CA1238593A - Microorganisms of the genus pseudomonas and process for the degradation of compounds containing methyl groups in aqueous solutions - Google Patents
Microorganisms of the genus pseudomonas and process for the degradation of compounds containing methyl groups in aqueous solutionsInfo
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- CA1238593A CA1238593A CA000455349A CA455349A CA1238593A CA 1238593 A CA1238593 A CA 1238593A CA 000455349 A CA000455349 A CA 000455349A CA 455349 A CA455349 A CA 455349A CA 1238593 A CA1238593 A CA 1238593A
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- Prior art keywords
- dmf
- nrrl
- dimethyl
- methyl
- formamide
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/32—Processes using, or culture media containing, lower alkanols, i.e. C1 to C6
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/38—Pseudomonas
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse
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- Water Supply & Treatment (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
- Physical Water Treatments (AREA)
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- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
4-14450/ZFO/+
Microorganisms of the genus Pseudomonas and process for the degradation of compounds containing methyl groups in aqueous solutions Abstract The present invention relates to novel facultatively methylotrophic microorganisms of the genus Pseudomonas or of a Pseudomonas-like genus, protein-containing biomass and to a process for the microbiological purification of aqueous solutions containing, as pollutants, dimethylformamide and possibly further organic substances, for example methanol.
Microorganisms of the genus Pseudomonas and process for the degradation of compounds containing methyl groups in aqueous solutions Abstract The present invention relates to novel facultatively methylotrophic microorganisms of the genus Pseudomonas or of a Pseudomonas-like genus, protein-containing biomass and to a process for the microbiological purification of aqueous solutions containing, as pollutants, dimethylformamide and possibly further organic substances, for example methanol.
Description
~L23~ 3 4-14450/ZFO/+
Microorganisms of the genus Pseudomonas and process for the degradation of compounds containing meth~l groups in aqueous solutions The present invention relates to novel facultatively methylotrophic microorganisms of the genus Pseudomonas or of a Pseudomonas-like genus, mixed cultures, a process .for the microbiological purifica-tion of aqueous solutions by degrading compounds containing methyl groups in the presence of these microorganisms, the biomass with its constituents which is produced by these microorganisms, and to the use of the biomass obtainable in accordance with the process~
The expression "methylotrophic microorganisms"
shall be understood as meaning those microorganisms which grow on nutrient media containing, as carbon source, compounds having only one carbon atom, for example methanol, or several carbon atoms without a direct C-C bond, for example dimethylamine [Patt, Cole and Hansen, International J. Systematic ~acteriology 26
Microorganisms of the genus Pseudomonas and process for the degradation of compounds containing meth~l groups in aqueous solutions The present invention relates to novel facultatively methylotrophic microorganisms of the genus Pseudomonas or of a Pseudomonas-like genus, mixed cultures, a process .for the microbiological purifica-tion of aqueous solutions by degrading compounds containing methyl groups in the presence of these microorganisms, the biomass with its constituents which is produced by these microorganisms, and to the use of the biomass obtainable in accordance with the process~
The expression "methylotrophic microorganisms"
shall be understood as meaning those microorganisms which grow on nutrient media containing, as carbon source, compounds having only one carbon atom, for example methanol, or several carbon atoms without a direct C-C bond, for example dimethylamine [Patt, Cole and Hansen, International J. Systematic ~acteriology 26
(2), 226-229 (1979)].
The expression "facultatively methylotrophic micro-organisms" shall be understood as meaning those micro-.. *
, .
organisms which grow on nutrient media containing, ascarbon source, compounds having only one carbon atom, for example methanol, and/or compounds having several carbon atoms with or without a C-C bond~ for example glucose or dimethylamine.
There are described in the literature facultatively methylotrophic microorganisms that, in aqueous solution, are able to degrade, or utilise as carbon source or as carbon and nitrogen source, one or more compounds selected from the ~ollowing group of organic compounds: methane, methanol, ethanol, acetates, for example sodium acetate, glucose, certain methylammonium compounds, for example methylammonium chloride, ethylammonium chloride or trimethylammonium chloride, or the free amines of these salts.
Such microorganisms are deposited in various culture collections, for example in the American Type Culture Collection (ATCC), in the Deutsche Sammlung von Mikroorganismen (nsM) or in the National Collection of Industrial Bacteria (NCI~), and are listed in the catalogues published by these depositories.
Environmental Protection ~gency (EPA) Report 2-79-163 by Dojlido J.R. Oe December 197g and the abridged publication in Chem. ~nd. 34 (1982), 750 [Biologische EntEernung von gtickstoffverbindungen aus ~bwassern (Biological ~emoval of Nitrogen Compounds Erom Waste Waters)~ mention the possihility of eliminating dimethylformamide in aqueous solutions with activated sludges. Neither publication, however, contains a general characterisation of the micro-organisms present in these activated sludges or a characterisation of microorganisms that are capable of degrading dimethylformamide.
The literature contains no information on the origin, isolation, identification or the depository of "
~3~3 a dimethyl~ormamide-degrading microorganism or of a mixed culture. Even the references in the literature mentioned do not contain any reference to the use of an isolated microorganism or an isolated mixed culture in a microbiological process for the purification of waste water containing dimethylformamide as pollutant.
Large-scale production in the chemical industry gives rise to the formation of aqueous solutions, for example waste waters, that contain, for example, dimethylformamide and/or formamide, methyl~ or ethyl-formamide, acetamide, formate or acetate salts, glucose, methanol, ethanol, methyl-, ethyl-, dimethyl-or trimethyl-ammonium chloride, dimethyl phosphite, trimethyl phosphite, acetonitrile or isobutyronitrile as pollutants. The purification of these waste waters poses problems because of the hitherto-customary incineration process, since the incineration of organic waste materials is regarded as an extremely unfavour-able method of elimination which involves great expense and also high levels of environmental pollution.
The underlying problem of the present invention is to find and isolate novel microorganisms or mixed cultures that, in aqueous solutions, exhibit especially good growth whilst consuming ~he mentioned compounds as completely as possible.
This problem is solved by the present invention which relates to novel facultatively methylotrophic microorganisms oE the genus Pseudomonas or oE a Pseudomonas-like genus and mixed cultures. The present invention relates also to a process or the micro-biological purification of aqueous solutions by degradation o~ dimethylformamide, ~ormamide, methyl- or ethyl-formamide, acetamide, methanol, ethanol, saccharides, lower alkanoates, methyl-, ethyl-, dimethyl- or trimethyl-ammonium halides, dimethyl ., .
.
:
~L~3~3 phosphite, trimethyl phosphite, hydantoin, creatine, creatinine, barbituric acid, choline, betaine, sarcosine, dimethylglycine, glycine, glyoxylic acid, N-formylamino acids, N-acetylamino acids, acetonitrile or isobutyronitrile or mixtures of these compounds in the presence of these novel microorganisms or mixed cultures, to the biomass with its constituents which is produced by these novel microorganisms or mixed cultures, and to the use of the biomass obtainable in accordance with the process.
In the description of the present invention, the general terms used hereinbefore and hereinafter preferably have the following meanings:
Saccharides are, for example, mono- or di-saccharides, for example hexoses, for example glucose or fructose.
Lower alkanoates are, for example, methyl or ethyl formate or acetate or salts, for example alkali metal salts, of formic acid, for example sodium or potassium formate, or salts, for example alkali metal salts, of acetic acid, for example sodium or potassium acetate.
Methyl~, ethyl-, dimethyl- or trimethyl-ammonium haliaes are, for example, methyl-, ethyl-, dimethyl or trimethyl-ammonium chloride and also bromide.
N-formylamino aclds are, Eor example, N-formyl-glycine, N-formylalanine, N-Eormylvaline, N-formyl-serine or N-formylthreonine.
N-acetylamino acids are, for example, N-acetyl-glycine, N-acetylalanine, N-acetylvaline, N-acetyl-serine or N-acetylthreonine.
~ iomass is, for example, all cell systems which are in the living state, for example in the state of replication or resting, in the state of partial or complete cell death, or already in a state of enzymatic decomposition or of decomposition by foreign cultures, ", ~ ':
.
gL;i~3135~;~
and which are formed by the microorganisms of the present Application.
Constituents oE this biomass are, or example, the enzymes present therein or the enzyme extracts obtainable from the biomass which also are capable of degrading the above-mentioned compounds or pollutants, for example dimethylformamide, in aqueous solution.
The present invention relates especially to facultatively methylotrophic microorganisms of the genus Pseudomonas or of a Pseudomonas-like genus, mixed cultures and to a process ~or the microbiological purification of aqueous solutions by degradation of dimethylformamide, methyl~ormamide, ethylformamide, formamide, methanol, ethanol, sodium or potassium formate or acetate, methyl-, ethyl-, dimethyl- or trimethyl-ammonium chloride,ltrimethyl phosphite, dimethyl phosphite, acetonitrile, isobutyronitrile or mixtures of these compounds.
The present invention relates more especially to microorganisms selected from the group oE the following strains: DMF 3/3 (NRRI.-B-15358), DMF 3/4 (NRRL-B-15359), DMF 3/5 (NRRL-B-15360), D~F 3/6 (NRRL-B-15361), ~MF 3/11 (NRRL-B-15362), ~MF 3/12 (NRRL-B-15363), DMF 4/4 (NRRL-B-15364), DMF 5/3 (NRRL-B-15365), DMF 5/5 (NRRL-B-15366), DMF 5/7 (NRRL-B-15367), D~F 5/8 (NRRL-B-15368), D~F 5/~ (NRRL-B-15369)~
DMF 5/10 (tNRRL-B-15370), mixed cultures of these strains, the mixed culture DMF/HW 1-5 (NRRL-B-15371) and processes or the microbiological purification o aqueous solutions by degradation of dimethyl~
formamide.
The novel microorganisms originate from the activated sludge of a waste water puriEication plant oE
Ciba-Geigy ~G Switzerland (WPP Ciba-Geigy) and from the activated sludge of a sludge pool (drying bed) at ~æ3~s~3 ~ 6 --Rairouan, Tunisia. The mixed culture DMF/~W 1-5 was isolated from the sediment of the Hallwilersee in Switzerland. ~11 of the microorganisms and the mixed culture were deposited with the ~gricultural Research Culture Collection in Peoria, Illinois 61604, USA, on 13th April, 1983.
The deposit number and origin of each individual strain and of the mixed cul~ure are indicated in the following Table 1:
Table 1 Internal re~erence ~eposit No. Origin -DMF 3/3 NRRL B-15358 WPP Ciba-Geigy DMF 3/4 NRRL B-15359 WPP Ciba-Geigy DMF 3/5 NRRL B-15360 WPP Ciba-Geigy DMF 3/6 NRRL B-15361 WPP Ciba-Geigy DMF 3/11 NRRL B-15362 WPP Ciba-Geigy DMF 3/12 NRRL B-15363 WPP Ciba-Geigy DMF 4/4 NRRL B-15364 Kairouan DMF 5/3 NRRL B-15365 Kairouan DMF 5/5 NRRL B-15366 Kairouan DMF 5/7 NRRL B-15367 Kairouan DMF 5/8 NRRL B-15368 Kairouan DMF 5/9 NRRL B-15369 Kairouan DMF 5/10 NRRL B-15370 Kairouan DMF/HW 1-5 (mixed NRRL B-15371 Hallwilersee culture) Characterisation of the novel microorganisms:
1. ~ethod of isolation:
A sample of waste water or a suspension of ....
activated sludge (1 g per 10 ml of sterile water) was introduced into a shaking flask containing 20 ml of sterile nutrient solution MV 7. Approximately 0.1 g of sterile-filtered dimethyl~ormamide and, to buffer the nutrient solution, 1 ml of lM phosphate buffer of pH 7 were added, and the shake culture was incubated at 2~C and 250 rpm for 7-14 days. 1 ml of this first enrichment culture was introduced into 20 ml of fresh nutrient solution MV 7 with 0.1 g of dimethylformamide and the batch was again incubated at 28 and pH 7 (shake culture 250 rpm, 7-14 days). 1 ml of the second enrichment culture was in turn added to 20 ml o~ fresh nutrient solution MV 7 with 0.1 g of dimethylformamide and incubation was carried out under the conditions mentioned. A fourth enrichment culture was produced under the conditions mentioned from 1 ml of the third enrichment culture, ayain with 20 ml of nutrient solution MV 7 and O.l g of dimethyl~ormamide. The enrichment cultures so produced were each plated onto sterile solid MV 7-agar nutrient substrates [nutrient solution MV 7 with the addition of 20 g/litre agar (Difco)] and incubated at 28C. Single colonies were carefully lifted and streaked again on the same medium. This procedure was repeated several times until pure isolates were obtained.
The nutrient solution MV 7 use~ contains, in one litre of water, the following constituents: 2 g Oe NH4NO3 (nitrogen source), 1.4 g of Na2HPO~, 0.6 g oE
KH2PO4 (buEfer and phosphorus source), 0.2 g of MgSO4 H20, n-01 g oE CaC12 . 2 H20, 0.001 g of Fe904 . 7 H2O and 1 ml oP trace element solution (consisting of 20 mg/litre each of Na2MoO4 . 2 H2O, Ma2B47 10 H2O~ MnSO4 H2O and CuSO4 5 H2O)-To prepare this nutrient solution, the salts are dissolved in distilled water, the solution is adjusted .. .
, ~23~5g3 to pH 7 with dilute aqueous sodium hydroxide solution while maintaining the pH at 7 and made up to 1 litre with distilled water. To prepare the solid nutrient substrate MV 7-agar, 20 g/litre agar (Difco) are also added to the nutrient solution MV 7. Sterilisation is carried out in an autoclave (2 atm, 120C).
2 General findings and microscopy .
All the strains listed in 'rable 1 and also the mixed culture grow under aerobic conditions at temperatures of up to approximately 37, optimally at from 28 to 30. The microorganisms are gram-negative (samples for gram-staining test taken from an MV 7-dimethylformamide culture) and oxidase-positive (samples for the test taken from nutrient-agar, MV 7-DMF-agar and MV 7-DMF-liquid culture). In liquid cultures containing dimethylformamide, it is observed that all the strains tend to have flaky growth.
Under microscopic examination (optical microscope), strains DMF 3/3, 3/4, 3/5, 3/6, 3/11 and
The expression "facultatively methylotrophic micro-organisms" shall be understood as meaning those micro-.. *
, .
organisms which grow on nutrient media containing, ascarbon source, compounds having only one carbon atom, for example methanol, and/or compounds having several carbon atoms with or without a C-C bond~ for example glucose or dimethylamine.
There are described in the literature facultatively methylotrophic microorganisms that, in aqueous solution, are able to degrade, or utilise as carbon source or as carbon and nitrogen source, one or more compounds selected from the ~ollowing group of organic compounds: methane, methanol, ethanol, acetates, for example sodium acetate, glucose, certain methylammonium compounds, for example methylammonium chloride, ethylammonium chloride or trimethylammonium chloride, or the free amines of these salts.
Such microorganisms are deposited in various culture collections, for example in the American Type Culture Collection (ATCC), in the Deutsche Sammlung von Mikroorganismen (nsM) or in the National Collection of Industrial Bacteria (NCI~), and are listed in the catalogues published by these depositories.
Environmental Protection ~gency (EPA) Report 2-79-163 by Dojlido J.R. Oe December 197g and the abridged publication in Chem. ~nd. 34 (1982), 750 [Biologische EntEernung von gtickstoffverbindungen aus ~bwassern (Biological ~emoval of Nitrogen Compounds Erom Waste Waters)~ mention the possihility of eliminating dimethylformamide in aqueous solutions with activated sludges. Neither publication, however, contains a general characterisation of the micro-organisms present in these activated sludges or a characterisation of microorganisms that are capable of degrading dimethylformamide.
The literature contains no information on the origin, isolation, identification or the depository of "
~3~3 a dimethyl~ormamide-degrading microorganism or of a mixed culture. Even the references in the literature mentioned do not contain any reference to the use of an isolated microorganism or an isolated mixed culture in a microbiological process for the purification of waste water containing dimethylformamide as pollutant.
Large-scale production in the chemical industry gives rise to the formation of aqueous solutions, for example waste waters, that contain, for example, dimethylformamide and/or formamide, methyl~ or ethyl-formamide, acetamide, formate or acetate salts, glucose, methanol, ethanol, methyl-, ethyl-, dimethyl-or trimethyl-ammonium chloride, dimethyl phosphite, trimethyl phosphite, acetonitrile or isobutyronitrile as pollutants. The purification of these waste waters poses problems because of the hitherto-customary incineration process, since the incineration of organic waste materials is regarded as an extremely unfavour-able method of elimination which involves great expense and also high levels of environmental pollution.
The underlying problem of the present invention is to find and isolate novel microorganisms or mixed cultures that, in aqueous solutions, exhibit especially good growth whilst consuming ~he mentioned compounds as completely as possible.
This problem is solved by the present invention which relates to novel facultatively methylotrophic microorganisms oE the genus Pseudomonas or oE a Pseudomonas-like genus and mixed cultures. The present invention relates also to a process or the micro-biological purification of aqueous solutions by degradation o~ dimethylformamide, ~ormamide, methyl- or ethyl-formamide, acetamide, methanol, ethanol, saccharides, lower alkanoates, methyl-, ethyl-, dimethyl- or trimethyl-ammonium halides, dimethyl ., .
.
:
~L~3~3 phosphite, trimethyl phosphite, hydantoin, creatine, creatinine, barbituric acid, choline, betaine, sarcosine, dimethylglycine, glycine, glyoxylic acid, N-formylamino acids, N-acetylamino acids, acetonitrile or isobutyronitrile or mixtures of these compounds in the presence of these novel microorganisms or mixed cultures, to the biomass with its constituents which is produced by these novel microorganisms or mixed cultures, and to the use of the biomass obtainable in accordance with the process.
In the description of the present invention, the general terms used hereinbefore and hereinafter preferably have the following meanings:
Saccharides are, for example, mono- or di-saccharides, for example hexoses, for example glucose or fructose.
Lower alkanoates are, for example, methyl or ethyl formate or acetate or salts, for example alkali metal salts, of formic acid, for example sodium or potassium formate, or salts, for example alkali metal salts, of acetic acid, for example sodium or potassium acetate.
Methyl~, ethyl-, dimethyl- or trimethyl-ammonium haliaes are, for example, methyl-, ethyl-, dimethyl or trimethyl-ammonium chloride and also bromide.
N-formylamino aclds are, Eor example, N-formyl-glycine, N-formylalanine, N-Eormylvaline, N-formyl-serine or N-formylthreonine.
N-acetylamino acids are, for example, N-acetyl-glycine, N-acetylalanine, N-acetylvaline, N-acetyl-serine or N-acetylthreonine.
~ iomass is, for example, all cell systems which are in the living state, for example in the state of replication or resting, in the state of partial or complete cell death, or already in a state of enzymatic decomposition or of decomposition by foreign cultures, ", ~ ':
.
gL;i~3135~;~
and which are formed by the microorganisms of the present Application.
Constituents oE this biomass are, or example, the enzymes present therein or the enzyme extracts obtainable from the biomass which also are capable of degrading the above-mentioned compounds or pollutants, for example dimethylformamide, in aqueous solution.
The present invention relates especially to facultatively methylotrophic microorganisms of the genus Pseudomonas or of a Pseudomonas-like genus, mixed cultures and to a process ~or the microbiological purification of aqueous solutions by degradation of dimethylformamide, methyl~ormamide, ethylformamide, formamide, methanol, ethanol, sodium or potassium formate or acetate, methyl-, ethyl-, dimethyl- or trimethyl-ammonium chloride,ltrimethyl phosphite, dimethyl phosphite, acetonitrile, isobutyronitrile or mixtures of these compounds.
The present invention relates more especially to microorganisms selected from the group oE the following strains: DMF 3/3 (NRRI.-B-15358), DMF 3/4 (NRRL-B-15359), DMF 3/5 (NRRL-B-15360), D~F 3/6 (NRRL-B-15361), ~MF 3/11 (NRRL-B-15362), ~MF 3/12 (NRRL-B-15363), DMF 4/4 (NRRL-B-15364), DMF 5/3 (NRRL-B-15365), DMF 5/5 (NRRL-B-15366), DMF 5/7 (NRRL-B-15367), D~F 5/8 (NRRL-B-15368), D~F 5/~ (NRRL-B-15369)~
DMF 5/10 (tNRRL-B-15370), mixed cultures of these strains, the mixed culture DMF/HW 1-5 (NRRL-B-15371) and processes or the microbiological purification o aqueous solutions by degradation of dimethyl~
formamide.
The novel microorganisms originate from the activated sludge of a waste water puriEication plant oE
Ciba-Geigy ~G Switzerland (WPP Ciba-Geigy) and from the activated sludge of a sludge pool (drying bed) at ~æ3~s~3 ~ 6 --Rairouan, Tunisia. The mixed culture DMF/~W 1-5 was isolated from the sediment of the Hallwilersee in Switzerland. ~11 of the microorganisms and the mixed culture were deposited with the ~gricultural Research Culture Collection in Peoria, Illinois 61604, USA, on 13th April, 1983.
The deposit number and origin of each individual strain and of the mixed cul~ure are indicated in the following Table 1:
Table 1 Internal re~erence ~eposit No. Origin -DMF 3/3 NRRL B-15358 WPP Ciba-Geigy DMF 3/4 NRRL B-15359 WPP Ciba-Geigy DMF 3/5 NRRL B-15360 WPP Ciba-Geigy DMF 3/6 NRRL B-15361 WPP Ciba-Geigy DMF 3/11 NRRL B-15362 WPP Ciba-Geigy DMF 3/12 NRRL B-15363 WPP Ciba-Geigy DMF 4/4 NRRL B-15364 Kairouan DMF 5/3 NRRL B-15365 Kairouan DMF 5/5 NRRL B-15366 Kairouan DMF 5/7 NRRL B-15367 Kairouan DMF 5/8 NRRL B-15368 Kairouan DMF 5/9 NRRL B-15369 Kairouan DMF 5/10 NRRL B-15370 Kairouan DMF/HW 1-5 (mixed NRRL B-15371 Hallwilersee culture) Characterisation of the novel microorganisms:
1. ~ethod of isolation:
A sample of waste water or a suspension of ....
activated sludge (1 g per 10 ml of sterile water) was introduced into a shaking flask containing 20 ml of sterile nutrient solution MV 7. Approximately 0.1 g of sterile-filtered dimethyl~ormamide and, to buffer the nutrient solution, 1 ml of lM phosphate buffer of pH 7 were added, and the shake culture was incubated at 2~C and 250 rpm for 7-14 days. 1 ml of this first enrichment culture was introduced into 20 ml of fresh nutrient solution MV 7 with 0.1 g of dimethylformamide and the batch was again incubated at 28 and pH 7 (shake culture 250 rpm, 7-14 days). 1 ml of the second enrichment culture was in turn added to 20 ml o~ fresh nutrient solution MV 7 with 0.1 g of dimethylformamide and incubation was carried out under the conditions mentioned. A fourth enrichment culture was produced under the conditions mentioned from 1 ml of the third enrichment culture, ayain with 20 ml of nutrient solution MV 7 and O.l g of dimethyl~ormamide. The enrichment cultures so produced were each plated onto sterile solid MV 7-agar nutrient substrates [nutrient solution MV 7 with the addition of 20 g/litre agar (Difco)] and incubated at 28C. Single colonies were carefully lifted and streaked again on the same medium. This procedure was repeated several times until pure isolates were obtained.
The nutrient solution MV 7 use~ contains, in one litre of water, the following constituents: 2 g Oe NH4NO3 (nitrogen source), 1.4 g of Na2HPO~, 0.6 g oE
KH2PO4 (buEfer and phosphorus source), 0.2 g of MgSO4 H20, n-01 g oE CaC12 . 2 H20, 0.001 g of Fe904 . 7 H2O and 1 ml oP trace element solution (consisting of 20 mg/litre each of Na2MoO4 . 2 H2O, Ma2B47 10 H2O~ MnSO4 H2O and CuSO4 5 H2O)-To prepare this nutrient solution, the salts are dissolved in distilled water, the solution is adjusted .. .
, ~23~5g3 to pH 7 with dilute aqueous sodium hydroxide solution while maintaining the pH at 7 and made up to 1 litre with distilled water. To prepare the solid nutrient substrate MV 7-agar, 20 g/litre agar (Difco) are also added to the nutrient solution MV 7. Sterilisation is carried out in an autoclave (2 atm, 120C).
2 General findings and microscopy .
All the strains listed in 'rable 1 and also the mixed culture grow under aerobic conditions at temperatures of up to approximately 37, optimally at from 28 to 30. The microorganisms are gram-negative (samples for gram-staining test taken from an MV 7-dimethylformamide culture) and oxidase-positive (samples for the test taken from nutrient-agar, MV 7-DMF-agar and MV 7-DMF-liquid culture). In liquid cultures containing dimethylformamide, it is observed that all the strains tend to have flaky growth.
Under microscopic examination (optical microscope), strains DMF 3/3, 3/4, 3/5, 3/6, 3/11 and
3/12 have the shape oE oblong rods, and strains DMF 4/4, 5/3, 5/5, 5/7, 5/8, 5/9 and 5/10 the shape of short rods or oval beads. The electron microscope image accords well with the observations made by optical microscopy. Two types oE cell in particular are observed:
a) oblong rods, in some cases with polar Elagella, 0.8 x 2 ~ in size (strain~ DMF 3/3, 3/4, 3/5, 3/6, 3/11, 3/12), b) short rods, in some cases with polar flagella, 0.7 x 1.1 ~ in size (strains DMF 4/4, 5/3, 5/5, 5j7, 5/8, 5/9 and 5/10). In the case of strains DMF 4/4 and 5/3, short, fine "hairs" on the cell wall are to be ~38~
observed.
3. Biochemical characterisation and classification of the novel microorganisms For general biochemical characterisation and classification of the strains listed in Table 1, two commercially available test systems are used.
a) "Oxi/Ferm Tube" test (Roche) This test system is used in the case of gram-negative rods with positive oxidase reaction. The parkicular kest is carried out in parallel twice in each case with inoculum of the relevant strain taken from dimethylformamide-agar and nutrient-agar. See the manufacturee's instructions for a description of the standardised biochemical tests and for experimental procedure. The test results are summarised in Table 2.
....
. . . ....... .
. ,:
~38S~3 Table 2: DMF Strains tested with "Oxi/Ferm Tube."
(48/28C) , . .
Internal biochemical test reference ~
s~ ~o O ~ ~ '~V~I
i~ ,~ .~ ~ ~ o ~1 ~ a) ~ ~ ~ ~ u o a) o ~ ,, o o a~
h ~ ~ 1~ ~H ~1 ~l1 ~ :' ~
_ ~ O . !a;N __ ~ O 1~1 U U
DMF 3/3 _ _ + _ + + _ +
DMF 3/4 _ _ + _ _ ~ + + _ DMF 3/5 _ _ + _ _ + + _ +
DMF 3/6 _ _ _~ _ _ + _ + +
DMF 3/11 _ _ + _ _ + + _ +
DMF 3/12 _ _ + _ _ * + _ _ _ ____ _ ~1 DMF 4/4 _ _ ~ + _ ~- + _ t DMF 5/3 .~ _ ~ ~ _ ~ _ + .
DMF 5/5 _ _ + ~ _ ~ _ _ D~F 5/7 _ _ .~ ~ _ _ _ _ DMF 5/8 _ _ ~ + _ ~ ~ ~ +
DMF 5/9 _ _ + + _ _ _ _ _ DMF 5/10 _ _ _ ~ _ + + _ +
.., ~
, ~
:
.
:~23~5~3 The findings show that the group of strains DMF
3/3, 3/4, 3/5, 3/6, 3/11 and 3/12 (H2S formation clearly negative and xylose cleavage clearly positive) diEfers biochemically ~rom the group of strains DMF
a) oblong rods, in some cases with polar Elagella, 0.8 x 2 ~ in size (strain~ DMF 3/3, 3/4, 3/5, 3/6, 3/11, 3/12), b) short rods, in some cases with polar flagella, 0.7 x 1.1 ~ in size (strains DMF 4/4, 5/3, 5/5, 5j7, 5/8, 5/9 and 5/10). In the case of strains DMF 4/4 and 5/3, short, fine "hairs" on the cell wall are to be ~38~
observed.
3. Biochemical characterisation and classification of the novel microorganisms For general biochemical characterisation and classification of the strains listed in Table 1, two commercially available test systems are used.
a) "Oxi/Ferm Tube" test (Roche) This test system is used in the case of gram-negative rods with positive oxidase reaction. The parkicular kest is carried out in parallel twice in each case with inoculum of the relevant strain taken from dimethylformamide-agar and nutrient-agar. See the manufacturee's instructions for a description of the standardised biochemical tests and for experimental procedure. The test results are summarised in Table 2.
....
. . . ....... .
. ,:
~38S~3 Table 2: DMF Strains tested with "Oxi/Ferm Tube."
(48/28C) , . .
Internal biochemical test reference ~
s~ ~o O ~ ~ '~V~I
i~ ,~ .~ ~ ~ o ~1 ~ a) ~ ~ ~ ~ u o a) o ~ ,, o o a~
h ~ ~ 1~ ~H ~1 ~l1 ~ :' ~
_ ~ O . !a;N __ ~ O 1~1 U U
DMF 3/3 _ _ + _ + + _ +
DMF 3/4 _ _ + _ _ ~ + + _ DMF 3/5 _ _ + _ _ + + _ +
DMF 3/6 _ _ _~ _ _ + _ + +
DMF 3/11 _ _ + _ _ + + _ +
DMF 3/12 _ _ + _ _ * + _ _ _ ____ _ ~1 DMF 4/4 _ _ ~ + _ ~- + _ t DMF 5/3 .~ _ ~ ~ _ ~ _ + .
DMF 5/5 _ _ + ~ _ ~ _ _ D~F 5/7 _ _ .~ ~ _ _ _ _ DMF 5/8 _ _ ~ + _ ~ ~ ~ +
DMF 5/9 _ _ + + _ _ _ _ _ DMF 5/10 _ _ _ ~ _ + + _ +
.., ~
, ~
:
.
:~23~5~3 The findings show that the group of strains DMF
3/3, 3/4, 3/5, 3/6, 3/11 and 3/12 (H2S formation clearly negative and xylose cleavage clearly positive) diEfers biochemically ~rom the group of strains DMF
4/4, 5/3, S/5, 5/7, 5/8, 5/9 and 5/10 ~H2S formation clearly positive, and xylose cleavage only in one case clearly negative). The biochemical differences within the two ~roups are slight. ~fter numerical evaluation of the test results of Table 2 guided by the instructions given by the manufacturer, it is possible to group the individual strains on the basis of their respective degrees of correspondence and approximately classify them taxonomically as Eollows:
Table 3: Taxonomic classification on the basis of numerical evaluation of the_"Oxi-Ferm Tube" Test Internal Taxonomic classification reference DMF 3/3 Pseudomonas stutzeri DMF 3/11 Pseudomonas-like, ~chromobacter sp.
DMF S/7 Pseudom~nas vesicularis DMF 5/8 no classiEication ~MF 5/9 DMF 5/10 _ "
~3 b) "API 20 E" Test This test s~stem is used specifically for distinguishing enterobacteriaceae and generally for detecting gram-negative bacteria. The standardised biochemical tests and the manner in which they are carried out are described in the manufacturer's guide.
The tests were likewise carried out in parallel twice in each case with inoculum of the relevant strain taken from dimeth~lformamide-agar and nutrient-agar.
,. . . : .
~23~S~3 Table 4: DMF Strains tested with API 20 E (API-Laborsystem GmbH2 (48h, 28C) _ . _ . Internal reference Biochemical test ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ O
~ ~ ~ ~ ~ ~ ~ In n u~ In ~ Ln ONPG: hydrolysis by + + + + ~ + ~ + ~ + + _ +
~-aalactosidase _ ADH~ arainine dihvdrolase _ _ _ _._ _ _ _ _ _ _ _ LDC, lysine decarboxylase _ _ _ _ _ _ _ _ _ _ _ ODC: ornithine decarboxylase _ _ ~ _ CIT: citrate utiLisation + + + + + + + + + ~ f + +
H2S: thiosulphate cleavage _ ~ ~ ~ - - - - _ URE: urease _ _ _ _ _ _ _ _ _ _ _ _ TDA: tryptophan desaminase _ _ ~ + ~ + _ _ _ _ _ _ _ IND: tryptophan degradation _ _ _ _ _ _ _ _ _ _ _ _ indole ~ormation VP: acetoin test _ +~+ + ~ ~ + + + +
GEL: gelatin liquefaction_ _ _ + ~ ~ - - - _ _ GLU: alucose utilisation _ +
MAN: mannitol utilisation INO: inositol utilisation _ _ _ - - - - - ------------~
SOR: sorbitol utilisation _ _ _ _ _ _ _ _ _ _ _ _ -- . ... . . .
RHA: rhamnose utilisation _ _ SAC: saccharose utilisation _ _ _ _ _ _ _ _ _ _ _ _ _ MEL: melibiose utilisation _ _ _ _ _ _ _ _ _ _ _ _ AMY: amygdal;.ne utilisation _ _ _ _ _ _ _ _ _ _ _ _ _ A~A- L-arabinose utilisation _ + _ _ _ _ _ ~ _ _ _ _ OX: oxidase .~ + ~ + + .~ + ~ +
_ , ~0 : + + -t + + -~ + ~ ~ + ~ + +
2 nitrate reduction _ 2 + + + + -~ + + ~ + + + + ~
LCAT: catalase + + + + + ~ + ~ +
. .~ .
'" .
.
~L~3~
The numerical evaluation of the results of the API 20 E test in accordance with the manuPacturer's instructions does not permit unequivocal taxonomical classification, so that the results shown in Table 4 can be used only for biochemical characterisation of the relevant strains.
Strains DMF 3/3, 3/4, 3/5, 3/6, 3/11 and 3/12 can be classified on the basis of morphological features (rods with polar flagella) and biochemical properties in the Oxi-Ferm test and API test as pseudomonads.
Strains 4/4, 5/3, 5/5, 5/7, 5/8, 5/9 and 5/10 have an oval cell form (very short, elagellate rods to spherical shape). Owing to a number of similarities in biochemical behaviour (exception: H2S formation in the Oxi-Ferm test) these strains also are referred to in the description oP the present invention as pseudomonads, the more so as oval cell forms are observed also in other Pseudomonas strains, for example Pseudomonas putida or Pseudomonas ovalis.
_ 4. Preservation oE the strains The following methods are suitable for preserving the strains given in Table 1:
a) adsorption Oe the cell material o~ the relevant strain onto glass beads in glycerol solution and subsequent storage at -20C, b) storing the cell material Oe the relevant strain on slant agar, and c) lyo-ampoul~s. The relevant culture is centrieuged ofP from the nutrient solution and the biomass is resuspended in Erom 1/4 to 1/3 parts by volume oP
15~ skim milk and lyophilised.
. .
,: , , .
.
~S93
Table 3: Taxonomic classification on the basis of numerical evaluation of the_"Oxi-Ferm Tube" Test Internal Taxonomic classification reference DMF 3/3 Pseudomonas stutzeri DMF 3/11 Pseudomonas-like, ~chromobacter sp.
DMF S/7 Pseudom~nas vesicularis DMF 5/8 no classiEication ~MF 5/9 DMF 5/10 _ "
~3 b) "API 20 E" Test This test s~stem is used specifically for distinguishing enterobacteriaceae and generally for detecting gram-negative bacteria. The standardised biochemical tests and the manner in which they are carried out are described in the manufacturer's guide.
The tests were likewise carried out in parallel twice in each case with inoculum of the relevant strain taken from dimeth~lformamide-agar and nutrient-agar.
,. . . : .
~23~S~3 Table 4: DMF Strains tested with API 20 E (API-Laborsystem GmbH2 (48h, 28C) _ . _ . Internal reference Biochemical test ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ O
~ ~ ~ ~ ~ ~ ~ In n u~ In ~ Ln ONPG: hydrolysis by + + + + ~ + ~ + ~ + + _ +
~-aalactosidase _ ADH~ arainine dihvdrolase _ _ _ _._ _ _ _ _ _ _ _ LDC, lysine decarboxylase _ _ _ _ _ _ _ _ _ _ _ ODC: ornithine decarboxylase _ _ ~ _ CIT: citrate utiLisation + + + + + + + + + ~ f + +
H2S: thiosulphate cleavage _ ~ ~ ~ - - - - _ URE: urease _ _ _ _ _ _ _ _ _ _ _ _ TDA: tryptophan desaminase _ _ ~ + ~ + _ _ _ _ _ _ _ IND: tryptophan degradation _ _ _ _ _ _ _ _ _ _ _ _ indole ~ormation VP: acetoin test _ +~+ + ~ ~ + + + +
GEL: gelatin liquefaction_ _ _ + ~ ~ - - - _ _ GLU: alucose utilisation _ +
MAN: mannitol utilisation INO: inositol utilisation _ _ _ - - - - - ------------~
SOR: sorbitol utilisation _ _ _ _ _ _ _ _ _ _ _ _ -- . ... . . .
RHA: rhamnose utilisation _ _ SAC: saccharose utilisation _ _ _ _ _ _ _ _ _ _ _ _ _ MEL: melibiose utilisation _ _ _ _ _ _ _ _ _ _ _ _ AMY: amygdal;.ne utilisation _ _ _ _ _ _ _ _ _ _ _ _ _ A~A- L-arabinose utilisation _ + _ _ _ _ _ ~ _ _ _ _ OX: oxidase .~ + ~ + + .~ + ~ +
_ , ~0 : + + -t + + -~ + ~ ~ + ~ + +
2 nitrate reduction _ 2 + + + + -~ + + ~ + + + + ~
LCAT: catalase + + + + + ~ + ~ +
. .~ .
'" .
.
~L~3~
The numerical evaluation of the results of the API 20 E test in accordance with the manuPacturer's instructions does not permit unequivocal taxonomical classification, so that the results shown in Table 4 can be used only for biochemical characterisation of the relevant strains.
Strains DMF 3/3, 3/4, 3/5, 3/6, 3/11 and 3/12 can be classified on the basis of morphological features (rods with polar flagella) and biochemical properties in the Oxi-Ferm test and API test as pseudomonads.
Strains 4/4, 5/3, 5/5, 5/7, 5/8, 5/9 and 5/10 have an oval cell form (very short, elagellate rods to spherical shape). Owing to a number of similarities in biochemical behaviour (exception: H2S formation in the Oxi-Ferm test) these strains also are referred to in the description oP the present invention as pseudomonads, the more so as oval cell forms are observed also in other Pseudomonas strains, for example Pseudomonas putida or Pseudomonas ovalis.
_ 4. Preservation oE the strains The following methods are suitable for preserving the strains given in Table 1:
a) adsorption Oe the cell material o~ the relevant strain onto glass beads in glycerol solution and subsequent storage at -20C, b) storing the cell material Oe the relevant strain on slant agar, and c) lyo-ampoul~s. The relevant culture is centrieuged ofP from the nutrient solution and the biomass is resuspended in Erom 1/4 to 1/3 parts by volume oP
15~ skim milk and lyophilised.
. .
,: , , .
.
~S93
5. ~egradable compounds A selection of those compounds (carbon sources) which can be degraded by the strains or the mixed culture mentioned in Table 1 upon the addition of a nitrogen source, for example ammonium nitrate, is given in Table 6.
~38593 Table 6:
_ ~
Internal reference C-sources ~ ~ ~ ~ . ~ ~ ~ ~ ~ ~ _ O _ _ _ _ _ _ _ _ _ _ dimethylform- ~¦ + + + + + + ~ + + ~ + ~ +
amlde (2 q/l~ _ _ _ _ _ _ _ formamide _ _ ~ + + + + + + + + + ~ +
(2 q/l) __ _ _ _ _ _ _ _ _ methylform- + + + + + ~ + + + + + ~ + +
amide (5 a/l) _ _ _ _ ~ _ _ _ ethylform- + + + + + + + + + + + + + +
amide (5 q/l) _ _ _ _ _ _ _ acetamide + ~- + + + + + + + + + + + +
( a/ ) __ _ _ _ _ _ _ _ methyl formate + + + + + + ~ + + ~ + ~ + +
~ q~ ~ _ _ _ _ _ __ __ __ sodium formate + + + + + + + + ~ + + + + +
~L~ __ _ _ _ _ __ sodium acetate + + + + + + + + + + + + ~ +
( q/ ) _ _ _ _ _ _ _ . _ ~+ + + + + + + ~ + + + + + +
methanol + ~ .~ ~ + + _ _ _ _ _ _ _ +
(2 q~l) _ _ _ ~_.
ethanol r+ _ + _ _ (2 q/l)_ _ _ _ _ _ _ _ _ _ _ methylammo-nium chloride .~ + t + + + + + + .~ + ~ + +
5~a/1) _ _ _ _ ~ _ _ _ _ _ _ _ _ _ dimethylammo-nium chloride + + ~ + + + + + + + + + + +
( a/ )_ _ _ _ _ _ __ _ _ _ trimethylammo-nium chloride + .~ ~ .~ + + + + + + + + + ~
(5 a/l)_ _ _ _ _ _ _ _ _ _ _ ethylammonium ~ + + + + + ~ + ~ ~ + + ~ +
chloride (5 a/l~ _ _ _ _ _ _ _ _ _ trimethyl phos- ~ + + + + + _ _ _ _ _ _ _ +
phit~l2 q/l~ _ _ _ _ _ _ _ _ _ _ ~"
' ;
Table 6 (continued) _ Internal reference C-sources _ _ _ _ _ _ _ o __ _ . ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ i~
n u~ u~ Lt~ u~ u~
, . __ _ _ _ _ _ _ _ _ hydantoin + + + + + + + ~ + + + + + +
_ _ _ _ _ _ _ _ . .
creatine + + + + + + + + ~ ~ + + + +
~ . _ . _ _ _ _ _ _ . _ __ creatinine _ _ + _ _ _ _ ~ ~ ~ ~ + +
barbituric ~ + + + + + + + + + + + + +
acid . _ _ _ _ _ _ _ _ _ _ _ ._ choline .~ + ~ + + + + + ~ + + ~ + ~
_ _ _ _ __ _ ; _ _ _ betaine + + ~ + + + + + + + + + + +
_ _ _ __ _ _ dimethyl- + + + + + ~ + + ~ + + + + +
glycine _ _ _ _ _ _ __ _ _ _ sarcosine + + + + .~ + + + + + ~ + + +
. _ _ _ ___ _ _ glycine + + + + + + + + + + + + + ~
_ . _ _ _ ~ _ _ _ _ __ glyoxylic + + + + .~ + + + + + + + + +
ac ld _ _ _ _ __ _ _ _. l .___ acetonltrile _ _ _ _ _ _ _ __ _ _ _ _ +
isobutyro- _ _ _ __ _ ~ + + + + + + + +
nitrile ~ _ _ _ _ __ ~ _. _ _ _ (~: good growth, + moderate growth, - only slight if any growth) . . , .
.. . .
, ~ , .
:
S~3 If the aqueous solution to be purified contains one of the nitrogen-containing compounds mentioned in Table 6, for example dimethylformamide, methyl-formamide, ethylformamide, hydantoin, creatinine, creatine or barbituric acid, it is not necessary in most cases to add a nitrogen source.
The experimental findings given in Table 6 were determined after preparing a shake culture by inoculation of the relevant DMF-containing preculture into nutrient solution MV 7 containing the relevant carbon or carbon and nitrogen source (composition described hereinbefore), and buffering to p~ 7, incubating over a period of 5 to 8 days at 28C and 250 rpm and measuring the optical density OD650 (moderate growth OD650 < 0.15, good growth OD
0.15) and optionally adding ammonium chloride.
From the strains listed in Table 1, mutants can form spontaneously (natural mutants) or artificial mutants can be produced which, like the natural strains, are capable of degrading the above-mentioned compounds and salts, especially dimethylformamide, in aqueous solution and produce biomass. Such mutants can be produced by chemical means, eor example by treatment with certain guanidine derivatives, ~or example N-methyl-N'-nitro-N-nitrosoguanicline or with an alkali nitrite, Eor example sodium nitrite, or by physical means, for example by ultra-violet, X-ray or radioactive radiation.
The microorganisms according to the invention are used in a process for the microbiological purification of aqueous solutions containing dimethylformamide, formamide, methylformamide, ethylformamide, acetamide, methanol, ethanol, saccharides, lower alkanoates, methyl-, ethyl-, dimethyl- or trimethyl-ammonium halides, dimethyl phosphite, trimethyl phosphite, .
. : ~ ... ''''''' :
iZ3~593 hydantoin, creatine, creatinine, barbituric acid, choline, betaine, sarcosine, dimethylglycine, glycine, glyoxylic acid, N-Eormylamino acids, N-acetylamino acids, acetonitrile or isobutyronitrile or mixtures of these compounds, which process is characterised in that there is cultured in such an aqueous solution a micro-organism of the genus Pseudomonas or of a Pseudomonas-like genus, which microorganism produces biomass in this solution, or a mutant, derived from this micro-organism, which is also capable of producing this biomass, in the presence of essential inorganic salts and optionally a nitrogen source, at from approximately 20C to approximately 40C and at a pH of from approximately 4 to approximately 7.5, under aerobic conditions, and, if desired, khe resulting biomass is isolated.
During the culturing or fermentation , the strains listed in Table 1 degrade the pollutants present in aqueous solution, for example in a waste water, for example dimethylformamide, formamide, methylformamide, ethylformamide, methanol, ethanol, sodium acetate, glucose, methyl-, ethyl-, dimethyl- or trimethyl-ammonium chloride, dimethyl phosphite, trimethyl phosphite, acetonitrile, isobutyronitrile or mixtures oE these compounds or salts and consume oxygen. ~uring the degradation process, the microorganisms produce biomass which is characterised, for example, by a multiple oE the approximate empirical formula C5HgNOx (x ~ 4-5) and a content oE approximately 37.0 % C, 6.2%.H and 9.6~ N (lyophilised). Further fermentation products that are ~ormed are carbon dioxide and, under neutral and acidic conditions, ammonium ions. ~uring fermentation, the pH o~ the solution to be purified is to be adjusted to values of between 4~0 and 7.5, preferably approximately 6 to 7, . ,,, :
,, ,, , :, ~
by the addition of buffer solution, for examp]e phosphate buffer solution, or aqueous bases, for example dilute, aqueous sodium or potassium hydroxide solution.
To purify aqueous solutions containing dimethyl-formamide, strains ~MF 3/3, 3/4, 3/5, 3/6, 3/11, 3/12, 4/4, 5/3, 5/5, 5/7, 5/~, 5/9 and 5/10 and the mixed culture VMF/HW 1-5 are preferably used. To purify aqueous solutions containing formamide, strains DMF 4/4, 5/3, 5/5, 5/7, 5/8, 5/9 and 5/10 and the mixed culture DMF/HW 1-5 are preerably used.
For degrading formate salts, ~or example sodium formate, there are suitable only strains DMF 4/4, 5/3, 5/5, 5/7, S/8, 5/9 and 5/10, for methanol only strains 3/3, 3/4, 3/5, 3/6, 3/11 and 3/12 and the mixed culture DMF/HW 1-5, and for dimethyl phosphite and trimethyl phosphite only strains DMF 3/3, 3/4, 3/6, 3/11 and 3/12 and the mixed culture DMF/HW 1-5.
Culturing is carried out in the presence of essential inorganic salts. ~uch salts are, for example, water-soluble halides, Eor example chlorides, carbonates, sulphates or phosphates of alkali metals, for example sodium or potassium, alkaline earth metals, for example calcium or magnesium, or transition metals, for example iron, manganese, tnolybdenum, copper or zinc.
PreEerred essentlal inorganic salts are, for example, the salts present in the nutrient solution MV 7, for example disodium or dipotassium hydrogen phosphate, sodium or potassium dihydrogen phosphate, ma~nesium and iron sulphate and potassium and calcium chloride. %inc, manganese and copper sulphate, sodium molybdate and borax may also be added in small amounts.
For purifying aqueous solutions that contain, ~or example, methanol, ethanol, acetate or glucose and no ~3~5~3i nitrogen-containing compounds, there are added as nitrogen source, for example, amino acids, peptides or proteins or their degradation products, such as peptone or tr~ptone, meat extracts, flours, for example of corn, whea-t or beans, ~or example o~ the soya bean, distillation residues of alcohol production, yeast extracts, ammonium salts, for example ammonium chloride, or nitrates, for example potassium or ammonium nitrate.
Some of the strains mentioned in Table 1 can, at the beginning of fermentation, grow only after the addition of a nitrogen source even if a nitrogen-containing compound to be ~egraded is already present in the aqueous solution. Therefore, a nitrogen source is added if, in aqueous solutions, dimethylformamide is to be degraded in the presence of strains DMF 5/5, 5/7, 5/8, 5/~ and 5/10 or formamide is to be degraded in the presence of strains DMF 3/3l 3/4, 3/5, 3/6, 3/11 and 3/12.
Culturing is eEfected un~er aerobic conditions, Eor example with oxygen or air being supplied, and while shaking or stirring in shaking Elasks or fermenters. When using the microorganisms according to the invention in purification platlts, culturing can be carried out in open or closed clearing basins also in the presence oE other microorganisms. Culturing can be carried out within a temperature range of from approximately 25 to approximately 35C, preEerably at Erom approximately 27 to approximately 28C.
Culturlng can be carried out batchwise, for example by single or repeated addition of nutrient solution, or continuously by continuous addition oE
nutrient solution.
Culturing is preferably e~ected in several stages by first preparing one or more precultures, for example , . .
.
' :
:
~ 385~3 in a liquid nutrient medium, for example MV 7, which are then transferred by inoculation to the actual main culture. A preculture can be prepared, for example, by transferring a sample with cell material of the micro-organism concerned, which is stored, for example, on slant agar, to a sterile nutrient solution, for example MV 7, with a suitable carbon source, for example dimethylformamide, and incubating for several days at 28C. Fresh nutrient solutions, for example MV 7, with the same carbon source are inoculated with this first preculture and again incubated for several days at 28C.
The course of the fermentation can be monitored analytically by taking samples, for example by measuring the pH of the culture or the optical density which is a measure of the growth of the particular strain, as well as by gravimetric analysis on the basis of the dry weight of the biomass formed.
The present invention relates also to the biomass.
This can he worlced up, for example according to one o the numerous methods described in European Patent SpeciEication 0 010 243, and converted, for example, into fertiliser.
The biomass is a valuable raw material that has a defined and reproducible composition. ~pproximate empirical Eormula: C5FIgN0x (X = 4-5); content (lyophilisate): 37.0 % C, 6.2~ H and 9.6% N.
The present invention relates also to the enzymes present in the blomass or the enzyme extract obtainable from the biomass. Enzymes can be extracted from the biomass in a manner known E~ se, for example as described in British Patent Application 2,019,390.
The biomass obtainable in accordance with the process can be used as single cell protein, for example as cattle feed additive. The biomass can also be used, ' ~
~Z38593 for example, in the form of a suspension or after being worked up, for example after dehydration or pasteurisation, as fertiliser. The biomass can also be used as starting material for the production of biogas with a high calorific value (composition: approximately 70 % methane, 29 ~ carbon dioxide and 1 % hydrogen;
calorific value: approximately 5500-6500 kcal/m3), for example by anaerobic fermentation in fermentation towers. The residue (digested sludge) from the process for the production of biogas is also a high-grade fertiliser which, compared with the original biomass, is highly enriched with nitrogen.
The following Examples illustrate the present invention. Temperatures are given in degrees Centigrade.
Example 1 (Preparation of the preculture):
A sample of the microorganism DMF 3/6 which is stored on slant agar is introduced into a shalcing flask containing 20 ml of nutrient solution MV 7 having the composition indicated above and containing dimethyl-formamide in a concentration of 5 g/litre, and the batch is incubated for 72 hours at 28 and 250 rpm.
5-7 ml of this first preculture are introduced into a second shaking flask containing 100 ml of nutrient solution MV 7 (without ammonium nitrate) and dimethyl-~ormamide in a concentration of 5 g/litre and 5 mmol of phosphate buffer (p~ 7), and the batch is incubated for 72 hours at 28 and 250 rpm.
Example 2:
Precultures of strains DMF 3/3, 3/4, 3/5, 3/6, 3/11, 3/12, 4/4, 5/3, 5/5, 5/7, 5/8, 5/9 and 5/10 and of the mixed culture DMF/HW 1-5 can be prepared analoyously to Example 1. To prepare precultures oE
strains DMF 5/5, 5/7, 5/8, 5/9 and 5/10, ammonium nitrate (2 g/litre) is added as nitrogen source.
Example 3:
In a laboratory fermenter, optionally heat-sterilLsed (sterLlisation 20 minutes at 120) nutrient solution MV 7 (without ammonium nitrate) is combined with an opkionally sterile-fi]tered, aqueous dimethylEormamide solution in such a manner that an approximate working volume of 10 litres with a concentration of approximately 5 g/litre dimethyl-formamide is obtained.
~ sample with approximately 500 ml of the second . ~ :
~3 preculture o-E strain ~MF 3/6 is added and the ~ollowing conditions are maintained in the ~ermenter: pH value of 7.0 which is maintained constant by the addition, as requiredj o~ aqueous 0.1N NaOH or 0.1N HCl solution, temperature of 28, air supply 0~25 litres/l minute and stirring speed of 400-700 rpm.
The strain grows in this solution containing dimethylformamide as the sole carbon and nitrogen source. After approximately 55 hours, the dimethyl-formamide used has been degraded. The resulting biomass is in the ~orm of a mixture of single cells or aggregates of dif~ering size which can be separated off by sedimentation or centri~ugation. -~xample 4:
DimethylEormamide can be degraded in aqueous solution analogously to Example 3 by culturing in a laboratory fermenter precultures or mixed cultures of strains DMF 3/3, 3/4, 3/5, 3/6, 3/ll, 3/12, 4/4, 5/3, 5/5, 5/7, 5/8, S/9 and 5/10 or the mixed culture DMF/H~ 1-5. When precultures of strains DMF 5/5, 5/7, 5/8, 5/9 and 5/10 are used, ammonium nitrate (2 g/litre) is added as nitrogen source.
Example 5:
In a laboratory eermenter, optionally heat-sterilised (~terilisation 20 minutes at 120) nutrient solution MV 7 ~without ammonium nitrate) is combined with an optLonally sterile~filtered waste-water solution containing dimethylformamide (composition: approximately 6.0% dimethylformamide, 7% methanol, 1.~% sodium sulphate, S.~% sodium dimethyl phosphate, 3.6% organic constituents and ?7% water) in . . ~ . .
: - :
~238~
such a manner that an approximate working volume of 8 litres with a concentration of approximately 5 g/litre dimethylformamide is obtained. A sample with approximately 500 ml of the second preculture of strain DMF 3/6 is added and the conditions mentioned in Example 3 are maintained in the fermenter. Degradation of dimethylformamide and methanol takes place within approximately 100 hours.
Example 6:
Dimethylformamide in optionally sterile-filtered waste water solutions can be degraded analogously to Example 5 by culturing in a laboratory fermenter precultures or mixed cultures of strains DMF 3/3, 3/4, 3/5, 3/6, 3/11, 3/12, 4/4, 5/3, 5/5, 5/7, 5/8, 5/9 and 5/10 or the mixed culture DMF/HW 1-5. When precultures of strains DMF 5/5, 5/7, 5/8, 5/9 and 5/10 are used, ammonium nitrate (2 g/litre) is added as nitrogen source.
~38593 Table 6:
_ ~
Internal reference C-sources ~ ~ ~ ~ . ~ ~ ~ ~ ~ ~ _ O _ _ _ _ _ _ _ _ _ _ dimethylform- ~¦ + + + + + + ~ + + ~ + ~ +
amlde (2 q/l~ _ _ _ _ _ _ _ formamide _ _ ~ + + + + + + + + + ~ +
(2 q/l) __ _ _ _ _ _ _ _ _ methylform- + + + + + ~ + + + + + ~ + +
amide (5 a/l) _ _ _ _ ~ _ _ _ ethylform- + + + + + + + + + + + + + +
amide (5 q/l) _ _ _ _ _ _ _ acetamide + ~- + + + + + + + + + + + +
( a/ ) __ _ _ _ _ _ _ _ methyl formate + + + + + + ~ + + ~ + ~ + +
~ q~ ~ _ _ _ _ _ __ __ __ sodium formate + + + + + + + + ~ + + + + +
~L~ __ _ _ _ _ __ sodium acetate + + + + + + + + + + + + ~ +
( q/ ) _ _ _ _ _ _ _ . _ ~+ + + + + + + ~ + + + + + +
methanol + ~ .~ ~ + + _ _ _ _ _ _ _ +
(2 q~l) _ _ _ ~_.
ethanol r+ _ + _ _ (2 q/l)_ _ _ _ _ _ _ _ _ _ _ methylammo-nium chloride .~ + t + + + + + + .~ + ~ + +
5~a/1) _ _ _ _ ~ _ _ _ _ _ _ _ _ _ dimethylammo-nium chloride + + ~ + + + + + + + + + + +
( a/ )_ _ _ _ _ _ __ _ _ _ trimethylammo-nium chloride + .~ ~ .~ + + + + + + + + + ~
(5 a/l)_ _ _ _ _ _ _ _ _ _ _ ethylammonium ~ + + + + + ~ + ~ ~ + + ~ +
chloride (5 a/l~ _ _ _ _ _ _ _ _ _ trimethyl phos- ~ + + + + + _ _ _ _ _ _ _ +
phit~l2 q/l~ _ _ _ _ _ _ _ _ _ _ ~"
' ;
Table 6 (continued) _ Internal reference C-sources _ _ _ _ _ _ _ o __ _ . ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ i~
n u~ u~ Lt~ u~ u~
, . __ _ _ _ _ _ _ _ _ hydantoin + + + + + + + ~ + + + + + +
_ _ _ _ _ _ _ _ . .
creatine + + + + + + + + ~ ~ + + + +
~ . _ . _ _ _ _ _ _ . _ __ creatinine _ _ + _ _ _ _ ~ ~ ~ ~ + +
barbituric ~ + + + + + + + + + + + + +
acid . _ _ _ _ _ _ _ _ _ _ _ ._ choline .~ + ~ + + + + + ~ + + ~ + ~
_ _ _ _ __ _ ; _ _ _ betaine + + ~ + + + + + + + + + + +
_ _ _ __ _ _ dimethyl- + + + + + ~ + + ~ + + + + +
glycine _ _ _ _ _ _ __ _ _ _ sarcosine + + + + .~ + + + + + ~ + + +
. _ _ _ ___ _ _ glycine + + + + + + + + + + + + + ~
_ . _ _ _ ~ _ _ _ _ __ glyoxylic + + + + .~ + + + + + + + + +
ac ld _ _ _ _ __ _ _ _. l .___ acetonltrile _ _ _ _ _ _ _ __ _ _ _ _ +
isobutyro- _ _ _ __ _ ~ + + + + + + + +
nitrile ~ _ _ _ _ __ ~ _. _ _ _ (~: good growth, + moderate growth, - only slight if any growth) . . , .
.. . .
, ~ , .
:
S~3 If the aqueous solution to be purified contains one of the nitrogen-containing compounds mentioned in Table 6, for example dimethylformamide, methyl-formamide, ethylformamide, hydantoin, creatinine, creatine or barbituric acid, it is not necessary in most cases to add a nitrogen source.
The experimental findings given in Table 6 were determined after preparing a shake culture by inoculation of the relevant DMF-containing preculture into nutrient solution MV 7 containing the relevant carbon or carbon and nitrogen source (composition described hereinbefore), and buffering to p~ 7, incubating over a period of 5 to 8 days at 28C and 250 rpm and measuring the optical density OD650 (moderate growth OD650 < 0.15, good growth OD
0.15) and optionally adding ammonium chloride.
From the strains listed in Table 1, mutants can form spontaneously (natural mutants) or artificial mutants can be produced which, like the natural strains, are capable of degrading the above-mentioned compounds and salts, especially dimethylformamide, in aqueous solution and produce biomass. Such mutants can be produced by chemical means, eor example by treatment with certain guanidine derivatives, ~or example N-methyl-N'-nitro-N-nitrosoguanicline or with an alkali nitrite, Eor example sodium nitrite, or by physical means, for example by ultra-violet, X-ray or radioactive radiation.
The microorganisms according to the invention are used in a process for the microbiological purification of aqueous solutions containing dimethylformamide, formamide, methylformamide, ethylformamide, acetamide, methanol, ethanol, saccharides, lower alkanoates, methyl-, ethyl-, dimethyl- or trimethyl-ammonium halides, dimethyl phosphite, trimethyl phosphite, .
. : ~ ... ''''''' :
iZ3~593 hydantoin, creatine, creatinine, barbituric acid, choline, betaine, sarcosine, dimethylglycine, glycine, glyoxylic acid, N-Eormylamino acids, N-acetylamino acids, acetonitrile or isobutyronitrile or mixtures of these compounds, which process is characterised in that there is cultured in such an aqueous solution a micro-organism of the genus Pseudomonas or of a Pseudomonas-like genus, which microorganism produces biomass in this solution, or a mutant, derived from this micro-organism, which is also capable of producing this biomass, in the presence of essential inorganic salts and optionally a nitrogen source, at from approximately 20C to approximately 40C and at a pH of from approximately 4 to approximately 7.5, under aerobic conditions, and, if desired, khe resulting biomass is isolated.
During the culturing or fermentation , the strains listed in Table 1 degrade the pollutants present in aqueous solution, for example in a waste water, for example dimethylformamide, formamide, methylformamide, ethylformamide, methanol, ethanol, sodium acetate, glucose, methyl-, ethyl-, dimethyl- or trimethyl-ammonium chloride, dimethyl phosphite, trimethyl phosphite, acetonitrile, isobutyronitrile or mixtures oE these compounds or salts and consume oxygen. ~uring the degradation process, the microorganisms produce biomass which is characterised, for example, by a multiple oE the approximate empirical formula C5HgNOx (x ~ 4-5) and a content oE approximately 37.0 % C, 6.2%.H and 9.6~ N (lyophilised). Further fermentation products that are ~ormed are carbon dioxide and, under neutral and acidic conditions, ammonium ions. ~uring fermentation, the pH o~ the solution to be purified is to be adjusted to values of between 4~0 and 7.5, preferably approximately 6 to 7, . ,,, :
,, ,, , :, ~
by the addition of buffer solution, for examp]e phosphate buffer solution, or aqueous bases, for example dilute, aqueous sodium or potassium hydroxide solution.
To purify aqueous solutions containing dimethyl-formamide, strains ~MF 3/3, 3/4, 3/5, 3/6, 3/11, 3/12, 4/4, 5/3, 5/5, 5/7, 5/~, 5/9 and 5/10 and the mixed culture VMF/HW 1-5 are preferably used. To purify aqueous solutions containing formamide, strains DMF 4/4, 5/3, 5/5, 5/7, 5/8, 5/9 and 5/10 and the mixed culture DMF/HW 1-5 are preerably used.
For degrading formate salts, ~or example sodium formate, there are suitable only strains DMF 4/4, 5/3, 5/5, 5/7, S/8, 5/9 and 5/10, for methanol only strains 3/3, 3/4, 3/5, 3/6, 3/11 and 3/12 and the mixed culture DMF/HW 1-5, and for dimethyl phosphite and trimethyl phosphite only strains DMF 3/3, 3/4, 3/6, 3/11 and 3/12 and the mixed culture DMF/HW 1-5.
Culturing is carried out in the presence of essential inorganic salts. ~uch salts are, for example, water-soluble halides, Eor example chlorides, carbonates, sulphates or phosphates of alkali metals, for example sodium or potassium, alkaline earth metals, for example calcium or magnesium, or transition metals, for example iron, manganese, tnolybdenum, copper or zinc.
PreEerred essentlal inorganic salts are, for example, the salts present in the nutrient solution MV 7, for example disodium or dipotassium hydrogen phosphate, sodium or potassium dihydrogen phosphate, ma~nesium and iron sulphate and potassium and calcium chloride. %inc, manganese and copper sulphate, sodium molybdate and borax may also be added in small amounts.
For purifying aqueous solutions that contain, ~or example, methanol, ethanol, acetate or glucose and no ~3~5~3i nitrogen-containing compounds, there are added as nitrogen source, for example, amino acids, peptides or proteins or their degradation products, such as peptone or tr~ptone, meat extracts, flours, for example of corn, whea-t or beans, ~or example o~ the soya bean, distillation residues of alcohol production, yeast extracts, ammonium salts, for example ammonium chloride, or nitrates, for example potassium or ammonium nitrate.
Some of the strains mentioned in Table 1 can, at the beginning of fermentation, grow only after the addition of a nitrogen source even if a nitrogen-containing compound to be ~egraded is already present in the aqueous solution. Therefore, a nitrogen source is added if, in aqueous solutions, dimethylformamide is to be degraded in the presence of strains DMF 5/5, 5/7, 5/8, 5/~ and 5/10 or formamide is to be degraded in the presence of strains DMF 3/3l 3/4, 3/5, 3/6, 3/11 and 3/12.
Culturing is eEfected un~er aerobic conditions, Eor example with oxygen or air being supplied, and while shaking or stirring in shaking Elasks or fermenters. When using the microorganisms according to the invention in purification platlts, culturing can be carried out in open or closed clearing basins also in the presence oE other microorganisms. Culturing can be carried out within a temperature range of from approximately 25 to approximately 35C, preEerably at Erom approximately 27 to approximately 28C.
Culturlng can be carried out batchwise, for example by single or repeated addition of nutrient solution, or continuously by continuous addition oE
nutrient solution.
Culturing is preferably e~ected in several stages by first preparing one or more precultures, for example , . .
.
' :
:
~ 385~3 in a liquid nutrient medium, for example MV 7, which are then transferred by inoculation to the actual main culture. A preculture can be prepared, for example, by transferring a sample with cell material of the micro-organism concerned, which is stored, for example, on slant agar, to a sterile nutrient solution, for example MV 7, with a suitable carbon source, for example dimethylformamide, and incubating for several days at 28C. Fresh nutrient solutions, for example MV 7, with the same carbon source are inoculated with this first preculture and again incubated for several days at 28C.
The course of the fermentation can be monitored analytically by taking samples, for example by measuring the pH of the culture or the optical density which is a measure of the growth of the particular strain, as well as by gravimetric analysis on the basis of the dry weight of the biomass formed.
The present invention relates also to the biomass.
This can he worlced up, for example according to one o the numerous methods described in European Patent SpeciEication 0 010 243, and converted, for example, into fertiliser.
The biomass is a valuable raw material that has a defined and reproducible composition. ~pproximate empirical Eormula: C5FIgN0x (X = 4-5); content (lyophilisate): 37.0 % C, 6.2~ H and 9.6% N.
The present invention relates also to the enzymes present in the blomass or the enzyme extract obtainable from the biomass. Enzymes can be extracted from the biomass in a manner known E~ se, for example as described in British Patent Application 2,019,390.
The biomass obtainable in accordance with the process can be used as single cell protein, for example as cattle feed additive. The biomass can also be used, ' ~
~Z38593 for example, in the form of a suspension or after being worked up, for example after dehydration or pasteurisation, as fertiliser. The biomass can also be used as starting material for the production of biogas with a high calorific value (composition: approximately 70 % methane, 29 ~ carbon dioxide and 1 % hydrogen;
calorific value: approximately 5500-6500 kcal/m3), for example by anaerobic fermentation in fermentation towers. The residue (digested sludge) from the process for the production of biogas is also a high-grade fertiliser which, compared with the original biomass, is highly enriched with nitrogen.
The following Examples illustrate the present invention. Temperatures are given in degrees Centigrade.
Example 1 (Preparation of the preculture):
A sample of the microorganism DMF 3/6 which is stored on slant agar is introduced into a shalcing flask containing 20 ml of nutrient solution MV 7 having the composition indicated above and containing dimethyl-formamide in a concentration of 5 g/litre, and the batch is incubated for 72 hours at 28 and 250 rpm.
5-7 ml of this first preculture are introduced into a second shaking flask containing 100 ml of nutrient solution MV 7 (without ammonium nitrate) and dimethyl-~ormamide in a concentration of 5 g/litre and 5 mmol of phosphate buffer (p~ 7), and the batch is incubated for 72 hours at 28 and 250 rpm.
Example 2:
Precultures of strains DMF 3/3, 3/4, 3/5, 3/6, 3/11, 3/12, 4/4, 5/3, 5/5, 5/7, 5/8, 5/9 and 5/10 and of the mixed culture DMF/HW 1-5 can be prepared analoyously to Example 1. To prepare precultures oE
strains DMF 5/5, 5/7, 5/8, 5/9 and 5/10, ammonium nitrate (2 g/litre) is added as nitrogen source.
Example 3:
In a laboratory fermenter, optionally heat-sterilLsed (sterLlisation 20 minutes at 120) nutrient solution MV 7 (without ammonium nitrate) is combined with an opkionally sterile-fi]tered, aqueous dimethylEormamide solution in such a manner that an approximate working volume of 10 litres with a concentration of approximately 5 g/litre dimethyl-formamide is obtained.
~ sample with approximately 500 ml of the second . ~ :
~3 preculture o-E strain ~MF 3/6 is added and the ~ollowing conditions are maintained in the ~ermenter: pH value of 7.0 which is maintained constant by the addition, as requiredj o~ aqueous 0.1N NaOH or 0.1N HCl solution, temperature of 28, air supply 0~25 litres/l minute and stirring speed of 400-700 rpm.
The strain grows in this solution containing dimethylformamide as the sole carbon and nitrogen source. After approximately 55 hours, the dimethyl-formamide used has been degraded. The resulting biomass is in the ~orm of a mixture of single cells or aggregates of dif~ering size which can be separated off by sedimentation or centri~ugation. -~xample 4:
DimethylEormamide can be degraded in aqueous solution analogously to Example 3 by culturing in a laboratory fermenter precultures or mixed cultures of strains DMF 3/3, 3/4, 3/5, 3/6, 3/ll, 3/12, 4/4, 5/3, 5/5, 5/7, 5/8, S/9 and 5/10 or the mixed culture DMF/H~ 1-5. When precultures of strains DMF 5/5, 5/7, 5/8, 5/9 and 5/10 are used, ammonium nitrate (2 g/litre) is added as nitrogen source.
Example 5:
In a laboratory eermenter, optionally heat-sterilised (~terilisation 20 minutes at 120) nutrient solution MV 7 ~without ammonium nitrate) is combined with an optLonally sterile~filtered waste-water solution containing dimethylformamide (composition: approximately 6.0% dimethylformamide, 7% methanol, 1.~% sodium sulphate, S.~% sodium dimethyl phosphate, 3.6% organic constituents and ?7% water) in . . ~ . .
: - :
~238~
such a manner that an approximate working volume of 8 litres with a concentration of approximately 5 g/litre dimethylformamide is obtained. A sample with approximately 500 ml of the second preculture of strain DMF 3/6 is added and the conditions mentioned in Example 3 are maintained in the fermenter. Degradation of dimethylformamide and methanol takes place within approximately 100 hours.
Example 6:
Dimethylformamide in optionally sterile-filtered waste water solutions can be degraded analogously to Example 5 by culturing in a laboratory fermenter precultures or mixed cultures of strains DMF 3/3, 3/4, 3/5, 3/6, 3/11, 3/12, 4/4, 5/3, 5/5, 5/7, 5/8, 5/9 and 5/10 or the mixed culture DMF/HW 1-5. When precultures of strains DMF 5/5, 5/7, 5/8, 5/9 and 5/10 are used, ammonium nitrate (2 g/litre) is added as nitrogen source.
Claims (8)
1. Biologically pure microorganisms of the genus Pseudomonas or of a Pseudomonas-like genus selected from the group of the following strains:
DMF 3/3 (NRRL-B-15358), DMF 3/4 (NRRL-B-15359), DMF 3/5 (NRRL-B-15360), DMF 3/6 (NRRL-B-15361), DMF 3/11 (NRRL-B-15362), DMF 3/12 (NRRL-B-15363), DMF 4/4 (NRRL-B-15364), DMF 5/3 (NRRL-B-15365), DMF 5/5 (NRRL-B-15366), DMF 5/7 (NRRL-B-15367), DMF 5/8 (NRRL-B-15368), DMF 5/9 (NRRL-B-15369) and DMF 5/10 (NRRL-B-15370), the mixed culture DMF/HW 1-5 (NRRL-B-15371), or other mixed cultures thereof, and mutants derived thereof, which produce biomass in aqueous solution containing dimethylformamide, formamide, methyl- or ethyl-formamide, acetamide, methanol, ethanol, saccharides, lower alkanoates, methyl-, ethyl-, dimethyl- or trimethyl-ammonium halides, dimethyl phosphite, trimethyl phosphite, hydantoin, creatine, creatinine, barbituric acid, choline, betaine, sarcosine, dimethylglycine, glycine, glyoxylic acid, N-formylamino acids, N-acetylamino acids, aceto-nitrile or isobutyronitrile or mixtures of these compounds.
DMF 3/3 (NRRL-B-15358), DMF 3/4 (NRRL-B-15359), DMF 3/5 (NRRL-B-15360), DMF 3/6 (NRRL-B-15361), DMF 3/11 (NRRL-B-15362), DMF 3/12 (NRRL-B-15363), DMF 4/4 (NRRL-B-15364), DMF 5/3 (NRRL-B-15365), DMF 5/5 (NRRL-B-15366), DMF 5/7 (NRRL-B-15367), DMF 5/8 (NRRL-B-15368), DMF 5/9 (NRRL-B-15369) and DMF 5/10 (NRRL-B-15370), the mixed culture DMF/HW 1-5 (NRRL-B-15371), or other mixed cultures thereof, and mutants derived thereof, which produce biomass in aqueous solution containing dimethylformamide, formamide, methyl- or ethyl-formamide, acetamide, methanol, ethanol, saccharides, lower alkanoates, methyl-, ethyl-, dimethyl- or trimethyl-ammonium halides, dimethyl phosphite, trimethyl phosphite, hydantoin, creatine, creatinine, barbituric acid, choline, betaine, sarcosine, dimethylglycine, glycine, glyoxylic acid, N-formylamino acids, N-acetylamino acids, aceto-nitrile or isobutyronitrile or mixtures of these compounds.
2. The mixed culture DMF/HW 1-5 (NRRL-B-15371) according to claim 1.
3. Process for the microbiological purification of aqueous solutions containing dimethylformamide, formamide, methyl- or ethyl-formamide, acetamide, methanol, ethanol, saccharides, lower alkanoates, methyl-, ethyl-, dimethyl-or trimethyl-ammonium halides, dimethyl phosphite, tri-methyl phosphite, hydantoin, creatine, creatinine, barbituric acid, choline, betaine, sarcosine, dimethyl-glycine, glycine, glyoxylic acid, N-formylamino acids, N-acetylamino acids, acetonitrile or isobutyronitrile or mixtures of these compounds, characterised in that there is cultured in such aqueous solution a biologically pure microorganism of the genus Pseudomonas or of a Pseudomonas-like genus selected from the group of the following strains:
DMF 3/3 (NRRL-B-15358), DMF 3/4 (NRRL-B-15359), DMF 3/5 (NRRL-B-15360), DMF 3/6 (NRRL-B-15361), DMF 3/11 (NRRL-B-15362), DMF 3/12 (NRRL-B-15363), DMF 4/4 (NRRL-B-15364), DMF 5/3 (NRRL-B-15365), DMF 5/5 (NRRL-B-15366), DMF 5/7 (NRRL-B-15367), DMF 5/8 (NRRL-B-15368), DMF 5/9 (NRRL-B-15369) and DMF 5/10 (NRRL-B-15370), or the mixed culture DMF/HW 1-5 (NRRL-B-15371), or a mutant derived thereof according to claim 1 and, if desired, the resulting biomass is isolated.
DMF 3/3 (NRRL-B-15358), DMF 3/4 (NRRL-B-15359), DMF 3/5 (NRRL-B-15360), DMF 3/6 (NRRL-B-15361), DMF 3/11 (NRRL-B-15362), DMF 3/12 (NRRL-B-15363), DMF 4/4 (NRRL-B-15364), DMF 5/3 (NRRL-B-15365), DMF 5/5 (NRRL-B-15366), DMF 5/7 (NRRL-B-15367), DMF 5/8 (NRRL-B-15368), DMF 5/9 (NRRL-B-15369) and DMF 5/10 (NRRL-B-15370), or the mixed culture DMF/HW 1-5 (NRRL-B-15371), or a mutant derived thereof according to claim 1 and, if desired, the resulting biomass is isolated.
4. Process according to claim 3 for the microbiological purification of aqueous solutions containing dimethyl-formamide, formamide, methanol, ethanol, sodium or potassium formate or acetate, methyl-, ethyl-, dimethyl-or trimethyl-ammonium chloride, diemethyl phosphite, tri-methyl phosphite, acetonitrile, isobutyronitrile or mixtures of these compounds, characterised in that there is cultured in such an aqueous solution a biologically pure microorganism of the genus Pseudomonas or of a Psaudomonas-like genus selected from the group of the following strains:
DMF 3/3 (NRRL-B-15358), DMF 3/4 (NRRL-B-15359), DMF 3/5 (NRRL-B-15360), DMF 3/6 (NRRL-B-15361), DMF 3/11 (NRRL-B-15362), DMF 3/12 (NRRL-B-15363), DMF 4/4 (NRRL-B-15364), DMF 5/3 (NRRL-B-15365), DMF 5/5 (NRRL-B-15366), DMF 5/7 (NRRL-B-15367), DMF 5/8 (NRRL-B-15368), DMF 5/9 (NRRL-B-15369) and DMF 5/10 (NRRL-B-15370), or the mixed culture DMF/HW 1-5 (NRRL-B-15371), and, if desired, the resulting biomass is isolated.
DMF 3/3 (NRRL-B-15358), DMF 3/4 (NRRL-B-15359), DMF 3/5 (NRRL-B-15360), DMF 3/6 (NRRL-B-15361), DMF 3/11 (NRRL-B-15362), DMF 3/12 (NRRL-B-15363), DMF 4/4 (NRRL-B-15364), DMF 5/3 (NRRL-B-15365), DMF 5/5 (NRRL-B-15366), DMF 5/7 (NRRL-B-15367), DMF 5/8 (NRRL-B-15368), DMF 5/9 (NRRL-B-15369) and DMF 5/10 (NRRL-B-15370), or the mixed culture DMF/HW 1-5 (NRRL-B-15371), and, if desired, the resulting biomass is isolated.
5. Process according to claims 3 and 4 characterized in that the microorganisms are cultured in the presence of essential inor-ganic salts and optionally a nitrogen source, at from approximately 20°C to approximately 40°C and at a pH of from approximately 4 to approximately 7.5, under aerobic conditions.
6. Process according to claim 5, characterized in that cul-turing is carried out at 28°C.
7. Process according to claim 5, characterized in that cul-turing is carried out continuously.
8. The biomass characterized by a multiple of the approximate empirical formula C5H9NOx (x = 4-5) and a content (lyophilisate) of 37.0 % C, 6.2 % H and 9.6 % N, whenever prepared according to the process of claim 3, or by an obvious chemical equivalent thereof.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CH2962/83-6 | 1983-05-31 | ||
| CH296283 | 1983-05-31 |
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| Publication Number | Publication Date |
|---|---|
| CA1238593A true CA1238593A (en) | 1988-06-28 |
Family
ID=4245550
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000455349A Expired CA1238593A (en) | 1983-05-31 | 1984-05-29 | Microorganisms of the genus pseudomonas and process for the degradation of compounds containing methyl groups in aqueous solutions |
Country Status (11)
| Country | Link |
|---|---|
| EP (1) | EP0127581B1 (en) |
| JP (1) | JPS59227294A (en) |
| AT (1) | ATE42969T1 (en) |
| CA (1) | CA1238593A (en) |
| DE (1) | DE3478124D1 (en) |
| DK (1) | DK268884A (en) |
| ES (1) | ES532914A0 (en) |
| FI (1) | FI842113A (en) |
| IL (1) | IL71949A (en) |
| PT (1) | PT78659B (en) |
| ZA (1) | ZA844108B (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0263900A1 (en) * | 1986-10-06 | 1988-04-20 | Celgene Corporation | Process for culturing methylophilus methylotrophus |
| AU583978B2 (en) * | 1986-03-10 | 1989-05-11 | Phillips Petroleum Company | Fermentation of bacteria at high productivities |
| JPH07102355B2 (en) * | 1987-11-19 | 1995-11-08 | 三菱瓦斯化学株式会社 | How to remove dimethylformamide |
| US5932456A (en) * | 1995-06-07 | 1999-08-03 | Ingram-Howell, L.L.C. | Production of ethanol and other fermentation products from biomass |
| US5677154A (en) * | 1995-06-07 | 1997-10-14 | Ingram-Howell, L.L.C. | Production of ethanol from biomass |
| ES2095193B1 (en) * | 1995-07-18 | 1997-09-01 | Univ Madrid Politecnica | USE OF BACTERIAL STRAINS OF THE GENE PSEUDOMONAS PCH3 AND GCH1 FOR SOIL BIORESTORATION AND WATER PURIFICATION, CONTAMINATED BY HERVICIDES OF THE ACETAMIDAS GROUP. |
| US5863750A (en) * | 1996-12-18 | 1999-01-26 | Cytec Tech Corp | Methods for the detoxification of nitrile and/or amide compounds |
| US6060265A (en) * | 1996-12-18 | 2000-05-09 | Cytec Technology Corporation | Methods for the detoxification of nitrile and/or amide compounds |
| EP1236689A1 (en) * | 2001-02-22 | 2002-09-04 | Realco 2001 S.A./N.V. | Method for the treatment of aqueous effluent |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4490471A (en) * | 1981-12-11 | 1984-12-25 | Ciba-Geigy Corporation | Microorganisms of the genus Pseudomonas and process for degrading compounds which contain methyl groups in aqueous solutions |
-
1984
- 1984-05-24 AT AT84810254T patent/ATE42969T1/en not_active IP Right Cessation
- 1984-05-24 DE DE8484810254T patent/DE3478124D1/en not_active Expired
- 1984-05-24 EP EP19840810254 patent/EP0127581B1/en not_active Expired
- 1984-05-25 FI FI842113A patent/FI842113A/en not_active Application Discontinuation
- 1984-05-29 IL IL7194984A patent/IL71949A/en not_active IP Right Cessation
- 1984-05-29 CA CA000455349A patent/CA1238593A/en not_active Expired
- 1984-05-29 ES ES532914A patent/ES532914A0/en active Granted
- 1984-05-29 PT PT7865984A patent/PT78659B/en unknown
- 1984-05-30 DK DK268884A patent/DK268884A/en not_active Application Discontinuation
- 1984-05-30 JP JP59108714A patent/JPS59227294A/en active Pending
- 1984-05-30 ZA ZA844108A patent/ZA844108B/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| ZA844108B (en) | 1985-01-30 |
| PT78659B (en) | 1986-09-08 |
| DK268884A (en) | 1984-12-01 |
| IL71949A0 (en) | 1984-09-30 |
| EP0127581B1 (en) | 1989-05-10 |
| DK268884D0 (en) | 1984-05-30 |
| ATE42969T1 (en) | 1989-05-15 |
| DE3478124D1 (en) | 1989-06-15 |
| PT78659A (en) | 1984-06-01 |
| ES8600171A1 (en) | 1985-10-01 |
| ES532914A0 (en) | 1985-10-01 |
| JPS59227294A (en) | 1984-12-20 |
| FI842113A0 (en) | 1984-05-25 |
| EP0127581A1 (en) | 1984-12-05 |
| IL71949A (en) | 1988-12-30 |
| FI842113A (en) | 1984-12-01 |
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