BACILLUS STRAINS WITH THE ABILITY TO DEGRADE INORGANIC NITROGEN COMPOUNDS

Abstract

The invention concerns new Bacillus strains which are able to degrade effectively inorganic nitrogen compounds and are further able to inhibit the growth of pathogens of aquatic animals.

Claims

1-17. (canceled)

18. A Bacillus strain or a preparation thereof, wherein the strain or preparation is able to degrade at least one inorganic nitrogen compound and is further able to inhibit the growth of at least one pathogen.

19. The Bacillus strain or preparation thereof of claim 18, wherein the at least one pathogen is a pathogen of an aquatic animal.

20. The Bacillus strain or preparation thereof of claim 19, wherein the pathogen is selected from the group consisting of: Vibrio harveyi, Vibrio parahaemolyticus, Aeromonas hydrophila and Streptococcus agalactiae.

21. The Bacillus strain or preparation thereof of claim 18, wherein the at least one inorganic nitrogen compound is selected from the group consisting of: ammonia; nitrite; and nitrate.

22. The Bacillus strain or preparation thereof of claim 18, wherein the Bacillus strain or preparation thereof are selected from the group consisting of: a) a Bacillus strain as deposited under one of the following numbers at the DSMZ: DSM 33349, DSM 33350, DSM 33351 and DSM 33352; b) a mutant of a Bacillus strain in paragraph a) with a sequence identity to the strain of at least 95%; c) a preparation of a strain according to paragraphs a) or b); and d) a preparation comprising an effective mixture of compounds as contained in a strain as listed in paragraphs a) or b) or as contained in the preparation of paragraph c).

23. The Bacillus strain or preparation thereof of claim 22, wherein the Bacillus strain or preparation thereof are selected from the group consisting of: a) a Bacillus strain as deposited under one of the following numbers at the DSMZ: DSM 33349, DSM 33350, DSM 33351 and DSM 33352; b) a mutant of a Bacillus strain of paragraph a) with a sequence identity to the strain of at least 99%; c) a preparation of a strain according to paragraphs a) or b);

24. The Bacillus strain or preparation thereof of claim 18, wherein the Bacillus strain has a 16S rDNA sequence with a sequence identity of at least 99% to a sequence according to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 11 or SEQ ID NO: 16.

25. The Bacillus strain or preparation thereof of claim 24, wherein the Bacillus strain or mutant thereof has a 16S rDNA sequence with a sequence identity of at least 99.8%, to a sequence according to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 11 or SEQ ID NO: 16.

26. The Bacillus strain of claim 18, wherein the strain is characterized by at least one of the following characteristics: a) an ability to grow anaerobically; b) an ability to grow in presence of 1 wt.-% of NaCl for at least one day; c) an ability to germinate and/or to degrade inorganic nitrogen compounds in C minimal media; d) an enzymatic activity selected from the group consisting of: cellulase activity; xylanase activity; protease activity; catalase activity; superoxide dismutase activity; e) an ability to grow in presence of 2 mM bile.

27. The Bacillus strain of claim 26, wherein the strain is characterized by at least three, of characteristics a) to e).

28. The Bacillus strain or a preparation thereof claim 26, wherein the Bacillus strain or preparation thereof are selected from the group consisting of: a) a Bacillus strain as deposited under one of the following numbers at the DSMZ: DSM 33349, DSM 33350, DSM 33351 and DSM 33352; or b) a mutant of a Bacillus strain as listed in paragraph a) with a sequence identity to said strain of at least 99%.

29. The Bacillus strain or preparation thereof of claim 20, wherein the at least one inorganic nitrogen compound is selected from the group consisting of: ammonia; nitrite; and nitrate.

30. The Bacillus strain or preparation thereof of claim 29, wherein the Bacillus strain or preparation thereof are selected from the group consisting of: a) a Bacillus strain as deposited under one of the following numbers at the DSMZ: DSM 33349, DSM 33350, DSM 33351 and DSM 33352; b) a mutant of a Bacillus strain as listed in (a) with a sequence identity to the strain as listed in paragraph a) of at least 95%; c) a preparation of a strain according to paragraphs a) or b); d) a preparation comprising an effective mixture of compounds as contained in a strain as listed in paragraphs a) or b) or as contained in the preparation of paragraph.

31. The Bacillus strain or preparation thereof of claim 30, wherein the Bacillus strain or mutant thereof has a 16S rDNA sequence with a sequence identity of at least 99% to a sequence according to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 11 or SEQ ID NO: 16.

32. A method of decreasing the amount of inorganic nitrogen compounds in an aqueous system and/or controlling the amount of inorganic nitrogen compounds in an aqueous system, the method comprising supplying the aqueous system with at least one strain and/or at least one preparation of claim 18.

33. The method of claim 32, wherein the inorganic nitrogen compounds are selected from the group consisting of ammonium, nitrite and nitrate.

34. The method of claim 33, wherein the aqueous system is rearing water.

35. A method of feeding an animal, treating an animal for a disease or condition or preventing a disease or condition in an animal, comprising providing the animal with at least one strain and/or at least one preparation of claim 18.

36. The method of claim 35, wherein the animal is an aquatic animal.

37. The method of claim 36, wherein the animal is a crustacean or finfish.

Description

WORKING EXAMPLES

Example 1: Capacity of Strains to Reduce Nitrogen

[0188] Bacillus strains from diverse environmental samples were screened regarding their water remediation characteristics. Various tests were carried out for determination of the capacity to reduce nitrogen compounds under broad conditions in order to receive superior strains for aquaculture bioremediation. Strains were tested under different salinities (0-3%) and varying C:N-ratios (20-2600). The four B. subtilis strains DSM 33351, DSM 33352, DSM 33349 and DSM 33350 showed very promising properties, which are described in the following.

[0189] All nitrite (NO.sub.2—N) measurements were carried out using the Nitrite cuvette test 0.015-0.6 mg/L NO.sub.2—N and Nitrite cuvette test 0.6-6.0 mg/L NO.sub.2—N(Hach Company, Loveland, Colo., USA). All ammonia (NH4.sub.+-N) concentrations were determined by a flow injection analyzer (FIA, applied system: FOSS FIAstar 5000). Within the system sample aliquots were alkalized in order to generate ammonia (NH3) from solvated ammonium ions (NH4+) quantitatively. Ammonia diffused via a semipermeable membrane (gas diffusion). Changes of the pH can then be monitored via an indicator-solution (photometric detection, applied indicator based on 5′,5″-Dibrom-o-kresolsulfonphthalein (“Bromkresolpurpur”, Sigma-Aldrich, St. Louis, Mo., USA). Extinction will increase non-linear with higher ammonia concentrations and needs to be calibrated properly. The method has a validated measuring area of 1-13 mg/L (linear range) and 10-130 mg/L (non-linear range).

[0190] All strains were tested for the capacity of ammonia reduction in C minimal medium containing only glucose as carbon source and 5 ppm ammonia as sole nitrogen source under different salinities. For each B. subtilis strain, 50 μl of a glycerol stock was inoculated to 10 ml C minimal medium with a C:N ratio of 68 (0.0352 g/l MnSO.sub.4×1H.sub.2O, 2.46 g/l MnSO.sub.4×7H.sub.2O, 0.2 KH.sub.2PO.sub.4, 0.6 g/l K2HPO.sub.4, 0.02 FeSO.sub.4×7H.sub.2O, 0.2792 g/l (NH.sub.4).sub.2SO.sub.4, 10 g/l glucose and 0 or 15 or 30 g/l NaCl) and incubated in 100 ml shaking flasks at 28° C. and 200 rpm. After 48 h of incubation, the concentration of ammonia was measured with the method described above. The percentage amount of ammonia reduced by each strain was calculated referring to the initial concentration, which was determined in the non-inoculated control for each salinity (Table 1.1). Strains DSM 33351, DSM 33352, DSM 33349 and DSM 33350 were able to efficiently remove ammonia in C minimal medium containing only glucose as carbon source and 5 ppm ammonia as nitrogen source, as maximum 0.3% of the initial ammonia concentration were still detectable after 48 h under all salinity conditions.

TABLE-US-00001 TABLE 1.1 Ammonia reduction in C minimal medium containing only glucose as carbon source and 5 ppm ammonia as nitrogen source under different salinities. *Removed ammonia [%]/**Ammonia concentration [mg/l] Strain* 0% NaCl 1.5% NaCl 3.0% NaCl DSM 33351 99.7 100.0 100.0 DSM 33352 99.7 100.0 99.7 DSM 33349 99.9 100.0 99.9 DSM 33350 99.7 100.0 99.7 Initial concentration** 4.45 4.53 4.47

[0191] All strains were further tested for their capacity of nitrite reduction in C minimal medium containing only glucose as carbon source and 5 ppm nitrite as nitrogen source. For each B. subtilis strain, 50 μl of a glycerol stock was inoculated to 10 ml C minimal medium with a C:N ratio of 2600 (0.0352 g/l MnSO.sub.4×1H.sub.2O, 2.46 g/l MnSO.sub.4×7H.sub.2O, 0.2 KH.sub.2PO.sub.4, 0.6 g/l K2HPO.sub.4, 0.02 FeSO.sub.4×7H.sub.2O, 0.0075 g/l NaNO.sub.2, 10 g/l glucose and 0 or 15 or 30 g/l NaCl) and incubated in 100 ml shaking flasks at 28° C. and 200 rpm. After 48 h of incubation, the concentration of nitrite was measured with the method described above. The percentage of nitrite reduced by each strain was calculated referring to the initial concentration, which was determined in the non-inoculated control for each salinity (Table 1.2). Under 0 and 15% salinity, strains DSM 33351, DSM 33352, DSM 33349 and DSM 33350 were able to efficiently remove all nitrite within 48 h. Under 3% salinity, strain DSM 33351 and DSM 33350 reduced nitrite to 0 mg/ml, while strain DSM 33352 and DSM 33349 removed 67 and 87% in reference to 5.15 mg/l as initial concentration respectively.

TABLE-US-00002 TABLE 1.2 Nitrite reduction in C minimal medium containing only glucose as carbon source and 5 ppm nitrite as nitrogen source under different salinities. *Removed nitrite [%]/**Nitrite concentration [mg/l] Strain* 0% NaCl 1.5% NaCl 3.0% NaCl DSM 33351 100.0 100.0 100.0 DSM 33352 100.0 100.0 67.09 DSM 33349 100.0 100.0 86.64 DSM 33350 100.0 100.0 100.0 Initial concentration** 5.24 5.19 5.19

[0192] All strains were tested for their capacity of ammonia and nitrite reduction in C minimal medium containing only glucose as carbon source and final concentration of 2.5 ppm of both nitrite and ammonia and a C:N ratio of 20. 10 ml of the respective media (0.0352 g/l MnSO.sub.4×1H.sub.2O, 2.46 g/l MnSO.sub.4×7H.sub.2O, 0.2 KH.sub.2PO.sub.4, 0.6 g/l K2HPO.sub.4, 0.02 FeSO.sub.4×7H.sub.2O, 0.1396 g/l (NH4).sub.2SO.sub.4, 0.00375 g/l NaNO.sub.2, 1.517 g/l glucose and 0 or 15 or 30 g/l NaCl) were inoculated with a single colony of strain DSM 33351, DSM 33352, DSM 33349 or DSM 33350 grown on TSA agar for 24 h at 37° C. Cultures were incubated at 28° C. at 200 rpm. After 48 h, the concentrations of ammonia and nitrite were determined with the above described methods. The amount of ammonia and nitrite reduced by each strain was calculated referring to the initial concentration, which was determined in the non-inoculated control for each salinity (Table 1.3). Strains DSM 33351, DSM 33352, DSM 33349 and DSM 33350 were able to simultaneously remove 99.7% of the initial ammonia and 100% of the initial nitrite after 48 h under all salinities in C minimal medium containing only glucose as carbon source and final concentration of 2.5 ppm nitrite and ammonia as nitrogen source.

TABLE-US-00003 TABLE 1.2 Ammonia and nitrite reduction in C minimal medium containing only glucose as carbon source and final concentration of 2.5 ppm nitrite and ammonia as nitrogen source under different salinities. *Removed ammonia *Removed nitrite [%]/**Ammonia [%]/**Nitrite concentration [mg/l] concentration [mg/l] 0% 1.5% 3.0% 0% 1.5% 3.0% Strain* NaCl NaCl NaCl NaCl NaCl NaCl DSM 33351 99.7 99.7 99.7 100.0 100.0 100.0 DSM 33352 99.7 99.7 99.7 100.0 100.0 100.0 DSM 33349 99.7 99.7 99.7 100.0 100.0 100.0 DSM 33350 99.7 99.7 99.7 100.0 100.0 100.0 Initial 2.12 2.13 2.24 2.27 2.20 2.06 concentration**

[0193] Overall, these data show that B. subtilis strains DSM 33351, DSM 33352, DSM 33349 and DSM 33350 can use both ammonia and nitrite as sole nitrogen source and efficiently remove both compounds within 48 h.

Example 2: Outgrowth of Spores in C Minimal Medium and Nitrogen Reduction

[0194] The ability of Bacillus strains to produces spores is a great advantage for receiving long-term stable product.

[0195] In order to produce spores of B. subtilis DSM 33351, DSM 33352, DSM 33349 and DSM 33350, the strains were pre-grown for 14 hours at 30° C. in 50 g of medium containing 43 g/kg soymeal and 22 g/kg of a sugar-solution (320 g/kg glucose, 90 g/kg fructose, 350 g/kg maltose and 100 g/kg maltotriose) and were shaken at 200 rpm. Next, 14 g of the previous culture was used to inoculate 200 g of spore-production-medium and were shaken at 200 rpm while incubated at 30° C. The spore-production-medium consisted of 21 g/kg of the abovementioned sugar-solution, 43 g/kg soymeal, 0.29 g/kg MgSO.sub.4×7 H.sub.2O, 0.032 g/kg MnSO.sub.4×H.sub.2O, 0.023 g/kg FeSO.sub.4×7H.sub.2O and 0.003 g/kg ZnSO.sub.4×7H.sub.2O. After 48 hours of incubation, the spores were harvested by centrifugation and resuspended in 20% glycerol in PBS buffer and stored at −80° C.

[0196] Spores DSM 33351, DSM 33352, DSM 33349 and DSM 33350 were tested regarding their potential to grow out in C-minimal medium with ammonia and nitrite as nitrogen source and regarding their capacity to reduce ammonia and nitrite efficiently.

[0197] For testing the outgrowth in a microtiter plate based assay, 10.sup.6 CFU/ml spores of strain DSM 33351, DSM 33352, DSM 33349 and DSM 33350 were inoculated to a final volume of 150 μl ml of the respective C-minimal medium with glucose as only carbon source and with ammonia and nitrite as nitrogen source (0.0352 g/l MnSO.sub.4×1H.sub.2O, 2.46 g/l MnSO.sub.4×7H.sub.2O, 0.2 KH.sub.2PO.sub.4, 0.6 g/l K2HPO.sub.4, 0.02 FeSO.sub.4×7H.sub.2O, 0.1396 g/l (NH.sub.4)2SO.sub.4, 0.00375 g/l NaNO.sub.2 and 10 g/l glucose (C:N ratio=20)) supplemented with 10% PrestoBlue™ (Thermo Fisher Scientific, Waltham, Mass., USA). Spores were incubated at 37° C. and orbital shaking at 350 rpm for 30 h in a microtiter plate reader (TECAN Infinite® M1000 Pro). The detection of outgrowth of the spores occurred via the metabolic activity of the outgrowing cells, which reduce the nonfluorescent resazurin of PrestoBlue™ to bright red fluorescent resorufin. The fluorescence was measured real-time during cultivation (excitation filter: 550-12 nm; emission filter: 590 nm). The resulting kinetics was used to calculate the timepoint of spore outgrowth based on a defined threshold. The hours until outgrowth for strains DSM 33351, DSM 33352, DSM 33349 and DSM 33350 are depicted in Table 2.1. All strains were able to grow out in C minimal medium containing only glucose as C source and final concentration of 2.5 ppm nitrite and ammonia within 23.35 h, while DSM 33349 grew out fastest with 18.89 h.

TABLE-US-00004 TABLE 2.1 Outgrowth of spores in C minimal medium containing only glucose as carbon source and final concentration of 2.5 ppm nitrite and ammonia as nitrogen source. Time until outgrowth Strain [h] DSM 33351 23.35 DSM 33352 21.14 DSM 33349 18.89 DSM 33350 20.00

[0198] Spores of strains DSM 33351, DSM 33352, DSM 33349 and DSM 33350 were also tested regarding their ability to remove ammonia and nitrite in C minimal medium containing only glucose as carbon source and final concentration of 2.5 ppm of each nitrite and ammonia. 10 ml of the respective media in 100 ml shaking flasks (0.0352 g/l MnSO.sub.4×1H.sub.2O, 2.46 g/l MnSO.sub.4×7H.sub.2O, 0.2 KH.sub.2PO.sub.4, 0.6 g/l K.sub.2HPO.sub.4, 0.02 FeSO.sub.4×7H.sub.2O, 0.1396 g/l (NH.sub.4)2SO.sub.4, 0.00375 g/l NaNO.sub.2, and 10 g/l Glucose (C:N ratio=20)) were inoculated with 10.sup.7 CFU/ml from the spore stocks. Cultures were incubated at 28° C. and 200 rpm. After 52 h, the concentrations of ammonia and nitrite were determined with the above described methods. The amount of ammonia and nitrite reduced by each strain was calculated referring to the initial concentration, which was determined in the non-inoculated control (Table 2.2). Ammonia and nitrite were removed efficiently from flasks inoculated with spores from strains DSM 33351, DSM 33352, DSM 33349 and DSM 33350. The strains were able to simultaneously remove minimum 99.6% of ammonia and 100% of nitrite within 52 h.

TABLE-US-00005 TABLE 2.2 Ammonia and nitrite reduction in C minimal medium containing only glucose as C source and final concentration of 2.5 ppm nitrite and ammonia as nitrogen source inoculated with spores. *Removed ammonia [%]/ *Removed nitrite [%]/ **Ammonia concentration **Nitrite concentration Strain* [mg/l] [mg/l] DSM 33351 99.7 100.0 DSM 33352 96.8 100.0 DSM 33349 98.8 100.0 DSM 33350 99.6 100.0 Initial 2.25 2.46 concentration**

[0199] These data show that spores of strains DSM 33351, DSM 33352, DSM 33349 and DSM 33350 are not only able to germinate and grow out in C minimal medium containing with 2.5 ppm nitrite and ammonia as sole nitrogen source, but they are also able to remove 2.5 ppm of each nitrite and ammonia within 52 h.

Example 3: Ability of Strains to Inhibit Pathogens

[0200] The ability to inhibit different pathogens important in aqua culture was analyzed using well diffusion antagonism tests (Parente et al., 1995). Pathogens tested in these assays were Vibrio harveyi DSM 19623, Vibrio parahaemolyticus DSM 10027, Aeromonas hydrophila DSM 30187, Streptococcus agalactiae DSM 2134 and Flavobacterium columnare DSM 25092.

[0201] Vibrios are known to be associated with disease and high mortality in shrimp, but can also infect fish (Chatterjee and Haldar, 2012). Tilapia infections can be associated with Streptococcus agalactiae, a widely distributed bacterium, causing e.g. hemorrhage or erratic swimming (Mishra et al., 2018). Columnaris disease is caused by Flavobacterium columnare affecting fresh water fish. Salmon and trout farms, for instance, report high annual losses (Pulkkinen et al., 2010).

[0202] The pathogens were grown under appropriate conditions in liquid medium until an optical density of OD600 of at least 1.0 was reached. Pathogens were plated with a sterile spatula on the surface of Caso Yeast agar plates. Holes of 9 mm diameter were cut into the dried plates. Strains DSM 33351, DSM 33352, DSM 33349 and DSM 33350 were grown in LB-Kelly (LB Media supplemented with trace elements solution of DSMZ media 1032) for 16 h at 37° C. and 200 rpm in 100 ml shaking flasks. The cut wells were filled with non-inoculated media as control and the 100 μl of OD 5.0 adjusted cultures from strains DSM 33351, DSM 33352, DSM 33349 and DSM 33350. The plates were incubated under suitable conditions and the zone of clearance in mm was determined measuring from the edge of the cut well to the border of the cleared lawn. This zone was measured horizontally and vertically, and the average was taken. A scoring in low (+), medium (++), high (+++) and very high (++++) inhibition was applied according to Table 3.1.

TABLE-US-00006 TABLE 3.1 Scoring of pathogen inhibition based on diameter of hole and clearing zone in well diffusion antagonism tests. Score Diameter of hole + clearing zone [mm] Low (+) ≤9.5 mm Medium (++), >9.5 ≤ 15  High (+++) >15 ≤ 25 Very high (++++) >25 ≤ 30

[0203] Results for the inhibition of the pathogens by strains DSM 33351, DSM 33352, DSM 33349 and DSM 33350 can be found in Tables 3.2 to 3.6 respectively.

TABLE-US-00007 TABLE 3.2 Inhibitory capacity of B. subtilis DSM 33351 against different pathogens in a well diffusion antagonism assays on Caso Yeast medium, inhibition intensity was scored according to Table 4.1. Pathogen Inhibition intensity Vibrio harveyi DSM 19623 + Vibrio parahaemolyticus DSM 10027 + Aeromonas hydrophila DSM 30187 ++ Streptococcus agalactiae DSM 2134 ++++

[0204] The data show that B. subtilis DSM 33351 can inhibit the growth of Vibrio harveyi DSM 19623, Vibrio parahaemolyticus DSM 10027, Aeromonas hydrophila DSM 30187 and Streptococcus agalactiae DSM 2134.

TABLE-US-00008 TABLE 3.3 Inhibitory capacity of B. subtilis DSM 33352 against different pathogens in a well diffusion antagonism assays on Caso Yeast medium, inhibition intensity was scored according to Table 5.1. Pathogen Inhibition intensity Aeromonas hydrophila DSM 30187 ++ Streptococcus agalactiae DSM 2134 +++

[0205] The data show that B. subtilis DSM 33352 can inhibit the growth of Aeromonas hydrophila DSM 30187 and Streptococcus agalactiae DSM 2134.

TABLE-US-00009 TABLE 3.4 Inhibitory capacity of B. subtilis DSM 33349 against different pathogens in a well diffusion antagonism assays on Caso Yeast medium, inhibition intensity was scored according to Table 6.1. Pathogen Inhibition intensity Vibrio harveyi DSM 19623 + Aeromonas hydrophila DSM 30187 ++ Streptococcus agalactiae DSM 2134 ++++ Flavobacterium columnare DSM 25092 +

[0206] The data show that B. subtilis DSM 33349 can inhibit the growth of Vibrio harveyi DSM 19623, Aeromonas hydrophila DSM 30187, Streptococcus agalactiae DSM 2134 and Flavobacterium columnare DSM 25092.

TABLE-US-00010 TABLE 3.4 Inhibitory capacity of B. subtilis DSM 33350 against different pathogens in a well diffusion antagonism assays on Caso Yeast medium, inhibition intensity was scored according to Table 7.1. Pathogen Inhibition intensity Vibrio harveyi DSM 19623 + Vibrio parahaemolyticus DSM 10027 + Aeromonas hydrophila DSM 30187 ++ Streptococcus agalactiae DSM 2134 ++++ Flavobacterium columnare DSM 25092 +

[0207] The data show that B. subtilis DSM 33350 can inhibit the growth of Vibrio harveyi DSM 19623, Vibrio parahaemolyticus DSM 10027, Aeromonas hydrophila DSM 30187, Streptococcus agalactiae DSM 2134 and Flavobacterium columnare DSM 25092.

LITERATURE

[0208] Parente, E., Brienza, C., Moles, M., & Ricciardi, A. (1995). A comparison of methods for the measurement of bacteriocin activity. Journal of microbiological methods, 22(1), 95-108. [0209] Chatterjee, S. and Haldar, S. (2012). Vibrio Related Diseases in Aquaculture and Development of Rapid and Accurate Identification Methods. J. Marine Sci. Res. Dev. S1:002. doi:10.4172/2155-9910.S1-002. [0210] Pulkkinen, K., Suomalainen, L.-R., Read, A. F., Ebert, D., Rintamäki, P. and Valtonen, E. T. (2010). Intensive fish farming and the evolution of pathogen virulence: the case of Columnaris disease in Finland. Proc. R. Soc. B. 277, 593-600. [0211] Mishra, A., Nam, G. H., Gim, J. A., Lee, H. E., Jo, A. and Kim, H. S. (2018). Current Challenges of Streptococcus Infection and Effective Molecular, Cellular, and Environmental Control Methods in Aquaculture. Mol. Cells. 41(6):495-505