Method for controlling growth of microorganisms and/or biofilms in an industrial process

Abstract

Disclosed is a method for controlling a biofilm, for removing a formed biofilm and/or for controlling a growth of microorganisms, preferably bacteria, in an aqueous environment of an industrial manufacturing process including cellulosic fibre material. In the method, a composition including a compound selected from a group consisting of 3-[(4-methylphenyl)sulphonyl]-2-propenenitrile and 4-amino-N-2-thiazolyl-benzenesulphonamide is administered to the aqueous environment of the process.

Claims

1. A method for controlling a biofilm, for removing a formed biofilm and/or for controlling growth of bacteria belonging to a genus of Meiothermus, Deinococcus and/or Pseudoxanthomonas, either alone or in any combination, in an aqueous environment of an industrial manufacturing process comprising a cellulosic fibre material, wherein the aqueous environment comprises the said bacteria, or the aqueous environment is in contact with a biofilm formed by any of said bacteria and said method comprising administering to the aqueous environment of the process a composition comprising a compound selected from a group consisting of 3-[(4-methylphenyl) sulphonyl]-2-propenenitrilex and 4-amino-N-2-thiazolyl-benzenesulphonamide.

2. The method according to claim 1, wherein the composition is administered to the aqueous environment in an amount of 0.01-100 ppm calculated as active compound.

3. The method according to claim 1, wherein the composition is administered to the industrial manufacturing process comprising the cellulosic fibre material, which is selected from manufacture of paper, board, pulp, tissue, moulded pulp, non-woven or viscose.

4. The method according to claim 3, wherein the aqueous environment comprises a residual of peroxide from about 0.01 to about 100 ppm.

5. The method according to claim 1, wherein the cellulosic fibres are lignocellulosic fibres.

6. The method according to claim 1, wherein the composition is administered periodically in the aqueous environment for 3-45 minutes for 6-24 times a day.

7. The method according to claim 1, wherein the composition is used in addition to other biocidal or antimicrobial agents.

8. The method according to claim 7, wherein the aqueous environment comprises a residual of active halogen in the range from about 0.01 to about 20 ppm, given as active chlorine.

9. The method of claim 2, wherein the composition is administered in an amount of 0.01-10 ppm, calculated as active compound.

10. The method according to claim 9, wherein the composition is administered in an amount of 0.01-2 ppm calculated as active compound.

11. The method according to claim 10, wherein the composition is administered to the aqueous environment in an amount of 0.01-1 ppm calculated as active compound.

12. The method according to claim 10, wherein the composition is administered in an amount of 0.01-0.5 ppm calculated as active compound.

13. The method according to claim 12, wherein the composition is administered in an amount of 0.01-0.3 ppm calculated as active compound.

14. The method of claim 1, wherein the aqueous environment comprises starch, inorganic mineral particles in form of fillers and/or paper coating materials, hemicelluloses, lignin and/or dissolved and colloidal substance.

15. The method according to claim 6, wherein the composition is administered periodically in the aqueous environment for 10-30 minutes for 12-24 times a day.

16. The method according to claim 3, wherein the industrial manufacturing process is manufacture of pulp, paper or board.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) Some embodiments of the invention are described more closely in the following non-limiting examples.

(2) Materials and Methods used in the Examples

(3) Pure cultures of Meiothermus silvanus, a microbe species commonly found in paper machine biofilms (Ekman J, Journal of Industrial Microbiology & Biotechnology 34:203-211) and Pseudoxanthomonas taiwanensis, another species commonly found in paper machine environments (Desjardins, E & Beaulieu, C, Journal of Industrial Microbiology & Biotechnology 30:141-145) were used to study the efficacy of various chemicals to prevent biofilm formation.

(4) Biofilm tests were done in either synthetic commercial R2-broth (Lab M Ltd, UK) or fibre-containing synthetic paper machine water, SPW (prepared according to Peltola, et al., J. Ind. Microbiol. Biotechnol. 2011, 38: 1719-1727) using 96-microwell plate wells with peg lids (Thermo Fischer Scientific Inc., USA). Plates were incubated at 45° C. with a rotary shaking (150 rpm) providing high flow in each well.

(5) 3-[(4-methylphenyl)sulfonyl]-2-propenenitrile, hereinafter called Corn pound A, was obtained from EMD Biosciences Inc, USA; purity ≥98%, E-isomer.

(6) 4-amino-N-2-thiazolyl-benzenesulphonamide, hereinafter called Compound B, was obtained from Sigma Aldrich Finland Oy.

(7) 2,2-dibromo-3-nitrilopropionamide, hereinafter called DBNPA, was obtained from Kemira Oyj (Fennosan R20, 20% active ingredient).

(8) Test Method for Prevention of Biofilm Formation

(9) For experiments of preventing biofilm formation wells of 96-microwell plates with peg-lids were filled with R2-broth or SPW, inoculated with the pure bacterial cultures and treated with different amounts of chemical compounds to be tested. Peg-lid was put on. After 24 hours the wells were emptied and a fresh solution of pure culture containing SPW or R2 broth with different amounts of test chemicals were added to the wells and the original peg-lid was put back in place. After an additional 24 hours, i.e. 48 hours after starting the test, the wells were emptied, rinsed and the peg lid and wells were left to dry.

(10) Test Method for Removal of Existing Biofilm

(11) For experiments of removing already existing (preformed) biofilm wells of 96-microwell plates with peg-lids were filled with SPW, inoculated with the pure bacterial cultures. Biofilm was grown for 24 hours without addition of any chemical compound to be tested. In some experiments after 24 hours the procedure was repeated by emptying the wells and by addition of a fresh solution of SPW inoculated with pure bacterial culture, again without any test chemical compound. The original peg-lid was put back in place and biofilm was allowed to grow for additional 24 h, i.e. in total 48 h.

(12) After 24 or 48 hours after starting the test, the wells were emptied and a fresh solution of SPW, inoculated with the pure bacterial cultures and with different amounts of chemical compounds to be tested were added and the original peg-lid was placed back in place. After an additional 2 or 24 hours the wells were emptied, rinsed and the peg lid and wells were left to dry.

(13) Quantification of Formed Biofilm

(14) The amount of biofilm formed on the microwells and peg surfaces was quantified with a staining solution by adding 200 μl of 1% Crystal Violet (Merck Millipore KGaA, Germany) in methanol to each well and placing the peg-lid back on. After 3 minutes the wells were emptied and the wells and pegs were rinsed 3 times with tap water. The attached Crystal Violet was dissolved into ethanol and the absorbance at 595 nm was measured. The values shown in the following tables are average absorbance from 8 replicate wells and pegs.

(15) All absorbance values in Examples 1-6 are given actual measured values. In calculation for biofilm reduction percentages it was taken in account that the SPW alone for 2 days without any bacterial inoculum gave a background value of 0.14.

Example 1

(16) Tables 1 and 2 demonstrate the ability of Compound A to prevent biofilm formation of Meiothermus silvanus and Pseudoxanthomonas taiwanensis. Test conditions simulated paper or board making process conditions (synthetic paper machine water, high temperature, fibres present, high flow) and Compound A was observed to control biofilms at a very low concentration. Already a dosage of 0.13 mg/I active Compound A gave over 90% biofilm reduction effect. For comparison, the conventional antimicrobial agent DBNPA required a dosage of 1 mg/I active compound to reach same biofilm reduction efficacy. The results for DBNPA are given in Tables 3 and 4.

(17) Table 1 shows the effect of Compound A dosing to Meiothermus silvanus biofilms in SPW at 45° C. and 150 rpm (high mixing). Biofilm was stained and quantified by absorbance measurement. Dosage given as active ingredient.

(18) TABLE-US-00001 TABLE 1 Dosage of Compound A Biofilm quantity after 48 h contact time [mg/l] Abs. at 595 nm Biofilm reduction [%] 0 0.98 — 0.01 0.80 21.4 0.03 0.75 27.4 0.08 0.58 47.6 0.13 0.22 90.5 0.20 0.15 98.8

(19) Table 2 shows the effect of Compound A dosing to Pseudoxanthomonas taiwanensis biofilms in SPW at 45° C. and 150 rpm (high mixing). Biofilm was stained and quantified by absorbance measurement. Dosage given as active ingredient.

(20) TABLE-US-00002 TABLE 2 Dosage of Compound A Biofilm quantity after 48 h contact time [mg/l] Abs. at 595 nm Biofilm reduction [%] 0 1.48 — 0.01 1.42 4.5 0.03 1.26 16.4 0.08 0.88 44.8 0.13 0.55 69.4 0.20 0.39 81.3

(21) Table 3 shows the effect of DPNPA dosing to Meiothermus silvanus biofilms in SPW at 45° C. and 150 rpm (high mixing). Biofilm was stained and quantified by absorbance measurement. Dosage given as active ingredient.

(22) TABLE-US-00003 TABLE 3 Dosage of DBNPA Biofilm quantity after 48 h contact time [mg/l] Abs. at 595 nm Biofilm reduction [%] 0 0.66 0.2 0.57 16.9 0.6 0.35 60.7 1 0.15 98.8

(23) Table 4 shows the effect of DPNPA dosing to Pseudoxanthomonas taiwanensis biofilms in SPW at 45° C. and 150 rpm (high mixing). Biofilm was stained and quantified by absorbance measurement. Dosage given as active ingredient.

(24) TABLE-US-00004 TABLE 4 Dosage of DBNPA Biofilm quantity after 48 h contact time [mg/l] Abs. at 595 nm Biofilm reduction, [%] 0 1.65 0.2 1.46 12.6 0.6 1.23 27.8 1 0.14 99.9

(25) Results in Tables 1-4 demonstrate that Compound A is capable to prevent biofilm formation of dominant industrial biofilm-formers under paper machine conditions at a very low dosage when compared to conventional biocide used in paper industry.

Example 2 (Reference)

(26) Tables 5 and 6 show effect of a well-known antibiotic Gramicidin against biofilm formation of Meiothermus silvanus and Pseudoxanthomonas taiwanensis. In a synthetic growth medium R2-broth Gramicidin was capable to prevent biofilm formation at clearly lower concentration than in conditions simulating paper or board making process (synthetic paper machine water, high temperature, fibres present, high flow).

(27) The results in Table 5 and 6 demonstrate expected behaviour of a clinical antimicrobial compound with deteriorating performance when exposed to non-clinical conditions. In contrary, Compound A was capable to control biofilms in paper machine water at a very low concentration as shown in Example 1.

(28) Table 5 shows the effect of Gramicidin dosing to Meiothermus silvanus biofilms in R2-broth and SPW. Biofilm was stained and quantified by absorbance measurement. Dosage given as active ingredient.

(29) TABLE-US-00005 TABLE 5 Biofilm quantity after 48 h Biofilm quantity after 48 h Dosage of contact time in R2-broth contact time in SPW Gramicidin Abs. at Biofilm Abs. at Biofilm [mg/l] 595 nm reduction, [%] 595 nm reduction, [%] 0 1.60 — 1.36 — 0.2 1.40 13.7 1.33 2.5 1 0.66 64.4 1.41 −4.1 3 0.17 97.9 0.45 74.6 10 0.14 100.0 0.19 95.9

(30) Table 6 shows the effect of Gramicidin dosing to Pseudoxanthomonas taiwanensis biofilms in R2-broth and SPW. Biofilm was stained and quantified by absorbance measurement. Dosage given as active ingredient.

(31) TABLE-US-00006 TABLE 6 Biofilm quantity after 48 h Biofilm quantity after 48 h Dosage of contact time in R2-broth contact time in SPW Gramicidin Abs. at Biofilm Abs at Biofilm [mg/l] 595 nm reduction, [%] 595 nm reduction, [%] 0 2.78 — 2.37 — 3 2.80 −0.8 2.25 5.4 10 2.55 8.7 2.41 −1.8 25 0.19 98.1 2.42 −2.2

Example 3

(32) Tables 7 and 8 demonstrate the ability of Compound B to prevent biofilm formation of Meiothermus silvanus and Pseudoxanthomonas taiwanensis. Test conditions are identical to test conditions of Example 1.

(33) Table 7 shows the effect of Compound B dosing to Meiothermus silvanus biofilms in SPW at 45° C. and 150 rpm (high mixing). Biofilm was stained and quantified by absorbance measurement. Dosage given as active ingredient.

(34) TABLE-US-00007 TABLE 7 Dosage of Compound B Biofilm quantity after 48 h contact time [mg/l] Abs. at 595 nm Biofilm reduction [%] 0 0.88 0.1 0.62 34.4 0.25 0.18 94.0 1 0.15 99.1 3 0.16 97.9 10 0.18 94.0

(35) Table 8 shows the effect of Compound B dosing to Pseudoxanthomonas taiwanensis biofilms in SPW at 45° C. and 150 rpm (high mixing). Biofilm was stained and quantified by absorbance measurement. Dosage given as active ingredient.

(36) TABLE-US-00008 TABLE 8 Dosage of Compound B Biofilm quantity after 48 h contact time [mg/l] Abs. at 595 nm Biofilm reduction [%] 0 2.41 0.25 2.35 2.6 1 2.04 16.3 3 0.84 69.3 10 0.54 82.4

(37) Results in Tables 7 and 8 demonstrate that Compound B can prevent biofilm formation of dominant industrial biofilm-formers under paper machine conditions.

Example 4

(38) Tables 9 and 10 demonstrate the ability of Compound A to remove already formed biofilm of Meiothermus silvanus and Pseudoxanthomonas taiwanensis. Test conditions simulated paper making process conditions (synthetic paper machine water, high temperature, fibres present, high flow). Compound A was observed to remove already formed biofilms. A single dosage of 0.5 mg/I active compound removed all of the biofilm formed during the 48-hour pre-growth time in 24 hours after addition of Compound A.

(39) Table 9 shows the effect of Compound A dosage to Pseudoxanthomonas taiwanensis biofilms in SPW at 45° C. and 150 rpm (high mixing). Biofilm was pre-grown for 48 h after which Compound A was added in given amount. After 24 hours the biofilm was stained and quantified by absorbance measurement. Compound A dosage is given as active compound.

(40) TABLE-US-00009 TABLE 9 Dosage of Biofilm quantity after 48 h pre-growth Compound A and 24 h contact time [mg/l] Abs. at 595 nm Biofilm reduction [%] 0 2.48 0.2 1.73 32.2 0.5 0.13 100.2

(41) Table 10 shows the effect of Compound A dosing to Meiothermus silvanus biofilms in SPW at 45° C. and 150 rpm (high mixing). Biofilm was pre-grown for 48 h after which Compound A was added in given amount. After 2 hours the biofilm was stained and quantified by absorbance measurement. Compound A dosage is given as active compound.

(42) TABLE-US-00010 TABLE 10 Dosage of Biofilm quantity after 48 h pre-growth Compound A and 2 h contact time [mg/l] Abs. at 595 nm Biofilm reduction [%] 0 1.30 0.5 1.20 8.0 1 1.11 16.3 2 0.99 26.6

Example 5

(43) Compound A was obtained and its E- and Z-isomers were separated from each other. Tables 11 and 12 demonstrate the ability of E- and Z-isomers of Compound A to prevent biofilm formation of Meiothermus silvanus and Pseudoxanthomonas taiwanensis. Test conditions are identical to test conditions of Example 1. It is seen that both isomers of Compound A prevent biofilm formation.

(44) Table 11 shows the effect of E- and Z-isomers of Compound A to Meiothermus silvanus biofilms in SPW at 45° C. and 150 rpm (high mixing). Biofilm was stained and quantified by absorbance measurement. Compound A dosage is given as active compound.

(45) TABLE-US-00011 TABLE 11 Biofilm quantity after 48 h Biofilm quantity after 48 h Dosage of contact time, E-isomer contact time Z-isomer Compound A Abs. at Biofilm Abs. at Biofilm [mg/l] 595 nm reduction [%] 595 nm reduction [%] 0 1.52 — 1.52 — 0.1 0.40 88.9 0.16 99.1 0.2 0.16 99.3 0.15 99.4

(46) Table 12 shows the effect of E- and Z-isomers of Compound A to Pseudoxanthomonas taiwanensis biofilms in SPW at 45° C. and 150 rpm (high mixing). Biofilm was stained and quantified by absorbance measurement. Compound A dosage is given as active compound.

(47) TABLE-US-00012 TABLE 12 Biofilm quantity after 48 h Biofilm quantity after 48 h Dosage of contact time, E-isomer contact time, Z-isomer Compound A Abs. at Biofilm Abs. at Biofilm [mg/l] 595 nm reduction [%] 595 nm reduction [%] 0 1.46 — 1.46 — 0.1 0.36 90.6 0.16 99.3 0.2 0.16 99.1 0.16 99.3

Example 6

(48) Compound A was obtained and its E- and Z-isomers were separated from each other. Table 13 demonstrates the ability E- and Z-isomers of Compound A to remove already formed biofilms of Meiothermus silvanus and Pseudoxanthomonas taiwanensis. Test conditions simulated paper making process conditions (synthetic paper machine water, high temperature, fibres present, high flow). It is seen that both isomers of Compound A are effective in removing of already formed biofilms.

(49) Table 13 shows the effect of E- and Z-isomers of Compound A to Meiothermus silvanus biofilms in SPW at 45° C. and 150 rpm (high mixing). Biofilm was pre-grown for 24 h after E- or Z-isomer of Compound A was added in amount indicated. After 24 hours the biofilm was stained and quantified by absorbance measurement. Compound A dosage is given as active compound.

(50) TABLE-US-00013 TABLE 13 Biofilm quantity after 24 h Biofilm quantity after 24 h pre-growth and 24 h contact pre-growth and 24 h contact Dosage of time, E-isomer time, Z-isomer Compound A Abs. at Biofilm Abs. at Biofilm [mg/l] 595 nm reduction [%] 595 nm reduction [%] 0 1.36 — — — 0.2 0.90 67.6 0.81 71.3 1 0.26 95.0 0.27 94.6

(51) Even if the invention was described with reference to what at present seems to be the most practical and preferred embodiments, it is appreciated that the invention shall not be limited to the embodiments described above, but the invention is intended to cover also different modifications and equivalent technical solutions within the scope of the enclosed claims.