WO1992000370A1 - Oxalate degrading bacterium - Google Patents

Abstract

A new oxalate degrading bacterial species, variant B-1, belonging to the genus Pseudomonas and characterized by deposit ATCC 53883 is provided.

Description

OXALATE DEGRADING BACTERIUM

FIELD OF THE INVENTION

This invention relates to microorganisms capable of degrading oxalates and to their use for bioremediation of oxalate-rich wastes. More particularly, this invention relates to a new oxalate degrading bacterial species belonging to the genus Pseudomonas, the species variant B- 1.

BACKGROUND OF THE INVENTION

As awareness of the need for environmental

protection increases, new methods are being developed for safe and efficacious decomposition of industrial wastes.

Oxalate-containing effluents are generated, for example, by metal working processes, including anodizing and preparation of metals to receive protective coatings, in the manufacture of textiles, leather and paper and in the mining industry, as an ingredient of froth flotation agents and in the processing of bauxite by the Bayer process to produce aluminum. The accumulation of oxalate ions in process effluents presents a disposal problem. The solid oxalates may be buried or may be burned in a kiln; the former method of disposal may not be

environmentally desirable and the latter is energyconsuming and expensive.

Relatively few microorganisms are known to decompose oxalates and bacterial strains with this ability are even fewer. If a bacterial strain is to be useful for bioremediation of oxalate-rich effluents in a variety of situations, it should be fully characterized and its safety and efficacy established.

Prior literature, which discloses various

investigations on microorganisms which are capable of degrading oxalates are listed below. For ease of

reference throughout the remainder of this specification, the following articles shall be referred to by number in parenthesis: 1. Dijkhuizen , L. , M. Wiersma and W. Harder (1977) "Energy Production and Growth of Pseudomonas oxalaticus Oxl on Oxalate and Formate" , Archives of Microbiology 115 : 229-236

2. Dijkhuisen, L. and W. Harder (1979) "Regulation of Autotrophic and Heterotrophic Metabolism in

Pseudomonas oxalaticus 0X1:Growth on Mixtures of Oxalate and Formate in Continuous Culture", Archives of Microbiology 123:55-63.

3. Dijkhuisen, L. , B. van der Werf and W. Harder (1980) "Metabolic Regulation in Pseudomonas oxalaticus 0X1.

Diauxic Growth on Mixtures of Oxalate and Formate or Acetate", Archives of Microbiology 124:261-268.

4. Quayle, J.R. and D.B. Keech (1959a). "Carbon

Assimilation by Pseudomonas oxalaticus 0X1. 1. Formate and Carbon Dioxide Utilization during Growth on Formate", Biochemistry Journal 72:623-630.

5. Quayle, J.R. and D.B. Keech (1959b) . "Carbon

Assimilation by Pseudomonas oxalaticus 0X1. 2. Formate and Carbon Dioxide Utilization by Cell-Free Extracts of the Organisms Grown on Formate", Biochemistry Journal 72:631-637.

6. Quayle, J.R. and D.B. Keech (1960) . "Carbon

Assimilation by Pseudomonas oxalaticus 0X1. 3. Oxalate Utilization during Growth on Oxalate", Biochemistry Journal 75:515-523.

7. Quayle, J.R., D.B. Keech, and G.A. Taylor (1961).

"Carbon Assimilation by Pseudomonas oxalaticus 0X1. 4. Metabolism of Oxalate in Cell-Free Extracts of the Organism Grown on Oxalate", Biochemistry Journal 78:225- 236.

8. Blackmore, M.A., I.R. Quayle and I.O. Walker (1968) "Choice Between Autotrophy and Heterotrophy in

Pseudomonas oxalaticus Utilization of Oxalate by Cells after Adaptation from Growth on Formate to Growth on Oxalate", Biochemistry Journal 107:699-704. 9. ATCC Catalogue of Strains, 15th Edition, 1982, P.

176.

10. Bhat, J.V. and Barker, H.A. (1948), J. Bacteriol . , Vol 55, pp. 359-368.

11. Jayasuriya, G.C.N. (1955) J. Gen. Microbiol . Vol 12; pp 419-428.

12. Chandra, T.S. and Shethna, Y.I. (1975), Antonie van Leeuwenhoek, Vol. 41, pp 101-111.

13. Chandra, T.S. and Shethna, Y.I. (1975), Antonie van Leeuwenhoek, Vol. 41, pp 465-477. 14. Ogle, J.W., Jandra, J.M., Woods, D.E. and Vasil,

M.L. (1987), J. Infec. Dis . Vol. 155, pp 119-126

The series of papers by Dijkhuizen et al and Quayle et al (references 1 to 8) refer to an oxalate-degrading bacterial species which they called Pseudomonas oxalaticus, but which is now renamed Alcaligenes oxalaticus species. The strains of this organism in the ATCC collection are described as having peritrichous flagellation (9), indicating that these strains were wrongly classified as pseudomonads.

Bhat and Barker (10) have isolated from soil a species, Vibrio oxaliticus which can decompose oxalate. An oxalate-decomposing bacterial strain identified as ODI by Jayasuriya (11) was partially characterized and appeared to be a species of Pseudonomas .

Chandra and Shethna (12, 13) have also isolated and partially characterized Pseudonomas strains which degrade oxalate.

SUMMARY OF THE INVENTION

According to an aspect of the invention, a

biologically pure culture of a novel Pseudomonas

microorganism is isolated from rhizosphere soil

surrounding a plant which produces oxalates. The

Pseudomonas microorganism is characterized by deposit ATCC 53883.

According to another aspect of the invention, a biologically pure culture of the microorganism Pseudomonas sp B-1 characterized by the deposit ATCC 53883.

According to another aspect of the invention, a biologically pure microorganism Pseudomonas sp B-1 is isolated from rhizosphere soil surrounding a Rhubarb plant or a Dieffenbachia plant and having the following taxonomic data:

i) the following characteristics common to

Pseudomonas strains:

Gram negative +

Aerobic metabolism +

Polar flagella +

Motility +

Oxidase weak and slow

Catalase +

ii) resembles Xylophilis ampelina

iii) differs from Xylophilis ampelina in the following distinct characteristics :

X. ampelina B-l

Utilization oxalate as

sole carbon source - + Acid from O-F arabinose +

Urease +

Growth Factor Requirements + BRIEF DESCRIPTION OF THE DRAWINGS

The invention, as exemplified by a preferred embodiment, is described with reference to the drawings in which:

Figure 1 shows a Transmission Electron Micrograph (TEM) of a negatively stained preparation of B-1;

Figure 2 shows a TEM of B-1;

Figure 3 shows a TEM of a negatively stained

preparation of B-1. The bar indicates a scale of 1.0 μm;

Figure 4 shows a TEM of a ruthenium red stained section of cells of B-1. The bar indicates a scale of 1.0 μm;

Figure 5 shows a high magnification TEM of a section of ruthenium red stained cells of B-1. The bar indicates a scale of 1.0 μm;

Figure 6 shows a diagram of several types of

bacterial cell wall;

Figure 7 shows a flowchart of taxonomic

characterization of B-1;

Figure 8 shows the absorption spectrum of a

methanol-chloroform extract of B-1;

Figure 9 shows tolerance of B-1 to high levels of oxalate;

Figure 10 shows oxalate degradation by B-1; and Figure 11 shows oxalate degradation by B-1 at half normal concentration of medium constituents.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Oxalates in industrial wastes are presenting an increasing problem from the standpoint of disposal into the environment. There is available an energy intensive process for ridding industrial waste of oxalates. The process involves the calcination of the industrial waste; that is burning of waste at a temperature in the rang2 of 800°C to yield an Na₂CO₃ or calcination at a temperature in the range of 500°C after reaction of the industrial waste with lime to yield CaCO₃. Both of these calcination processes are energy intensive and require a special type of kiln capable of handling fine solids. Although there is a variety of industrial wastes which include oxalates, as described above, one important source of oxalates is derived from the Bayer process which relates to the processing of bauxite for the production of alumina.

Further details of the Bayer process and its resultant production of industrial waste including oxalates is disclosed in applicant's copending Canadian patent application entitled "Biodegradation of Oxalate Ions in Aqueous Solution" S.N. 574,176 filed August 9, 1988.

The microorganism, according to this invention, which is particularly suited to degrading oxalates in a variety of industrial wastes to thereby provide an oxalate-reduced or oxalate-free waste stream was

discovered from soils surrounding oxalate producing plants. The isolated microorganism was stressed in an oxalate rich media and then subjected to a purification process. The microorganism, according to this invention, under appropriate conditions degrades oxalates in various types of wastes by the oxalate providing a carbon source for the metabolism of the microorganism. As the

microorganism metabolizes the oxalates, the oxalates are transformed into biomass and possible carbon dioxide. By way of taxonomical characterization, it has been

discovered that the microorganism as isolated in

accordance with the above procedure, is a novel

microorganism. It is thought that this microorganism exists in soils surrounding any form of oxalate producing plant, such as Dieffenbachia picta (Dieffenbachia), Jatropha padogrica; Monstera ssp; Philodendron spp; Bheum rhaponticum

(Rhubarb). Preferably the microorganism of this

invention is isolated from soils surrounding the Rhubarb or Dieffenbachia plant. Particular details of the manner in which the microorganism of this invention was isolated from ground surrounding Rhubarb and Dieffenbachia plants is set out in the following Examples.

By way of detailed taxonomical characterization, it was found that the microorganism is of the Pseudomonas genus, but it not similar to any known species of

Pseudomonas . The species Of Pseudomonas has therefore been identified as species B-1. The microorganism is on deposit at ATCC under No. 53883, deposited March 27, 1989 in accordance with the requirements of the Budapest

Treaty.

In use the microorganisms of this invention may be introduced to an oxalate containing waste under

conditions which allow the microorganism to grow and by metabolism degrade the oxalates into biomass and possibly carbon dioxide. A particular example of the manner in which the microorganism of this invention may be employed in degrading oxalates is disclosed in applicant's

aforementioned co-pending Canadian patent application SN 574,176. It is appreciated, however, that in a variety of other oxalate rich effluents, the microorganisms of this invention may be employed, it being appreciated by those skilled in the art that such other forms of oxalate containing effluents may require pre-treatment to avoid any other types of contaminants killing off the

microorganisms. It is appreciated that, in accordance with standard culture techniques, the oxalate containing sample to be treated by the microorganism of this

invention may be cultured on an experimental basis in the newly considered effluent to determine its growth

characteristics in that particular effluent. As set out in the following Examples, details of the taxonomical characteristics of the microorganism are provided to distinguish it from all other types of known Pseudomonas microorganisms. EXAMPLE 1 - Isolation of Oxalate-Decomposing Species

Pseudomonas Variant B-1

The oxalate degrading bacteria were isolated from the oxalate-rich rhizosphere soil of two different types of plants, Rhubarb (Ru) and Dieffenbachia (DE) . One gram of soil from each plant root zone was added to a sterile 250 mL flask containing 100 mL of Oxa-1 medium adjusted to 7.0 with NaOH. The flasks were incubated for 72 hours at 28°C on a rotary shaker at 150 rpm. This culture was then used to inoculate subsequent flasks containing the same medium in order to enrich for oxalate degrading microorganisms. After the third passage, a mixed culture of bacteria was obtained from each soil type that grew in oxalic acid medium.

The Ru mixed culture was then used to inoculate a rotating biological contactor which was operated using oxalic acid medium. The above culture was then

inoculated onto nutrient agar, and oxalate plates from a dilution series to obtain approximately 100 colonies per plate. The plates were then incubated at 28°C for 48 hours. After the incubation period, colonies were picked according to their morphology and color and streaked out on oxalic acid plates and nutrient agar plates and incubated at 28°C for 48 hours to ensure pure cultures. Material was stored in a conventional manner and used in further characterization studies.

EXAMPLE 2

Three one gram soil samples were obtained: Rhubarb soil from Embrun, Ontario (RuE), Rhubarb soil from

Richmond, Ontario (RuR), and Dieffenbachia soil from Ottawa, Ontario (DE) and were placed in 100 mL of Oxa-1 medium containing NaH₂PO₄.H₂O, 0.5 g/L; MgS0₄.7H₂O, 0.1 g/L; (NH₄)₂S0₄.7H₂O, 0.5 g/L; FeSO₄.7H₂O, 0.05 g/L; Yeast

Extract, 0.1 g/L; oxalic acid 5 g/L; Distilled water,

1000 mL. The medium had been autoclaved, cooled, and the oxalic acid was added at room temperature. The medium was adjusted to pH 7.0 with sterile NaOH. The flasks containing the medium and the soil sample were incubated at 28°C and 150 rpm for 48 hours. After 48 hours, the cultures obtained were subcultured into Oxa-1 media using a 5% inoculum. After two serial transfers into Oxa-1 medium, the resultant oxalate degrading cultures were plated out on solid Oxa-1 medium (3% agarose), at various dilutions, to obtain the individual oxalate degrading bacteria (Table 1). The results show that at the higher dilutions, all of the organisms present on the Oxa-1 plates resemble B-1. At the lower dilutions, the

percentage of B-1 like organisms decreases. This was probably due to other scavenging organisms taking up space on the plates.

Isolates were observed under a light microscope for cellular morphology and Gram staining characteristics and physiological and biochemical tests were performed using Hoffmann-LaRoche diagnostic tests for Gram-negative bacteria. The data obtained are set out in Table 2.

Isolates RuE and DE are identical to variant B-1.

Isolate RuR differed from B-1 in its growth on dulcitol and on medium used to detect the deamination of

phenylalanine to pyruvate.

TABLE 1

BACTERIAL COUNTS FROM ISOLATION

Yellow Total Yellow

Dil. Bacteria Bacteria Bacteria

Sample 10^-x Cells/mL Cells/mL Percent

RuE- 1a 8 9.00 × 10⁸ 9.00 × 10⁸ 100.0

RuE- 1b 8 1.10 × 10⁹ 1.80 × 10⁹ 61.1

RuE-2a 7 5.10 x 10⁸ 7.30 × 10⁸ 69.9

RuE-2b 7 7.90 × 10⁸ 1.03 × 10⁹ 76.7

RuE-3a 6 1.07 × 10⁸ 5.01 × 10⁸ 21.3

RUE- 3b 6 1.16 × 10⁸ 4.39 × 10⁸ 26.4

RuR- 1a 8 4.00 × 10⁸ 4.00 × 10⁸ 100.0

RuR-1b 8 3.00 × 10⁸ 3.00 × 10⁸ 100.0

RuR-2a 7 1.80 × 10⁸ 3.90 × 10⁸ 46.1

RuR-2b 7 2.30 × 10⁸ 4.00 × 10⁸ 57.5

RuR- 3 a 6 1.00 × 10⁷ 3.33 × 10⁸ 3.0

RuR-3b 6 1.30 × 10⁷ 3.48 × 10⁸ 3.7

DE-1a 8 1.00 × 10⁸ 1.00 × 10⁸ 100.0

DE-1b 8 2.00 × 10⁸ 2.00 × 10⁸ 100.0

DE-2a 7 1.30 × 10⁸ 1.30 × 10⁸ 100.0

DE-2b 7 2.30 × 10⁸ 2.70 × 10⁸ 85.2

DE-3a 6 2.20 × 10⁷ 1.26 × 10⁸ 17.5

DE-3b 6 2.20 × 10⁷ 1.03 × 10⁸ 21.3

SUBSTITUTE SHEET TABLE 2

PHYSIOLOGY AND BIOCHEMISTRY OF ISOLATES

Isolates Tests B-1 RuE RUR DE Yellow + + + +

Rod (0.5 um in diameter) + + + +

Oxalate + + + +

Gram Staining

Characteristics - - - - Oxidase - - - -

Glucose - - - -

Gas from Glucose - - - -

Lysine Decarboxylase - - - - Ornithine Decarboxylase - - - - Arginine Dihydrolase - - - - Hydrogen sulfide - - - -

Indole - - - -

Adonitol - - - -

Lactose - - - - Arabinose - - - -Sorbitol - - - -

Dulcitol - - + -

Phenylalanine Deaminase - - + - Urea - - - - Citrate - - - -

Xylose

- - - -

EXAMPLE 3

Two soils from Alberta (garden loam from University of Alberta greenhouse and overburden from the dumpsite at St. Claire) and one forest soil from North Carolina were enriched with oxalate by blending in powdered sodium oxalate, and then were agitated intermittently ove a two week period.

The soils were extracted with Phosphate Buffered Saline (PBS) and the supernatants from these extractions were diluted in PBS blanks and plated on oxalate medium. Yellow colonies predominated in all of the highest dilutions showing growth on the oxalate medium. When these yellow colonies were purified and subjected to the traditional taxonomic analysis (carbon source,

morphology, enzyme activity) they proved to be identical to variant B-1.

From the studies of Examples 2 and 3, Pseudomonas variant B-1 is seen to be a ubiquitous organism which can be consistently isolated from oxalate-enriched soils.

EXAMPLE 4

Cultures of B-1, isolated as in Example 1, were grown up, cells were chemically fixed and embedded by standard techniques and thin sections of ruthenium red stained cells were examined by Transmission Electron Microscopy (TEM) and their cell envelope structure was compared with that of several Pseudomonas species (P.

aeruginosa, P. cepacia,. P . fluorescens) . The B-1 cells were found to be rod-shaped with single polar flagella

(Figures 1, 2 and 3) and to have the gracilocutes

structures typical of Gram-negative bacteria (Figures 4 to 6). Detailed examination of the fine structure of their cell envelopes showed the presence of a discernible peptidoglycan layer (Figures 2, 4 to 6) that is never seen in the cell wall of any of the Pseudomonas species listed above. The peptidoglycan layer of the cell wall of B-1 is indicated by the arrows in Figures 4 and 5. B 1 is a Gram-negative rod-shaped organism not identical with Pseudonomas aeruginosa, P . cepacia or P . fluorescens.

EXAMPLE 5

Cultures of B-1, isolated as in Example 1, were grown up and used for a variety of characterization studies. The results of some of these studies are shown in Tables 3 to 6 and Figure 7. Tables 4, 5 and 6 set out data obtained by the American Type Culture Collection. (ATCC). Note that while the ATCC found no growth at 37°C, the inventors found consistent growth at this temperature.

These characterization studies on variant B-1 indicate that it belongs to the genus Pseudomonas, but does not correspond to any of the known pathogenic species of this genus listed in Figure 7.

The biochemical, physiological and nutritional characteristics of B-1 are not in agreement with

published data or ATCC strain data from any known species of Pseudomonas.

B-1 is able to utilize oxalate as its sole carbon source, as seen in Table 3.

TABLE 3

GROWTH OF B-l ON VARIOUS CARBON SOURCES

SOURCE GROWTH SOURCE GROWTH

D-glucose - L-malate + lactose - pelargonate - maltose - propionate - sucrose - succinate +

D-fructose + L-tartrate -

D-ribose + DL-alanine +

L-rhamnose - betaine -

L-arabinose - glycine - cellobiose - L-histidine -

D-mannitol - DL-norleucine -

D-sorbitol - L-proline - ethanol - L-valine - methanol - DL-arganine - glycerol - benzylamine - adonitol - butylamine - benzoate - puterescine -

D-gluconate - D-tryptopan -

M-hydroxybenzoate - L-tryptophane -

2-ketogluconate + mesaconate -

DL-lactate + DL-glycerate +

oxalate +

TABLE 4

COMPARISON OF B-l WITH TYPICAL PSEUDOMONAS

Pseudomonas B=1

Gram negative + +

Aerobic metabolism + +

Polar flagella + +

Motility + +

Oxidase + or - weak and slow Catalase + +

TABLE 5

COMPARISON OF B-1 WITH XYLOPHILIS AMPELINA

X . ampelina Isolate B-1

Flagella monotrichous + +

Growth at 4°C, 37°C - -

Yellow pigment + +

Very slow sparse growth + +

Gelatin - -

Nitrate reduction - -

Phenylalanine deamination N.G. N.G.

Catalase + +

Oxidase - slow & weak

Arginine, ornithine,

lysine decarboxylation - -

Acid from:

O-F D-xylose - -

D-ribose - -

L-rhamnose - -

D-glucose - -

D-mannose - -

D-fructose - - sucrose - - trehalose - - cellobiose - - lactose - - maltose - - dulcitol - -

D-mannitol - -

D-sorbitol - -

Utilization of Sole Carbon Sources

benzoate - _ gluconate - - propionate - - malonate - -

DL-malate + + succinate - -

Summary of Significant Differences Between the Organisms:

Utilization of oxalate as

sole carbon sources - +

Acid from O-F arabinose + -

Urease + -

Growth Factor Requirements + - N.G. = no growth TABLE 6

CHARACTERISTICS OF VARIANT B-1 Physiology and Biochemistry

Gram positive -

Gram negative +

Gram variable - Motile at RT +

Flagella peritrichous -

Flagella lophotrichous -

Flagella monotrichous +

Flagella lateral - 4°C growth -

25°C growth +

30°C growth +

37°C growth -

4l°C growth - Fluorescein produced -

Pyocyanine produced -

Diffusible orange -

Diffusible yellow -

Diffusible purple - Non-diffusible green -

Other non-diff. pigments Y

Melanin pigment produced - pH 6.0 growth +

3% NaCl growth +

6.5% NaCl growth -

MacConkey agar growth +

Skim milk agar growth W

Aesculin hydrolysis -

Casein hydrolysis - Starch hydrolysis -

Gelatinase -

Tween 20 hydrolysis +

Tween 80 hydrolysis -

Indole - Simmons citrate growth W

Urease -

Nitrate to nitrite -

Nitrite reduction -

Nitrite to nitrogen gas - Hydrogen sulfide (TSI)* -

Lead acetate strip * +

Lysine decarboxylase -

Arginine (Mollers) -

Ornithine decarboxylase - Phenylalanine deamination NG

Phosphatase -

Catalase +

Oxidase slow & weak +

Gluconate oxidation - Growth on Malonate as SCS -

Tyrosine degradation - d1-hydroxybutyrate growth - growth on 0.05% cetrimide - Growth on acetate as SCS - Testosterone deg. - Mucoid growth on glucose

agar

0.1% TTC growth - 0.02% TTC growth + Litmus Milk acid - Litmus milk peptonized -

W = weakly positive

Y = yellow

TTC = Triphenyl Tetrazolium chloride

Reactions in Hugh & Leifson O-F Medium

Acid from:

L-arabinose K cellobiose K ethanol K

D-fructose K

D-glucose A02 K

D-glucose An02 -

Alkaline pH in D-glucose +

Acid from:

glycerol K i-inositol K lactose K maltose K

D-mannitol K

D-mannose K

L-rhamnose K

D-ribose - sucrose . K trehalose K

D-xylose K adonitol K dulcitol K

D-galactose K inulin K salicin K

D-sorbitol K

Control K

K= alkaline

- = no change

+ = acid Sole Carbon Sources in Stanier's Mineral Base: (Growth factors not required)

L-arabinose - cellobiose -

D-fructose +

D-glucose - lactose - maltose - D-mannitol -

L-rhamnose -

D-ribose +

D-sorbitol - sucrose - trehalose -

D-xylose - adonitol - erythritol - glycerol - ethanol - geranitol - inositol - sebacic acid - acetamide - adipate - benzoate - butyrate - citraconate -

D-gluconate - M-hydroxybenzoate -

2-ketogluconate +

DL-lactata +

L-malate +

pelargonate - propionate - quinate - succinate +

L-+-tartrate - valerate - B-alanine -

D-A-Alanine +

betaine - glycine -*

L-histidine - DL-norleucine -

L-proline -

D-tryptophan -

L-valine -

DL-arginine - benzylamine - butylamine - putrescine - mesaconate -

DL-glycerate +

L-tryptophan - methanol - Oxalate +

W = weakly positive

* = became positive after 4 weeks

EXAMPLE 6

B-1 was grown on an oxalate medium with and without 0.01% yeast extract for analysis by the PLFAME method in which phospholipid fatty acids are extracted from the bacteria, methyl esters of these prepared and the esters were analyzed by mass spectroscopy-gas chromatography.

90% of the phospholipid fatty acids were 16:1w7c, 16:0 and I8:lw7c which is characteristic of Gram-negative bacteria. The presence of 16:lw7c and 18:1w7c, which are end products of the anaerobic desaturation biosynthetic pathway, suggests that B-1 may use this pathway for biosynthesis. Only hydroxyl fatty acids were detected in the extracts of B-1, a further indication that the cells have a conventional Gram-negative lipopolysaccharide structure. Taken together, these PLFAME data indicate that B-1 belongs to the genus Pseudomonas. When B-1 phospholipid extracts were analyzed using the

computerized Microbial Identification System (MIS) , which has a reference bank including all presently named species of Pseudomonas genus, there was no "make" for B-1, indicating that B-1 is a member of the genus Pseudomonas that does not correspond with any currently named species of that genus.

Table 7 shows the PLFAME profile of B-1.

TABLE 7

B-1 ISOLATES GROWN WITH AND WITHOUT YEAST EXTRACT

pmol/mgdw Mole %

PLFAME B1 B1+YE B1 B1+YE

14:0 0.00 0.89 0.00 0.09

15:1 0.86 0.45 0.18 0.04

15:0 1.08 1.05 0.23 0.10

16:lw7c 163.96 268.73 34.72 25.84

16:lw7t 3.26 6.08 0.69 0.58

16:0 130.21 451.47 27.58 43.42

17 : lw8 1.52 1.21 0.32 0.12 cyl7:0 6.13 4.37 1.30 0.42

17: 0 14.79 13.51 3.13 1.30

POLY18 0.00 6.21 0.00 0.60

18:3w6 0.00 3.15 0.00 0.30

18:lw9c 0.92 0.43 0.19 0.04

18 : 1w7c 140.15 260.53 29.68 25.05

18:lw7t 0.00 0.73 0.00 0.07

18:0 9.32 16.71 1.97 1.61

BR19 : 1 0.00 1.27 0.00 0.12

POLY20 0.00 3.08 0.00 0.30

TOTAL 472.20 1039.87 100.0 100.0 pmol/mgdw Mole %

OH-FA B1 B1+YE B1 B1+YE

3-OH10:0 0.00 1.23 0.00 30.52

3-OH12.0 5.11 0.00 68.50 0.00

BR-OH13 : 0 1.73 0.00 23.19 0.00

3-OH14:0 0.62 0.00 8.31 0.00

3-OH16:0 0.00 2.80 0.00 69.48

TOTAL 7.46 4.03 100.00 100.0

SUBSTITUTE SHEET EXAMPLE 7 - Comparison of B-1 and Previously Reported

Oxalate-Decomposinα Pseudomonads

A pure culture of isolate B-1 was grown in Oxa-1 medium modified to contain (COOH)₂.2H₂O, 7.0 g/L and adjusted to pH 7.2 with a NaOH. When B-1 reached mid- to late exponential phase of growth in a rotary shaker incubator at 28°C and 150 rpm, it was used to inoculate the various media.

Nitrogen Source Testing:

Ammonium sulfate in (Oxa-1) medium was substituted with either ammonium nitrate, sodium nitrate, L-asparagine or peptone. The experiment was carried out in duplicate in 105 mL flasks at 28°C and 150 rpm.

Carbon Source Testing:

Oxalic acid in oxalic acid medium (Oxa-1) was substituted with 0.1 and 1.0% glycolate, formaldehyde, acetate, ethylene glycol, starch, dextrin or formate.

The experiment was carried out in duplicate in 250 mL flasks at 28°C and 150 rpm.

Temperature Tolerance:

Oxalic acid medium (Oxa-1) was inoculated with B-1 and incubated at 37°C and 40°C. The experiment was carried out in duplicate in 250 mL flasks at 150 rpm.

Absorption Spectrum of Insoluble Yellow Pigment:

Three liters of Oxalic acid medium (Oxa-1) was inoculated with B-1. The culture was grown to late logarithmic phase in a rotary shaker incubator at 28°C and 150 rpm and centrifuged in a Sorvall RC-5B

refrigerated superspeed (DuPont Instruments) centrifuge at 10,000 g for 10 minutes. Once the biomass was recovered (8.19 g wet weight), a chloroform:methanol (1:1) extraction was performed such that there was 10:1 solution to biomass ratio. To enhance the color of the extract, the solvent was evaporated down to 20 mL in a rotoevaporator for one minute at 60°C and 60 rpm under vacuum. Absorbance of the extract was then recorded on a Varian DMS 200 UV-Visible spectrophotometer over the wavelength range from 200 to 600 nm.

Table 8 compares the characteristics of B-1 with those reported for Pseudomonas strain ODl by Jayasuriya (11) and for Pseudomonas strain YOx by Chandra et al (12) . Table 9 summarizes the differences between B-1 and these organisms. Organism YOx appeared to be a larger organism than B-1 and showed a significant difference with respect to oxidase, H₂S production and Litmus milk reaction, particularly in pH. These tests alone show that YOx is a different species from isolate B-1. The absorption spectrum of the methanol:chloroform extracted pigment of B-1 (Figure 8) shows a further difference between B-1 and Yox. Organism OD1 appeared to be slightly larger than B1; however, the difference is not very substantial.

Unlike B-1, 0D1 failed to grow on Jayasuriya's oxalate medium at 37°.

B-1 is able to grow on a minimal medium containing oxalate. This has economic implications, in that it means that inclusion of expensive additives will not be necessary to support B-1 in a treatment system in which it is employed.

On the basis of all taxonomic and characterization studies presented, it can be seen that variant B-1 is a pseudomonad which is different from any previously known named species within the genus Pseudomonas .

TABLE 8

Characteristics Jayasuriya Chandra & and Tests B1 OD1 Shethna

YOx

Aerobic + + +

Rod + + +

Size (micrometer) 0.4 X 1.0 0.5 X 1.5 0.75x1.5

Monotrichous + + +

Motile + + +

Gram Staining - -

Catalase + +

Oxidase - +

Insoluble Pigment yellow yellow yellow

Absorption Peaks (run) 261,321,425 375,440

Max. Temp °C 37 <37 37

Nitrogen sources

- (NH,)₂HPO, + +

- NH₄NO₃ + +

- NaNO₃ + +

- glycine + +

- L-asparagine + +

- Peptone + +

Carbon Sources:

- Glycollate + +

- DL-lactate + +

- DL-malate + +

- succinate + +

- methanol - -

- formaldehyde - -

- formate - -

- ethanol - - -

- acetate - - -

- ethylene-glycol - -

- glycine - -

- glycerol - -

- propionate - -

- n-butyrate - -

- citrate - - -

- glucose - - -

- mannose - -

- sucrose - - TABLE 8 Continued

- maltose - -

- lactose - -

- starch - -

- dextrin - -

- urea - -

- mannitol -

- alanine -

H₂S prod. -

Nitrite Prod. -

Optimum Oxalate (g/L) 5 - 10 10

Max. Oxalate (g/ 'L) 20 20

Tolerated

pH range 6 - 9 6.5 - 9

Litmus milk was

alkaline + +

Acid or gas prodc.

from: glucose, mannose

sucrose, maltose,

lactose, starch or - - dextrine

TABLE 9

SIGNIFICANT DIFFERENCES BETWEEN THE ORGANISMS

Characteristics Jayasuriya Chandra & and Tests B1 OD1 Shethna

YOx

Size (micrometer) 0.4×1. 0 0.5X1.5 0.75X1 . 5

Oxidase -

Absorption Peaks (nm) 261 , 321 , 425 375 , 440 of Pigment

Max. Temp. °C 37 <37 37

H₂S Prod. - +

Litmus milk was + + no change alkaline

SHEET EXAMPLE 8

Cells of B-1 were used for preparation of DNA which was probed with a 741 base pair Pstl-Nrul fragment derived from the upstream region of the Pseudomonas

aeruginosa exotoxin A structural gene, using standard methodology. This probe is capable of distinguishing P. aeruginosa from other Pseudomonas spp . when used as a probe in Southern hybridizations (14).

Strain B-1 gave negative results in the Southern hybridization with the above-described probe, indicating that it is genetically distinct from P. aeruginosa and lacks the genetic material required to produce exotoxin A. EXAMPLE 9

Filtrates of 24 and 48 hour cultures of variant B-1 grown on either nutrient broth or oxalate medium were applied to tissue culture preparations of Vero (monkey kidney) HeLa (human cervical carcinoma) and CHO (Chinese hamster ovary) cells lines for assessment of

toxigenicity.

Weak, non-specific cytotoxicity was observed in all cell culture systems only with the 8 hour oxalate

filtrate. These effects were not observed with the nutrient broth filtrates.

EXAMPLE 10

Culture filtrates as in Example 9 were employed in a suckling mouse test [Dean, A. G. et al, "Test for

Escherichia coli enterotoxin using Infant Mice: Application in a study of Diarrhia in Children in Honolulu", The Journal of Infectious Disease 125(4) : 407-411] using 32 day- old CFI mice, performed at the National Laboratory for Enteic Pathogens, Bureau of Microgiology, Ottawa, Canada, in accordance with Dean, A.G, et al (1972) J. Infec.

Diseases , V. 125, pp 407-411. The intestinal weight/remaining body weight ratios (IW/BW) observed in the suckling mice after 4 hours were 0.060 for oxalate cultures and 0.053 for nutrient broth. Ratios under 0.090 are considered negative for microbial enterotoxins reactive in this bioassay.

EXAMPLE 11

The culture supernatant of an exponential phase culture of B-1 was instilled in the right eye of 8 male New Zealand Rabbits (2.5 - 3.0 Kg) while sterile

Phosphate-Buffered Saline (PBS) was instilled in their left eyes, as a control, in the standard rabbit eye toxicity test. The eyes were examined by a veterinary surgeon, at daily intervals for 7 days, and one animal was sacrificed each day and both eyes were removed and prepared for routine light microscope histology. The culture supernatant of B-1 produced no gross signs of inflammation in any of the treated eyes, and no abnormal histological changes were seen in any of the treated eyes that were examined histologically. The histological specimens were scored according to the schedule of Table 10 and all scores were 0. These data confirm the DNA probe data, which indicated that B-1 lacked the genetic ability to produce exotoxin A, and establish that B-1 produced no toxins that react in the standard rabbit eye test.

TABLE 10

HISTOPATHOLOGICAL SCORING CRITERIA EDEMA

0 = Normal

1 = Minimal Edema

2 = Mild Edema

3 = Moderate Edema

4 = Severe Edema

INFLAMMATION

0 = No Inflammation

1 = Occasional Inflam. Cells

2 = New Inflam. Cells

3 = Moderate Inflam. Cells

4 = Abundant Inflam. Cells (Occlude the Architecture)

EPITHELIAL LINING

0 = Intact

1 = Occasional Loss of Superficial Urothelium

2 = Widespread Loss of Superficial Urothelium

3 = Full Thickness Ulcer

4 = Complete Loss of Urothelium

CONGESTION

0 = No Congestion of Vessels

1 = Mild Congestion

2 = Moderate Congestion

3 = Severe Dilation of Vessels

4 = Severe Dilation of Vessels with Destruction of

Vascular Endothelium

EXAMPLE 12

Cells of B-1 were grown in sodium oxalate medium (NH₄H₂P0₄, 0.5 g/L; MgSO₄.7H₂O, 0.1 g/L; Yeast Extract, 0.1 g/L; FeSO₄.7H₂, 0.05 g/L; and (COONa)₂.H₂O, 7 g/L)

centrifuged, resuspended in PBS to a concentration of 1 × 10⁸ cells/ml, and either injected into the peritoneum of mice or instilled into the lower left lobe of the lungs of rats using a bead-tipped needle. 0.1 ml was instilled in each case and the animals were sacrificed 4 hours after this instillation, and their peritoneal fluid or their homogenized lung tissue were centrifuged at low speed (200Xg) to remove cellular debris, before plating on oxalate medium to detect living cells of B-1. This plating analysis of the peritoneal fluid of 20 treated mice and 8 treated rats yielded negative data indicating that cells of B-1 do not even persist for 4 hours when introduced into the peritoneum of the mouse or into the lung of the rat. These results indicate that B-1 is nonpathogenic in that its culture supernatant contains no exotoxins and in that it fails to persist in animal tissues in which pathogenic species of Pseudomonas are known to cause infections.

EXAMPLE 13 - TOLERANCE OF B-1 TO HIGH LEVELS OF OXALIC

ACID

Oxalic acid medium, Oxa-1, was made up such that duplicate 250 mL flasks contained concentrations of

10000, 12000, 14000, 16000, 18000 and 20000 ppm oxalate ion. Each flask was inoculated with a 5% inoculum from a mid-exponential phase culture of B-1 and incubated at 28°C and 150 rpm. At time zero, a sample was taken and an oxalate assay was performed to measure the initial oxalate concentration; samples taken at regular intervals over 145 hours were analyzed for oxalate. The results are shown in Figure 9 (points represent average of duplicate values) and demonstrate that the B-1 isolate

SUBSTITUTE SHEET can tolerate and degrade high concentrations of oxalic acid.

EXAMPLE 14 - SODIUM OXALATE DEGRADATION BY ISOLATE B-l A 5% inoculum of isolate B-1 was added to three 250 mL flasks containing sodium oxalate medium (NH₄H₂PO₄, 0.5 g/L; MgS0₄.7H₂O, 0.1 g/L; Yeast Extract, 0.1 g/L;

FeSO₄.7H₂, 0.05 g/L; and (COONa)₂.H₂O, 7 g/L) and incubated at 28°C and 150 rpm. The initial oxalate concentration and turbidity of the sample were recorded at time zero followed by sampling at 10, 13, 16, 19, and 23 hours. The turbidity of the culture was measured

spectrophotometrically at 540 nm on Varian DMA 200 US-VIS spectrophotometer. The oxalate concentration of the samples was determined by the indole method. The results are shown in Figure 10 (points represent average of triplicate values). In another experiment, sodium oxalate (Na-Ox) medium constituents were reduced to half normal concentration. Duplicate sets of 250 mL flasks containing either normal or half normal medium

concentration were inoculated and incubated at 28°C and 150 rpm. Oxalate degradation was monitored over 40 hours. Half concentration medium constituents were found to be sufficient to maintain the same rate of oxalate degradation as normal medium as seen in Figure 11 (points represent average of duplicate values).

Although preferred embodiments of the invention are described herein in detail, it will be understood by those skilled in the art that variations may be made thereto without departing from the spirit of the

invention or the scope of the appended claims.

Claims

WE CLAIM:

1. A Pseudomonas microorganism as isolated from

rhizosphere soil surrounding a plant which produces oxalate, said Pseudomonas microorganism being characterized by deposit ATCC 53883.

2. A biologically pure culture of the microorganism Pseudomonas sp B-1 characterized by the deposit ATCC 53883.

3. A biologically pure microorganism Pseudomonas

sp B-1 isolated from rhizosphere soil surrounding a

Rhubarb plant or a Dieffenbachia plant and having the following toxonomic data:

i) the following characteristics common to

Pseudomonas strains:

Gram negative +

Aerobic metabolism +

Polar flagellum +

Motility +

Oxidase weak and slow

Catalase +

ii) resembles Xylophilis ampelina

iii) differs from Xylophilis ampelina in the following distinct characteristics:

X. ampelina B-1

Utilization of oxalate as

sole carbon source - +

Acid from O-F arabinose + -

Urease + - Growth Factor Requirements + -

4. A biologically pure microorganism of claim 3 wherein said microorganism is further characterized by the physiological structure of Figure l having a single polar flagellum.

5. A biologically pure culture of the microorganism of claim 1, 2 or 3 in a culture system comprising

metabolically assimilable nutrient sources of carbon, nitrogen and phosphorus.

6. A biologically pure culture of claim 5 wherein said carbon source is an oxalate.

7. A biologically pure culture of the micororganism Pseudomonas spB-1 wherein the cell wall has a peptidoglycan layer.

8. A biologically pure culture of the microorganism Pseudomonas sp B-1 having the following characteristics:

B1 OD1 YOX

Size (micrometer) 0.4x1.0 0.5×1.5 0.75×1.5 Oxidase - +

Absorption Peaks (nm) 261,321,425 375,440 of Pigment Max. Temp. ºC 37 <37 37

H₂S Prod. - +

Litmus milk was + + no change alkaline