Poultry farm practices

Abstract

The present invention relates to methods of improving the environment within a poultry farming facility including reducing ammonia production in a poultry facility, inhibiting urease enzymes in poultry litter, reducing levels of pathogenic bacteria in poultry litter, improving productivity of poultry farms, reducing or preventing pododermatitis in poultry reared in mass production poultry facilities and controlling pests in poultry litter. Compositions, suitable for use in such methods, comprising at least one microorganism of the genus Bacillus and at least one biosurfactant wherein the biosurfactant is present in an amount of 2 mg/L to 7000 mg/L are also described.

Claims

1. A method for preparing a composition comprising endospores of Bacillus subtilis and at least one biosurfactant, the method comprising: a) culturing Bacillus subtilis RSA-203 (ATCC Accession No. PTA-13451) in a culture broth so as to encourage formation of a foamate enriched with the at least one biosurfactant; b) collecting and condensing the foamate from the culture broth to form a collected and condensed foamate; c) causing a culture comprising Bacillus subtilis to undergo endospore formation to produce a culture broth comprising the endospores of Bacillus subtilis; and d) combining the culture broth comprising the endospores with (i) an amount of the collected and condensed foamate comprising the at least one biosurfactant; and (ii) at least one anionic, cationic, non-ionic or zwitterionic surfactant, other than the at least one biosurfactant present in the foamate; thereby forming the composition; wherein the at least one biosurfactant in the composition is present in an amount of 2 mg/L to 7000 mg/L; and wherein the amount of the at least one biosurfactant in the composition is higher than in the culture broth comprising endospores.

2. The method according to claim 1, wherein the Bacillus subtilis in the composition is present in an amount of 110.sup.4 cfu/mL to 110.sup.13 cfu/mL.

3. The method according to claim 1, wherein the at least one biosurfactant is selected from surfactin, lichenysin, fengycin, iturin and mixtures thereof.

4. The method according to claim 3, wherein the at least one biosurfactant is selected from surfactin, lichenysin and mixtures thereof.

5. The method according to claim 4, wherein the at least one biosurfactant is surfactin.

6. The method according to claim 1, wherein the at least one biosurfactant is present in an amount of 50 to 7000 mg/L.

7. The method according to claim 1, wherein the surfactant has a hydrophilic lipophilic balance of 12 or greater.

8. The method according to claim 1, wherein the surfactant is an alkylbenzene sulfonic acid surfactant.

9. The method of claim 7, wherein the surfactant has a hydrophilic lipophilic balance of 12 to 14.

10. The method of claim 1, wherein the endospores of Bacillus subtilis comprise endospores of a microorganism other than Bacillus subtilis RSA-203.

Description

BRIEF DESCRIPTION OF THE FIGURE

(1) FIG. 1 is a graphical representation showing the growth of microorganisms over time with varying amounts of sulfate ions.

EXAMPLES

Example 1: Production of Surfactin Biosurfactant

(2) Bacillus subtilis NRRL B-3383 strain (originally obtained from the United States Department of Agriculture) from bacterial culture was transferred at a 2% volume by volume inoculum into 4 L shake flasks containing 2.5 L of 10% vinasse based MMS broth. The vinasse based MMS broth containing:

(3) TABLE-US-00001 component quantity vinasse 100 mL ammonium nitrate 4.1 g sodium phosphate dibasic 5.68 g tetrasodium tetrahydrate EDTA 1.8 mg manganese sulfate 6.8 mg autoclaved deionized water to 1 L

(4) The flasks were placed on orbital shakers (SKC 6100, Jeio Tech) at 150 rpm while incubating at 30 C. (MCO-801C Incubator, Sanyo). After 72 hours, flasks were removed from the incubator and the biomass removed from the crude culture broth by centrifugation at 8,500 rpm for 20 min at 4 C. (Sorvall Evolution RC).

(5) The pH of the resulting supernatant was brought to a pH of 2.0 using HCl which resulted in precipitation of surfactin arid the supernatant stored overnight at 4 C. to ensure complete precipitation. The precipitate was collected by centrifugation at 8,500 rpm for 20 minutes at 4 C. Approximately 2.5 g/L of crude material was collected in the pellet. The pellet was suspended in deionized water and the pH adjusted to 8.0 using 1 M NaOH. The aqueous solution was extracted with an equal volume of dichloromethane. The dichloromethane layer was separated and allowed to evaporate to provide purified crystalline surfactin in an amount of 50 mg/L to 750 mg/L.

(6) The samples of crystalline surfactin were examined for purity against a standard composition of pure surfactin (Sigma Aldrich, 98% pure). Analysis of the standard composition by LC-MS showed peaks with retention times at 1.03, 1.23, 1.61, 1.74, 2.15 and 2.93 minutes. Purity was calculated based on peak area.

(7) Four samples tested for purity using the above method were found to be 80%, 56%, 58% and 61% pure.

Example 2: Production of Blends of Surfactin and Lichenysin

(8) Bacillus subtilis and Bacillus lichenifbrmis were used to inoculate 4 L shake flasks containing 10% molasses based MMS broth. The molasses based MMS broth containing:

(9) TABLE-US-00002 component quantity molasses 100 mL ammonium nitrate 4.1 g sodium phosphate dibasic 5.68 g tetrasodium tetrahydrate EDTA 1.8 mg manganese sulfate 6.8 mg autoclaved deionized water to 1 L

(10) The flasks were placed on orbital shakers (SKC 6100, Jeio Tech) at 150 rpm while incubating at 30 C. (MCO-801C Incubator, Sanyo). After 72 hours, flasks were removed from the incubator and the biomass removed from the culture broth by centrifugation at 8,500 rpm for 20 min at 4 C. (Sorvall Evolution RC).

Example 3

(11) In a 1 liter bioreactor a buffer solution was prepared containing 800 mL demineralized water, monopotassium phosphate (20.4 g), disodium phosphate (28.4 g) and yeast extract (5 g). The buffer was mixed and the pH adjusted to 7. The buffer was then autoclaved. In a 1 L bioreactor, 200 mL of the buffer solution was mixed with magnesium sulfate (2 mL of 12 g/100 mL solution), calcium chloride (1 mL, 0.1 M), ferrous sulfate heptahydrate (1 mL, 15.7 g/100 mL solution), manganese sulfate monohydrate (1 mL, 3.8 g/100 niL solution), sodium EDTA (1 mL, 0.18 g/100 mL solution) and glucose (20 mL, 20 g/80 g deionized water) and ammonium nitrate (10 mL, 82 g/200 mL solution). Bacillus species spores were inoculated into the bioreactor at 2% wt. The biomass was agitated at 300 rpm for 12 hours at 35 C. The Bacillus was incubated until the cell density was to 110.sup.14 cfu per mL and the OD.sub.600 nm was 2.2.

Example 4: Coproduction of Bacillus Species and Biosurfactant

(12) 2200 lbs (998 kg) of demineralized water was added to a 560 gallon (2200 L) stainless steel reactor and warmed to 35 C. 8.7 lbs (3.95 kg) of monopotassium phosphate and 13.3 lbs (6 kg) of disodium phosphate were added and the composition mixed. Magnesium sulfate (240 g), calcium chloride (33 g), EDTA (2.2 g) dissolved in 100 mL of water, ferrous sulfate (104 g), manganese sulfate (41.7 g), dextrose monohydrate (10.7 lbs, 4.85 kg), autoclaved yeast extract (2.14 lbs, 0.97 kg), ammonium chloride (6.1 lbs, 2.77 kg) and sodium nitrate (9.6 lbs, 4.35 kg) were added and mixing was continued at a temperature of 35 C. 23 L of Bacillus innoculum having an OD.sub.600 nm of 2.2 and a surface tension greater than 50 dynes/cm was added. The mixture was mixed and maintained at 35 C. and aerated to a dissolved oxygen level of 50% with filtered air. The culture was stopped when the culture medium reached an optical density of 1.7 to 2.0 Absorbence units.

Example 5: Composition

(13) A composition for use in the methods of the invention was then prepared by further dilution of the culture medium produced in Example 4. In a 1000 gallon stainless steel dilution reactor was added 2200 lbs (998 kg) of demineralized water and monosodium phosphate (5 lbs, 2.27 kg) was added with agitation. Sodium hydroxide (5-60 lbs, 2.27-27.2 kg) and <1% wt non-ionic dodecylbenzenesulfonic acid was added. The pH was adjusted to 6-8 with sodium hydroxide. The cultured bacteria and medium were then added and the composition mixed. Surface tension was checked and additional concentrated surfactin foam condensate was added to provide a surface tension of 27-35 dynes/cm.

Example 6: Composition

(14) The method of Example 5 was repeated with the exception that the pH was adjusted to 3.5 to 5 with citric acid.

Example 7: Ammonia Production in Chicken Litter

(15) Chicken litter containing chicken faeces was divided into two cubic yard boxes in equal amounts. One box was monitored for ammonia production without, further treatment (control). The other box was treated by spraying the chicken litter with a composition comprising microbes of the Bacillus species including B. subtilis, vinasse residue and surfactin. The composition was derived from culturing process and diluted to 5-25% per litre with water and the litter was treated at a rate of 1 L per 100 square metres.

(16) Results:

(17) The control box had an ammonia concentration of 20 ppm. The treated box had an ammonia concentration of 8 ppm.

Example 8: Ammonia Production in Poultry Barns

(18) Four poultry barns (broilers), each having an area of 22,000 square feet (2044 m.sup.2) and housing 22,300 birds were used. In all four houses, the poultry litter was treated 5 days before the placement of poultry chicks in the barn.

(19) Barn One was treated with the composition of Example 4.12.5 (47.3 L) gallons of the composition of Example 5 was mixed with 185 gallons (700 L) of water then blended and sprayed on the poultry litter comprising sawdust and compost in half the barn. This process was repeated with another 12.5 gallons (47.3 L) of composition in 185 gallons (700 L) of water to treat the other half of the barn.

(20) Barn Two was treated with the composition of Example 5.12.5 gallons (47.3 L) of the composition of Example 5 was mixed with 185 gallons (700 L) of water then blended and sprayed on the poultry litter in half the barn. This process was repeated with another 12.5 gallons (47.3 L) of composition in 185 gallons (700 L) of water to treat the other half of the barn.

(21) Barns Three and Four were treated with aluminium bisulphate, a pH adjustment substance. In each barn, 1650 pounds (748 kg) of aluminium bisulphate was blended in 500 gallons (1893 L) of water, and sprayed on the poultry litter.

(22) In each barn the aqueous treatment was applied by a motorized 6 nozzle sprayer equipped with and fed from a 200 gallon (757 L) tank, mounted on a four wheeled vehicle. After application, the poultry litter was windrowed by tractor.

(23) After treatment three samples of litter were taken from each of the Barns and analysed for ammonia gas.

(24) The poultry chicks were placed in the barns and air samples were taken at days 1, 5, 16, 21 and 39 and analysed for ammonia concentrations using Drager tubes (Drager Safety Inc., Pittsburgh, Pa. USA). The results are shown in Table 1.

(25) TABLE-US-00003 TABLE 1 Day Day twenty one Day five Day sixteen one Day 39 Barn One 10 10 20 20 12 Barn Two 10 10 20 20 15 Barn Three 0 20 18 >25 Full fans Barn Four 0 20 18 >25 Full fans

(26) Full fans indicates that mechanical ventilation of the barn was required to control ammonia concentrations.

(27) The poultry in all four barns were assessed for health, morbidity and weight. The poultry in all four barns were in good health.

(28) The feet of the poultry were analysed by paw grading; healthy paws graded as 1, through to infected and blistered paws graded as 4. In Barns One and Two the paw grading was 1. No birds had blisters on their feet.

(29) Morbidity levels in Barns One and Two was reduced by an average of 133 birds compared to Barns Three and Four.

(30) Bird weights increased in Barns One and Two by 15 to 20 points compared to Barns Three and Four. This equates to a total of 11,000 lbs of additional weight in 35 days in Barns One and Two.

Example 9

(31) On day 21 of the poultry farm trial of Example 8, a sample of poultry litter was taken and the litter was analysed by plating on agar plates. The plates were incubated and then stained by Gram staining. Only Gram positive species were present.

Example 10: Control of Salmonella Pathogens

(32) Chicken manure was sterilized by autoclaving at a minimum temperature of 121 C. for 35 minutes, to ensure that the samples included no competing bacteria. After sterilization, the chicken manure was inoculated with Salmonella enterica and allowed to incubate for 24 hours. After incubation the non-control samples of chicken manure were treated with a composition of Example 6 by spraying approximately 100 L of composition on the chicken manure (25 g). Control samples included sterilized chicken manure and sterilized chicken manure inoculated with Salmonella enterica and incubated for 24 hours but these controls were not treated with the composition of Example 6.

(33) The samples were analysed for bacterial growth, both Salmonella enterica and Bacillus subtilis on growth media (ATCC medium 3, nutrient agar). The analysis of the controls were performed after sterilization (time 0), after inoculation and incubation for 24 hours (time 0) and the analysis of the test samples was carried out at 3, 6, 24, 48 and 96 hours after application of the composition of Example 6.

(34) A supplemental Triple Sugar Iron (TSI) test, was used to distinguish between the growth of Bacillus spp. and Salmonella spp. Salmonella enterica produces hydrogen sulphide resulting in a dark colour in this test whereas the Bacillus spp. does not.

(35) The presence of Salmonella enterica was assessed over a period of four days. After the four day period the test samples were re-innoculated with Salmonella enterica and incubated for 24 hours. The samples were then evaluated for a further period of four days. The samples for re-inoculation had been initially inoculated with Salmonella enterica incubated for 24 hours, treated with the composition of Example 5, incubated for 4 days, then reinoculated with Salmonella enterica and incubated for 24 hours. A sample was then analysed at time zero (control) then at 3, 6, 24, 48 and 96 hours. The results are shown in Table 2.

(36) TABLE-US-00004 TABLE 2 Time Sample Description (hrs) Results Comments Control Sterilized chicken manure 0 No 1 growth Control Sterilized chicken manure 0 Growth Growth positive 2 inoculated with S. enterica for S. enterica by TSI test Sample Sterilized chicken manure 3 Growth TSI test negative 1 inoculated with S. enterica for Salmonella and treated with 6 Growth TSI test negative composition of Example 5 for Salmonella 24 Growth TSI test negative for Salmonella 48 Growth TSI test negative for Salmonella 96 Growth TSI test negative for Salmonella Control Treated sample 0 Growth TSI test negative 3 reinnoculated with for Salmonella S. enterica Sample Sample 1 re-innoculated 3 Growth TSI test negative 2 with S. enterica for Salmonella 6 Growth TSI test negative for Salmonella 24 Growth TSI test negative for Salmonella 48 Growth TSI test negative for Salmonella 96 Growth TSI test negative for Salmonella

(37) There was no growth of pathogenic Salmonella enterica observed in any of the test samples treated with the composition of Example 6 from 3 hours to 96 hours. Furthermore, no growth of Salmonella enterica in the re-innoculated samples over 3 hours to 96 hours.

(38) This demonstrates that the composition comprising a microorganism of the genus Bacillus and biosurfactant is effective against S. enterica found in chicken manure. The treatment is effective within 3 hours and has a long-lasting effect.

Example 11: Pest Control

(39) 25 gallons (95 L) of the composition of Example 6 was added to 400 gallons (1514 L) of water and sprayed on 21,000 square feet of poultry litter. The litter was then windrowed. The litter was checked for darkling beetle larvae and any larvae found were collected and monitored. The darkling beetle larvae expired within 2 hours of exposure to the litter treatment. No live darkling beetle larvae were found in the treated litter 24 hours after treatment.

Example 12: Control of Campylobacter jejuni Pathogen

(40) Chicken manure was sterilized by autoclaving at a minimum temperature of 121 C. for 35 minutes, to ensure the samples included no competing bacteria. After sterilization, the chicken manure was inoculated with Campylobacter jejuni. The non-control samples of chicken manure was treated with a composition of Example 6 by spraying approximately 100 L of composition on the chicken manure (25 g). Controls included sterilized chicken manure and sterilized chicken manure inoculated with Campylobacter jejuni but neither sample was treated with a composition of Example 6. The samples were incubated at 42 C.

(41) The samples were analysed for bacterial growth on Campylobacter jejuni media. The analysis of the controls were performed after sterilization (time 0), after inoculation and incubation for 24 hours (time 0) respectively, and the analysis of the test samples was carried out at 3, 6, 24, 48 and 96 hours after application of the composition of Example 6.

(42) After the 96 hour test period, the test samples were reinoculated with Campylobacter jejuni. A sample (control 4) was assessed at time 0 to confirm viability of the bacteria. The reinoculated samples were then incubated for 96 hours and assessed again. The results are shown in Table 3.

(43) TABLE-US-00005 TABLE 3 Times Sample Description (hrs) Results Control 1 Sterilized chicken manure 0 No growth Control 2 Sterilized chicken manure inoculated 0 Growth with C. jejuni Control 3 Sample of Example 5 0 No growth Sample 1 Sterilized chicken manure inoculated 3 No Growth with C. jejuni and treated with 6 No Growth composition of Example 5 24 No Growth 48 No Growth 96 No Growth Control 4 Treated chicken manure reinoculated 0 Growth with C. jejuni Sample 2 Treated chicken manure with 96 No Growth C. jejuni

(44) There was no pathogenic Campylobacter jejuni observed in any of the test samples treated with a composition of Example 6 from 3 hours to 96 hours. Furthermore, no growth was observed in the reinoculated sample after 96 hours incubation.

(45) This demonstrates that the composition comprising a microorganism of the genus Bacillus and biosurfactant is effective against C. jejuni found in chicken manure. The treatment was effective within 3 hours and has a long-lasting effect.

Example 13: Control of Ammonia in Poultry Egg Laying Facility

(46) A four storey egg laying facility having birds in cages and manure falling onto conveyor belts was assessed for ammonia concentration. In the facility the manure from the top storeys is delivered by conveyor belt to a v-shaped platform door separating top and bottom floors. When the door opens, the manure (16000 lbs) is deposited onto a windrow approximately 6 feet high.

(47) The windrow of manure was misted with a composition of Example 5 at 4 locations.

(48) At locations 1 and 2, the composition was applied neat. At locations 3 and 4 the composition of Example 5 was applied diluted 1:16 with water. No fans were used if the ammonia levels remained below 50 ppm.

(49) When the manure was left untreated and fans were not used to disperse the ammonia produced, ammonia levels in the facility reached 500 ppm. When fans were used, ammonia levels were reduced to <50 ppm.

(50) The ammonia levels were sampled at intervals after treatment and the results are shown in Table 4.

(51) TABLE-US-00006 TABLE 4 Loca- Loca- Date Time tion 1 tion 2 Location 3 Location 4 Location 5 30/10 9.21 am 19 ppm 18 ppm 23 ppm 23 ppm 25 ppm 4/11 8.03 am 46 ppm 44 ppm 50 ppm* 52 ppm* 56 ppm* 10/11 3.10 pm 30 ppm 21 ppm 30 ppm 31 ppm 41 ppm 12/11 9.36 am 11 ppm 6 ppm 13 ppm 11 ppm 19 ppm 15/11 10.05 am 26 ppm 22 ppm 42 ppm 40 ppm 40 ppm 20/11 3.30 pm 29 ppm 24 ppm 36 ppm 41 ppm 45 ppm 22/11 8.29 am 39 ppm 28 ppm 35 ppm 36 ppm 41 ppm 24/11 9.40 am 31 ppm 29 ppm 36 ppm 40 ppm 44 ppm 28/11 7.51 am 29 ppm 39 ppm 47 ppm 53 ppm 55 ppm *ineffective spraying resulted in increased ammonia levels but dropped upon reapplication correctly.

(52) The results show that application of the composition of the invention to poultry manure was able to maintain ammonia levels below 50 ppm for more than 28 days.

(53) In a variation, the manure may be treated by misting with a continuous low dosage while being deposited on the platform door or while located on the platform door to provide contact with more surface area.

Example 14: Alternative Biosurfactant Production

(54) TABLE-US-00007 TABLE 5 Amount/ Amount/ 150 L 1000 L KH.sub.2PO.sub.4 306 gm 2.04 Kg 15 mM Monopotassium Phosphate (MW 136.09) Na.sub.2HPO.sub.4 581.25 gm 3.87 Kg 27.3 mM Disodium Phosphate (MW 141.96) MgSO.sub.47H.sub.2O 88.7 gm 592 gm 2.4 mM Magnesium Sulfate Heptahydrate (MW 246.48) CaCl.sub.26H.sub.2O 0.23 gm 1.5 gm 7 M Calcium Chloride Hexahydrate (MW 219.08) C.sub.10H.sub.14N.sub.2Na.sub.2O.sub.82H.sub.2O 0.22 gm 1.5 gm 4 M Disodium EDTA Dihydrate (MW 372.24) FeSO.sub.47H.sub.2O 0.34 gm 2.3 gm 8 M Ferrous Sulfate Heptahydrate (MW 284.05) MnSO.sub.4H.sub.2O 226 gm 1.51 Kg 10 mM Manganese SulfateMonohydrate (MW 151.0) C.sub.6H.sub.12O.sub.6H2O 6.6 Kg 44 Kg 44 gm/L Glucose Monohydrate Yeast extract 1% w/v 150 gm 1 Kg NH.sub.4Cl 400 gm 2.67 Kg 50 mM Ammonium Chloride (MW 53.49) NaNO.sub.3 638 gm 4.25 Kg 50 mM Sodium Nitrate (MW 84.99)

(55) An appropriate amount of filtered water was added to the bioreactor and bought to 35 C. The water was aerated continuously from this point. The nutrients listed above in Table 5 were added in the order given. Addition of ammonium and nitrate sources were added just before inoculation of the preparation to prevent any contaminating bacteria from growing because of lack of nitrogen. The innoculum was added in an amount to achieve an initial OD.sub.66 nm of 0.15. The culture conditions and process were monitored by optical density measurements at OD.sub.66 nm.

(56) Biosurfactant production was monitored by surface tension measurements. The surface tension of the broth can be titrated by measuring the culture fluid neat and making dilutions to determine the level of biosurfactant is above the Critical Micelle Concentration (CMC). The surface tension will not increase until the surfactin is diluted below the CMC.

(57) The strain of microorganism may be monitored by culturing samples on MMS-Y and Blood Agar plates. If a mixed culture is used and the colony types can be differentiated on an agar plate, the inoculums and the final culture should be serially diluted to perform plate counts of each strain. This can not only determine the purity of the culture but also if one strain out competes the others.

Example 15: The Effect of Sulfate at Different Concentrations on Culture

(58) The effect of sulfate on culture broth was tested by removing all sources of sulfate from the media and replacing them with chloride salts. The culture broth contained monopotassium phosphate/dipotassium phosphate buffer adjusted to pH 7 with potassium hydroxide. Samples were then spiked with varying concentrations of sodium sulfate (1.8 M) at 1 mL/L, 0.8 mL/L, 0.6 mL/L, 0.4 mL/L and 0.2 mL/L. Every half hour the optical density, pH and surface tension was evaluated. This test was done with the RSA-203 bacterial strain.

(59) The results are shown in FIG. 1. The results show that the microorganisms grow equally well with chloride salts as they do with sulfate salts. In all samples, the surface tension had dropped and stabilized around 27 dynes by 5 hours.