FI70665C - FOERFARANDE FOER ELIMINERING AV BAKTERIER I PROCESSVATTEN AV PAPPERSFABRIKER OCH ANVAENDNING AV BAKTERIOFAGER FOER DETTA AENDAMAOL - Google Patents

Description

1 70665

This invention relates to the control of microbes which interfere with the paper industry, and in particular to the control of bacteria which form mucus and impair the quality of food paper or board in the process waters of paper mills.

Process waters in the paper industry provide very favorable conditions for the growth of bacteria and fungi. Temperature, acidity value and nutrient concentrations are in many cases almost optimal for microbes. As a result, paper mills may occasionally experience mucus disturbances caused by microbes that grow into clumps. Often this is still accompanied by strong mucus formation. Bacteria or bacterial spores (eg Bacillus) that remain on food paper or board form their own microbiological problem.

Mucus disorders have traditionally been controlled with the help of chemicals. In the past, mercury compounds were used, which were abandoned because of their toxicity in the late 1960s, even before their use was banned. Today, organic compounds that are considered second class toxins are used. Each anti-fouling chemical has a certain biological half-life in the process water, which varies from a few tens of minutes to several days. Some degradation products may also be bacteriostatic.

As a completely new possibility, a natural microbiological mucus control agent has now been developed. It is based on the utilization of bacteriophages. Bacteriophages are bacterial viruses that multiply only in their host bacteria, nowhere else.

They are completely harmless to the environment and to humans. According to the latest research, they may be suitable for e.g. in the treatment of bacterial inflammatory diseases in humans (Slopek, S. et al. 1981). Results of bacteriophage treatment of suppurative bacterial 2 70665 infections. II. Detailed evaluation of the results. -Arch. Immun.Ther.Exp. 31: 293-327).

The basic idea in the use of bacteriophages according to the invention is to look for their own phages for the mucus bacteria of paper mills. The phage infects its host bacterium, which begins to amplify it. Once a sufficient number of phages have amplified within the bacterial cell, they disrupt the cell wall of the host bacterium using the enzyme lysozyme. The result is the destruction of the host bacterium and the release of new phages. These, in turn, can infect new host bacteria. There is a limited “chain reaction.” Similar phage-induced but completely uncontrolled bacterial pests are known from dairy acid cultures.

The taxonomic assay of the bacteriophages of the invention is described in Intervirology 3: 201-219 (1974) and belongs to the A or B series of tail bacteriophages.

Bacteriophages are intended to be used primarily to prevent the growth of mucus bacteria and to destroy food-grade bacteria in the process waters of the paper industry, but also to treat the newly formed mucus disorder.

The advantages of bacteriophages compared to chemicals are e.g. their non-toxicity to humans and the environment, their specificity and their ability to fit into a small space. Some phages can be produced in high concentrations (10 -10 pcs / ml). According to preliminary experiments, a dose of this size class (10-10) is sufficient for the treatment of process water with a volume of about 1000 m 3 and a concentration of up to 10,107 bacteria / ml of the bacteria to be controlled.

The invention also relates to a method for destroying precipitating bacteria which cause precipitation and / or impair the quality of food-grade paper and board in the process waters of paper mills by adding phage of this bacterium to these process waters.

3 70665

Phage of the bacterium Klebsiella pneumoniae is preferably added to the process water of the paper mill, but alternatively or in addition, phages which destroy the following bacteria may be added; Bacillus sp., Pseudomonas sp., Sphaerotilus natans, Acinetobacter sp., Enterobacter sp. and / or Alcaligenes sp.

Said phages are preferably added in such an amount, for example as an aqueous suspension, that the paper mill nr 'race water initially contains at least 1 phage for every 1000 bacteria to which the phage specifically acts.

As can be seen from the above description, the isolation and growth of phages to the desired concentration is a procedure familiar to those skilled in the art that has previously been applied in medicine (Slopek et al.)

The invention is described in more detail below by means of examples.

Example 1 a. Bacteria

The model bacterium has been isolated from the process water of a newsprint mill. The bacterium has been isolated using a selective mFCIC medium at 37 ° C for 24 h. It has been identified as a species of Klebsiella pneumoniae using the Enterotube system.

This species is a typical bacterium in paper mills, which is almost always present in mucus samples (Harju-Jeantv, Pontus and Väätänen, Pentti 1984. -Paper and Wood / Papper och Trä 1984 (3): 245-259 ). The bacterial strain has been stored in tryptone-yeast extract medium in a refrigerator.

b) Bacteriophage

The phage of the above-mentioned Klebsiella bacterium has been isolated in Finland from the activated sludge of the Pätti treatment plant in the city of Vaasa on 31 May 1984. Isolation was performed by so-called using the double layer method. Hard agar (approximately 20 ml of tryptone-yeast extract agar) was first poured into a sterile petri dish. A 45 ° C mixture of active Klebsiella pneumoniae (1 ml), molten 1% soft agar (5 ml) and phage 4,70665 sample (1 ml) was poured on top of this. Some dilutions of the phage sample were also cultured. After soft agar coagulated, petri dishes were incubated for 24 h at 37 ° C.

The appearance of clear circles, i.e. plaques, in the otherwise uniform turbid bacterial growth indicated the presence of the phages in question. plaque. Phage were produced by inoculating 1 plaque into fresh Klebsiella suspension (5 ml) and incubating the suspension for 4 h at 37 ° C. In the next step, a 100 ml batch of phage was then prepared, followed by a 100 ml batch. phage

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the density was finally about 10 pcs / ml. The bacteria were filtered off from the suspension, and 1% chloroform was added thereto. The phagusus pension was stored in the refrigerator.

o. Tehokoe

The experimental combination was the aforementioned Klebsiella bacterium and its phage. The experiments were performed in sterile, cardboard mill process water (consistency about 1%). The controls used process water from which the pulp fibers had been filtered off. The purpose was to test the effects of fibers on phage potency. The test system was as follows:

Combination Fibers Klebsiella Bacteriophage A + + + B + + C - + + D - +

The suspensions were prepared in 250 ml Ellenmeyer flasks and shaken at about 30 ° C. Bacteriophage was added to a concentration of 7 x 10 / ml. The potency of the phages was determined by determining the Klebsiella content of the suspensions using mFCIC medium.

d. Score

The results are shown in Table 1.

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table 1

Effect of bacteriophage on Klebsiella in process water samples at shaking at + 30 ° C

Combination _Test time, h_ 0 2 4 6 8 _ Bacterial concentration pcs / ml A 3.6x104 3.3x104 5.3x104 l, 3x104 5.1x103 B 4.0x104 2.9x104 1.3χ105 Ι, ΙχίΟ6 4.0xl06 C 2.9x104 2.3x104 2.4x104 4.6x104 1.8x104 D 4.0x104 2.1x104 6.7x105 4.6x105 4.4x106

The table shows that the bacteriophage prevents bacterial growth in the fiber suspension (A), even destroying some of the bacteria. In control (B), bacterial growth begins after 2 hours and has reached a concentration of almost 1000 times the phage suspension (B) at 8 hours. The fibers had no significant effect on phageLe efficacy.

Example 2

At the same bacterial and phage combination, the effect of phage dilutions on Klebsiella bacterial lysis was tested. The experiments were performed in tryptone-yeast extract-glucose broth at 28 ° and 37 °. Phage potency was assessed as tube clarifications compared to sterile control. The Klebsiella concentration at the beginning of the experiment was about g 10 pcs / ml and the density of the undiluted phage suspension was also

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10 pcs / ml. The results are shown in Table 2.

Table 2

Effect of dilution on the potency of bacteriophages at 28 ° C in broth. Results reported as tube turbidities.

Phage density _Time, h _ * Ρ1 / ια1 o 2 1/2 6 8 3/4 19 _Sameus_ 10® + + + 0 0 0 + 7 10 + + + + 0 0 0 106 +++ ++ (+) + + o 10® +++ +++ ++ ++ + 104 +++ +++ +++ ++ (+) ++

Control +++ +++ +++ +++ +++ 0 = clear + + + = completely cloudy 6 70665

Table 3

Effect of dilution on the potency of bacteriophages at 37 ° C in broth. Results reported as tube turbidities.

Phage density __Time, h_

pcs / ml “Ö 1 1/4 2 1/2 6 8 3/4 W

_ _Sameus_ lo8 + + + ++ O 0 0 + + (+) 7 10 + + + + + O 0 0 + + lo6 + + + + + + + 0 + + + lo5 + + + + + + + + + + + + + ++ 4 10 +++ + + +++ + + + +++ + + (+)

Comparison + + + + + + +++ +++ +++ +++

Phage are effective at up to 1/100 dilution at 28 ° C and remain effective for up to 19 hours. At 37 ° C, iaagi works faster than at 28 ° C, but the effect is maintained for a shorter time than at 28 ° C.

Example 3 a. Bacteria

Model bacterium 2 has been isolated from the process water of a fine paper mill. It has been identified as Enterobacter cloacae using the Enterotube II system. The proportion of isolated bacteria in the process water bacteria of the factory in question was considerable, about 30%.

b) Bacteriophage

The phage of the above-mentioned Enterobacter species have been isolated from the activated sludge of the Vaasa city treatment plant. Isolation has been described previously in the patent application.

c. Tehokoe

The experimental combination was the above-mentioned Enterobacter bacterium and its phage.

The test was performed in lean broth (10% tryptone yeast extract broth) with shaking at 37 ° C. It had previously been found that cellulose fibers did not significantly affect the efficacy of bacteriophages, which is why they were excluded from the test combination.

7 70665

The effect of phages on bacteria was monitored by ATP (adenosine tri-phosphate) measurements to assess bacterial biomass. The starting concentration of the bacterium grown in the exponential phase (fresh bacterium) was about 10 “* pcs / ml.

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Phages were added to a concentration of 10 at the beginning of the experiment.

d. Score

The results are shown in Table 4.

Table 4

Effect of phage on the growth of Enterobacter cloacae in 10% broth at 37 ° C. The bacterial concentration at the beginning of experiment 5 7 is about 10 pcs / ml and the phage concentration is about 10 pcs / ml.

Test period Bacterial biomass ng ATP / ml t control phage 0 6.6 6.6 115 20 3.3 145 56 1.2 3 360 2.6 4 420 2.2 5 920 2.1 6 1 800 4.7 1820 2 500 40 1910 2 400 43 1950 1 900 44 2040 2 800 37 2140 3 300 51 23 2 000 75 24 2 300 200 2440 3 200 520

The results show that the bacteriophage inhibits the bacterial growth for approximately 24 hours. In the early stages of the experiment, even slight bacterial destruction is observed in the presence of phages.

8 70665

Example 4 a. Bacteria

Model bacterium 3 has been isolated from process water in a cardboard box. The bacterial strain belongs to the genus Pseudomonas.

b) Bacteriophage

The phage of the above-mentioned Pseudonomas strain has been isolated from the residential wastewater of the Tuovila agglomeration in the municipality of Musta Island. The method previously described in the patent application has been used for isolation.

c. Tehokoe

The experimental combination was the above-mentioned Pseudomonas bacterium and its phage.

The test was performed in dilute broth (10% tryptone-yeast extract broth) with shaking at 37 °.

The effect of phages on bacteria was monitored by ATP measurements. The starting concentration of the freshly grown bacterium was about 10 pcs / ml. Phages were added to a concentration of 2 x 10 μl / ml at the beginning of the experiment.

d. Score

The results of the experiment are shown in Table 5.

The results show that the phage we isolated have inhibited the growth of the host bacterium for more than 18 h. In the control without phages, bacterial growth has started within about an hour of the start of the experiment.

Table 5

Effect of phage on growth of Pseudomonas strain in 10% broth in 37. The initial bacterial concentration is about 10 pcs / ml and the phage concentration is 2 x 10 pcs / ml.

9 70665

Test time Bacterial biomass ng ATP / ml

Control Phage O00 1.8 4.1 125 14 3.0 32 4.9 226 46 3.2 245 70 3.6 1 0 3 υ 140 2.4 45 3 3 510 3.2 415 770 2.5 450 770 3.3 535 660 2.4 630 1 280 6.1 650 1 050 5.9 1225 2 600 3.6 1400 3 300 2.6 1505 4 700 3.0 1625 1 640 2.5 1715 1 700 2.5 1815 980 4.6 1855 980 7.0 1925 890 7.6 40 19 840 8.8

Example 5 a. Bacteria

The bacterium is the same Enterobacter cloacae as the bacterium mentioned in Example 3, which is derived from the process water of a fine paper mill.

b) Phage

The bacteriophage is the same as the phage mentioned in Example 3.

c. Efficacy test The aim of this experiment was to determine whether bacteriophages can be used to control the growth of an exponentially advanced bacterium.

10 70665

The experimental combination was the above-mentioned E. cloacae bacterium and its phage.

The experiment was performed in a fermentor in a volume of 14 liters at 37 ° using aeration (about 80% O 2) with stirring varying from 150 to 600 rpm. between. Bacterial biomass was measured as £ ATP values. The bacterial concentration at the beginning of the experiment was about 10 pcs / ml. Phages were added for 2 h 20 min. to a concentration of 4 x 106 pcs / ml.

d. Score

The results are shown in Table 6.

It is observed that about 2 1/2 hours after phage addition, the ATP concentration of the bacteria begins to decrease. This means the breakdown of bacterial cells. At the end of the experiment, the ATP level in the phage vessel was less than 10% of the control. Bacterial lysis was caused by bacteriophages, the number of which increased approximately 200-fold during the experiment.

Table 6

Ability of phages to repel exponentially growing Enterobacter cloacae in a fermentor at 37 °. The initial concentration of bacteria is about 10 μl / ml. Phages added 2 h 20 min.

for β to a concentration of 4 x 10 pcs / ml.

Test time Bacterial biomass ng ATP / ml

Phages Control3- without phages 000 3.1 6.6 O30 8.6 105 31 20 145 170 56 210 290 23 ^ phage addition 250 660 360 1 5 3 508 330 540 345 550 11 70665 4 455 420 1 5 4 410 30 4JU 270 445 270 5 230 920 530 230 45 5 270 55 5 3 180 6 1 800 23 2 000 24 2 300 a Made separately by shaking in a volume of 100 ml

Claims (4)

70665 1. The use of A or B series of tailed bacteriophages in paper mill process water which cause the formation of mucus-forming bacteria and / or which degrade bacteria which degrade the quality of organ paper or board. 2. A method for destroying slime-forming bacteria which cause precipitation and / or degrade the quality of food paper or board in paper mill process water, characterized in that a series A or B tail bacteriophage of that bacterium is added to the paper mill process water. Method according to Claim 2, characterized in that phage of the bacterium Klebsiella pneumoniae is added to the process water of the paper mill. Method according to Claim 2 or 3, characterized in that the phages are added in such a way that the process water of the paper mill initially contains at least 1 phage for every 1000 bacteria affected by the phage.