 and having ATCC Accession No. PTA-1457; A
Rhizobium leguminosarum bv. phaseoli strain designated B5 and having ATCC Accession No. PTA-1458; A Rhizobium leguminosarum bv. trifolii strain designated T10 and having ATCC
Accession No. PTA-1459; A Rhizobium meliloti strain designated M8 and having ATCC No. 700899; A Rhizobium leguminosarum bv.
viceae strain designated P1 and having ATCC No. 700900; and A
Rhizobium leguminosarum bv, viceae strain designated P2 and having ATCC No. 700901.

27. A process as defined in any one of claims 12 to 16 or 24 to 26 wherein said chemicals are chosen from the group consisting of PCBs, TNT, atrazine and nitrate.

28. The method of claim 17 wherein said chemicals are chosen from the group consisting of PCBs, TNT, atrazine and nitrate.