DE2103071C3 - Process for increasing the growth of microorganisms - Google Patents

Description

15th

The invention relates to a method for increasing the growth of microorganisms.

The cultivation of microorganisms is used today in many technical forms and for different ones Purposes and carried out for the extraction of products of great importance. It is common in the Used to diagnose pathogens. Breeding can also be used for the purpose of modification the composition of the culture medium, as is the case with the purification of waste water and other waste products caused by microorganisms.

Many important products are obtained as by-products in the cultivation of microorganisms. Typical examples include the manufacture of antibiotics, a number of which are natural Products of the metabolism of specific microorganisms are the production of enzymes that are excreted in large quantities by certain microorganisms, producing lactic acid from whey and the production of ethyl alcohol from carbohydrates.

Microorganisms are currently grown in sterile, defined culture media, the minimum Include amounts of impurities and the usually fully sufficient amounts of contain known desired nutritional substances, such as. B. Corn steeple, sugar and other substances in forms accessible to the microorganism. It is also common to have many such Ventilate mixtures well with gases containing oxygen. In other areas of application, for example When treating or processing wastewater, the culture medium is not in its composition regulated, however, the growth of the cleaning microorganisms is controlled by known ones Working methods reinforced.

DT-PS 3 26 439 describes a method for destruction or weakening of the functions of microorganisms described, substances which micro-organic or fermentative as well as enzymatic decomposition can fall permanently can be sterilized by holding them under nitrogen oxide at a pressure of more than 25 atm.

With reference to the publication of Ha 11 o n, "Journal of the Chemical Society", vol. 39 (1881), pp. 247 to 259, in particular pp. 253, 254, wildly stated in DT-PS 3 26 439, ... let bacteria im Meat juice is better to live in a nitrogen oxide atmosphere than in air. It's wild, however, on p. 254 the publication of the Journal of the Chemical Society clearly stated that the germs through the gas were not affected. A measure to increase the growth of microorganisms can neither the DT-PS 3 26 439 nor the publication 1. c. can be removed.

The purpose of the invention is to create a measure for increasing the growth process of Microorganisms.

This object is achieved according to the invention by creating a method for increasing the growth of microorganisms, which is characterized in that a culture medium containing the microorganism _{is brought into contact with gaseous N 2} O, NO ₂ , NO or a mixture thereof.

The metabolic processes are thus significantly increased. Finally, the desired products resulting from the cultivation of the microorganisms can be separated from the culture medium. It is not known how the added compounds actually work during cultivation and growth. The growth acceleration to be achieved according to the invention is reliably applicable to bacteria, fungi and other microorganisms.

The nitrogen supplied in the form of gaseous nitrogen oxides obviously does not act as a nutrient medium, and there can also be no relationship between the amounts of metabolically converted nitrogen and the amounts supplied in gaseous form according to the invention are determined. The culture media, each inoculated with a certain type of microorganism were made from commercially available Sources received and with a view to achieving an extent of growth that at least as high as what is normally available. Such culture media contain completely and also contain sufficient amounts of nitrogen in a form accessible to the microorganism all nutrients necessary for good growth in adequate quantities and in suitable proportions. Nevertheless, treatment of such culture media resulted in during the breeding or cultivation with the mentioned gaseous nitrogen oxides significantly better results.

For detection, a microorganism which was cultured according to the invention with one of the highest degrees of success was substantially the same Way grown with the modification that nitral ions (in a group of experiments) and Nitrite ions (in a second group of experiments) in the solution instead of the gaseous nitrogen oxides in stoichiometrically equivalent amounts to the doses of the gaseous nitrogen oxides mentioned were introduced. The purpose of these tests was to determine whether the same results were achieved upon introduction can be realized by nitrogen in the form of salts. But the results clearly showed that no increase or activation corresponding to that due to the nitrogen gases was realized by using the nitrogen salts occurred.

The invention is explained in more detail below with the aid of the examples.

In each of the examples below, the culture medium was on the microorganisms to be grown Voted. Test tubes with a capacity of 15 ml, designed for use in one Opacimeter suitable were at 118 bis

_{c p} in an autoclave sterilized. The culture media were inoculated as usual.

ι Dependence on the requirements of the

h, 6 or 7 ml of the culture medium containing the Mikrooreanismen, Incoming in each test tube ht F ^alls initial plate counts performed

they were earthen vorgemen at this time The sample glasses were with rubber caps ⁿ ° rschlossen. _Be ; _a n _en examples were the OF INVENTION Hunessemäß-introduced gases in sizes of parts Milli the usual separately supplied gases zugeführlen The gases were added under Verg a syringe through the rubber-Ttouf "therethrough. gases in glasses

rden _{jn t} he} amount of the sample gases are removed, after which the sample gases were introduced. Foremost glasses were subjected to essentially the same gas shipment, with the modification that the gases used did not contain any of the gaseous nitrogen oxide mentioned. * °

The gases and mixtures that were used were of the purest quality available, with all material analyzed after manufacture was to check the purity and concentration of the nitrogen oxides to check. => 5

All glasses were in a mechanical one Anparaiur introduced them continuously during the whole breed shook. The cloudiness, piatteiuühlungen or both depending on the experiment were recorded at selected time intervals before and after the gas metering in order to reduce the Observe effect of gases on growth. The growth of the microorganisms occurs as an increase in size of the individual microorganisms or as an increased multiplication of the microorganisms or in Form of both effects.

In many examples in which a named nitric oxide was added, the gases were diluted with helium. Helium was chosen to create a non-reactive or inactive atmosphere for NO, which would be subject to decomposition upon dilution with oxygen or ordinary air. In the same series of experiments, the stable oxides of nitrogen (e.g., N ₂ O and NO ₂ ) were often similarly diluted with helium to ensure that comparable results were obtained.

An increased haze that occurs in the examples is due to the increase in size of each Microorganisms and justified by the need to reproduce. The opacimeter is working based on the determination of the light transmitted through the sample glasses. The regulations were carried out by means of a conventional, commercially available apparatus.

The increase in the number of plates (vital numbers) is directly related to the increased number of organisms that appear as a result of growth.

To perform the peacock count, 1 ml of the culture solution is placed in Sabouraud's agar substrate in the valley of the Cultivation of fungi and the other fillings placed in a trypticase-glucose agar.

At spi e! 1

An alpha hemolytic streptococcus was found in a thioglycollate medium specific for streptococci inoculated. The medium was divided into 7 ml portions, and each portion was placed in individual sample glasses. The readings in the opacimeter were performed at time intervals as indicated below 1:

Comments on the table below:

Column 1 - before the introduction of gas
Column 2–4 hours after introduction
Column 3 - 24 hours after introduction
Column 4 _ 34 hours after introduction
Column 5 - 52 hours after introduction

Feed mix

A 0.5 ml 65 ppm NO ₂ , remainder He

B 0.5 ml 22 ppm NO ₂ , remainder He

C 0.5 ml 15 ppm NO ₂ , remainder He

D 0.5 ml 9 ppm NO ₂ , remainder He

E 0.5 ml 4.9 ppm NO ₂ , remainder He

F 0.5 ml 0.9 ppm NO ₂ , remainder He

G 0.5 ml helium alone (control)

136 136 152 222 340 136 138 156 228 340 137 136 266 300 355 135 139 270 305 375 135 136 264 298 360 137 137 154 197 315 136 136 146 183 296

The opacimeter reading was taken at the intervals given below. Example 2

Notes on the table below:

The Bacillus Paracolon was in a for Bacilli 65 column 1 - before the introduction of gas specific peptone colloid medium inoculated. The medium was divided into 7 ml portions, and each Part was placed in a separate test glass.

SÖalte 2 - 24 hours after introduction

Column 3 - 36 hours after introduction and column 4 - 52 hours after introduction

Opacimeter

Introductory mix

A 0.5 ml 65 ppm NO ₂ , remainder He

B 0.5 ml 22 ppm NO., Remainder He

C 0.5 ml 15 ppm NO ,, remainder He

D 0.5 ml 9 ppm NO ₂ , remainder He

E 0.5 ml 4.9 ppm NO ₂ , remainder He

I '0.5 ml 0.9 ppm NO ₂ , remainder He

G 0.5 ml helium alone (control)

204 202 210 188 198 208 190 196 206 170 190 210 172 197 214 250 260 260 150 185 200

Example 3

A hemolyzing Staphylococcus aureus was inoculated in trypticase soy broth. The broth was divided into 7 ml portions and each portion was placed in a separate test tube suitable for opacimeter readings. Readings were taken over a period of 48 hours at the specified time intervals.

Notes on the table below:

Column 1 - before the introduction of gas Column 2 - 24 hours after the introduction and Column 3 - 48 hours after introduction

Turbidity meter

liin introductory mixture

A 0.5 ml 75 ppm N ₂ O, remainder He

B 0.5 ml 60 ppm N ₂ O, remainder He

C 0.5 ml 31 ppm N ₂ O, remainder He

D 0.5 ml 12 ppm N ₂ O, remainder He

E 0.5 ml 5.1 ppm N ₂ O, remainder He

F 0.5 ml 1.5 ppm RO, remainder He

G 0.5 ml helium alone (control)

78 75 250 78 110 230 76 74 254 75 73 240 76 71 228 75 70 200 77 74 78

Example 4

A hemolyzing Staphylococcus aureus was inoculated into trypticase-glucose agar. Disk counts were before the introduction before · gas and after 48 hours in the manner described below recorded.

Mixture of perfumes

Λ 0.5 ml 75 ppm N ₂ O, remainder Hc

B 0.5 ml (> 0 ppm N ₂ O, remainder He

C 0.5 ml 31 ppm N ₂ O, remainder Hc

O 0.5 ml 12 ppm N ₂ O, remainder He

E 0.5 ml 5.1 ppm N ₂ O, remainder He

! 0.5 ml 1.5 ppm N ₂ O, remainder He

(i 0.5 ml helium alone (control:

l'laHen / älilung

before the 48 hours after yield

Gascin- the gas lead

guide

400 11,400,000 2 S 500 200 6,600,000 33,000 500 1: 600,000 25 200 200 12,000,000 60,000 200 900,000 4,500 100 720,000 7 200 300 <P 400 28

The yields were from the 48 hour plate counts calculated essentially in the same way as in Example 3 for the various samples.

Example 5

A culture of Micrococcus albus was transformed into a Trypticasc soy broth inoculated. The broth was divided into 7 ml aliquots and each aliquot was divided into one separate test glass introduced for readings

is suitable in an opacimeter. Readings were taken in at the intervals given below.

Notes on the table below:

Column 1 - before the introduction of gas

Column 2 - 24 hours after introduction and

Column 3 - 48 hours after introduction

Opacimeter

Introductory mix

A 0.5 ml ppmN;, O, RcstHe

B 0.5 ml 60 ppm N ₂ O, remainder Hc

C 0.5 ml 31 ppm N ₂ O, remainder He

D 0.5 ml 12 ppm N ₂ O, remainder He

E 0.5 ml 5.1 ppm N ₂ O, remainder Hc

F 0.5 ml 1.5 ppm N ₂ O, remainder He

G 0.5 ml helium alone (control)

80 80 99 81 77 100 79 79 95 82 80 93 83 78 94 85 80 97 83 81 90

B e i s ρ i e 1 6

A culture of Staphylococcus aureus (hemolytic, coagulase positive) was inoculated into trypticase soy broth. Quantities of 7 ml lines were pipetted into individual test tubes. The test tubes were capped and kept in the refrigerator overnight to stop growth. The test tubes were then removed from the refrigerator and mixtures of NO in helium in the amount of V2 ^m 'were applied. Counting was carried out before gas injection and 24 hours after gas injection using trypticase glucose extract agar.

The samples with the same prefix come from the same culture batch.

Introductory mix

Plate Count Plate Count Yield

before gas after gas supply

feed

A-I 75 ppm NO. Rest hey

B-I 75 ppm NO, remainder Hc

C-I 75 ppm NO, remainder He

A-2 50 ppm NO, remainder He

B-2 50 ppm NO, remainder He

C-2 50 ppm NO, remainder He

A-3 25 ppm NO, remainder He

B-3 25 ppm NO, remainder He

C-3 25 ppm NO, remainder He

A-4 V ₂ ml helium alone (control)

B-4 V ₂ ml helium alone (control)

40 / mi 3,000,000 75,000 60 / ml 420,000 7000 40 / ml 2,400,000 60,000 60 / ml 3,000,000 50,000 40 / ml 2,400,000 60,000 20 / ml 370,000 18 500 40 / ml 270,000 6 750 40 / ml 130000 3 250 40 / ml 260,000 6 500 40 / ml 60,000 1,500 60 / ml 110000 1,833

Example 7

A culture of Ncisseria (gram positive) was inoculated in thioglycolate medium, followed by 7 ml portions were pipetted into sterile sample glasses, which were suitable for readings in the opacimeter. One Counting was carried out before and after the gas injection using trypticase glucose extract agar. Readings were taken at 22 and 26 hour intervals as indicated below.

709 619/138

ίο

Notes on the table below:

Column 1 - reading in the opacimeter before the gas supply

Column 2 - reading in the opacimeter 22 hours after the (ias / ufführung

Column 3 - reading in the opacifier 26 hours after the gas supply

Column 4 - Plate counting before the material feed

Column 5 - plan counting 22 hours after the gas supply

Column 6 - plate count 26 hours after gas injection

Feed mix

A 0.5 ml

B 0.5 ml

C 0.5 ml

D 0.5 ml

E 0.5 ml

F 0.5 ml

G 0.5 ml

H 0.5 ml

I 0.5 ml

75 ppm N ₂ O + He
75 ppm N ₂ O + Hc
75 ppm NO + He
75 ppm NO - | - He
15 ppm NO ₂ + He
15 ppm NO ₂ -I- He
Helium alone (control)
Helium alone (control)

70.4 ppm NO »+ 65 ppm
N ₂ O

Turbidity measurement 2 3 Plan counting 5 6th Ausoeuie 1 200 4th 126,000,000 51 212 760 114,000,000 166,000 52 230 840 108,000,000 135 800 51 234 760 138,000,000 142,000 53 222 76Ü 120,000,000 181500 59 228 800 120,000,000 150,000 52 212 760 84,000,000 157 900 55 214 720 96,000,000 116 800 56 226 880 114,000,000 108 900 55 760 150,000

Example 8

A culture of Penicillium was placed in 600 ml of Sabouraud liquid medium to be shipped a very small platinum wire that had been inserted into the medium in an inclined position. The medium was used for the detection of mold and yeast contamination from pharmaceutical Particularly suitable for products.

Observation of the growth of Penicillium is through the formation of clumps or colonies impaired. However, in general, consistent and usable results have been obtained by a seaworthy Dilute the culture during the rest of the standard procedure plate count readings enables. For all counts, 1 ml of the culture medium was transferred to a sterile Pctri dish applied, and then 9 ml of Sabouraud dextrose agar in test tubes over the Medium poured and mixed. The mixture was then diluted 1: 100. All plate counts were arbitrarily designed so as not to be affected by the gas supplied or the sample glasses used were influenced.

Initially, 5 plate counts were taken from any sample of the 600 ml inoculation medium. This Pre-treatment counts were as follows:

Pre-count 1 plate count 270

Pre-count 2 plate count 240

Pre-count 3 plan count 300

Pre-count 4 plate count 270

Precount 5 plate count 240

60

This gives an average of 264 ml, which is taken as the starting or starting level can be.

36 sample glasses were each aseptically loaded with 6 ml of the 600 ml starting vaccination medium. The trial lasers were closed with sterile rubber stoppers if treatment with air is indicated below is this treatment with pre-cleaned

Air of essentially atmospheric composition carried out.

The test glasses were divided into 3 sets A, B and C and numbered from 1 to 12. Every test glass was treated according to the assigned number in the following way:

1 - Vo ml 75 ppm N ₂ O, remainder He

2 - '/ ο ml 75 ppm N "0, remainder He

Vo ml 75 ppm NO, remainder He

V ₂ ml 75 ppm NO, remainder He

V ₂ ml 65 ppm NO ₂ , remainder He

V ₂ ml 65 ppm NO ₂ , remainder He

7 - 1 ml 70.4 ppm NO ₂ -f 65 ppm N ₂ O,

Rest air

8 - 1 ml 70.4 ppm NO ₂ + 65 ppm N ₂ O,

ReEt air

9 - 1 ml air (control)

10 - 1 ml air (control)

11 - Ά ml He (control)

12 - V ₂ ml He (control)

The samples were then shaken for the time indicated below. The observed Results are shown below.

Group A - 70 hours

Plate count A-I 4600 A-2 4800 A-3 4800 A-4 5200 A-5 4800 A-6 4600 A-7 5600 A-8 6700 A-9 2100 A-10 2300 A-Il 2100 A-12 2400

Group B -% hours

Plan counting

12th

Group C - 118 hours

Plate count

BI 5700 Example 9 C-I 6700 B-2 4300 C-: 6700 B-3 3700 c -: - 5200 B-4 5900 C-4 4800 B-5 5200 C-5 4200 B-6 6100 C-6 6100 B-7 4700 C-7 5600 B-S 4800 C-8 5200 B-9 2700 C-9 2900 B-IO 2600 C-IO 2700 B-Il 2900 C-Il 1900 B-12 2700 C-12 2200

A Rhodotorula yeast was inoculated into Sabouraud liquid medium. The medium was divided into 7 ml portions and each portion was placed in a separate test tube suitable for readings in the turbidity meter. Readings were taken for up to 30 hours at the time intervals indicated below.

Notes on the table below:

Column 1 - before the gas supply

Column 2 - 20 hours after the gas call was made

and
Column 3 - 30 hours after the gas supply

Feed mix

Turbidity measurement

1 2

A 0.5 ml 65 ppm NO ₂ . Rest hey

B 0.5 ml 22 ppm XOj, remainder He

C 0.5 ml 15 ppm NO ₂ . Rest hey

D 0.5 ml 9 ppm NO ₂ , remainder Hc

E 0.5 TiI 4.9 ppm NO ₂ , remainder He

F 0.5 ml 0.9 ppm "NO _2. Remainder He

G 0.5 ml helium alone {control)

7S 192 218 7S 187 216 79 2OS 220 74 218 230 77 175 200 78 173 200 78 174 195

Claims (1)

Claim: Process for increasing the growth of microorganisms. characterized in that a culture medium containing the microorganism _{is brought into contact with gaseous N 2} O, NO ₂ or NO or a mixture thereof.