CA2447749A1 - Gram-positive fatty acid degrader - Google Patents
Gram-positive fatty acid degrader Download PDFInfo
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- CA2447749A1 CA2447749A1 CA002447749A CA2447749A CA2447749A1 CA 2447749 A1 CA2447749 A1 CA 2447749A1 CA 002447749 A CA002447749 A CA 002447749A CA 2447749 A CA2447749 A CA 2447749A CA 2447749 A1 CA2447749 A1 CA 2447749A1
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
- C02F3/343—Biological treatment of water, waste water, or sewage characterised by the microorganisms used for digestion of grease, fat, oil
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/32—Processes using, or culture media containing, lower alkanols, i.e. C1 to C6
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/11—Bacillus megaterium
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
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Abstract
The invention discloses a Gram-positive microorganism, Bacillus megaterium that effectively and efficiently degrades fats, oils and grease. A composition comprising said microorganism and a method for degrading fatty acids and grease are also disclosed. Availability of glycerol to the biodegrading microorganism was discovered to enhance biodegradation.
Description
GRAM-POSITIVE FATTY ACID DEGRADER
This is a continuation in part of pending U. S. application serial number 091861,142 filed May 18, 2001.
FIELD OF THF~ INVENTION
The present invention is related to identifying a Gram positive organism that degrades fats, oils and grease. More particularly, the present invention is related to providing a non-pathogenic, spore-forming Gram-positive, lipophilic bacterial strain that produces extracellular lipase and also efficiently oxidizes or degrades fatty acids and grease. The invention is further related to a liquid and dry formulations comprising said Gram-positive organism and a method for enhancing fatty acid degradation employing glycerol, BACKGROUND
Most food service establishments are required to have a device that prevents grease from flowing directly from the kitchen or food preparation area into the sewer or to an on-site waste disposal system. Commonly called grease traps, these devices function to physically prevent oils and grease from flowing directly into the sanitary sewer and to store the separated grease solid for eventual solid waste disposal.
Many municipalities place restrictions and surcharges based on the.
biological oxygen demand (BOD) and oil and grease (O&G) levels in the effluents' from grease traps. In addition to wastewater treatment costs and surcharges, the grease solids from the traps must also be periodically,removed and. disposed.
The food service establishment then faces two ~ recurring charges for wastewater treatment, one a municipal treatment cost and secondly a grease disposal cost.
However, the frequency of pumping the accumulated grease solids can be quite variable, ranging from several weeks to several months. If traps are not cleaned on a regular basis, grease clogs may occur causing wastewater to back up into the food preparation area causing malodors and requiring the establishment to SUBSTITUTE SHEET (RULE 26) close until the problem is corrected. In addition to providing physical means to trap O&G, grease traps can function to ~biologicaUy rnediate-a reduction of BOD and O&G in the bulk liquid resulting in cleaner effluent wastewater. This reduction of BOD and O&G is dependent upon the hydraulic retention time, which is dependent S on the size of the grease trap and wastewater flow. Other factors Chat affect biological activity within a grease trap include pH, temperature and whether or not the' facility practices bioaugmentation.
Bioaugmentation, the addition of commercial bacterial products that increase the biological activity in the system, has been used to reduce the BOD and O&G in the elrluenis from grease traps. This has helped to reduce surcharges that the food establishments must pay to municipalities for wastewater services.
Additionally, bioaugmentation has been used to decrease the pumping frequency of grease traps, to keep drain lines open and to reduce malodors.
In addition to grease traps, bioaugmentation has also been used to help remove grease from lift stations, drain lines, septic tames, waste treatment facilities and other situations where grease accumulation can cause flow problems and malodors.
Bioaugmentation products can be either liquid or dry. Because of ease of handling, liquid products are generally preferred and can be added by a liquid metering pump drawing on a container that is replenished on a periodic basis.
However, dry formulations are preferred for other applications such as waste treatment facilities.
Strains used in bioaugmentation of grease applications produce an important - v ~ extracellular- enzyme, ~ lipase. This enzyme hydrolyzes. and breaks the ester- bond.
between the glycerol. backbone and the fatty acid moieties making up the grease.
The glycerol is quickly disposed by biodegradation. However, the fatty acids are di~cult to degrade and can persist causing pH drops, clogging and malodors.
When Gram-negative microorganisms are used for bioaugmentation in liquid products, they are present as vegetative cells and as such, they may be killed by chemicals, such as surfactants and preservatives, which are often used in such SUBSTITUTE SHEET (RULE 26) formulations. Therefore, products containing Gram-negative organisms cannot contain biocides .and surfactants. Then, unpreserved liquid products may develop severe malodors from microbial contaminants growing in the product. Some of these contaminants may be undesirable in a food service environment.
Furthermore, ~unpreserved products may ~aIso suffer from ~ decreased shelf Life and ~e8icacy.
. , , Clearly, while Gram-negative : ~croorganisms have an advantage in fatty acid degradation, their use in residential and food service products have serious . . , drawbacks.
Dry Gram-negative products, on the other hand, do have an advantage of improved shelf life over liquid Gram-negative formulations. however, this advantage is modest and varies significantly from bacterial strain to bacterial strain in the product. Although dry products can be rehydrated with water and applied like liquid products, the disadvantages of using unpreserved liquids containing Gram negative microorganisms still apply to rehydrated dry materials.
. Many Gram-negative microorganisms are known to have the ability to biodegrade fatty acids generated by the action of lipase. This ability to oxidize and degrade fatty acids is generally not found in Gram-positive, spore-forming microorganisms, specifically members of the genus Bacillus.
Accordingly, there is a need to develop bioaugmentation formulations that, can effectively and e~ciently degrade or oxidize fats, oil and grease without causing .
malodors or other undesirable conditions, such as occurs with Gram-negative .
organisms.
Specifically, there is a need to find non-pathogenic, spore-forming Gram . positive, lipophilic bacterial strain that produces extracellular.lipase and efficiently , .oxidizes or breaks down.fatty acids and grease. ~ Heretofore, such ~a Gram-positive organism and a formulation containing the same have not been identified or produced.
Tf fatty acid degrading microorganisms are ident~ed, there is a further need to augment or enhance the fatty acid degrading activity of such microorganisms in order to maximize the effectiveness of products based thereon.
This is a continuation in part of pending U. S. application serial number 091861,142 filed May 18, 2001.
FIELD OF THF~ INVENTION
The present invention is related to identifying a Gram positive organism that degrades fats, oils and grease. More particularly, the present invention is related to providing a non-pathogenic, spore-forming Gram-positive, lipophilic bacterial strain that produces extracellular lipase and also efficiently oxidizes or degrades fatty acids and grease. The invention is further related to a liquid and dry formulations comprising said Gram-positive organism and a method for enhancing fatty acid degradation employing glycerol, BACKGROUND
Most food service establishments are required to have a device that prevents grease from flowing directly from the kitchen or food preparation area into the sewer or to an on-site waste disposal system. Commonly called grease traps, these devices function to physically prevent oils and grease from flowing directly into the sanitary sewer and to store the separated grease solid for eventual solid waste disposal.
Many municipalities place restrictions and surcharges based on the.
biological oxygen demand (BOD) and oil and grease (O&G) levels in the effluents' from grease traps. In addition to wastewater treatment costs and surcharges, the grease solids from the traps must also be periodically,removed and. disposed.
The food service establishment then faces two ~ recurring charges for wastewater treatment, one a municipal treatment cost and secondly a grease disposal cost.
However, the frequency of pumping the accumulated grease solids can be quite variable, ranging from several weeks to several months. If traps are not cleaned on a regular basis, grease clogs may occur causing wastewater to back up into the food preparation area causing malodors and requiring the establishment to SUBSTITUTE SHEET (RULE 26) close until the problem is corrected. In addition to providing physical means to trap O&G, grease traps can function to ~biologicaUy rnediate-a reduction of BOD and O&G in the bulk liquid resulting in cleaner effluent wastewater. This reduction of BOD and O&G is dependent upon the hydraulic retention time, which is dependent S on the size of the grease trap and wastewater flow. Other factors Chat affect biological activity within a grease trap include pH, temperature and whether or not the' facility practices bioaugmentation.
Bioaugmentation, the addition of commercial bacterial products that increase the biological activity in the system, has been used to reduce the BOD and O&G in the elrluenis from grease traps. This has helped to reduce surcharges that the food establishments must pay to municipalities for wastewater services.
Additionally, bioaugmentation has been used to decrease the pumping frequency of grease traps, to keep drain lines open and to reduce malodors.
In addition to grease traps, bioaugmentation has also been used to help remove grease from lift stations, drain lines, septic tames, waste treatment facilities and other situations where grease accumulation can cause flow problems and malodors.
Bioaugmentation products can be either liquid or dry. Because of ease of handling, liquid products are generally preferred and can be added by a liquid metering pump drawing on a container that is replenished on a periodic basis.
However, dry formulations are preferred for other applications such as waste treatment facilities.
Strains used in bioaugmentation of grease applications produce an important - v ~ extracellular- enzyme, ~ lipase. This enzyme hydrolyzes. and breaks the ester- bond.
between the glycerol. backbone and the fatty acid moieties making up the grease.
The glycerol is quickly disposed by biodegradation. However, the fatty acids are di~cult to degrade and can persist causing pH drops, clogging and malodors.
When Gram-negative microorganisms are used for bioaugmentation in liquid products, they are present as vegetative cells and as such, they may be killed by chemicals, such as surfactants and preservatives, which are often used in such SUBSTITUTE SHEET (RULE 26) formulations. Therefore, products containing Gram-negative organisms cannot contain biocides .and surfactants. Then, unpreserved liquid products may develop severe malodors from microbial contaminants growing in the product. Some of these contaminants may be undesirable in a food service environment.
Furthermore, ~unpreserved products may ~aIso suffer from ~ decreased shelf Life and ~e8icacy.
. , , Clearly, while Gram-negative : ~croorganisms have an advantage in fatty acid degradation, their use in residential and food service products have serious . . , drawbacks.
Dry Gram-negative products, on the other hand, do have an advantage of improved shelf life over liquid Gram-negative formulations. however, this advantage is modest and varies significantly from bacterial strain to bacterial strain in the product. Although dry products can be rehydrated with water and applied like liquid products, the disadvantages of using unpreserved liquids containing Gram negative microorganisms still apply to rehydrated dry materials.
. Many Gram-negative microorganisms are known to have the ability to biodegrade fatty acids generated by the action of lipase. This ability to oxidize and degrade fatty acids is generally not found in Gram-positive, spore-forming microorganisms, specifically members of the genus Bacillus.
Accordingly, there is a need to develop bioaugmentation formulations that, can effectively and e~ciently degrade or oxidize fats, oil and grease without causing .
malodors or other undesirable conditions, such as occurs with Gram-negative .
organisms.
Specifically, there is a need to find non-pathogenic, spore-forming Gram . positive, lipophilic bacterial strain that produces extracellular.lipase and efficiently , .oxidizes or breaks down.fatty acids and grease. ~ Heretofore, such ~a Gram-positive organism and a formulation containing the same have not been identified or produced.
Tf fatty acid degrading microorganisms are ident~ed, there is a further need to augment or enhance the fatty acid degrading activity of such microorganisms in order to maximize the effectiveness of products based thereon.
SUBSTITUTE SHEET (RULE 26) SUMMARY OF THE INVENTION - -It is, therefore, an object of the present invention to provide a non-pathogenic, spore-forming Gram positive, lipophilic bacterial strain that produces extracellular lipase and also efficiently hydrolyzes or degrades fatty acids and grease or a mixture of fatty acid and grease . - ~ -;: . . , .
- It is a further object of the present invention to provide liquid and dry compositions comprising a non-pathogenic, spore-forming Gram-positive, lipophilic bacterial strain that produces extracellular lipase and efficiently hydrolyzes or degrades fats, oils and grease.
An additional object of the present invention is to provide a method for degrading fatty acid and grease using a Gram positive strain ofBacillus species.
Yet another object of the present invention is to enhance the biodegrading activity of Grampositive strain of8aeillus species.
A further object of the invention is to provide a method for augmenting fatty acid degrading activity of microorganisms employing glycerol as an activity enhancer.
Various other objects and advantages of the present invention will become evident from a brief description of the drawings and detailed description of the invention.
Additional advantages and novel features of the invention will be set forth in part in the description that follows, and~in part will become more apparent to those.
skilled in the art upon examination of the following or upon learning by practice of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
2S - - In the drawings, -FIG. -1-shows the e~oacy of various microorganisms to degrade grease and FIGS. 2 through 7 show the effect of glycerol on biodegrading activity of SB3112 and various microorganisms.
- It is a further object of the present invention to provide liquid and dry compositions comprising a non-pathogenic, spore-forming Gram-positive, lipophilic bacterial strain that produces extracellular lipase and efficiently hydrolyzes or degrades fats, oils and grease.
An additional object of the present invention is to provide a method for degrading fatty acid and grease using a Gram positive strain ofBacillus species.
Yet another object of the present invention is to enhance the biodegrading activity of Grampositive strain of8aeillus species.
A further object of the invention is to provide a method for augmenting fatty acid degrading activity of microorganisms employing glycerol as an activity enhancer.
Various other objects and advantages of the present invention will become evident from a brief description of the drawings and detailed description of the invention.
Additional advantages and novel features of the invention will be set forth in part in the description that follows, and~in part will become more apparent to those.
skilled in the art upon examination of the following or upon learning by practice of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
2S - - In the drawings, -FIG. -1-shows the e~oacy of various microorganisms to degrade grease and FIGS. 2 through 7 show the effect of glycerol on biodegrading activity of SB3112 and various microorganisms.
SUBSTITUTE SHEET (RULE 26) DETAILED DESCRIPTION OF THE INVENTION
The above and various other objects and advantages of the present invention are achieved by a biologically pure culture of a Gram-positive microorganism, ~ Bacillus megaterium, strain SB3112, having the identifying characteristics of ATCC
deposit number PTA 3142, and a composition comprising.the same. The deposit shall be maintained in viable condition at the ATCC during the entire term of the issued patent and shall be made available to any person or entity for non-commercial use without restriction, but in accordance with the provisions of the law governing the deposit.
It should be understood that unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and rnatenials similar or equivalent to those described herein can be used in the 1 S practice or testing of the present invention, the methods and materials described herein are preferred. Unless mentioned otherwise, the techniques employed or contemplated herein are standard methodologies well known to one of ordinary skill in the art. The materials, methods and examples are only exemplary and not limiting.
The term "biodegradation'; "biodegraded", or "biodegrading" as used herein means that the substrate is broken down, oxidized or degraded by tk~e microorganism And the term "activity enhancement" as used herein means that the biodegradation activity of the . microorganism is increased by the presence or addition of a particular component, said component being designated as "activity enhancer", or "activator"~.
By utilizing a Gram-positive, spore-forming organism that can oxidize fatty acids, one can obtain the advantages of an improved preserved liquid product, and stable, easy to use dry products. Unlike Grate-negative containing products, the preserved, spore-based Gram-positive containing product can contain preservatives and surfactants to aid in the biodegradation of fats, oils or grease, because the SUBSTITUTE SHEET (RULE 26) spores . are relatively resistant to tiiocides and surfactants. Furthermore, these products may also contain micronutrients promoting the growth of the microorganisms. Thus, a Gram positive product comprising lipase producing, fatty acid degrading, spore-forming microorganism in a preserved liquid formulation - offers various advantages -required for efficacious degradation of oil and grease.
r " . , ~ ~ i Dry products ,also benefit from the activity of SB3112'. -Liquid and dry products formulated in accordance with the present invention for grease traps, or other similar uses where- fatty -acid or grease needs to be degraded, may also contain in addition to surfactants, biocides, growth promoting non-toxic amounts of inorganic nutrients and micronutrients, certain activity enhancers, stabilizers, viscosifiers, enzymes, fillers, preservatives and the like. Table 1 lists examples of various components that a liquid formulation may contain in addition to SB3112 in accordance with the present invention.
Other inclusions in both liquid and-dry formulations are exemplified below:
(A) Other microorganisms may be selected from the group consisting of the genera Acinetobacter, Aspergillus, Azospirillum, Burkholderia, Bacillus, Ceriporiopsis, Enterobacter, Escherichia, Lactobacillus, Paenebacillus, Paracoccus, Pseudomonas, Rhodococcus, Syphingomonas, Streptococcus, Thiobacillus, Trichoderma, and Xanthomonas.
(B) Within Bacillus genera, the microorganism may be selected from the group consisting of Bacillus liclienifvrrreis, Bacillus subtilus, Bacillus amyoliquofaciehs, Bacillus laeuolacticus, and Bacillus pasteurii and Bacillus megaterium other than strain SB3112 and a combination thereof, The -. -- - preservative; is ,selected from, the group- consisting of 1,2-benzisothiazol-in-3-one;5-chloro-2-methyl-4-isothiazolin-3-one, 2-methyl-4-isothiazolin-3-one;
quaternium-15; phenol; sodium o-phenylphenate; o-phenylphenol; 6-acetoxy-2,4-dimethyl-m-dioxane; chlorhexidine;
Iris(hydroxymethyl)nitromethane; hexahydro-1,3,5-tris(2-hydroxyethyl)-s-triazine; p-hydroxybenzoic acid or its methyl, ethyl, propyl, or butyl esters;
The above and various other objects and advantages of the present invention are achieved by a biologically pure culture of a Gram-positive microorganism, ~ Bacillus megaterium, strain SB3112, having the identifying characteristics of ATCC
deposit number PTA 3142, and a composition comprising.the same. The deposit shall be maintained in viable condition at the ATCC during the entire term of the issued patent and shall be made available to any person or entity for non-commercial use without restriction, but in accordance with the provisions of the law governing the deposit.
It should be understood that unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and rnatenials similar or equivalent to those described herein can be used in the 1 S practice or testing of the present invention, the methods and materials described herein are preferred. Unless mentioned otherwise, the techniques employed or contemplated herein are standard methodologies well known to one of ordinary skill in the art. The materials, methods and examples are only exemplary and not limiting.
The term "biodegradation'; "biodegraded", or "biodegrading" as used herein means that the substrate is broken down, oxidized or degraded by tk~e microorganism And the term "activity enhancement" as used herein means that the biodegradation activity of the . microorganism is increased by the presence or addition of a particular component, said component being designated as "activity enhancer", or "activator"~.
By utilizing a Gram-positive, spore-forming organism that can oxidize fatty acids, one can obtain the advantages of an improved preserved liquid product, and stable, easy to use dry products. Unlike Grate-negative containing products, the preserved, spore-based Gram-positive containing product can contain preservatives and surfactants to aid in the biodegradation of fats, oils or grease, because the SUBSTITUTE SHEET (RULE 26) spores . are relatively resistant to tiiocides and surfactants. Furthermore, these products may also contain micronutrients promoting the growth of the microorganisms. Thus, a Gram positive product comprising lipase producing, fatty acid degrading, spore-forming microorganism in a preserved liquid formulation - offers various advantages -required for efficacious degradation of oil and grease.
r " . , ~ ~ i Dry products ,also benefit from the activity of SB3112'. -Liquid and dry products formulated in accordance with the present invention for grease traps, or other similar uses where- fatty -acid or grease needs to be degraded, may also contain in addition to surfactants, biocides, growth promoting non-toxic amounts of inorganic nutrients and micronutrients, certain activity enhancers, stabilizers, viscosifiers, enzymes, fillers, preservatives and the like. Table 1 lists examples of various components that a liquid formulation may contain in addition to SB3112 in accordance with the present invention.
Other inclusions in both liquid and-dry formulations are exemplified below:
(A) Other microorganisms may be selected from the group consisting of the genera Acinetobacter, Aspergillus, Azospirillum, Burkholderia, Bacillus, Ceriporiopsis, Enterobacter, Escherichia, Lactobacillus, Paenebacillus, Paracoccus, Pseudomonas, Rhodococcus, Syphingomonas, Streptococcus, Thiobacillus, Trichoderma, and Xanthomonas.
(B) Within Bacillus genera, the microorganism may be selected from the group consisting of Bacillus liclienifvrrreis, Bacillus subtilus, Bacillus amyoliquofaciehs, Bacillus laeuolacticus, and Bacillus pasteurii and Bacillus megaterium other than strain SB3112 and a combination thereof, The -. -- - preservative; is ,selected from, the group- consisting of 1,2-benzisothiazol-in-3-one;5-chloro-2-methyl-4-isothiazolin-3-one, 2-methyl-4-isothiazolin-3-one;
quaternium-15; phenol; sodium o-phenylphenate; o-phenylphenol; 6-acetoxy-2,4-dimethyl-m-dioxane; chlorhexidine;
Iris(hydroxymethyl)nitromethane; hexahydro-1,3,5-tris(2-hydroxyethyl)-s-triazine; p-hydroxybenzoic acid or its methyl, ethyl, propyl, or butyl esters;
SUBSTITUTE SHEET (RULE 26) benzoic, ascorbic, citric; or sorbic acid; imidazolidinyl urea; diazolidinyl urea; dirnethylol dimethylhydantoin; methylene . bistliiocyanate; 2-bromo-2-nitropropane-1,3-diol; 1,2-benzisothiazoline-3-one; methyl anthranilate and a mixture thereof.
S (C) The surfactant is selected from the group consisting of trideceth-3;t 3 mole ethylene oxide adduct of a linear, primary C12-14 alcohol; 7 mole ethylene oxide adduct of a linear, primary C12-14 alcohol;. sodium lauryl sulfate;
ammonium lauryl sulfate; dodecyl benzene sulfonic acid; ammonium Iauryl sulfate; sodium xylene sulfonate; sodium lauryl sulfate; cocamide IO diethanolamine; lauramine oxide; sodium alphasulfo methyl C12-18 ester and disodium alphasulfo CI2-I8 fatty acid salt; sodium dodecylbenzene sulfonate; alkyl polyglycoside; nonylphenoxypoly (ethyleneoxy) ethanol, branched; noriylphenoxypoly (ethyleneoxy) ethanol, branched; alkoxylated linear alcohol; blend of ethoxylates of linear, primary I2-14 carbon number 15 alcohol; linear alkyl sulfate; alkanolamide; octylphenoxypolyethoxyethanol absorbed on magnesium carbonate; sodium dodecylbenzene sulfonate and isopropyl alcohol; poe (6) tridecyl alcohol; poly (oxy-1,2-ethanediyl), alpha (nonylphenyl)-omega hydroxy, branched, or other surfactants known in the art, and a mixture thereof.
20 ~ (D) The nutrients are selected from the group consisting of disodium phosphate;
monosodium phosphate, sodium thiosulfate, ammonium sulfate, magnesium sulfate, manganese sulfate, ferrous sulfate, potassium phosphate, dipotassium phosphate, monocalcium phosphate, magnesium chloride,. .
calcium . chloride, chelated ~ iron, manganese . chloride, ~ sodium molybdate, 25 cobalt chloride, sodium nitrate, bakers yeast, yeast extract, soy peptone, skim milk powder, beef extract, sarsaponin, whey, gelatin, and urea and others known in the art, and a mixture thereof.
S (C) The surfactant is selected from the group consisting of trideceth-3;t 3 mole ethylene oxide adduct of a linear, primary C12-14 alcohol; 7 mole ethylene oxide adduct of a linear, primary C12-14 alcohol;. sodium lauryl sulfate;
ammonium lauryl sulfate; dodecyl benzene sulfonic acid; ammonium Iauryl sulfate; sodium xylene sulfonate; sodium lauryl sulfate; cocamide IO diethanolamine; lauramine oxide; sodium alphasulfo methyl C12-18 ester and disodium alphasulfo CI2-I8 fatty acid salt; sodium dodecylbenzene sulfonate; alkyl polyglycoside; nonylphenoxypoly (ethyleneoxy) ethanol, branched; noriylphenoxypoly (ethyleneoxy) ethanol, branched; alkoxylated linear alcohol; blend of ethoxylates of linear, primary I2-14 carbon number 15 alcohol; linear alkyl sulfate; alkanolamide; octylphenoxypolyethoxyethanol absorbed on magnesium carbonate; sodium dodecylbenzene sulfonate and isopropyl alcohol; poe (6) tridecyl alcohol; poly (oxy-1,2-ethanediyl), alpha (nonylphenyl)-omega hydroxy, branched, or other surfactants known in the art, and a mixture thereof.
20 ~ (D) The nutrients are selected from the group consisting of disodium phosphate;
monosodium phosphate, sodium thiosulfate, ammonium sulfate, magnesium sulfate, manganese sulfate, ferrous sulfate, potassium phosphate, dipotassium phosphate, monocalcium phosphate, magnesium chloride,. .
calcium . chloride, chelated ~ iron, manganese . chloride, ~ sodium molybdate, 25 cobalt chloride, sodium nitrate, bakers yeast, yeast extract, soy peptone, skim milk powder, beef extract, sarsaponin, whey, gelatin, and urea and others known in the art, and a mixture thereof.
SUBSTITUTE SHEET (RULE 26) (E) The dry fillers -are selected from the group consisting of starch, sodium chloride, glucose, sodium bicarbonate, corncobs, bran, and various ground grains.
(F) The enzymes are selected from the group consisting of protease, amylase, lipase andr cellulase and others known in the art, and a mixture thereof.
(G) The range ~(wlw) in which various components may be included in the dry pioduct composition in accordance with the present invention are as follows:
(i) SB3112 ranging from about 1 x 105 to.about . 5 x 10" CFU/g (ii) glycerol ranging from about 0 to about 10%
(iii) surfactant ranging from 0 to 10%
(iv) enzymes ranging from 0 to 1 (v) preservative ranging from 0 to 1.0%
(vi) nutrients ranging from about .0 to 20%
(vii) filler ranging from 0 to 95%
(H) The range (w/v) in which various components may be included in the liquid product composition in accordance with the present invention are as follows:
(i) SB3112 ranging from about 1 x 105 to about 5 x 10" CFU/ml (ii) glycerol ranging from about 0 to about 10%
(iii) surfactant ranging from 0 to 10°%
(iv) preservative ranging from 1 ppm to 1.0%
(v) color ranging from 0 to 1 (vi) fragrance ranging from 0 to L0%
(vii) viscosifier ranging from 0 to S%
(viii) nutrients ranging from 0 to 20%
(ix) enzymes ranging from 0 to 1% .
SUPERIORITY OF SB3112:
In order to show unexpectedly significant superiority of SB3112 over other Bacillus megaterium strains, the following side-by-side comparative study was performed using three other Bacillus megaterium strains having designations Strain 1, Strain 2 and Strain 3. Stearic acid (SA) and glycerol (G) were used as substrates.
(F) The enzymes are selected from the group consisting of protease, amylase, lipase andr cellulase and others known in the art, and a mixture thereof.
(G) The range ~(wlw) in which various components may be included in the dry pioduct composition in accordance with the present invention are as follows:
(i) SB3112 ranging from about 1 x 105 to.about . 5 x 10" CFU/g (ii) glycerol ranging from about 0 to about 10%
(iii) surfactant ranging from 0 to 10%
(iv) enzymes ranging from 0 to 1 (v) preservative ranging from 0 to 1.0%
(vi) nutrients ranging from about .0 to 20%
(vii) filler ranging from 0 to 95%
(H) The range (w/v) in which various components may be included in the liquid product composition in accordance with the present invention are as follows:
(i) SB3112 ranging from about 1 x 105 to about 5 x 10" CFU/ml (ii) glycerol ranging from about 0 to about 10%
(iii) surfactant ranging from 0 to 10°%
(iv) preservative ranging from 1 ppm to 1.0%
(v) color ranging from 0 to 1 (vi) fragrance ranging from 0 to L0%
(vii) viscosifier ranging from 0 to S%
(viii) nutrients ranging from 0 to 20%
(ix) enzymes ranging from 0 to 1% .
SUPERIORITY OF SB3112:
In order to show unexpectedly significant superiority of SB3112 over other Bacillus megaterium strains, the following side-by-side comparative study was performed using three other Bacillus megaterium strains having designations Strain 1, Strain 2 and Strain 3. Stearic acid (SA) and glycerol (G) were used as substrates.
SUBSTITUTE SHEET (RULE 26) Stearic acid degradation was determined by standard respirometric (oxygen uptake) measurement and sterile techniques were used in all studies.
A CES AER-200 respirometer unit was used. The S00 ml respirometer bottles contained SSCs minimal salt medium and 20 mM MOPS adjusted to pH 7.2.
S Respirometer bottles were prepared by adding SSC3 mineral salt media to each bottle. This media contained the following chemicals per. liter of water:
NH4Cl, 0.8g; MgS04~7Ha0, 0.2. ~g; CaCl2~2Ha0, 10 mg, FezNazEthylene diamine tetraacetic acid, 1S mg; KH2POa, 3.06 g; Fe~SOa~'IHzO, 28 ~~g; ZnS04~7Ha0, 140 p.g; MnS04~H20, 84 pg; CoCl2~6H20, 24 pg; CuSOa ~ SH20, 2S ug;
NaMo04~2H20, 24 pg. The buffer, 3-(N-morpholino) propane sulfonic acid (MOPS), was added 4.28 g per liter. The pH of the medium was adjusted to 7.0 prior to autoclaving. The bottles containing SSC3 media were autoclaved for two hours.
The target substrate, 1000 ppm stearic acid (SA), was added as indicated 1 S below. The SA was weighed and added individually into the appropriate reactor prior to autoclaving. Glycerol, 500 ppmv, was added to all bottles. Glycerol was frst diluted SO% v/v with deionized water to reduce the viscosity and 0.5 ml was added to each reactor prior to autoclaving. A pure culture of microorganism was used as inoculum. The optical density (OD) of an overnight culture in plate count broth (PCB) was determined in order to estimate the bacterial count. The target dose into each reactor was 1 x 1106 CFUImI based on the optical density measurement of the broth culture. Following inoculation, a caustic trap containing S rnl of 30% KOH to remove carbon dioxide was suspended in the bottle and the septum cap ~ was secured tightly. MacConkey streaks were performed .on ~ the 2S ~ inoculum prior to its use to ensure the absence of Gram-negative contamination.
MacConkey and standard methods agar (SMA) plate streaks were also performed on each bottle's contents at the end of the experiment (MacConkey agar plates were used to detect Gram negative contamination; SMA plates were used to test for pure cultures).
A CES AER-200 respirometer unit was used. The S00 ml respirometer bottles contained SSCs minimal salt medium and 20 mM MOPS adjusted to pH 7.2.
S Respirometer bottles were prepared by adding SSC3 mineral salt media to each bottle. This media contained the following chemicals per. liter of water:
NH4Cl, 0.8g; MgS04~7Ha0, 0.2. ~g; CaCl2~2Ha0, 10 mg, FezNazEthylene diamine tetraacetic acid, 1S mg; KH2POa, 3.06 g; Fe~SOa~'IHzO, 28 ~~g; ZnS04~7Ha0, 140 p.g; MnS04~H20, 84 pg; CoCl2~6H20, 24 pg; CuSOa ~ SH20, 2S ug;
NaMo04~2H20, 24 pg. The buffer, 3-(N-morpholino) propane sulfonic acid (MOPS), was added 4.28 g per liter. The pH of the medium was adjusted to 7.0 prior to autoclaving. The bottles containing SSC3 media were autoclaved for two hours.
The target substrate, 1000 ppm stearic acid (SA), was added as indicated 1 S below. The SA was weighed and added individually into the appropriate reactor prior to autoclaving. Glycerol, 500 ppmv, was added to all bottles. Glycerol was frst diluted SO% v/v with deionized water to reduce the viscosity and 0.5 ml was added to each reactor prior to autoclaving. A pure culture of microorganism was used as inoculum. The optical density (OD) of an overnight culture in plate count broth (PCB) was determined in order to estimate the bacterial count. The target dose into each reactor was 1 x 1106 CFUImI based on the optical density measurement of the broth culture. Following inoculation, a caustic trap containing S rnl of 30% KOH to remove carbon dioxide was suspended in the bottle and the septum cap ~ was secured tightly. MacConkey streaks were performed .on ~ the 2S ~ inoculum prior to its use to ensure the absence of Gram-negative contamination.
MacConkey and standard methods agar (SMA) plate streaks were also performed on each bottle's contents at the end of the experiment (MacConkey agar plates were used to detect Gram negative contamination; SMA plates were used to test for pure cultures).
SUBSTITUTE SHEET (RULE 26) The Treatments ~ imn duplicate> were as follows:
Glycerol + SB3112 Glycerol + Stearic acid + S$3112 Glycerol + Strain 1 ~ Glycerol + Stearic acid + Strain 1 Glycerol'+ Strain 2 ,Glycerol +~ Stearic acid + Strain Z
Glycerol + Strain 3 Glycerol + Stearic acid + Strain 3 .
Z O As indicated above, all cultures were tested with stearic acid plus glycerol. A
glycerol only control was also monitored for each individual strain.
The values for stearic acid degradation lag phase, oxygen uptake rate, and the total cumulative oxygen uptake for each strain are presented in Table 2.
These oxygen uptake rates were calculated using a three-point average and the maximum values are shown. The data clearly indicate surprising results, showing that is superior, demonstrating the highest activity on stearic acid by having the shortest lag phase, the fastest uptake rate and the greatest, cumulative oxygen uptake, compared to all other strains tested.
Etlicacv of SB3112 for Degrading various fatty acids Having determined the superiority of SB3112 compared to other Bacillus megaterium strains, the following study was performed to determine the efficacy Qf SB3112 for degrading laity acids. For this purpose, a number of fatty acids (99%
or greater purity) were obtained from commercial sources as listed below:
oleic acid, C-18:1 .
stearic acid, C-18 palmitic acid, C-16 valeric acid, C-5 butyric acid, C-4 acetic acid, C-2 SUBSTITUTE SHEET (RULE 26) Respirometric (oxygen uptake) activity was used to measure the ability of to degrade fatty acids.
A CES AER-200 respirometer unit was used to monitor oxygen uptake in 500 ml bottles maintained at 25°C. Each 500 ml bottle contained SSC3 minimal salt ~ medium and 20 mM MOPS buffer (pH adjusted to 7.2). Glycerol, 500 p~mv, was added to all bottles. Glycerol was first diluted 50% v/v with deionized water to reduce the viscosity and 0.5 ml was added to each reactor. High molecular weight fatty acids (C-16 and C-18) were weighed individually and~added to the appropriate bottles to a 1000 mg/L concentration except for oleic acid, which was tested at 200 mg/L. The low molecular weight fatty acids (C-2, C-4 and C-S) were flter sterilized using chemical resistant filters (0.2 Vim) and were added . to the appropriate autoclaved bottles to provide 1000 mg/L concentration.
A pure culture of SB3112 was used as inoculum The optical density (OD) of an overnight culture in plate count broth (PCB) was determined in order to 1 S estimate the bacterial count. The target dose into each reactor was 1 x 106 CFU/ml based on the optical density measurement of the broth culture. MacConkey streaks were performed on the inoculum at the time of use to detect Gram negative contamination. Sterile techniques were used in all experiments.
Treatments (numbers show triplicates as indicated below) 1,2,3. Glycerol only control 4,5,6. C-18 7,8,9. C-16 I 0, I 1,12. C-5 13,14,15. . . C-4 ~ ~ 16,17,18. ~ . C-2 19,20,21. C-18:1 (C represents carbon chain length) MacConkey and standard methods agar (SMA) agar streaks were performed on each bottle's contents at the end of the experiment. The MacConkey agar plates SUBSTITUTE SHEET (RULE 26) were used to detect Gram-negative contamination. The SMA plates were used to test for pure culture. No contamination was detected Oxygen uptake response attributed to the individual fatty acid was calculated by subtracting the glycerol data from the fatty acid plus glycerol data, in order to observe the amount of oxygen uptake attributed to the oxidation of the fatty acid alone. The pH of each reactor at the end'~of the experiment was within they range of 6.8 to 7.2. Standard Methods Agar (SMA) plates were streaked from each bottle. All bottles showed single colorry morphologies on SMA plates at the end of the experiment indicating that pure culture testing was achieved. Final MacConkey streaks of all bottles were negative (no Gram-negative contamination).
Table 3 shows the activity of SB3112 for degrading various fatty acids. The oxygen uptake rates were calculated using a three-point running average and the maximum value is shown in the Table. The results indicate that SB3112 degrades (oxidizes) all these fatty acids (oleic, stearic, palmitic, valeric, butyric and acetic acids). Tt may be noted that valeric and butyric acids are particularly odorous, and because SB3112 degrades these compounds, SB3112 may be useful in reducing odors attributable to these and similar volatile fatty acids.
Ef'ficncy of SB3112 to degrade ;~,rease The following study was performed to show the efficacy of SB3112 to degrade grease.
Respirorneter bottles were prepared by adding 250 mt of SSCs mineral salt media to each bottle. The pH of the medium was adjusted to 7.0 prior to autoclaving. The bottles containing SSC3 media were autoclaved for two hours.
. .. ~ - Waste kitchen ,grease was ~autoclaved~ separately to kill unwanted.
microorganisms..
Following autoclaving . of the grease, 2 ~ ml of the ~ hot sterile grease was added to each cooled 2S0 ml respirometer bottle. The inoculum for the respirometer bottles was an overnight broth culture (plate count broth) of each strain, or the 9002 blend of microorganisms. Each bottle was inoculated to contain initially ~ 3 x 105 CFUImI. Following inoculation a caustic trap containing 5 ml of 30% I~OH to SUBSTITUTE SHEET (RULE 26) remove carbon dioxide was suspended in the bottle and the septum cap was secured tightly.
To show superiority of SB3112, a comparative study was performed using two other inoculum samples. Thus, the three inoculum were as follows:
S 1. Bacillus megaterium strain SB3112.
2. Inoculum X002, representing a, blend of four spores (Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus pasteurii, and Bacillus laevolacticus) that produce extracellular enzymes lipase, amylase and protease. This mixture is known to affect the degradation of kitchen waste materials, but does not degrade fatty acids.
3. Inoculum SB3012 and SB3013 representing a blend of Gram negative microorganisms known for their ability to degrade fatty acids.
Following inoculation, the bottles containing the SSC3 media, waste kitchen grease, and the inoculum were placed into~the respirometer water bath (23°C).
Each bottle was then connected to a respirometer port. Consumption of oxygen was monitored continuously by the respirometer.
The results of this experiment are shown in Figure 1. The data show that the Gram-negative microorganism mixture (SB3012 and SB3013) began consuming oxygen almost immediately at a rate of 1.5 mg Oa/L/hr. The 9002 spore blend also began consuming oxygen quickly, but the rate of 1.2 mg OZ~L/hr was slightly lower than SB3012 and SB3013. Strain SB3112 had a lag of SO hr before oxygen consumption began at the rate ofØ5 mg OZ/L/hr. At about 180 hr, began a rapid rise in oxygen consumption surpassing both the spore blend 9002 and . -, wthe Gram-negative blend. The rate of oxygen.consurnption by SB3112 after 180,hr.
was 4.4 mg Oa/L/hr compared ~to 0.7 and 0.8 mg Oz/L/hr for SB3012 plus SB3013 and 9002, respectively, during the same time period. Uninoculated controls did not show oxygen uptake (data not shown).
Effect of Glycerol on Biode~radin~ Activity of SB3112 As shown by the following study, it was discovered that the presence of glycerol enhances the biodegrading activity of SB3112.
SUBSTITUTE SHEET (RULE 26) SB3112 culture was obtained from retained slant and plated on agar by standard method. A single colony was picked then ~grovim ovenaight in standard plate count broth Based on the measured optical density, 1 x 106 CFU/ml (target) of SB3112 was added to each reactor. Each substrate (stearic acid and glycerol) S was added to the reaction vessel prior to autoclaving. The reactor size was S00 rnl containing SSCa autoclaved medium.
Treatments (in duplicate) l, 2. Stearic acid, 0.2%, 3, 4. Stearic acid, 0.2% + 0.1 % glycerol 5, 6. Glycerol, 0.1 %.
Final MacConkey streaks were performed to detect ~ any Gram-negative contamination. No contamination was found. The results are shown in Figure 2.
The data clearly indicate that fatty acid degrading activity of SB3112 is significantly enhanced by the presence or addition of glycerol.
1S Comparative Study of Glycerol Level on Fatty Acid Desradation by Various Microorganisms.
Comparisons of biodegrading activity of various microorganisms as a function of various glycerol levels were performed to show the effect of glycerol as an activator. Long chain fatty acids (LCFA) were used as target substrates and an aqueous serial dilution of glycerol (G) was used to determine the effective concentration range of glycerol as an activator. Measurement of LCFA
degradation was determined by the respirometric oxygen uptake and the rate of biodegrading' activity of a microorganism was indicated by the rate of oxygen uptake, the . ~ , methods and materials v employed being. similar to . those described for various :.
2S experiments herein above. However, the specifics of various studies have been noted for each study set forth below. The Bacillus megaterium strains were obtained from ATCC.
Common methods for each of the following experiments:
A CES AER-200 respiromeriy system was used. The S00 ml respirometer bottles (reactors) contained SSC3 minimal salts medium and 20 mM MOPS adjusted SUBSTITUTE SHEET (RULE 26) to a pH of 7.2. The target' substrates, LCFA, were , added as described in the individual experimental methods below Glycerol was diluted 50% (w/w) with de-ionized water to reduce the viscosity and then pipetted (v/v) into the appropriate reactor. Reactors were ~ sterilized by autoclaving and sterile techniques were used in each experime~.t. .
MacConkey streaks were performed on each inoculum at time of use to detect Gram negative contamination.: ~ MacConkey and standard methods agar (SMA) agar plate streaks were performed on each bottle's contents at the end of the experiment.
The SMA plates were used to test for pure cultures.
Specific methods employed in each of the following four studies are detailed below.
STUDY 1: Glycernl Ladder This study was undertaken to determine the effective concentration of glycerol for the enhancement of degradation of a LCFA as measured by the increased rate of oxygen uptake.
Specific methods: Palmitic acid was added to each reactor at 500 mg per 500 ml (w/v). Diluted glycerol was added by pipette (v/v) as described in Table 4.
A pure culture of SB3112 was used as the inoculum. The optical density (OD) of an overnight culture in plate count broth (PCB) was determined in order to estimate the bacterial Count. The target dose into each reactor was based on the optical density measurement of the broth cultur$.
Figure 3 shows the averaged data ~.s a function of serial dilution of glycerol using palmitic acid as a substrate. All concentrations of glycerol (IO ppm to ppm) showed a significant enhancement of the degradation of palmitic acid.. .
Without the ~ addition of glycerol, ~ no significa~it oxygen uptake was observed indicating no degradation of paltnitic acid.
Figure 4 shows the oxygen uptake as a function of serial addition of glycerol in the absence of palmitic acid. These values were subtracted from the glycerol plus palnnitic acid data shown in Figure 3 (note the differences in the Y axis values between Figures 3 and 4). Figure S shows the result of the subtraction and thus SUBSTITUTE SHEET (RULE 26) shows only the oxygen uptake attributed to the degradation of palrnitic acid by sBS 1 ~ i.
It is clear from the data presented that glycerol at a concentration as low as ppm will stimulate the degradation of the LCFA,, palmitic acid. It is important to 5 ~ note that glycerol at a concentration ~of 10 ppm does not produce a significant increase in bacterial cell number STUDY 2: Other Bacillus megaterlum This study was undertaken to compare the 'effect of glycerol on the degradation of the LCFA, Palmitic acid, using various Bacillus megaterium strains.
Glycerol + SB3112 Glycerol + Stearic acid + S$3112 Glycerol + Strain 1 ~ Glycerol + Stearic acid + Strain 1 Glycerol'+ Strain 2 ,Glycerol +~ Stearic acid + Strain Z
Glycerol + Strain 3 Glycerol + Stearic acid + Strain 3 .
Z O As indicated above, all cultures were tested with stearic acid plus glycerol. A
glycerol only control was also monitored for each individual strain.
The values for stearic acid degradation lag phase, oxygen uptake rate, and the total cumulative oxygen uptake for each strain are presented in Table 2.
These oxygen uptake rates were calculated using a three-point average and the maximum values are shown. The data clearly indicate surprising results, showing that is superior, demonstrating the highest activity on stearic acid by having the shortest lag phase, the fastest uptake rate and the greatest, cumulative oxygen uptake, compared to all other strains tested.
Etlicacv of SB3112 for Degrading various fatty acids Having determined the superiority of SB3112 compared to other Bacillus megaterium strains, the following study was performed to determine the efficacy Qf SB3112 for degrading laity acids. For this purpose, a number of fatty acids (99%
or greater purity) were obtained from commercial sources as listed below:
oleic acid, C-18:1 .
stearic acid, C-18 palmitic acid, C-16 valeric acid, C-5 butyric acid, C-4 acetic acid, C-2 SUBSTITUTE SHEET (RULE 26) Respirometric (oxygen uptake) activity was used to measure the ability of to degrade fatty acids.
A CES AER-200 respirometer unit was used to monitor oxygen uptake in 500 ml bottles maintained at 25°C. Each 500 ml bottle contained SSC3 minimal salt ~ medium and 20 mM MOPS buffer (pH adjusted to 7.2). Glycerol, 500 p~mv, was added to all bottles. Glycerol was first diluted 50% v/v with deionized water to reduce the viscosity and 0.5 ml was added to each reactor. High molecular weight fatty acids (C-16 and C-18) were weighed individually and~added to the appropriate bottles to a 1000 mg/L concentration except for oleic acid, which was tested at 200 mg/L. The low molecular weight fatty acids (C-2, C-4 and C-S) were flter sterilized using chemical resistant filters (0.2 Vim) and were added . to the appropriate autoclaved bottles to provide 1000 mg/L concentration.
A pure culture of SB3112 was used as inoculum The optical density (OD) of an overnight culture in plate count broth (PCB) was determined in order to 1 S estimate the bacterial count. The target dose into each reactor was 1 x 106 CFU/ml based on the optical density measurement of the broth culture. MacConkey streaks were performed on the inoculum at the time of use to detect Gram negative contamination. Sterile techniques were used in all experiments.
Treatments (numbers show triplicates as indicated below) 1,2,3. Glycerol only control 4,5,6. C-18 7,8,9. C-16 I 0, I 1,12. C-5 13,14,15. . . C-4 ~ ~ 16,17,18. ~ . C-2 19,20,21. C-18:1 (C represents carbon chain length) MacConkey and standard methods agar (SMA) agar streaks were performed on each bottle's contents at the end of the experiment. The MacConkey agar plates SUBSTITUTE SHEET (RULE 26) were used to detect Gram-negative contamination. The SMA plates were used to test for pure culture. No contamination was detected Oxygen uptake response attributed to the individual fatty acid was calculated by subtracting the glycerol data from the fatty acid plus glycerol data, in order to observe the amount of oxygen uptake attributed to the oxidation of the fatty acid alone. The pH of each reactor at the end'~of the experiment was within they range of 6.8 to 7.2. Standard Methods Agar (SMA) plates were streaked from each bottle. All bottles showed single colorry morphologies on SMA plates at the end of the experiment indicating that pure culture testing was achieved. Final MacConkey streaks of all bottles were negative (no Gram-negative contamination).
Table 3 shows the activity of SB3112 for degrading various fatty acids. The oxygen uptake rates were calculated using a three-point running average and the maximum value is shown in the Table. The results indicate that SB3112 degrades (oxidizes) all these fatty acids (oleic, stearic, palmitic, valeric, butyric and acetic acids). Tt may be noted that valeric and butyric acids are particularly odorous, and because SB3112 degrades these compounds, SB3112 may be useful in reducing odors attributable to these and similar volatile fatty acids.
Ef'ficncy of SB3112 to degrade ;~,rease The following study was performed to show the efficacy of SB3112 to degrade grease.
Respirorneter bottles were prepared by adding 250 mt of SSCs mineral salt media to each bottle. The pH of the medium was adjusted to 7.0 prior to autoclaving. The bottles containing SSC3 media were autoclaved for two hours.
. .. ~ - Waste kitchen ,grease was ~autoclaved~ separately to kill unwanted.
microorganisms..
Following autoclaving . of the grease, 2 ~ ml of the ~ hot sterile grease was added to each cooled 2S0 ml respirometer bottle. The inoculum for the respirometer bottles was an overnight broth culture (plate count broth) of each strain, or the 9002 blend of microorganisms. Each bottle was inoculated to contain initially ~ 3 x 105 CFUImI. Following inoculation a caustic trap containing 5 ml of 30% I~OH to SUBSTITUTE SHEET (RULE 26) remove carbon dioxide was suspended in the bottle and the septum cap was secured tightly.
To show superiority of SB3112, a comparative study was performed using two other inoculum samples. Thus, the three inoculum were as follows:
S 1. Bacillus megaterium strain SB3112.
2. Inoculum X002, representing a, blend of four spores (Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus pasteurii, and Bacillus laevolacticus) that produce extracellular enzymes lipase, amylase and protease. This mixture is known to affect the degradation of kitchen waste materials, but does not degrade fatty acids.
3. Inoculum SB3012 and SB3013 representing a blend of Gram negative microorganisms known for their ability to degrade fatty acids.
Following inoculation, the bottles containing the SSC3 media, waste kitchen grease, and the inoculum were placed into~the respirometer water bath (23°C).
Each bottle was then connected to a respirometer port. Consumption of oxygen was monitored continuously by the respirometer.
The results of this experiment are shown in Figure 1. The data show that the Gram-negative microorganism mixture (SB3012 and SB3013) began consuming oxygen almost immediately at a rate of 1.5 mg Oa/L/hr. The 9002 spore blend also began consuming oxygen quickly, but the rate of 1.2 mg OZ~L/hr was slightly lower than SB3012 and SB3013. Strain SB3112 had a lag of SO hr before oxygen consumption began at the rate ofØ5 mg OZ/L/hr. At about 180 hr, began a rapid rise in oxygen consumption surpassing both the spore blend 9002 and . -, wthe Gram-negative blend. The rate of oxygen.consurnption by SB3112 after 180,hr.
was 4.4 mg Oa/L/hr compared ~to 0.7 and 0.8 mg Oz/L/hr for SB3012 plus SB3013 and 9002, respectively, during the same time period. Uninoculated controls did not show oxygen uptake (data not shown).
Effect of Glycerol on Biode~radin~ Activity of SB3112 As shown by the following study, it was discovered that the presence of glycerol enhances the biodegrading activity of SB3112.
SUBSTITUTE SHEET (RULE 26) SB3112 culture was obtained from retained slant and plated on agar by standard method. A single colony was picked then ~grovim ovenaight in standard plate count broth Based on the measured optical density, 1 x 106 CFU/ml (target) of SB3112 was added to each reactor. Each substrate (stearic acid and glycerol) S was added to the reaction vessel prior to autoclaving. The reactor size was S00 rnl containing SSCa autoclaved medium.
Treatments (in duplicate) l, 2. Stearic acid, 0.2%, 3, 4. Stearic acid, 0.2% + 0.1 % glycerol 5, 6. Glycerol, 0.1 %.
Final MacConkey streaks were performed to detect ~ any Gram-negative contamination. No contamination was found. The results are shown in Figure 2.
The data clearly indicate that fatty acid degrading activity of SB3112 is significantly enhanced by the presence or addition of glycerol.
1S Comparative Study of Glycerol Level on Fatty Acid Desradation by Various Microorganisms.
Comparisons of biodegrading activity of various microorganisms as a function of various glycerol levels were performed to show the effect of glycerol as an activator. Long chain fatty acids (LCFA) were used as target substrates and an aqueous serial dilution of glycerol (G) was used to determine the effective concentration range of glycerol as an activator. Measurement of LCFA
degradation was determined by the respirometric oxygen uptake and the rate of biodegrading' activity of a microorganism was indicated by the rate of oxygen uptake, the . ~ , methods and materials v employed being. similar to . those described for various :.
2S experiments herein above. However, the specifics of various studies have been noted for each study set forth below. The Bacillus megaterium strains were obtained from ATCC.
Common methods for each of the following experiments:
A CES AER-200 respiromeriy system was used. The S00 ml respirometer bottles (reactors) contained SSC3 minimal salts medium and 20 mM MOPS adjusted SUBSTITUTE SHEET (RULE 26) to a pH of 7.2. The target' substrates, LCFA, were , added as described in the individual experimental methods below Glycerol was diluted 50% (w/w) with de-ionized water to reduce the viscosity and then pipetted (v/v) into the appropriate reactor. Reactors were ~ sterilized by autoclaving and sterile techniques were used in each experime~.t. .
MacConkey streaks were performed on each inoculum at time of use to detect Gram negative contamination.: ~ MacConkey and standard methods agar (SMA) agar plate streaks were performed on each bottle's contents at the end of the experiment.
The SMA plates were used to test for pure cultures.
Specific methods employed in each of the following four studies are detailed below.
STUDY 1: Glycernl Ladder This study was undertaken to determine the effective concentration of glycerol for the enhancement of degradation of a LCFA as measured by the increased rate of oxygen uptake.
Specific methods: Palmitic acid was added to each reactor at 500 mg per 500 ml (w/v). Diluted glycerol was added by pipette (v/v) as described in Table 4.
A pure culture of SB3112 was used as the inoculum. The optical density (OD) of an overnight culture in plate count broth (PCB) was determined in order to estimate the bacterial Count. The target dose into each reactor was based on the optical density measurement of the broth cultur$.
Figure 3 shows the averaged data ~.s a function of serial dilution of glycerol using palmitic acid as a substrate. All concentrations of glycerol (IO ppm to ppm) showed a significant enhancement of the degradation of palmitic acid.. .
Without the ~ addition of glycerol, ~ no significa~it oxygen uptake was observed indicating no degradation of paltnitic acid.
Figure 4 shows the oxygen uptake as a function of serial addition of glycerol in the absence of palmitic acid. These values were subtracted from the glycerol plus palnnitic acid data shown in Figure 3 (note the differences in the Y axis values between Figures 3 and 4). Figure S shows the result of the subtraction and thus SUBSTITUTE SHEET (RULE 26) shows only the oxygen uptake attributed to the degradation of palrnitic acid by sBS 1 ~ i.
It is clear from the data presented that glycerol at a concentration as low as ppm will stimulate the degradation of the LCFA,, palmitic acid. It is important to 5 ~ note that glycerol at a concentration ~of 10 ppm does not produce a significant increase in bacterial cell number STUDY 2: Other Bacillus megaterlum This study was undertaken to compare the 'effect of glycerol on the degradation of the LCFA, Palmitic acid, using various Bacillus megaterium strains.
10 Specific Methods:
Palmitic acid was added 500 mg per 500 ml volume reactor (1000 ppmv). Glycerol was added 50 mg per 500 m1 reactor (100 ppmv). Pure culture of each strain was used as the inoculum. The optical density (OD) of an overnight culture in plate count broth (PCB) was determined in order to estimate the bacterial count. The target dose into each reactor was based on the optical density measurement of the broth culture. The treatments for each reactor are shown in Table 5.
Figure 6 shows the averaged oxygen uptake data for various Bacillus megaterium strains for the degradation of palmitic acid in the presence of glycerol.
Strain SB3112 initiated oxygen uptake, sooner, produced a high rate of uptake and showed a higher total cumulative oxygen uptake compared to Strain 3. Without the presence of glycerol there was no degradation of palinitic acid in this study as indicated by the absence of oxygen uptake.
'fhe .ATCC strains 2 and 1 showed no oxygen uptake on palmitic acid alone or on palrnitic acid with the addition of'glycerol. In fact, the degradation of glycerol was inhibited in the presence of PA The amount of oxygen uptake expected from 50 mg of glycerol should be approximately 70 mg.
SUBSTITUTE SHEET (RULE 26) In short, SB3112 degraded palmitic acid to a. much greater extent than strain 3 with the addition of glycerol. ATCC strains 2 and 1 did not degrade palmitic acid with or without glycerol Study 3: Other species of bacteria ~ This study was undertaken to .compare a commercially availablei product containing a Gxam-positive mixture of 5 to 7 strains with the fully formulated product of the present invention containing SB3112. All strains were in spore form including the SB3112.
Specific methods are as follows:
The fully formulated products were diluted into the appropriate reactor to achieve an initial spore count in the reactors of about 5.4 x~ 10~ CFU/ml. The treatments for each reactor are shown in Table 6.
Figure 7 shows the response to the addition of glycerol to LCFA substrate by the two products. The data indicate that whereas SB3112 containing product readily consumed the palmitic acid, the commercially available mixture . did not significantly degrade the palinitic acid. All products and reactors were free of Gram-negative contamination as indicated by no growth when streaked on MacConkey agar.
The data indicate that the product containing SB3112 degraded the paltnitic acid whereas the commercially available product did not significantly degrade the palmitic acid. Both products were able to degrade glycerol in the presence of the.
LCFA
Study 4: Effect of Glycerol on Biodegradation . ~ ~ ~ _ : 'This study was. undertaken to demonstrate the effect of glycerol..on, .
oxidation of fatty acids of various chain lengths.
Specific methods are as follows: Respirornetry was used to test each of a number of carboxylic acids ranging in chain length from 2 (acetic acid) to 18 (oleic acid). 1000 mg/L of each acid was tested except oleic which was tested at 200 mg/L. Glycerol was used at a concentration of 500 ppm The study was conducted for a period of 10 days.
SUBSTITUTE SHEET (RULE 26) Table 7 shows the utilization of long chain fatty acids by SB3112 spores in the presence of glycerol (glycerol uptake subtracted out). When compared with the data in the absence of glycerol the results obtained in the presence of glycerol demonstrate that glycerol significantly enhances degradation of long-chain (fatty) ~ acids.
Various embodiments of the present invention have_now been described in accordance with the objects. and advantages noted above. It will be appreciated that these examples are merely illustrative and not limiting of the invention. Of course, many variations and modifications of the present invention. will be apparent and suggested to those of ordinary skill in the art and all such variations and modifications are included within the purview and scope of the_claims.
SUBSTITUTE SHEET (RULE 26) Table 1. Example Components in a Liquid Composition' according to present invention.
i...,,y t~ t".. 7: ,: ~.1:"i,~
,. i, ~'~.n t .:~, a"..,~ AII10 utP.~r''~~p~'.' . 11 ~'Lti .'ref ~ " ,. .u rl,), l,ar~j,,v '~ .:.,,t,~.,:,', ,,a~[. .1, t",t,t ~y~Y:;',~~ ej. ~~
~;~r'k Compori;entH,,s ~
~, ..~e.,,~s,,~, , ~~ ,l,;j,1~;:~,,~Qu~hty Range , F,, Surfactants Linear Alkylbenzene O.S-S% ~ ~ 1-2%
. ~
Sul honic Acid ' Ethoxylated alcohol 0.S-S% O.S-1:S%
.
Preservatives . .
1,2-benzisothiazolin-3-one0.025-0.2%~ 0.045-0.1%
Methyl anthranilate 20-40 ppm 2S-3S ppm Microorganisms Bacillus licheniformas1 x 106 to S x 106 to 1 x 109 CFU/ml1 x 108 CFU/ml Bacillus amyoliquefaciens1 x 10 to S x 10" to 1 x 109 CFU/ml1 x 108 CFU/ml Bacillus pasteurii 1 x 10 to S x 10 to I X 109 CFU/ml1 x 108 CFU/ml Bacillus laevolacticus1 x 10 to S x 10" to 1 x 109 CFU/ml1 x 108 CFU/ml Activator Glycerol 0 -10% 0.001%-4%
Other ingredients Tetrasodium 0.1-1.0% 0.2-O.S%
ethylenediamine tetraacetate stabilizer' Fragrance 0 - O.S% 0.2-0.3%
Other spore forming 0 to 1 x 10 5 x 106 to 1 x microor anisms CFU/ml 1 O8 CFU/ml Viscosifier . 0 - O..S% 0.2-0.3%
S
SUBSTITUTE SHEET (RULE 26) Table 2. Stearic acid utilisation by various Bacillus tnegaterium strains.
' ' ' ~ P a ~ ~ ~ ~~ Stiain:!~ ~ ,y.Strain~:,~, meter 'o ~ v B3112n ,, 2 ,st ,~ 4''i y; .;',; ~ ~ ' Strait ~" ~ 3 ,:;
, ~ y ,,: , ; ,, n,S4")S ~r.tn.ky.y<iiSSc .e~lr;.~, .,!rtw., ., J. .n ,_,l,uu?if"n.Untl.e~..<v'.!..'':n:N~y~~un.l~;fln i.i~'"Ul ,,,..,.,411 ....a a,.,. , ~,'l Lag phase 20 72 60 100 nours Oxygen uptake~ 1.9 ~ 1.1 0. 8 ~ 0.9 ~
rate (ri ) Total 180 140 110 90 .
cumulative !
oxygen uptake ' , Each reactor volume was 0.5 liters.
Table 3: Degradation of fatty acids by Bacillus megaterium strain SB3112.
Fatty acid Oxygen Total , , , ' used ,~r uptake ' curiiulative ' ' .; rate'' ' , ' ox3%gen . intake'' (mg/hr)' '~'~
~
~ i > c rtn> ,a ' pf ,rr Ip J
a , "9, .4 . ~ ~:,,A "..n, ltll , 1 ,.:~
.
oleic acid, 4.9 185 C-18:1 stearic acid,1.75 230 palmitic 2.87 480 acid, valeric acid,2.37 342 butyric acid,1.96 308 acetic acid,1.7 247 :
C-2 . . . :. .~
:..-:-.:
v .
Each reactor volume was 0.5 liters.
Oleic acid was 100 mg per reactor and all other substrates were 500 m er reactor.
SUBSTITUTE SHEET (RULE 26) Table 4. Glycerol Ladder Treatments.
ReactorGlycerol; Palmih~ acid volume of d~luted,glycerol, ,, ppm r ~ 3l W .",t t ' '..
r , k d ,f v v.r. 5 , a n . v .
t ~ i . . , ., , f , p ,.~""~. ..~ er 500 ml , 500 rn ~ er reactor F .' , ' reactor ~~ ' , , 1,2,3. ;~to glycerolyes ~, t 0 4,5. 10 ppm yes ~ 10 pl ~ .
6,7. 25 ppm No ~ 25 p,l , 8,9,10.25 ppm yes 25 ul 11,12. 100 ppm No 100 pl 13,14,15.100 ppm yes 100 ul 16,17. 200 ppm No 0.20 ml 18,19,20.200 ppm yes 0.20 ml ~
Table 5. Other Bacillus megaterium Treatments.
h Reactor Glyceroh ppm Strain o ) .'.'.;, ; 5 S , ,~.~,.y ., ,~.~3 a ~ ' t t ' 1,3. No glycerol SB3112 4,5. 100 ppm SB3112 6,7. No glycerol Strain 2 8,9. 100 ppm Strain 2 14,15. No glycerol Strain 1 16,17. ~ 00 ppm ~ Strain 1 .
22,23. No glycerol Strain 3 ~ ~ ~
24. ~ 100 ppm Strain 3 SUBSTITUTE SHEET (RULE 26) Table 6. Other species of bacteria Treatments Reactor ' Palmitic product , # ~ r ayd ~as Y Y ''' '" ~ ~ f , , ~.t,i., hr.;:.,y,.1, 1,.,1 n.l'.... 1"l..,n.f,t ,1).... ~ ' :..tp..,in . I.
rielv ..
1,2,3 Yes Commercially available roduct 4,5,6 Yes SB3112 roduct 7,8 No Commercially available roduct 9,10 No SB3112 roduct Table 7. Growth rates of SB3112 on various carboxylic acids.
,_ iy s - , ,-~s~-, ~~ ~ a ~'~ ~
; C ' . Ye W G
k - , :.
With Gl Without cerol .~ Gl cerol f x ~,.
Fa O . . eri ~ Total " ' O '.~f Y ~
~ acid ii take cumulative ~ en ~'otal ' ' ' ~ ~ a ~:
ttY ?~'g p ?~'g . , . . , i ~ f t,., ' ~~ rate m . , '':~u f ~
/L%hr ~ r~'' o ' ~ ' take cuinulatne en a ~ take , ;',~
' g :~
' . p .
~3 , g , p, , f , , , '' , : ~ ',r " . late o eti a ' ' , , a - a take ~m~ ';~ t ~ Yg '~ ~t .' p ' ~ ~ '' ~
, , . . . ;;, ,..:.x ,:; . , . ..
~~ , t ~ e ~ ' a ~, "
I, v , .~, , r ~ , m ;,,;
, /L/hr ~
, ~~ ~
J. m ; /L
, .
;
. ., . , .
, . .
, ..
, oleic acid9.8 370 0 0 , C-18:1 stearic 3.5 460 0 0 acid, palmitic . , 5.7 . 960. . 0 0 acid, C-16 ~ ... ~:.:.;:., .... , . - ..-- -.
Y .v _ . ~
Glycerol uptake subtracted out Oleic acid was 200 mg per reactor while stearic and palmitic were 500 mg per 500 ml reactor.
"~~No uptake was measured for the above fatty acids prior to 150 hours of treatment without the presence of glycerol. -.
SUBSTITUTE SHEET (RULE 26)
Palmitic acid was added 500 mg per 500 ml volume reactor (1000 ppmv). Glycerol was added 50 mg per 500 m1 reactor (100 ppmv). Pure culture of each strain was used as the inoculum. The optical density (OD) of an overnight culture in plate count broth (PCB) was determined in order to estimate the bacterial count. The target dose into each reactor was based on the optical density measurement of the broth culture. The treatments for each reactor are shown in Table 5.
Figure 6 shows the averaged oxygen uptake data for various Bacillus megaterium strains for the degradation of palmitic acid in the presence of glycerol.
Strain SB3112 initiated oxygen uptake, sooner, produced a high rate of uptake and showed a higher total cumulative oxygen uptake compared to Strain 3. Without the presence of glycerol there was no degradation of palinitic acid in this study as indicated by the absence of oxygen uptake.
'fhe .ATCC strains 2 and 1 showed no oxygen uptake on palmitic acid alone or on palrnitic acid with the addition of'glycerol. In fact, the degradation of glycerol was inhibited in the presence of PA The amount of oxygen uptake expected from 50 mg of glycerol should be approximately 70 mg.
SUBSTITUTE SHEET (RULE 26) In short, SB3112 degraded palmitic acid to a. much greater extent than strain 3 with the addition of glycerol. ATCC strains 2 and 1 did not degrade palmitic acid with or without glycerol Study 3: Other species of bacteria ~ This study was undertaken to .compare a commercially availablei product containing a Gxam-positive mixture of 5 to 7 strains with the fully formulated product of the present invention containing SB3112. All strains were in spore form including the SB3112.
Specific methods are as follows:
The fully formulated products were diluted into the appropriate reactor to achieve an initial spore count in the reactors of about 5.4 x~ 10~ CFU/ml. The treatments for each reactor are shown in Table 6.
Figure 7 shows the response to the addition of glycerol to LCFA substrate by the two products. The data indicate that whereas SB3112 containing product readily consumed the palmitic acid, the commercially available mixture . did not significantly degrade the palinitic acid. All products and reactors were free of Gram-negative contamination as indicated by no growth when streaked on MacConkey agar.
The data indicate that the product containing SB3112 degraded the paltnitic acid whereas the commercially available product did not significantly degrade the palmitic acid. Both products were able to degrade glycerol in the presence of the.
LCFA
Study 4: Effect of Glycerol on Biodegradation . ~ ~ ~ _ : 'This study was. undertaken to demonstrate the effect of glycerol..on, .
oxidation of fatty acids of various chain lengths.
Specific methods are as follows: Respirornetry was used to test each of a number of carboxylic acids ranging in chain length from 2 (acetic acid) to 18 (oleic acid). 1000 mg/L of each acid was tested except oleic which was tested at 200 mg/L. Glycerol was used at a concentration of 500 ppm The study was conducted for a period of 10 days.
SUBSTITUTE SHEET (RULE 26) Table 7 shows the utilization of long chain fatty acids by SB3112 spores in the presence of glycerol (glycerol uptake subtracted out). When compared with the data in the absence of glycerol the results obtained in the presence of glycerol demonstrate that glycerol significantly enhances degradation of long-chain (fatty) ~ acids.
Various embodiments of the present invention have_now been described in accordance with the objects. and advantages noted above. It will be appreciated that these examples are merely illustrative and not limiting of the invention. Of course, many variations and modifications of the present invention. will be apparent and suggested to those of ordinary skill in the art and all such variations and modifications are included within the purview and scope of the_claims.
SUBSTITUTE SHEET (RULE 26) Table 1. Example Components in a Liquid Composition' according to present invention.
i...,,y t~ t".. 7: ,: ~.1:"i,~
,. i, ~'~.n t .:~, a"..,~ AII10 utP.~r''~~p~'.' . 11 ~'Lti .'ref ~ " ,. .u rl,), l,ar~j,,v '~ .:.,,t,~.,:,', ,,a~[. .1, t",t,t ~y~Y:;',~~ ej. ~~
~;~r'k Compori;entH,,s ~
~, ..~e.,,~s,,~, , ~~ ,l,;j,1~;:~,,~Qu~hty Range , F,, Surfactants Linear Alkylbenzene O.S-S% ~ ~ 1-2%
. ~
Sul honic Acid ' Ethoxylated alcohol 0.S-S% O.S-1:S%
.
Preservatives . .
1,2-benzisothiazolin-3-one0.025-0.2%~ 0.045-0.1%
Methyl anthranilate 20-40 ppm 2S-3S ppm Microorganisms Bacillus licheniformas1 x 106 to S x 106 to 1 x 109 CFU/ml1 x 108 CFU/ml Bacillus amyoliquefaciens1 x 10 to S x 10" to 1 x 109 CFU/ml1 x 108 CFU/ml Bacillus pasteurii 1 x 10 to S x 10 to I X 109 CFU/ml1 x 108 CFU/ml Bacillus laevolacticus1 x 10 to S x 10" to 1 x 109 CFU/ml1 x 108 CFU/ml Activator Glycerol 0 -10% 0.001%-4%
Other ingredients Tetrasodium 0.1-1.0% 0.2-O.S%
ethylenediamine tetraacetate stabilizer' Fragrance 0 - O.S% 0.2-0.3%
Other spore forming 0 to 1 x 10 5 x 106 to 1 x microor anisms CFU/ml 1 O8 CFU/ml Viscosifier . 0 - O..S% 0.2-0.3%
S
SUBSTITUTE SHEET (RULE 26) Table 2. Stearic acid utilisation by various Bacillus tnegaterium strains.
' ' ' ~ P a ~ ~ ~ ~~ Stiain:!~ ~ ,y.Strain~:,~, meter 'o ~ v B3112n ,, 2 ,st ,~ 4''i y; .;',; ~ ~ ' Strait ~" ~ 3 ,:;
, ~ y ,,: , ; ,, n,S4")S ~r.tn.ky.y<iiSSc .e~lr;.~, .,!rtw., ., J. .n ,_,l,uu?if"n.Untl.e~..<v'.!..'':n:N~y~~un.l~;fln i.i~'"Ul ,,,..,.,411 ....a a,.,. , ~,'l Lag phase 20 72 60 100 nours Oxygen uptake~ 1.9 ~ 1.1 0. 8 ~ 0.9 ~
rate (ri ) Total 180 140 110 90 .
cumulative !
oxygen uptake ' , Each reactor volume was 0.5 liters.
Table 3: Degradation of fatty acids by Bacillus megaterium strain SB3112.
Fatty acid Oxygen Total , , , ' used ,~r uptake ' curiiulative ' ' .; rate'' ' , ' ox3%gen . intake'' (mg/hr)' '~'~
~
~ i > c rtn> ,a ' pf ,rr Ip J
a , "9, .4 . ~ ~:,,A "..n, ltll , 1 ,.:~
.
oleic acid, 4.9 185 C-18:1 stearic acid,1.75 230 palmitic 2.87 480 acid, valeric acid,2.37 342 butyric acid,1.96 308 acetic acid,1.7 247 :
C-2 . . . :. .~
:..-:-.:
v .
Each reactor volume was 0.5 liters.
Oleic acid was 100 mg per reactor and all other substrates were 500 m er reactor.
SUBSTITUTE SHEET (RULE 26) Table 4. Glycerol Ladder Treatments.
ReactorGlycerol; Palmih~ acid volume of d~luted,glycerol, ,, ppm r ~ 3l W .",t t ' '..
r , k d ,f v v.r. 5 , a n . v .
t ~ i . . , ., , f , p ,.~""~. ..~ er 500 ml , 500 rn ~ er reactor F .' , ' reactor ~~ ' , , 1,2,3. ;~to glycerolyes ~, t 0 4,5. 10 ppm yes ~ 10 pl ~ .
6,7. 25 ppm No ~ 25 p,l , 8,9,10.25 ppm yes 25 ul 11,12. 100 ppm No 100 pl 13,14,15.100 ppm yes 100 ul 16,17. 200 ppm No 0.20 ml 18,19,20.200 ppm yes 0.20 ml ~
Table 5. Other Bacillus megaterium Treatments.
h Reactor Glyceroh ppm Strain o ) .'.'.;, ; 5 S , ,~.~,.y ., ,~.~3 a ~ ' t t ' 1,3. No glycerol SB3112 4,5. 100 ppm SB3112 6,7. No glycerol Strain 2 8,9. 100 ppm Strain 2 14,15. No glycerol Strain 1 16,17. ~ 00 ppm ~ Strain 1 .
22,23. No glycerol Strain 3 ~ ~ ~
24. ~ 100 ppm Strain 3 SUBSTITUTE SHEET (RULE 26) Table 6. Other species of bacteria Treatments Reactor ' Palmitic product , # ~ r ayd ~as Y Y ''' '" ~ ~ f , , ~.t,i., hr.;:.,y,.1, 1,.,1 n.l'.... 1"l..,n.f,t ,1).... ~ ' :..tp..,in . I.
rielv ..
1,2,3 Yes Commercially available roduct 4,5,6 Yes SB3112 roduct 7,8 No Commercially available roduct 9,10 No SB3112 roduct Table 7. Growth rates of SB3112 on various carboxylic acids.
,_ iy s - , ,-~s~-, ~~ ~ a ~'~ ~
; C ' . Ye W G
k - , :.
With Gl Without cerol .~ Gl cerol f x ~,.
Fa O . . eri ~ Total " ' O '.~f Y ~
~ acid ii take cumulative ~ en ~'otal ' ' ' ~ ~ a ~:
ttY ?~'g p ?~'g . , . . , i ~ f t,., ' ~~ rate m . , '':~u f ~
/L%hr ~ r~'' o ' ~ ' take cuinulatne en a ~ take , ;',~
' g :~
' . p .
~3 , g , p, , f , , , '' , : ~ ',r " . late o eti a ' ' , , a - a take ~m~ ';~ t ~ Yg '~ ~t .' p ' ~ ~ '' ~
, , . . . ;;, ,..:.x ,:; . , . ..
~~ , t ~ e ~ ' a ~, "
I, v , .~, , r ~ , m ;,,;
, /L/hr ~
, ~~ ~
J. m ; /L
, .
;
. ., . , .
, . .
, ..
, oleic acid9.8 370 0 0 , C-18:1 stearic 3.5 460 0 0 acid, palmitic . , 5.7 . 960. . 0 0 acid, C-16 ~ ... ~:.:.;:., .... , . - ..-- -.
Y .v _ . ~
Glycerol uptake subtracted out Oleic acid was 200 mg per reactor while stearic and palmitic were 500 mg per 500 ml reactor.
"~~No uptake was measured for the above fatty acids prior to 150 hours of treatment without the presence of glycerol. -.
SUBSTITUTE SHEET (RULE 26)
Claims (18)
1. Bacillus megaterium strain SB3112, having the characteristics of ATCC
deposit number PTA-3142.
deposit number PTA-3142.
2. A composition comprising Bacillus megaterium strain SB3112 having the characteristics of ATCC deposit number PTA-3142.
3. The composition of claim 2, further comprising additional species of microorganisms..
4. The composition of claim 3, wherein additional species of microorganisms is selected from the group consisting of genera: Acinetobacter, Aspergillus, Azospirillum, Burkholderia, Bacillus, Ceriporiopsis, Enterobacter, Escherichia, Lactobacillus, Paenebacillus, Paracoccus, Pseudomonas, Rhodococcus, Syphingomonas, Streptococcus, Thiobacillus, Trichoderma, and Xanthomonas.
5 The composition of claim 3, wherein said microorganism is selected from the group consisting of Bacillus licheniformis, Bacillus amyoliguofaciens, Bacillus laevolacticus, Bacillus pasteurii, Bacillus subtilus, Bacillus megaterium other than strain SB3112 and a combination thereof.
6 The composition of claim 4, additionally comprising components selected from the group consisting of a non-toxic nutrient formulation, surfactant, activator, preservative, filler, stabilizer, fragrance, viscosifier, enzymes and a combination thereof.
7. The composition of claim 6, wherein said activator is glycerol.
8. The composition of claim 6, wherein said preservative is selected from the group consisting of 1,2-benzisothiazolin-3-one; 5-chloro-2-methyl-4 isothiazolin-3-one, 2-methyl-4-isothiazolin-3-one; quaternium-15; phenol;
sodium o-phenylphenate; o-phenylphenol; 6-acetoxy-2,4-dimethyl-m-dioxane; tris(hydroxymethyl)nitromethane; hexahydro-1,3,5-tris(2-hydroxyethyl)-s-triazine; chlorhexidine; p-hydroxybenzoic acid or its methyl, ethyl, propyl, or butyl esters; benzoic, ascorbic, citric, or sorbic acid;
imidazolidinyl urea; diazolidinyl urea; dimethylol dimethylhydantoin;
methylene bisthiocyanate; 2-bromo-2-nitropropane-1,3-diol; 1,2-benzisothiazoline-3-one; methyl anthranilate., and a mixture thereof.
sodium o-phenylphenate; o-phenylphenol; 6-acetoxy-2,4-dimethyl-m-dioxane; tris(hydroxymethyl)nitromethane; hexahydro-1,3,5-tris(2-hydroxyethyl)-s-triazine; chlorhexidine; p-hydroxybenzoic acid or its methyl, ethyl, propyl, or butyl esters; benzoic, ascorbic, citric, or sorbic acid;
imidazolidinyl urea; diazolidinyl urea; dimethylol dimethylhydantoin;
methylene bisthiocyanate; 2-bromo-2-nitropropane-1,3-diol; 1,2-benzisothiazoline-3-one; methyl anthranilate., and a mixture thereof.
9. The composition of claim 6, wherein said surfactant is selected from the group consisting of trideceth-3; 3 mole ethylene oxide adduct of a linear, primary C12-14 alcohol; 7 mole ethylene oxide adduct of a linear, primary C12-14 alcohol; sodium lauryl sulfate; ammonium lauryl sulfate; dodecyl benzene sulfonic acid; ammonium lauryl sulfate; sodium xylene sulfonate; sodium lauryl sulfate; cocamide diethanolamine; lauramine oxide; sodium alphasulfo methyl C12-18 ester and disodium alphasulfo C12-18 fatty acid salt; soduim dodecylbenzene sulfonate; alkyl polyglycoside;
nonylphenoxypoly (ethyleneoxy) ethanol, branched; nonylphenoxypoly (ethyleneoxy) ethanol, branched; alkoxylated linear alcohol; blend of ethoxylates of linear, primary 12-14 carbon number alcohol; linear alkyl sulfate; alkanolamide; octylphenoxypolyethoxyethanol absorbed on magnesium carbonate; sodium dodecylbenzene sulfonate and isopropyl alcohol; poe (6) tridecyl alcohol; poly(oxy-1,2-ethanediyl), alpha (nonylphenyl)-omega hydroxy, branched, and a mixture thereof.
nonylphenoxypoly (ethyleneoxy) ethanol, branched; nonylphenoxypoly (ethyleneoxy) ethanol, branched; alkoxylated linear alcohol; blend of ethoxylates of linear, primary 12-14 carbon number alcohol; linear alkyl sulfate; alkanolamide; octylphenoxypolyethoxyethanol absorbed on magnesium carbonate; sodium dodecylbenzene sulfonate and isopropyl alcohol; poe (6) tridecyl alcohol; poly(oxy-1,2-ethanediyl), alpha (nonylphenyl)-omega hydroxy, branched, and a mixture thereof.
10. The composition of claim 2, further comprising components for a dry formulation as follows:
(i) SB3112 ranging from about 1 x 10 5 to about 5 x 10 11 CFU/g (ii) glycerol ranging from 0 to about 10%
(iii) surfactant ranging from 0 to 10%
(iv) enzyme ranging from 0 to 1.0%
(v) preservative ranging from 0 to 1.0%
(vi) nutrients ranging from about 0 to 20%
(vii) filler ranging from 0 to 95%;
all percentages being w/w.
(i) SB3112 ranging from about 1 x 10 5 to about 5 x 10 11 CFU/g (ii) glycerol ranging from 0 to about 10%
(iii) surfactant ranging from 0 to 10%
(iv) enzyme ranging from 0 to 1.0%
(v) preservative ranging from 0 to 1.0%
(vi) nutrients ranging from about 0 to 20%
(vii) filler ranging from 0 to 95%;
all percentages being w/w.
11. The composition of claim 2, further comprising components for a liquid formulation as follows:
(i) SB3112 ranging from about 1 x 10 5 to about. 5 x 10 11 CFU/ml (ii) glycerol ranging from about 0 to about 10%
(iii) surfactant ranging from 0 to 10%
(iv) preservative ranging from 1 ppm to 1.0%
(v) color ranging from 0 to 1%
(vi) fragrance ranging from 0 to 1.0%
(vii) nutrients ranging from 0 to 20%
(viii) enzymes ranging from 0 to 1%
(vii) viscosifer ranging from 0 to 5%;
all percentages being w/v.
(i) SB3112 ranging from about 1 x 10 5 to about. 5 x 10 11 CFU/ml (ii) glycerol ranging from about 0 to about 10%
(iii) surfactant ranging from 0 to 10%
(iv) preservative ranging from 1 ppm to 1.0%
(v) color ranging from 0 to 1%
(vi) fragrance ranging from 0 to 1.0%
(vii) nutrients ranging from 0 to 20%
(viii) enzymes ranging from 0 to 1%
(vii) viscosifer ranging from 0 to 5%;
all percentages being w/v.
12. A method for degrading fatty acid or grease, comprising exposing fatty acid, grease, or fatty acid and grease to a composition comprising Bacillus megaterium strain SB3112 having the characteristics of ATCC deposit number PTA-3142.
13. The method of claim 12, adding a biodegrading activator to the composition.
14. The method of claim 13, wherein said activator is glycerol in a range of about 0.001 % to about 10% (v/v).
15. The method of claim 12, adding a surfactant to the mixture to enhance bioavailabiliiy of fatty acids and grease.
16. A method for enhancing the biodegrading activity of a microorganism, comprising making glycerol available in an effective amount to enhance biodegradation activity of a microorganism employed for biodegradation.
17. The method of claim 16, wherein the amount of glycerol is in a range of about 5 ppm to about 1% (w/v).
18. The method of claim 17, wherein said microorganism belongs to Bacillus species.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US09/861,142 US6649401B2 (en) | 2001-05-18 | 2001-05-18 | Gram-positive fatty acid degrader |
| US09/861,142 | 2001-05-18 | ||
| PCT/US2002/013454 WO2002094181A2 (en) | 2001-05-18 | 2002-04-26 | Gram-positive fatty acid degrader |
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| Publication Number | Publication Date |
|---|---|
| CA2447749A1 true CA2447749A1 (en) | 2002-11-28 |
| CA2447749C CA2447749C (en) | 2013-01-15 |
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|---|---|---|---|
| CA2447749A Expired - Fee Related CA2447749C (en) | 2001-05-18 | 2002-04-26 | Gram-positive fatty acid degrader |
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|---|---|
| US (3) | US6649401B2 (en) |
| EP (1) | EP1392218B1 (en) |
| JP (1) | JP3975441B2 (en) |
| AT (1) | ATE346910T1 (en) |
| AU (1) | AU2002303532B2 (en) |
| CA (1) | CA2447749C (en) |
| DE (1) | DE60216448T2 (en) |
| ES (1) | ES2278026T3 (en) |
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| US7879593B2 (en) | 1999-12-16 | 2011-02-01 | Whiteman G Robert | Fermentation systems, methods and apparatus |
| US9969633B2 (en) * | 1999-12-16 | 2018-05-15 | Robert Whiteman | Systems and methods for treating oil, fat and grease in collection systems |
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| EP0569140A1 (en) * | 1992-04-16 | 1993-11-10 | Sybron Chemical Holdings Inc. | Drain opener formulation |
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| US6083737A (en) * | 1999-04-14 | 2000-07-04 | Roebic Laboratories, Inc. | Enzyme-producing strain of Bacillus pumilus |
| US6162635A (en) * | 1999-04-14 | 2000-12-19 | Roebic Laboratories, Inc. | Enzyme-producing strain of Bacillus bacteria |
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2001
- 2001-05-18 US US09/861,142 patent/US6649401B2/en not_active Expired - Lifetime
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2002
- 2002-04-23 US US10/128,050 patent/US6818211B2/en not_active Expired - Lifetime
- 2002-04-26 AU AU2002303532A patent/AU2002303532B2/en not_active Ceased
- 2002-04-26 EP EP02731558A patent/EP1392218B1/en not_active Expired - Lifetime
- 2002-04-26 CA CA2447749A patent/CA2447749C/en not_active Expired - Fee Related
- 2002-04-26 JP JP2002590902A patent/JP3975441B2/en not_active Expired - Fee Related
- 2002-04-26 MX MXPA03010552A patent/MXPA03010552A/en active IP Right Grant
- 2002-04-26 ES ES02731558T patent/ES2278026T3/en not_active Expired - Lifetime
- 2002-04-26 DE DE60216448T patent/DE60216448T2/en not_active Expired - Lifetime
- 2002-04-26 IL IL15891502A patent/IL158915A0/en unknown
- 2002-04-26 NZ NZ530191A patent/NZ530191A/en not_active IP Right Cessation
- 2002-04-26 WO PCT/US2002/013454 patent/WO2002094181A2/en active IP Right Grant
- 2002-04-26 AT AT02731558T patent/ATE346910T1/en not_active IP Right Cessation
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2003
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| JP3975441B2 (en) | 2007-09-12 |
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| EP1392218A2 (en) | 2004-03-03 |
| EP1392218A4 (en) | 2004-12-08 |
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| ATE346910T1 (en) | 2006-12-15 |
| US20030008382A1 (en) | 2003-01-09 |
| MXPA03010552A (en) | 2005-09-08 |
| WO2002094181A2 (en) | 2002-11-28 |
| US20050036990A1 (en) | 2005-02-17 |
| US6818211B2 (en) | 2004-11-16 |
| CA2447749C (en) | 2013-01-15 |
| AU2002303532B2 (en) | 2005-07-07 |
| WO2002094181B1 (en) | 2003-10-23 |
| NZ530191A (en) | 2005-07-29 |
| US20030049832A1 (en) | 2003-03-13 |
| WO2002094181A3 (en) | 2003-07-24 |
| ES2278026T3 (en) | 2007-08-01 |
| DE60216448T2 (en) | 2007-09-20 |
| JP2004528852A (en) | 2004-09-24 |
| IL158915A (en) | 2009-09-22 |
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