CA1273887A - Method of submersely growing pseudomonas aeruginosa bacterial strains - Google Patents
Method of submersely growing pseudomonas aeruginosa bacterial strainsInfo
- Publication number
- CA1273887A CA1273887A CA000499448A CA499448A CA1273887A CA 1273887 A CA1273887 A CA 1273887A CA 000499448 A CA000499448 A CA 000499448A CA 499448 A CA499448 A CA 499448A CA 1273887 A CA1273887 A CA 1273887A
- Authority
- CA
- Canada
- Prior art keywords
- bacterial strains
- pseudomonas aeruginosa
- nutrient medium
- aeruginosa bacterial
- grown
- Prior art date
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- Expired - Lifetime
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-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/104—Pseudomonadales, e.g. Pseudomonas
-
- C—CHEMISTRY; METALLURGY
- 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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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Epidemiology (AREA)
- Biochemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Public Health (AREA)
- Biomedical Technology (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Mycology (AREA)
- Veterinary Medicine (AREA)
- General Engineering & Computer Science (AREA)
- Immunology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Farming Of Fish And Shellfish (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
In a method of submersely growing certain H-type producing Pseudomonas aeruginosa bacterial strains of the taxonomic family of Pseudomonadaceae, the bacterial strains are grown in an aequeous nutrient medium free from antigens and proteins and containing a carbon source, a nitrogen source and mineral salts.
Description
1;~738~7 The invention relates to a method of submersely growing Pseudomonas aeruginosa bacterial strains of the taxonomic familiy of the Pseudomonadaceae.
Bacterium Pseudomonas aeruginosa is opportunistic pathogen which often occurs with hospital infections mainly patients having a weakened immune defense, such as patients suffering from burns, persons suffering from cystic fibrosis or having defective organic functions, and pa-tients suffering from tumours. Due to the occurrence of resistances, antibiotics are active against Pseudomonas infections to a limited extent only, and therefore attempts are made to fight infections caused by Pseudomonas aerugi-nosa by immunological methods.
Infections may be triggered by a plurality of strains producing 0-group antigens and H-antigens. According to the H-antigen pattern according to Ansorg (ZBl. Bakt. Hyg. I.
Abt. Orig. A 242, 228-238 (1978)), by using the indirect immunofluorescence technique, with Pseudomonas aeruginosa there is differentiated between a complex flagellar antigen a having the partial antigens ao, al, a2~ a3/ a4, and a uniform flagellar antigen _. The partial factors ao - a4 are independent determinants, a flagellar antigen pattern having several H-types resulting 0-groups and H-type show free combinations.
It is already known to produce Pseudomonas vaccines for the prophylaxis against Pseudomonas infections, wherein as starting materials either Pseudomonas aeruginosa bac-terial masses themselves and/or culture filtrates which were obtained by growing the micro-organisms on surface cultures or submersely in complex nutrient media are used.
~;~73&~7 With these complex nutrient media beside a carbon and energy source (mostly carbohydrates) and essential nutri-tive salts, various extracts and/or hydrolysates of animal, microbial or vegetable proteins (so-called peptones) were used. Such nutritive solution supplements are not defined as to their exact composition, and furthermore they are variable from lot to lot. Beside amino acids, they also contain incompletely degraded protein fragments and un-defined complexes of the same and serve substantially for meeting the amino acid and growth promoting substance de-mands. Culture supernatants, therefore, are always rich in substances of non-bacterial origin, which has the disadvan-tage that for preparing a flagellar antigen of Pseudomonas aeruginosa always several separating steps, which are to follow the cultivation step, are necessary in order to free an immunogenically effective flagellar antigen as far as possible from impurities stemming from the nutrient medium.
A further disadvantage of the hitherto used growing methods in lots or batches consists in that the bacteria are subjected to a condition of continuous physiological change from the time of their inoculation to the time of their separation from the culture. The trigger moment for this physiological and thus functional differentiation of the bacterial biomass is caused by the cellular growth itself and by the temporal change of the composition of the nutrient medium caused in turn by the cellular growth. The visible result of this spontaneous physiological differen-tiation is already recognizable by the various phases of the growth curve of a batch culture. As a consequence of the described direct relationship between the physiological ' .
~3~387 activity of the cellular mass on the one hand and its surrounding environment on the other hand, a preferred enrichment in various intra and extra cellular metabolic products depending on the growth phase and differentiation phase is connected therewith. Therefore, the culture su-pernatants of a batch culture contain an integrated mixture of all the metabolic products formed temporally over indi-vidual differentiation phases.
The invention aims at avoiding these difficulties and disadvantages and has as its object to provide a method of submersely growing strains of Pseudomonas aeruginosa bac-teria which gives, in much higher yields than has hitherto been the case, a bacterial biomass for a recovery of anti-gens whose physiological state remains constant during the total period of cultivation; in which a synthetic nutrient medium is used from which there result no additional impurities caused by autoclaving; and which enables an easier chemical working up of the bacterial biomass to flagellar antigen.
According to the invention, this object is achieved in that the following H-type antigen producing Pseudomonas aeruginosa bacterial strains strain H-type 1 170001 - b M-2 - b
Bacterium Pseudomonas aeruginosa is opportunistic pathogen which often occurs with hospital infections mainly patients having a weakened immune defense, such as patients suffering from burns, persons suffering from cystic fibrosis or having defective organic functions, and pa-tients suffering from tumours. Due to the occurrence of resistances, antibiotics are active against Pseudomonas infections to a limited extent only, and therefore attempts are made to fight infections caused by Pseudomonas aerugi-nosa by immunological methods.
Infections may be triggered by a plurality of strains producing 0-group antigens and H-antigens. According to the H-antigen pattern according to Ansorg (ZBl. Bakt. Hyg. I.
Abt. Orig. A 242, 228-238 (1978)), by using the indirect immunofluorescence technique, with Pseudomonas aeruginosa there is differentiated between a complex flagellar antigen a having the partial antigens ao, al, a2~ a3/ a4, and a uniform flagellar antigen _. The partial factors ao - a4 are independent determinants, a flagellar antigen pattern having several H-types resulting 0-groups and H-type show free combinations.
It is already known to produce Pseudomonas vaccines for the prophylaxis against Pseudomonas infections, wherein as starting materials either Pseudomonas aeruginosa bac-terial masses themselves and/or culture filtrates which were obtained by growing the micro-organisms on surface cultures or submersely in complex nutrient media are used.
~;~73&~7 With these complex nutrient media beside a carbon and energy source (mostly carbohydrates) and essential nutri-tive salts, various extracts and/or hydrolysates of animal, microbial or vegetable proteins (so-called peptones) were used. Such nutritive solution supplements are not defined as to their exact composition, and furthermore they are variable from lot to lot. Beside amino acids, they also contain incompletely degraded protein fragments and un-defined complexes of the same and serve substantially for meeting the amino acid and growth promoting substance de-mands. Culture supernatants, therefore, are always rich in substances of non-bacterial origin, which has the disadvan-tage that for preparing a flagellar antigen of Pseudomonas aeruginosa always several separating steps, which are to follow the cultivation step, are necessary in order to free an immunogenically effective flagellar antigen as far as possible from impurities stemming from the nutrient medium.
A further disadvantage of the hitherto used growing methods in lots or batches consists in that the bacteria are subjected to a condition of continuous physiological change from the time of their inoculation to the time of their separation from the culture. The trigger moment for this physiological and thus functional differentiation of the bacterial biomass is caused by the cellular growth itself and by the temporal change of the composition of the nutrient medium caused in turn by the cellular growth. The visible result of this spontaneous physiological differen-tiation is already recognizable by the various phases of the growth curve of a batch culture. As a consequence of the described direct relationship between the physiological ' .
~3~387 activity of the cellular mass on the one hand and its surrounding environment on the other hand, a preferred enrichment in various intra and extra cellular metabolic products depending on the growth phase and differentiation phase is connected therewith. Therefore, the culture su-pernatants of a batch culture contain an integrated mixture of all the metabolic products formed temporally over indi-vidual differentiation phases.
The invention aims at avoiding these difficulties and disadvantages and has as its object to provide a method of submersely growing strains of Pseudomonas aeruginosa bac-teria which gives, in much higher yields than has hitherto been the case, a bacterial biomass for a recovery of anti-gens whose physiological state remains constant during the total period of cultivation; in which a synthetic nutrient medium is used from which there result no additional impurities caused by autoclaving; and which enables an easier chemical working up of the bacterial biomass to flagellar antigen.
According to the invention, this object is achieved in that the following H-type antigen producing Pseudomonas aeruginosa bacterial strains strain H-type 1 170001 - b M-2 - b
2 5940 - aol a2
3 5939 - ao, a3
4 5933 _ ao, al, a2 1210 - ao~ al, a2 16990 - ao~ al, a2 ~3~87 170018 - ao, a3, a4 are grown in an aqueous nutrient medium, the aqueous nutrient medium being free from antigens and free from proteins and containing a nitrogen source and mineral salts as well as succinate or urea as carbon source.
The carbon source mentioned has proved to be particu-larly growth-promoting.
For the strains 170001, 5940, 5939, 5933 and 170018 the classification according too Ansorg is used. The allo-cation of the strains M-2, 1210 and 16990, not classified in types, to the corresponding H-serotype is effected on the basis of comparative examinations of the molecular weights and serological cross reactions by Montie et al.
Preferably, the Pseudomonas aeruginosa bacterial strains are grown continuously.
According to a further preferred working method, the Pseudomonas aeruginosa bacterial strains may be grown tur-bidostatically, the nutrient medium having an oxygen con-tent of from 5 to 20 %, preferably approximately 10 %, a cellular density of 2-3 x 109 cells/ml being maintained and a substrate dilution rate of from 0.1 ~ to 0.3 ,u being maintained by supplying fresh nutrient medium.
The growing of Pseudomonas aeruginosa bacterial strains according to the invention is effected under agi-tation and airing of an "open c~llture" in a manner that they are caused to grow maximally and form the flagellas.
The nutritive substrate is supplied in dependence on the growth rate of the culture or on the state of equilibrium reached by the culture. The regulation or control of the fresh nutrient medium supply is effected in dependence on a .
1~3~7 parameter that is proportionate to the growth of the cul-ture, preferably the temporal change of the cellular densi-ty. However, also other parameters which are proportionate to the development of the culture, such as the breathing activity, C02 production, change in the hydrogen ion con-centration, change in the carbon source concentration, respiration quotient, nitrogen consumption, oxygen con-sumption and heat tone may be used. Furthermore, the supply of fresh nutrient medium may be effected in dependence on the amount of the antigen formed.
With the continuous growing method according to the invention, culture liquid is withdrawn from the growing vessel simultaneously with the supply of fresh nutrient medium, so that between the growth and the physiologic activity, in particular the forming of cells on the one hand and the surrounding environment of the cells on the other hand, there adjusts a flow equilibrium state that is precisely definable and reproducible at any time and which can be maintained practically for an unlimited period of time.
As nutrient medium, a synthetic medium free from pro-teins is used, which exclusively contains chemically as well as physico-chemically qualitatively and quantitatively determinable components. An inorganic nutrient solution containing all essential elements is suitable.
With the preferred embodiment according to the inven-tion, for the fermentative production of the germs, the chemostatic principle of the continuous culture, in which the physiological activity of the culture is controlled by a limiting substrate, preferably the carbon and energy ~2'7~8~7 source, as well as the turbidostatic principle of the continuous culture, with which the dilution rate (micron) and thus the physiological activity of the culture is externally controlled via a growth parameter, preferably via the instantaneous cellular density of the culture, are applicable.
Preferably, the turbidostatic continuous cultivation is used.
In order to promote a maximal bacterial growth, also the temperature and the pH value are of importance; thus, suitably, while growing of the Pseudomonas aeruginosa bac-terial strains takes place, a temperature of from 20 to 35C, preferably 30C, and a pH of from 6.6 to 7.S, pref-erably 7.0, are to be maintained.
In detail, the method according to the invention, with the turbidostatic working manner, is carried out in a fermentor, in that at first the nutrient medium is inoccu-lated with a bacterial strain and it is proceeded under maintenance of the conditions stated, until the cellular density stated, of 2 - 3 x 109 cells/ml is achieved in the logarithmic final phase. As soon as this is the case, bacterial suspension is continuously withdrawn from the fermentor and substituted by fresh nutrient solution.
Suitably, the dilution rate shall not be more than 0.3 preferably of from 0.1 to 0.2 ~u.
Under the conditions stated, after achieving the flow equilibrium state, 1.5 to 2.0 g/l wet cells are obtained, which corresponds to a turbidity value of the suspension of from 0.5 to 0.7, prior to centrifugation at 590 nm, ac-cording to the method by Tyndall.
1~3~
The method according to the invention shall now beexplained in more detail by way of the following example:
Example:
A nutrient solution of the following composition was used:
Disodium succinate 4.05 g/l, Dipotassium monohydrogen phosphate 7 g/l, Potassium dihydrogen phosphate 3 g/l, Ammonium hydrogen phosphate 1 g/l, Magnesium sulfate . 7 H2O 0 05 g/l, Ferric chloride 0.0025 g/l, the pH was adjusted to 7.0 and the temperature was brought to 30C. The nutrient solution was inocculated with the strain Pseudomonas aeruginosa M-2 and introduced into a 15 1 fermentor. During a fermentation period of 96 hours, 140 1 of the nutrient medium were passed through, the air flow rate amounting to 10 % P02 (oxygen partial pressure).
The dilution rate was 0.1 tU. The cellular density amounted to 2-3 x 109 cells/ml after the flow equilibrium state had been reached.
Under the same conditions, the strains 170001 and 1210 were cultivated, the following table giving the cul-ture conditions, dilution rate, oxygen partial pressure in % and cell yield in g/l wet weight after centrifuging twice at 15,000 x g. Furthermore, the turbidity values of the suspension prior to centrifuging are given.
~73~
Culture Strain conditions Cell yield Dilution Oxygen Turbidity value g/l wet rate content at 590 nm;d=lcm cells _ ~
170001 0.1 ~ 10 % PO2 0.6 - 0.67 1.7 M-2 0.1 y 10 % PO2 0.6 - 0.7 1.6 - 1.7 1210 0.1 ~ 10 % PO2 0.55 - 0.65 1.5 - 1.6 1210 0.2 ~ 10 % PO2 0.6 1.5 - 1.6
The carbon source mentioned has proved to be particu-larly growth-promoting.
For the strains 170001, 5940, 5939, 5933 and 170018 the classification according too Ansorg is used. The allo-cation of the strains M-2, 1210 and 16990, not classified in types, to the corresponding H-serotype is effected on the basis of comparative examinations of the molecular weights and serological cross reactions by Montie et al.
Preferably, the Pseudomonas aeruginosa bacterial strains are grown continuously.
According to a further preferred working method, the Pseudomonas aeruginosa bacterial strains may be grown tur-bidostatically, the nutrient medium having an oxygen con-tent of from 5 to 20 %, preferably approximately 10 %, a cellular density of 2-3 x 109 cells/ml being maintained and a substrate dilution rate of from 0.1 ~ to 0.3 ,u being maintained by supplying fresh nutrient medium.
The growing of Pseudomonas aeruginosa bacterial strains according to the invention is effected under agi-tation and airing of an "open c~llture" in a manner that they are caused to grow maximally and form the flagellas.
The nutritive substrate is supplied in dependence on the growth rate of the culture or on the state of equilibrium reached by the culture. The regulation or control of the fresh nutrient medium supply is effected in dependence on a .
1~3~7 parameter that is proportionate to the growth of the cul-ture, preferably the temporal change of the cellular densi-ty. However, also other parameters which are proportionate to the development of the culture, such as the breathing activity, C02 production, change in the hydrogen ion con-centration, change in the carbon source concentration, respiration quotient, nitrogen consumption, oxygen con-sumption and heat tone may be used. Furthermore, the supply of fresh nutrient medium may be effected in dependence on the amount of the antigen formed.
With the continuous growing method according to the invention, culture liquid is withdrawn from the growing vessel simultaneously with the supply of fresh nutrient medium, so that between the growth and the physiologic activity, in particular the forming of cells on the one hand and the surrounding environment of the cells on the other hand, there adjusts a flow equilibrium state that is precisely definable and reproducible at any time and which can be maintained practically for an unlimited period of time.
As nutrient medium, a synthetic medium free from pro-teins is used, which exclusively contains chemically as well as physico-chemically qualitatively and quantitatively determinable components. An inorganic nutrient solution containing all essential elements is suitable.
With the preferred embodiment according to the inven-tion, for the fermentative production of the germs, the chemostatic principle of the continuous culture, in which the physiological activity of the culture is controlled by a limiting substrate, preferably the carbon and energy ~2'7~8~7 source, as well as the turbidostatic principle of the continuous culture, with which the dilution rate (micron) and thus the physiological activity of the culture is externally controlled via a growth parameter, preferably via the instantaneous cellular density of the culture, are applicable.
Preferably, the turbidostatic continuous cultivation is used.
In order to promote a maximal bacterial growth, also the temperature and the pH value are of importance; thus, suitably, while growing of the Pseudomonas aeruginosa bac-terial strains takes place, a temperature of from 20 to 35C, preferably 30C, and a pH of from 6.6 to 7.S, pref-erably 7.0, are to be maintained.
In detail, the method according to the invention, with the turbidostatic working manner, is carried out in a fermentor, in that at first the nutrient medium is inoccu-lated with a bacterial strain and it is proceeded under maintenance of the conditions stated, until the cellular density stated, of 2 - 3 x 109 cells/ml is achieved in the logarithmic final phase. As soon as this is the case, bacterial suspension is continuously withdrawn from the fermentor and substituted by fresh nutrient solution.
Suitably, the dilution rate shall not be more than 0.3 preferably of from 0.1 to 0.2 ~u.
Under the conditions stated, after achieving the flow equilibrium state, 1.5 to 2.0 g/l wet cells are obtained, which corresponds to a turbidity value of the suspension of from 0.5 to 0.7, prior to centrifugation at 590 nm, ac-cording to the method by Tyndall.
1~3~
The method according to the invention shall now beexplained in more detail by way of the following example:
Example:
A nutrient solution of the following composition was used:
Disodium succinate 4.05 g/l, Dipotassium monohydrogen phosphate 7 g/l, Potassium dihydrogen phosphate 3 g/l, Ammonium hydrogen phosphate 1 g/l, Magnesium sulfate . 7 H2O 0 05 g/l, Ferric chloride 0.0025 g/l, the pH was adjusted to 7.0 and the temperature was brought to 30C. The nutrient solution was inocculated with the strain Pseudomonas aeruginosa M-2 and introduced into a 15 1 fermentor. During a fermentation period of 96 hours, 140 1 of the nutrient medium were passed through, the air flow rate amounting to 10 % P02 (oxygen partial pressure).
The dilution rate was 0.1 tU. The cellular density amounted to 2-3 x 109 cells/ml after the flow equilibrium state had been reached.
Under the same conditions, the strains 170001 and 1210 were cultivated, the following table giving the cul-ture conditions, dilution rate, oxygen partial pressure in % and cell yield in g/l wet weight after centrifuging twice at 15,000 x g. Furthermore, the turbidity values of the suspension prior to centrifuging are given.
~73~
Culture Strain conditions Cell yield Dilution Oxygen Turbidity value g/l wet rate content at 590 nm;d=lcm cells _ ~
170001 0.1 ~ 10 % PO2 0.6 - 0.67 1.7 M-2 0.1 y 10 % PO2 0.6 - 0.7 1.6 - 1.7 1210 0.1 ~ 10 % PO2 0.55 - 0.65 1.5 - 1.6 1210 0.2 ~ 10 % PO2 0.6 1.5 - 1.6
Claims (4)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS.
1. A method of submersely growing Pseudomonas aeruginosa bacterial strains of the taxonomic family of Pseudo-monadaceae, wherein the following H-type antigen pro-ducing Pseudomonas aeruginosa bacterial strains are grown in an aqueous nutrient medium, said aqueous nutrient medium being free from antigens and free from proteins and containing a nitrogen source and mineral salts as well as a carbon source selected from the group consisting of succinate and urea.
2. A method as set forth in claim 1, wherein said Pseudo-monas aeruginosa bacterial strains are grown continuous-ly.
3. A method as set forth in claim 1 or 2, wherein said Pseudomonas aeruginosa bacterial strains are grown tur-bidostatically, said nutrient medium having an oxygen content of from 5 to 20 %, a cellular density of 2 - 3 x 109 cells/ml being maintained and a substrate dilutiond rate of from 0.1 µ to 0.3 µ being maintained by supplying fresh nutrient medium.
4. A method as set forth in claim 1, wherein said Pseudo-monas aeruginosa bacterial strains are grown at a tem-perature of from 20 to 35°C, a pH of from 6.5 to 7.5 being maintained.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000499448A CA1273887A (en) | 1985-01-14 | 1986-01-13 | Method of submersely growing pseudomonas aeruginosa bacterial strains |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AT0007385A AT384238B (en) | 1985-01-14 | 1985-01-14 | METHOD FOR SUBMERSE BREEDING OF PSEUDOMONAS AERUGINOSA BACTERIA |
| ATA73/85 | 1985-01-14 | ||
| CA000499448A CA1273887A (en) | 1985-01-14 | 1986-01-13 | Method of submersely growing pseudomonas aeruginosa bacterial strains |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA1273887C CA1273887C (en) | 1990-09-11 |
| CA1273887A true CA1273887A (en) | 1990-09-11 |
Family
ID=3480817
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000499448A Expired - Lifetime CA1273887A (en) | 1985-01-14 | 1986-01-13 | Method of submersely growing pseudomonas aeruginosa bacterial strains |
Country Status (9)
| Country | Link |
|---|---|
| EP (1) | EP0211014B1 (en) |
| JP (1) | JPH07114690B2 (en) |
| AT (2) | AT384238B (en) |
| CA (1) | CA1273887A (en) |
| DE (1) | DE3674502D1 (en) |
| DK (1) | DK421386A (en) |
| ES (1) | ES8706820A1 (en) |
| FI (1) | FI84280C (en) |
| WO (1) | WO1986004086A1 (en) |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1201638A (en) * | 1969-04-02 | 1970-08-12 | Chinese Petroleum Corp | Microbiological production of high protein compositions |
| US3987164A (en) * | 1970-10-20 | 1976-10-19 | Yuzuru Homma | Method for prevention of pseudomonas aeruginosa infections |
| US3928565A (en) * | 1971-10-19 | 1975-12-23 | Yuzuru Homma | Pharmaceutical preparation of pseudomonas aeruginosa bacterial component possessing anti-tumor and anti-infection properties |
| US4444888A (en) * | 1979-06-22 | 1984-04-24 | Sybron Corporation | Microorganism for decolorizing pulp and paper mill wastewater |
| US4482632A (en) * | 1981-05-07 | 1984-11-13 | Spraker Philip W | Microbiological process for removing oleaginous material from wastewater and microbiological combination capable of same |
| JPS5991879A (en) * | 1982-11-16 | 1984-05-26 | Hideaki Yamada | Method for cultivating bacterium of genus pseudomonas |
| BR8400054A (en) * | 1983-01-10 | 1984-08-14 | Nitto Chemical Industry Co Ltd | PROCESS TO GROW PSEUDOMONA BACTERIES |
-
1985
- 1985-01-14 AT AT0007385A patent/AT384238B/en not_active IP Right Cessation
-
1986
- 1986-01-13 AT AT86900708T patent/ATE56988T1/en not_active IP Right Cessation
- 1986-01-13 EP EP86900708A patent/EP0211014B1/en not_active Expired - Lifetime
- 1986-01-13 JP JP61500682A patent/JPH07114690B2/en not_active Expired - Fee Related
- 1986-01-13 DE DE8686900708T patent/DE3674502D1/en not_active Expired - Lifetime
- 1986-01-13 ES ES550832A patent/ES8706820A1/en not_active Expired
- 1986-01-13 CA CA000499448A patent/CA1273887A/en not_active Expired - Lifetime
- 1986-01-13 WO PCT/AT1986/000003 patent/WO1986004086A1/en active IP Right Grant
- 1986-09-03 DK DK421386A patent/DK421386A/en not_active Application Discontinuation
- 1986-09-11 FI FI863678A patent/FI84280C/en not_active IP Right Cessation
Also Published As
| Publication number | Publication date |
|---|---|
| ES8706820A1 (en) | 1987-07-01 |
| DE3674502D1 (en) | 1990-10-31 |
| DK421386D0 (en) | 1986-09-03 |
| ES550832A0 (en) | 1987-07-01 |
| CA1273887C (en) | 1990-09-11 |
| ATE56988T1 (en) | 1990-10-15 |
| EP0211014A1 (en) | 1987-02-25 |
| JPS62501330A (en) | 1987-06-04 |
| JPH07114690B2 (en) | 1995-12-13 |
| ATA7385A (en) | 1987-03-15 |
| FI863678A0 (en) | 1986-09-11 |
| WO1986004086A1 (en) | 1986-07-17 |
| FI84280C (en) | 1991-11-11 |
| FI863678A (en) | 1986-09-11 |
| DK421386A (en) | 1986-09-03 |
| AT384238B (en) | 1987-10-12 |
| FI84280B (en) | 1991-07-31 |
| EP0211014B1 (en) | 1990-09-26 |
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