DD279498A1 - PROCESS FOR MICROBIAL REMOVAL OF PHOSPHONO- BZW. PHOSPHINOVERBINDUNGEN - Google Patents

Abstract

The invention relates to a process for the microbial degradation of phosphono or phosphino compounds, wherein the phosphorus released from these substances is added to the metabolism of the bacteria. The two strains used Pseudomonas spec. GS and Alcaligenes spec. GL are also capable of producing toxic phosphonates, e.g. Glyphosate or Buminafos. The use of phosphonate-degrading microorganisms is of potential importance, for example, for the degradation of herbicidal phosphonates in agriculture and for the degradation of phosphonates in polluted effluents (for example detergent liquors). According to the invention, the phosphonic or phosphino compounds are added to the nutrient medium as a source of phosphorus and the nutrient medium subsequently inoculated with bacteria, or phosphonate-containing media of any desired composition are admixed with a sufficient amount of precultured bacteria. For the fermentation unsuitable media are modified by the addition of a carbon and a nitrogen source and by adjusting a physiological pH so that a degradation of the phosphonates contained therein is possible. This degradation also occurs in the presence of inorganic phosphate. {Microbial phosphonate degradation; Bacteria; Pseudomonas; Alcaligenes; glyphosate; buminafos; Culture Media; Phosphorus source; wastewater treatment; Fermentation conditions}

Description

Field of application of the invention

The invention relates to a process for the microwave degradation of phosphono or phosphino compounds (collectively referred to in this specification as phosphonates) with the release of inorganic phosphate. The ability to cleave the carbon-phosphorus bond allows the degrading organisms to use such substances to cover their phosphate needs for growth and replication. In the case of pesticidal properties of phosphonate-based xenobiotics, the organisms can both protect themselves (resistance) and help detoxify their environment.

The use of phosphonate-degrading microorganisms is of potential importance, for example, for the degradation of herbicidal phosphonates in agriculture and for the degradation of phosphonates in polluted effluents (eg detergent liquors).

Characteristic of the known state of the art

Egg; It is known that the most diverse microorganisms are able to access phosphono or phosphino compounds in order to meet their phosphate requirement (Wackett et al., J. Bacteriol 16911987], 710-717, Cook et al., J. Bacteriol 133 [19781, 85-90, Cordeiro et al., J. Am., Al., 10811986], 332-334 However, this degradation activity is largely due to the conversion of alkyl phosphonates or other non-toxic species that are readily accessible to degradation Pesticidal substances, such as N-phosphono-methylglycine (glyphosate) or phosphonomycin, can only be used by some microorganisms as the source of phosphate since the carbon-phosphorus bond has a high biochemical stability Glyphosate is degraded in the soil by the interaction of different species of microorganisms, the primary detoxification toxin being the formation of a non-toxic phosphonate (aminomethylphosphonic acid) b which represents a biochemically relatively readily available phosphate source (Roslucky, E.P. Soil. Bio !. Bioc'nem. 14 [1982], 87-92). This cometabolism of various species can not be used for targeted use in biotechnological processes for controlled phosphonate degradation.

On the other hand, the complete metabolism of glyphosai has also been described for various microorganisms (Jacob et al., J. Biol. Chem. 262 [1987], 1552-1 557, Pipke et al., Eur. J Biochem. 165 [1987], 267- 273, FEBS Letters 236 [1988] 135-138). The cited authors were able to detect the compounds sarcosine, glycine and aminomethylphosphonic acid (AMPA) as degradation products of glyphosate in microwave cultures.

The strain Pseudomonas PG 2982 (Jscob et al., 1987) was isolated from a Pseudomonas aeruginosa strain as a contaminant, with glyphosate being the only source of phosphorus. A fundamental disadvantage of Pseudomonas PG 2982 represents what is necessary to Glyphosatverwertung culture medium as ^r, which must contain as a carbon source the expensive D-gluconate and as vitamin thiamine hydrochloride. The utilization of I m / Iql glyphosate takes the relatively long period of 96 hours to complete. '

From glyphosate wastewater a microorganism of the genus Flavobacterium could be isolated. Also in this case glyphosate was offered as the only source of phosphorus.

A detailed characterization of this strain is not known (Balthazor et al., Appl. Environ. Microbiol. 51 [1986] 432-

The strain Arthrobacter spec. GLP-1 (Pipke et al., 1987) was isolated from enrichment cultures in glyphosate containing media (5-10 mMol / L) and is characterized by high glyphosate tolerance. This and the strain Arthrobacter atrocyaneus ATCC 13752 have a reduced specific growth rate in comparison to inorganic phosphate as Phosphorquello with glyphosate (Pipke et al., 1987, 1988).

That by Waukett et al. (1987) isolated Agrobacterium radiobacter shows C-PLyase activity with broad substrate specificity. The specific growth trajectory with glyphosate as the source of phosphorus is drastically reduced compared to inorganic phosphate.

Talbot et al. (Cun · Microbiol., 10 [19841, 255-260] succeeded in isolating 21 unspecified glyphosate-degrading strains from environmental probes. While the isolated Alcaligenes strain is resistant to 50mMol / L glyphosate, it also grows much more slowly on glyphosate than on inorganic phosphate.

All previously known from the literature glyphosate-degrading strains are inhibited by inorganic phosphate in their degradability.

Object of the invention

It is the object of the invention to develop a microbial process for the cleavage of carbon-phosphorus bonds, which allows an economic use in industry and agriculture and creates conditions for the purification of polluted wastewater.

Explanation of the essence of the invention

The invention has for its object to develop a method for the degradation of phosphonates by such microorganisms that can utilize a wide range of phosphonates without affecting the growth behavior.

According to the invention for the Phosphonatabbau the strains Alcalioenes spec. GL or Psaudorr onas spec. GS or their variants or mutants used. Diu strains Alcaligenes spec. Gl and Pseudomonas spec. GS are deposited in the IMET depository at the Central Institute for Microbiology and Experimental Therapy Jena under the designations IMET 11314 and IMET 11315.

These are strains which cleave phosphonates with the release of inorganic phosphate and utilize the liberated phosphate for their own growth.

In the following v / erder, the strains GL and GS characterized in detail:

Tribe description Alcaligenes spec. GL. IMET11314

Root forms on nutrient agar white, margined, round, slimy colonies. In the microscopic picture, Ko 'ken is present. The Bcc.eißelurig is polar monotrich.

Further details are listed in tabular form.

Gram reaction: negative Catalan: negative Growth at 40 ^° C: negative Growth at pH 4.5: negative Formation of fluorescent dyes: negative Cytchromoxidase: positive Arginine dihydrolase: negative Starch hydrolysis: negative gelatinase: positive urease: positive Reduction of nitrate to nitrite: positive DNase: positive Phenyliilanindeamiricsi ?: positive Äskulinhydiolase: positive H ₂ S image |: negative Sarcosine dehydrogenase: negative Indolbiidung: negative Atmonij: aerobic Utilization of glucose oxide / furm .: +/- Utilization of lactate: negative Utilization of pyruvate: positive Utilization ν η mannitol: negative Recycling of acetate: negative Recovery of Adonite: negative Rejection of inositol: negative Recycling of sucrose: negative Recovery of sarcosine: negutiv Utilization of D-xylose: negative

Utilization of various phosphonates as phosphorus source: Utilization of N-phosphonomethylglycine (glyphosate): positive Utilization of aminomethylphosphinic acid (AMPA): positive

C ₂ H ₅ O _x ? "" ₊ H

U ^c ι *

C ₂ H ₅

?, H

CHo-P-CH ₉ -N-CH ₉ COOH positive

OH

Antibiotic resistance:

Streptomycin: resistant

Rifampicin: msist

Tetracycline: sensitive from 25 mg / 1

Kanamycin: msist

Ampicillin: sensitive from 100 mg / ml

Chloramphenicol: sensitive from 50 mg / ml

Polymyxin B: sensitive

Nalidixin: sensitive from 500 mg / ml

Stammbeschreibung Pseudomon & spec. GS. IMET11315

Stem forms on Nähi agar white ganzrandige, round, slimy colonies. In the microscopic picture he is present as a movable rod. The Flagellation is monotrich polar, z.T. lateral. Further details are listed in tabular form.

Gram reaction: negative Spore color: negative catalase: positive Growth at 40 "C: positive Growth at 42 ° C: negative Growth at pH4.5 ° C negative Formation of fluorescent dyes: negative cytochrome oxidase: positive Arginine dihydrolase: delayed Starch hydrolysis: negative gelatinase: positive urease: positive Reduction of nitrate to nitrite: positive DNase: negative Phenylalanindeaminase: negative lysine decarboxylase: negative ornithine decarboxylase: negative H ₂ S formation: negative Sarcosine dehydrogenase: positive indole: negative Breathing: aerobic Utilization of glucose oxid./ferm .: +/- Utilization of lactate: positive Utilization of Pvruvat: positive Recycling of acetate: positive Recovery of Adonite: positive, delayed Recovery of inositol: negative Utilization of glycerine: positive Utilization of D-xylose: positive Recycling of sucrose positive Utilization of sarcosine a! S N source: positive

Utilization of different phosphonates as phosphorus source: N-phosphoriomethylglycine (glyphosate positive

Aminomethylphosphonic acid (AMPA) positive

N-Isopropylphosphinomothylglycine positive

2-amino-3-phosphonopropiolic acid positive

C ₂ H ₅ O

₊ ^H

η CH ₃ -P-CH ₂ -N-CH ₂ COOH

OH

positive

^PDBitiv

Antibiotic resistance:

streptomycin

rifampicin

tetracycline

kanamycin

ampicillin

chloramphenicol

nalidixic

polymyxins

sensitive from 200 mg / 1

sensitive

sensitive

sensitive from 1 000 mg / 1 sensitive from 500 mg / 1 sensitive from 30 mg / 1

sensitive

sensitive

Both strains can use a wide range of phosphonates as the source of phosphate and also tolerate high concentrations of these phosphonates. Furthermore, the two strains are characterized by the fact that their growth behavior in the presence of phosphonates is indistinguishable from that in the presence of inorganic phosphate. It is advantageous that these strains only make small demands on the cultivation medium (glucose or sucrose as source of C, vitamin supplementation). If one uses as Inokulum the strains Alcaligene'j spec. GL or Pseudomonas spec. GS or their mutants or variants, the phosphonate-rich nutrient media must contain a nitrogen source and mineral salts in addition to a well-usable carbon source. This growth is limited in or on diosen media by the available Phosphonatangebot. If the entire biomass of a preculture is used as the inoculum, the phosphonate-containing media need not have nutrient solution quality, but the metabolic capacity of the bacteria must be maintained over the period required for the degradation process.

The cultivation is carried out at a pH of 5-8, but preferably at pH 7-7.5. Phosphonate degradation also occurs in the presence of inorganic phosphate. As phosphonate-containing media are used, for example, agricultural or industrial wastewater. In order to allow the microorganisms an efficient phosphonate degradation, it is expedient to enrich these media with nutrients, especially carbohydrates and assimilable nitrogen.

The phosphonate degradation by the process according to the invention can be carried out either in a batch process or in a continuous culture, wherein under the latter conditions, depending on the dilution rate, phosphonates are not completely removed from the nutrient solution. Near-complete degradation of even toxic phosphonates (eg, Buminafos) is possible when the strains are pre-cultured in a medium with readily utilizable phosphonates (eg, glyphosate 5-10 mM / L) for biomass production and subsequent depletion be resuspended in P-free medium in a new medium containing the phosphoric or phosphino derivatives to be degraded.

embodiments example 1

A phosphonate-containing liquid nutrient medium (for example containing 1 mM glyphosate) is mixed with an aliquot of a phosphate-depleted culture of the strain Pseudomonas spec. GS inoculated (1 ml inoculum for 200 ml nutrient solution). Within 36 hours, the bacteria reach the stationary growth phase and produce with 1mM glyphosate as a phosphorus source the same dry biomass, which is achieved with inorganic phosphate as a source of phosphorus.

The used glyphosate is completely metabolized. ¹⁴ C ₃ glyphosate · metabolism studies have shown that 12% of the radioactivity used is CO; 1% are found in the aqueous cell extract, 11.5% are incorporated into the protein and 40% in membrane components. 36% of the radioactivity was excreted as Glyphosatmetabolite in the nutrient medium again.

Composition of the liquid nutrient medium:

Carbon source: 7g sodium pyruvate or

4 g of sucrose t- 0.1 g of glycerol

- Nitrogen source: 0.9 g sarcosine or 1 g NH ₄ NO ₃

(1OmM)

Phosphorus source: 0.169g Glyphosate or O, 136g KH ₂ PO ₄

(1mM) (1mM)

6.05 g Tris 15 mg CaCl ₂

200 mg MgSO ₄ .7H ₂ O

- 40 mg KCl

1 mg of FeSO ₄ .7H ₂ O

2 ml of micronutrient solution

adjust to pH 7.5 with HCl, ad 11H ₂ O dest. Trace element solution: 500 mg H ₃ BO ₃

400mg MnSO ₄ x H ₂ O 400mg ZnSO, χ H ₂ O 200mg Na ₂ MoO ₄ x 2H ₂ O 100mg CuSO ₄ χ 5H ₂ O 100mg KJ ad 11 H ₂ O dest.

Example 2

With bacteria of strain GS, a nutrient solution is inoculated with 1 mM glyphosate as the only phosphorus source. After growth, the biomass is harvested by centrifugation and suspended in a phosphate depletion medium (i.e., without a phosphorus source). After 24 hours, the entire biomass is harvested and placed in a medium containing the phosphonate to be degraded. In the case of glyphosate 100% of the substance is degraded in another 24 h. Composition of the liquid nutrient medium as in the example

Example 3

With bacteria of strain GL, a nutrient solution is inoculated with 5 mM glyphosate as the only source of phosphorus. After 48 hours of growth, the biomass is harvested by centrifugation and suspended in a phosphate depletion medium (i.e., without a phosphorus source). After 24 hours, the entire biomass is harvested and placed in a medium containing the phosphonate to be degraded. In the case of glyphosate, 30-80% of the substance is degraded in a further 48 h incubation (30% for a glyphosate use of 90: nM / l, 80% for a glyphosate use of 2 mM / l).

Composition of the N.ihrmedi'jrns:

50mM / ITris / HCIpH 7.5 20mM / INaCl 4mM / l NH ₄ Cl 0.4mM / l CaCl ₂ 1mM / l MgSO ₄ 0.8% glucose

Example 4

Industrial effluents containing phosphonates are made suitable by addition of 8 g / l glucose and 0.2 g / l NH ₄ Cl and by correcting the pH with NaOH to pH 7.5 as a medium for phosphonate-degrading bacteria. Inoculation of phosphate-depleted biomass according to Example 3 results in degradation of 50-80% phosphonate (eg, Buminafos) within 24-28 hours.

Claims (9)

-1- £ .'9 498 Claims: 1. A method for microwave degradation of phosphono or phosphino compounds, characterized in that the strains Alcaligenes spec. GL or Pseudomonas spec. GS or their mutants or variants used. 2. The method according to claim 1, characterized in that one uses these microorganisms as an inoculum for phosphonate-containing culture media wo'oei these nutrient media contain a well usable carbon source, a Stiv-kstoffquelle and mineral salts, and the phosphate requirement of the microorganisms during growth in or on This media is limited by the available Phosphonatangebot. 3. The method according to claim 1, characterized in that one uses as the inoculum, the entire biomass of a preculture, wherein the phosphonate medium need not have nutrient solution quality, but the metabolic capacity of the bacteria must be maintained over the necessary period for the degradation process. 4. The method according to any one of claims 1-3, dadu'ch characterized in that one carries out the cultivation at a pH of -8. 5. The method according to claim 4, characterized in that one carries out the cultivation at pH 7-7.5. 6. The method according to claim 1 and 3, characterized in that the medium contains phosphonates, which are toxic to the bacteria. 7. The method according to any one of claims 1,3 or 6, characterized in that one carries out the Phosphonatabbau in the presence of inorganic phosphate. 8. The method according to any one of claims 1,3, 6 or 7, characterized in that one uses as phosphonate-containing media agricultural or industrial wastewater. 9. The method according to claim 8, characterized in that enriches these media with nutrients before the addition of microorganisms.