JP2003038169A - Gram-positive fatty acid-degrading microorganism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide microorganisms capable of effectively and efficiently degrading a fat, an oil and a grease, and further to provide a composition having the microorganisms and a method for degrading a fatty acid and the grease. SOLUTION: The gram-positive fatty acid-degrading microorganisms capable of producing an extracellular lipase, and capable of simultaneously and efficiently degrading the grease or a mixture of the fatty acid and the grease, and the composition having the microorganisms are provided. The method for enhancing biological degradation uses glycerol with the biodegrading microorganisms.

Description

Detailed Description of the Invention

This application relates to pending US patent application Ser. No. 09 / 861,142 filed May 18, 2001.
It is a partial continuation application of the issue.

[0002]

FIELD OF THE INVENTION The present invention relates to identifying fats, oils and Gram-positive organisms that degrade fats and oils. More particularly, the present invention relates to providing a non-pathogenic sporulating Gram-positive lipophilic bacterial strain that produces extracellular lipase and at the same time efficiently oxidizes or degrades fatty acids and oils. The invention further relates to liquid and dry formulations containing the above Gram-positive organisms and methods for enhancing fatty acid degradation with glycerol.

[0003]

BACKGROUND OF THE INVENTION Most food service establishments need to be equipped with devices to prevent oils and fats from flowing directly from the kitchen or food preparation area into the sewer or waste treatment system within the establishment. These devices, commonly referred to as fat traps, physically prevent oils and fats from flowing directly into the sanitary sewer and function to store the separated fat solids for final solid waste treatment.

Many municipalities have a biological oxygen demand (B
OD) and limits and impositions based on oil and fat (O & G) levels in the effluent from the fat trap.
In addition to the cost and charges of wastewater treatment, fat solids from traps must also be regularly removed and disposed of. Thus, food service facilities face two recycling burdens for wastewater treatment, one for administrative treatment costs and second for fat and oil disposal costs.

However, the frequency with which accumulated fat and oil solids are pumped out is extremely variable, and may range from several weeks to several months. If you don't clean the trap regularly,
Occasionally, oil clogs can cause wastewater to flow back into the food preparation area, causing a foul odor and requiring the facility to be closed until the problem is corrected. In addition to providing a physical means of capturing O & G, the fat trap functions to biologically mediate the reduction of BOD and O & G in the bulk liquor, making effluent effluent cleaner. it can. This decrease in BOD and O & G depends on the hydraulic fluid retention time, which depends on the size of the fat and oil trap and the flow rate of the wastewater. Other factors affecting bioactivity within a fat trap include pH, temperature and whether the facility is performing bio-augmentation.

Bio-augmentation with the addition of commercial bacterial products that increase bioactivity in the system has been used to reduce BOD and O & G in the effluent from fat traps. This has helped reduce the levies that food facilities have to pay to municipalities for wastewater treatment services. In addition, bio-augmentation has been used to reduce the pumping frequency of fat traps and keep drain lines open to reduce malodor.

[0007] In addition to capturing fats and oils, bio-augmentation involves lift stations, drain lines, septic tanks, waste treatment facilities, and other lifts and other locations where accumulation of fats and oils can cause flow problems and malodors. It has been used to help remove fat from oil.

Bio-augmentation products are either liquid or dried. Liquid products are generally preferred for ease of handling, and some come with a liquid metering pump with a container that is replenished on a regular basis. But,
Dry formulations are preferred for other applications, such as waste disposal facilities.

The strains used in bio-augmentation for oil and fat applications produce lipase, a key extracellular enzyme. This enzyme hydrolyzes and destroys the ester bonds between the glycerol skeleton and the fatty acid moieties that make up fats and oils. Glycerol is rapidly processed by biodegradation. However, fatty acids are difficult to decompose and can remain to cause pH drop, clogging and malodor.

When using Gram-negative microorganisms for bio-augmentation in liquid products, they exist as vegetative cells and are therefore often used in such formulations such as surfactants and preservatives. May be killed by a substance. Therefore, products containing Gram-negative organisms cannot contain fungicides or surfactants. Therefore, liquid products that do not contain preservatives
May give off strong odor from microbial contaminants growing in the product. Some of these contaminants can be harmful in the food service environment. Moreover, preservative-free products can also result in reduced shelf life and reduced effectiveness. Clearly, Gram-negative microorganisms have an advantage in the breakdown of fatty acids,
There are significant drawbacks to their use in homes and food service products.

On the other hand, dried Gram-negative products have the advantage of improved shelf life compared to liquid Gram-negative formulations. However, this advantage is slight and there are significant variations depending on the bacterial strain in the product. Although the dry product can be rehydrated with water and applied like a liquid product, the disadvantages of using preservative-free products containing Gram-negative microorganisms also apply to rehydrated dry substances.

It is known that many Gram-negative microorganisms have the ability to biodegrade fatty acids produced by the action of lipases. This ability to oxidize and degrade fatty acids is not generally seen in Gram-positive spore-forming microorganisms, especially members of the genus Bacillus.

Accordingly, it is desirable to develop a bio-augmentation formulation capable of effectively and efficiently degrading or oxidizing fats, oils and fats without causing the malodor or other undesirable conditions as occurs with Gram-negative microorganisms. Is required.

In particular, it is necessary to find a non-pathogenic sporulating Gram-positive lipophilic bacterial strain which produces extracellular lipase and efficiently oxidizes or degrades fatty acids and fats. To date, such Gram-positive microorganisms and formulations containing them have not been identified or produced. Once a microorganism that degrades fatty acids is identified, there is a further need to enhance or enhance the fatty acid degrading activity of such a microorganism in order to maximize the effectiveness of the product based on it.

[0015]

Therefore, a non-pathogenic, spore-forming Gram-positive lipophilic agent that produces extracellular lipase and at the same time efficiently hydrolyzes or degrades fatty acids and fats or mixtures of fatty acids and fats. It is an object of the present invention to provide a sex bacterial strain.

[0016] To provide a liquid and dry composition comprising a non-pathogenic sporulating Gram-positive lipophilic bacterial strain that produces extracellular lipase and efficiently hydrolyzes or degrades fatty acids, oils and fats. It is a further object of the invention.

A further object of the present invention is to provide a method of degrading fatty acids and oils using a Gram-positive strain of Bacillus sp.

Yet another object of the present invention is to enhance the biodegradation activity of Gram-positive strains of Bacillus sp.

It is a further object of the present invention to provide a method of using glycerol as an activity enhancer to enhance microbial fatty acid degrading activity.

Various other objects and advantages of the present invention will be made apparent from the brief description of the drawings and the detailed description of the invention.

Additional advantages and novel features of the present invention will be set forth in part by those skilled in the art, in part in the description that follows, and in part based on the discussion below or through learning by practicing the invention. Will be.

[0022]

These and various other objects and advantages of the present invention include: Gram-positive microorganism, ATCC Deposit No. PTA-
A biologically pure culture of a Bacillus subtilis SB-3112 strain having the identifying characteristics of 3142, and a composition comprising the same. The deposited strain is maintained in viability conditions at the ATCC for the entire term of the issued patent and is provided to any person or institution for non-commercial purposes without limitation, but in accordance with the provisions of the law to which the deposited strain is governed. Shall be.

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 materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the methods and materials described herein are preferred. Unless otherwise noted, the techniques used or considered herein are standard techniques well known to those of skill in the art. The test materials, test methods and examples are illustrative only and not limiting.

The terms "biodegradable", "biodegraded" or "biodegradable" herein mean that the substrate is destroyed, oxidized or degraded by the microorganism. And when used here, the term "activity-enhancing"
It means that the biodegradation activity of the microorganism is enhanced by the presence or addition of certain components, such components being termed "activity enhancers" or "activators".

The use of Gram-positive spore forming organisms capable of oxidizing fatty acids offers the advantage of improved storage liquid products and stable, easy-to-use dry products. Unlike gram-negative bacteria-containing products, stored spore-based gram-positive bacteria-containing products are preservatives that aid in the breakdown of fats, oils or fats, because spores are relatively resistant to fungicides and surfactants. And a surfactant.
In addition, these products may also contain micronutrients that promote microbial growth. Therefore, Gram-positive products containing lipase production, fatty acid degradation, sporulating microorganisms in stored liquid formulations provide the various benefits required for effective degradation of oils and fats. Dried products also benefit from the activity of SB3112.

Liquid and dry products formulated in accordance with the present invention for fat traps or other similar applications where fatty acids or other fats need to be broken down may also include surfactants,
In addition to fungicides, non-toxic growth promoting inorganic nutrients and micronutrients, certain active enhancers, stabilizers, thickeners, enzymes, fillers, preservatives and the like may also be included. Table 1 lists examples of various ingredients that liquid formulations may include in addition to SB3112 according to the present invention.

Other components contained in the liquid and dry preparations are exemplified below: (A) Other microorganisms include Acinetobacter, Aspergillus, Azospirillum, Burkholderia, Bacillus, Ceripolyopsis, It may be selected from the group consisting of Enterobacter, Escherichia, Lactobacillus, Pembacillus, Paracoccus, Pseudomonas, Rhodococcus, Siphingomonas, Streptococcus, Thiobacillus, Trichoderma, and Xanthomonas.

In the genus Bacillus, the microorganism is Bacil
lus licheniformis, Bacillus subtilis, starch liquefied Bacillus, Bacillus laevolac
ticus, Bacillus pastureii,
It may be selected from the group consisting of giant bacteria other than the SB3112 strain, and combinations thereof.

The preservative (B) is 1,2-benzisothiazolin-3-one, 5-chloro-2-methyl-4-isothiazolin-3-one, 2-methyl-4-isothiazolin-3-one, quarter Nium-15, phenol, o
-Sodium phenylphenolate, o-phenylphenol, 6-acetoxy-2,4-dimethyl-m-dioxane, chlorhexidine, tris (hydroxymethyl) nitromethane, hexahydro-1,3,5-tris (2-hydroxyethyl)- s-triazine, chlorhexidine, p-hydroxybenzoic acid, or methyl thereof,
Ethyl, propyl or butyl ester, ascorbic acid, citric acid, or sorbic acid, imidazolidinyl urea, diazolidinyl urea, dimethylol dimethylhydantoin, methylene bisthiocyanate, 2-bromo-2-
It is selected from the group consisting of nitropropane-1,3-diol, 1,2-benzisothiazolin-3-one, methyl anthranilate, and mixtures thereof.

(C) The surfactant is trideceth-3, an ethylene oxide 3 of a linear primary C12-14 alcohol.
Molar adduct, linear primary C12-14 alcohol ethylene oxide 7 molar adduct, sodium lauryl sulfate,
Ammonium lauryl sulfate, dodecylbenzene sulfonic acid, ammonium lauryl sulfate, sodium xylene sulfonate, sodium lauryl sulfate, cocamide diethanolamine, lauramin oxide, α-sulfomethyl C1
2-18 sodium ester and α-sulfo C12-18
Fatty acid disodium salt, sodium dodecylbenzenesulfonate, alkyl polyglycoside, nonylphenoxypoly (ethyleneoxy) ethanol, branched, nonylphenoxypoly (ethyleneoxy) ethanol, branched, alkoxylated linear alcohol, linear primary Mixtures of C12-14 alcohol ethoxylates, linear alkyl sulfates, alkanolamides, octylphenoxypolyethoxyethanol adsorbed on magnesium carbonate, sodium dodecylbenzenesulfonate and isopropyl alcohol, poe (6) tridecyl alcohol, poly (oxy) -1,2-ethanediyl), α (nonylphenyl)
Selected from the group consisting of ω-hydroxy, branched, or other surfactants known in the art, and mixtures thereof.

(D) Nutrients are disodium phosphate,
Monosodium phosphate, thorium thiosulfate, ammonium sulfate, magnesium sulfate, manganese sulfate, ferrous sulfate, potassium sulfate, dipotassium phosphate, monocalcium phosphate, magnesium chloride, calcium chloride, chelated iron, manganese chloride , Sodium molybdate, cobalt chloride, sodium nitrate, baker's yeast, yeast extract, soy peptone, skim milk powder, beef extract, salsaponin,
It is selected from the group consisting of whey, gelatin and urea and others known in the art, and mixtures thereof.

(E) The dry filler is selected from the group consisting of starch, sodium chloride, glucose, sodium bicarbonate, corn cobs, bran, and various ground cereals.

The enzyme (F) is selected from the group consisting of proteases, amylases, lipases, cellulases and others known in the art, and mixtures thereof.

(G) The range (w / w) in which the various components can be included in the composition according to the invention is as follows: (i) from about 1 × 10 ⁵ to about 5 × 10 ¹¹ CFU. SB3112 (ii) ranging from 0 to 10% glycerol (iii) 0 to 10% surfactant (iv) 0 to 1.0% enzyme (v) 0 Preservatives in the range of 1.0% (vi) Nutrients in the range of about 0 to 20% (vii) Fillers in the range of 0 to 95% (H) Various ingredients in the liquid product composition according to the invention (I) SB3112 in the range of about 1 × 10 ⁵ to about 5 × 10 ¹¹ CFU / ml (ii) in the range of about 0 to about 10%. Glycerol (iii) 0-10% range surfactant (iv) 1 ppm-1.0% range. Preservatives (v) 0 to 1% colorants (vi) 0 to 1.0% fragrances (vii) 0 to 5% thickeners (viii) 0 to 20% Range of nutrients (ix) 0 to 1% range of enzyme SB3112 superiority: SB31 compared to other giant strains
The following parallel comparison tests were carried out using three other giant strains with the names strain 1, strain 2 and strain 3 because they show 12 unexpectedly important superiorities. Stearic acid (S
A) and glycerol (G) were used as substrates. Degradation of stearic acid was measured by standard respirometer (oxygen uptake) measurements and aseptic technique was used in all tests.

The CES AER-200 respirometer unit
used. SSC into a 500 ml respirometer bottle_Threeminimum
Add 20 mM MOPS adjusted to pH 7.2 with salt medium.
It was SSC in each bin_ThreeRespirometer by adding inorganic salt medium
A bottle was prepared. This medium is
Contains chemicals: NH_FourCl, 0.8 g; MgSO _Four
・ 7H_TwoO, 0.2g; CaCl_Two・ 2H_TwoO, 10m
g; Fe_TwoNa_TwoEthylenediaminetetraacetic acid, 15 mg;
KH_TwoPO_Four, 3.06 g; FeSO_Four・ 7H _TwoO, 2
8 μg; ZnSO_Four・ 7H_TwoO, 140 μg; MnSO
_Four・ H_TwoO, 84 μg; CoCl2.6H_TwoO, 24μ
g; CuSO_Four・ 5H_TwoO, 25 μg; NaMoO_Four・
2H_TwoO, 24 μg. Buffer solution, 3- (N-morpholino)
Add propanesulfonic acid (MOPS) 4.28g / l
It was The pH of the medium was adjusted to 7.0 before autoclaving. S
SC_ThreeThe bottle containing the medium was autoclaved for 2 hours.

Stearic acid (SA) 10 as a target substrate
00 ppm was added as shown below. Weigh SA,
Each was added to the appropriate reactor and autoclaved. Glycerol 500 ppmv was added to all bottles. After first diluting glycerol to 50% v / v with deionized water to reduce the viscosity and adding 0.5 ml to each reactor,
It was autoclaved. A pure culture of the microorganism was used as the inoculum. Plate count broth (PCB) to evaluate bacterial counts
The optical density (OD) of overnight cultures in was measured. The target dose for each reactor is based on the optical density measurement of broth culture,
It was 1 × 10 ⁶ CFU / ml. After inoculation, a caustic trap containing 5 ml of 30% KOH to remove carbon dioxide was suspended in the bottle and the septum stopper was tightened. The inoculum was MacConkey streak inoculated prior to use to ensure the absence of Gram-negative contaminants. At the end of the experiment, MacConkey and standard method agar (SMA) plate streak inoculations were also performed on the contents of each bottle (MacConkey agar plates were used for detection of Gram-negative contaminants; SMA plates were for pure cultures). Used to test).

The treatments (done twice) were: Glycerol + SB3112 Glycerol + Stearic acid + SB3112 Glycerol + Strain 1 Glycerol + Stearic acid + Strain 1 Glycerol + Strain 2 Glycerol + Stearic acid + Strain 2 Glycerol + Strain 3 Glycerol + stearic acid + strain 3 All cultures were tested for stearic acid plus glycerol as indicated above. A control of glycerol alone was also monitored for each strain.

Table 2 shows the stearic acid decomposition lag phase, oxygen uptake rate, and total cumulative oxygen uptake for each strain. These oxygen uptake rates were calculated using the average of three time points, and the maximum value is shown. The data clearly show surprising results, due to the superiority of SB3112, the shortest lag phase, the fastest uptake rate and the highest cumulative oxygen uptake compared to all other strains tested. It has been clarified that it shows the highest activity against.

SB311 in decomposing various fatty acids
Effect of 2 Since the superiority of SB3112 over other giant strains was confirmed, the following test was performed to investigate the effect of SB3112 in degrading fatty acids. To this end, some fatty acids (99% or higher purity) as listed below were obtained from commercial sources: oleic acid, C-18: 1 stearic acid, C-18 palmitic acid, C- 16 Respirometer (oxygen uptake) activity was used to measure SB3112's ability to degrade valeric acid, C-5 butyric acid, C-4 acetic acid, C-2 fatty acids.

Oxygen uptake was monitored in a 500 ml bottle maintained at 25 ° C using a CES AER-200 respirometer unit. Each 500 ml bottle is SS
C ₃ minimal salt medium and 20 mM MOPS buffer (pH adjusted to 7.2) were included. Glycerol, 500ppm
v was added to all bins. 50 glycerol first
Dilute with% v / v deionized water to reduce viscosity, 0.5
ml was added to each reactor. High molecular weight fatty acids (C-16 and C-18) are weighed individually and added to the appropriate bottles for 10
The concentration was set to 00 mg / L. Chemical resistance filter (0.
2 mg) to filter sterilize low molecular weight fatty acids (C-2, C-4 and C-5) and add to suitable autoclave bottles to remove 1000 mg except oleic acid tested at 200 mg / L / L.

A pure culture of SB3112 was used as inoculum. Plate counting broth (PC
The optical density (OD) was measured by overnight culture in B). The target dose for each reactor was 1 × 10 ⁶ CFU / ml based on the optical density measurement of broth culture. At the time of use, the inoculum was MacConkey streak inoculated to detect Gram-negative bacterial contamination. Aseptic technique was used in all experiments.

Treatment (numbers represent triplicates as shown below) 1, 2, 3. Glycerol alone control 4, 5, 6. C-18 7,8,9. C-16 10, 11, 12. C-5 13, 14, 15. C-4 16,17,18. C-2 19, 20, 21. C-18: 1 (C represents carbon chain length) At the end of the experiment, the contents of each bottle were filled with MacConkey and standard agar (SM
A) Striations were carried out. MacConkey agar plates were used to detect Gram-negative bacterial contamination. SMA plates were used for tests on pure culture. No contamination was detected.

To investigate the oxygen uptake attributable to fatty acid-only oxidation, the oxygen uptake response attributable to individual fatty acids was calculated by subtracting the glycerol data from the fatty acid plus glycerol data. The pH of each reactor at the end of the experiment was in the range of 6.8 to 7.2. Standard method agar (SMA) plates were inoculated with striae from each bottle. At the end of the experiment all bins showed a single colony morphology on SMA plates, suggesting that a pure culture test was performed. The final MacConkey streak inoculation in all bottles was negative (no Gram-negative bacterial contamination).

Table 3 shows S for decomposing various fatty acids.
The activity of B3112 is shown. Oxygen uptake rates were calculated using a three-point running average and the maximum values are shown in the table. Result is,
It is shown that SB3112 decomposes (oxidizes) all fatty acids (oleic acid, stearic acid, palmitic acid, valeric acid, butyric acid and acetic acid). Although valeric acid and butyric acid have been found to have particularly strong odors, SB3112 decomposes these compounds, and therefore SB3112 is useful in reducing the odor attributable to these and similar volatile fatty acids. Conceivable.

The efficacy of SB3112 for degrading fats and oils To demonstrate the efficacy of SB3112 for degrading fats and oils, the following experiment was conducted.

SSC in each bin_ThreeInorganic salt medium 250m
1 was added to prepare a respirometer bottle. Medium before autoclaving
PH was adjusted to 7.0. SSC_ThreeA bottle containing medium
It was autoclaved for 2 hours. Separately pressurizing kitchen waste oil and fat
Sterilization killed unwanted microorganisms. Pressurization of oils and fats
After sterilization, cool each 250 ml respirometer bottle to high temperature.
2 ml of fungal fat was added. Inoculum for respirometer bottles
Is the overnight broth culture of each strain (plate broth) or microorganisms
9002 mixture. First ~ 3x10 ⁵CFU
Each bottle was inoculated to contain / ml. Dioxide after inoculation
Caustic containing 5 ml of 30% KOH to remove carbon
Suspend the trap in a bottle and tighten the septum cap.
It was

To show the superiority of SB3112, the other two
A comparative study was performed using one inoculum sample.
That is, the three inoculums were: Giant bacterium SB3112 strain.

2. Four spores (Bacillus) that produce extracellular enzymes, lipase, amylase and protease
s licheniformis, Bacillus
amyoliquefaciens, Bacillus
pasteurii, and Bacillus lae
inoculum 90, which is a mixture of volatiles)
02. This mixture is known to affect the decomposition of kitchen waste, but fatty acids do not.

3. Inoculum SB3012, a mixture of Gram-negative microorganisms known for their ability to degrade fatty acids
And SB3013.

After inoculation, the bottle containing SSC ₃ medium, kitchen waste oil and inoculum was placed in a respirometer water bath (23 ° C). Each bottle was then connected to a respirometer port. Oxygen consumption was continuously monitored by a respirometer.

The results of this experiment are shown in FIG. Data is,
Gram-negative microbial mixture (SB3012 and SB301
3) shows that it started consuming oxygen almost immediately at a rate of 1.5 mg O ₂ / L / hr. The 9002 spore mixture also began to rapidly consume oxygen, but at a rate of 1.2 mg O ₂ / L / hr was slightly below SB3012 and 3013.
The SB3112 strain had a 50 hour delay before oxygen consumption began at a rate of 0.5 mg O ₂ / L / hr. About 1
At 80 hours, SB3112 began to spike oxygen consumption, surpassing both spore mixture 9002 and Gram-negative bacteria mixture. The oxygen consumption rate of SB3112 after 180 hours was 4.4 mgO ₂ / L / hr, while 0.73 and 0.8 mgO ₂ / L / hr for SB3012 plus SB3013 and 9002 respectively during the same period. The uninoculated control showed no oxygen consumption (data not shown).

Effect of Glycerol on the Biodegradation Activity of SB-3112 The presence of glycerol showed the presence of SB31 as shown in the following test.
It was discovered to enhance the biodegradation activity of 12.

SB3112 cultures were obtained from the holding slope cultures and plated on agar by standard methods. One colony was picked and grown overnight in standard plate counting broth. 1 × 10 ⁶ CFU / ml based on the measured optical density
(Target) SB3112 was added to each reactor. Each substrate (stearic acid and glycerol) was added to the reaction vessel and then autoclaved. The reactor size was 500 ml containing SSC ₃ autoclaved medium.

Treatment (duplicate) 1, 2. Stearic acid, 0.2%, 3, 4. Stearic acid, 0.2% + 0.1% glycerol 5,6. Glycerol, 0.1%.

A final MacConkey streak inoculation was performed to detect Gram-negative bacterial contamination. No contamination was observed. The results are shown in Figure 2. The data clearly show that the fatty acid degrading activity of SB3112 is significantly enhanced by the presence or addition of glycerol.

Comparative Test of Glycerol Levels for Fatty Acid Degradation by Different Microorganisms To demonstrate the effect of glycerol as an activator, a comparison of the biodegradation activity of different microorganisms as a function of different glycerol levels was performed. 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. The measurement of LCFA degradation was determined by respiratory oxygen uptake and the rate of microbial biodegradation activity was indicated by oxygen uptake. The methods and materials used were similar to those for the various experiments described above. However, details of various tests are described for each test described below. Giant strain was obtained from ATCC.

General method for each of the following experiments: CES
An AER-200 respirometer system was used. 500 ml
Respirometer bottle (reactor) with SSC ₃ minimal salt medium and pH
20 mM of MOPS adjusted to 7.2 was added. The target substrate, LCFA, was added as described in the individual experimental methods below.

Glycerol in deionized water at 50% (w /
w) Diluted to reduce viscosity and then pipetted into the appropriate reactor. The reactor was autoclaved and aseptic technique was used in each experiment.

Macconkey streak inoculation was performed on each inoculum at the time of use to detect Gram-negative contaminants. At the end of the experiment, MacConkey and standard method agar (SMA) plate streak inoculations were also performed on the contents of each bottle. SMA plates were used for testing pure cultures.

The specific method used in each of the following four tests is described below.

Test 1: Glycerol Ladder This test was performed to determine the effective concentration of glycerol for enhancing the degradation of LCFA as measured by the rate of increase in oxygen uptake.

Specific method: 50% palmitic acid was added to each reactor.
It was added at a rate of 0 mg / 500 ml (w / v). Diluted glycerol was pipetted (v / v) as described in Table 4. A pure culture of SB3112 was used as inoculum. To assess bacterial counts, the optical density (OD) of overnight cultures in plate-count broth (PCB) was measured. The target dose for each reactor was based on the optical density measurement of broth culture.

FIG. 3 shows the averaged data as a function of serial dilution of glycerol using palmitic acid as the substrate. All concentrations of glycerol (10 pp
m-200 ppm) showed a significant enhancement of palmitic acid degradation. When glycerol was not added, no significant oxygen uptake was observed, indicating no palmitic acid degradation.

FIG. 4 shows oxygen uptake as a function of serial dilution of glycerol in the absence of palmitic acid. These values were subtracted from the glycerol plus palmitic acid data shown in Figure 3 (note that the values on the Y axis are different in Figures 3 and 4). FIG. 5 shows the result of the subtraction and thus only the oxygen uptake attributable to palmitic acid degradation by SB3112.

It is clear from the data presented that glycerol stimulates the degradation of LCFA, palmitic acid at concentrations as low as 10 ppm. It should be noted that glycerol at a concentration of 10 ppm does not cause a significant increase in bacterial count.

Test 2: Other Bacteria This test uses LCF strains of various B.
A, carried out to compare the effect of glycerol on the degradation of palmitic acid.

Specific method: Palmitic acid was added in an amount of 500 mg per reactor having a capacity of 500 ml (1000 ppm).
v). 50 mg of glycerol per 500 ml reactor
Added (1000 ppmv). Pure cultures of each strain were used as inoculum. To assess bacterial counts, the optical density (OD) of overnight cultures in plate-count broth (PCB) was measured. The target dose for each reactor was based on the optical density measurement of broth culture. The treatment for each reactor is shown in Table 5.

FIG. 6 shows the mean oxygen uptake data for various Bacillus megaterium strains with respect to the degradation of palmitic acid in the presence of glycerol. The SB3112 strain started to take up oxygen earlier and produced a higher uptake rate, showing a higher total cumulative oxygen uptake compared to strain 3. In the absence of glycerol, no degradation of palmitinsan occurred in this test as indicated by the absence of oxygen uptake.

ATCC strains 2 and 1 showed no oxygen uptake with palmitic acid alone or palmitic acid supplemented with glycerol. Indeed, the presence of PA inhibited the degradation of glycerol. The expected amount of oxygen uptake from 50 mg glycerol should be about 70 mg.

Briefly, SB3112 degraded palmitic acid to a much greater extent than strain 3 supplemented with glycerol. ATCC strains 2 and 1 did not degrade palmitic acid with or without the presence of glycerol.

Test 3: Other species of bacteria This test was carried out to compare the fully formulated product of the invention containing SB3112 with a commercial product containing a gram positive mixture of 5-7 strains. All strains, including SB3112, were in spore form.

The specific method is as follows: The fully formulated product was placed in a suitable reactor and diluted to an initial spore count in the reactor of about 5.4 × 10 ⁴ CFU / ml. .
The treatment for each reactor is shown in Table 6.

FIG. 7 shows the response to glycerol addition to the LCFA substrate by the two products. The data is S
It shows that the product containing B3112 consumed palmitic acid immediately, whereas the commercial mixture did not significantly degrade palmitic acid. All products and reactors
Gram-negative contaminants were absent, as shown by the fact that no growth was observed when the filament was ingested on MacConkey agar.

The data show that the product containing SB3112 degraded palmitic acid and the commercial product did not significantly degrade palmitic acid. Both products are LCF
Glycerol could be degraded in the presence of A.

Test 4: Effect of Glycerol on Biodegradation This test was performed to demonstrate the effect of glycerol on the oxidation of fatty acids of various chain lengths.

The specific method is as follows: A respirometer was used to test each number of carboxylic acids in the chain length range of 2 (acetic acid) to 18 (oleic acid). 200 mg / L
1000mg of each acid except oleic acid tested in
/ L was tested. Glycerol was used at a concentration of 500 ppm. The test was carried out for 10 days.

Table 7 shows SB31 in the presence of glycerol.
Shows utilization of long chain fatty acids by 12 spores (minus glycerol uptake). Compared to the data in the absence of glycerol, the results obtained in the presence of glycerol
It has been shown that glycerol significantly enhances the degradation of long chain (fatty) acids.

Various embodiments of the invention have been described in accordance with the objects and advantages described above. It will be clear that these examples are merely illustrative and are not a limitation of the present invention. Of course, many variations and modifications of this invention will be apparent to and will be apparent to those of ordinary skill in the art, and all such variations and modifications are within the scope and spirit of the claims.

[Brief description of drawings]

1 shows in the drawing the efficacy of various microorganisms for degrading fats and oils.

FIG. 2 shows the effect of glycerol on the biodegradation activity of SB-3112 and various microorganisms.

FIG. 3 shows the effect of glycerol on the biodegradation activity of SB-3112 and various microorganisms.

FIG. 4 shows the effect of glycerol on the biodegradation activity of SB-3112 and various microorganisms.

FIG. 5 shows the effect of glycerol on the biodegradation activity of SB-3112 and various microorganisms.

FIG. 6 shows the effect of glycerol on the biodegradation activity of SB-3112 and various microorganisms.

FIG. 7 shows the effect of glycerol on the biodegradation activity of SB-3112 and various microorganisms.

[Table 1]

[Table 2]

[Table 3]

[Table 4]

[Table 5]

[Table 6]

[Table 7]

   ─────────────────────────────────────────────────── ─── Continued front page    (71) Applicant 502178344             Jonathan Leader             United States Virginia 24122               Montvar Goose Creek             Array Road 3801 (71) Applicant 502178355             Domenique Anthony Paone             United States Virginia 24018               Lonoke Forest Creek             7992 (72) Inventor Jesse Lindthinger             United States Virginia 24091               Freud Penlord 1424 (72) Inventor David Joseph Drahos             United States Virginia 24018               Lonoke Scottford Coun             To 6014 (72) Inventor Jonathan Leader             United States Virginia 24122               Montvar Goose Creek             Array Road 3801 (72) Inventor Domenique Anthony Paone             United States Virginia 24018               Lonoke Forest Creek             7992 F term (reference) 4B065 AA15X AC20 BB06 BB40                       BD12 BD27 CA31 CA54

Claims (18)

[Claims] 1. A giant strain SB-3112 having the characteristics of ATCC Deposit No. PTA-3142. 2. A composition comprising a Bacillus megaterium SB-3112 strain having the characteristics of ATCC Deposit No. PTA-3142. 3. The composition of claim 2, further comprising an additional species of microorganism. 4. A microorganism of an additional species is Acinetobacter, Aspergillus, Azospirillum, Burgholderia, Bacillus, Ceripolyopsis, Enterobacter, Escherichia, Lactobacillus, Penbacillus, The composition according to claim 3, which is selected from the group consisting of Paracoccus, Pseudomonas, Rhodococcus, Siphingomonas, Streptococcus, Thiobacillus, Trichoderma, and Xanthomonas. 5. The microorganism is Bacillus li.
cheniformis, starch liquefied Bacillus, Ba
cillus laevolacticus, Baci
illus pasteurii, Bacillus subtilis, SB3112
The microorganism according to claim 3, which is selected from the group consisting of giant bacteria other than strains and combinations thereof. 6. A non-toxic nutritional preparation, a surfactant, an activator, an antiseptic, a filler, a stabilizer, an aromatic, a thickener, an enzyme,
The composition of claim 4, additionally comprising a composition selected from the group consisting of: and a combination thereof. 7. The composition of claim 6, wherein the activator is glycerol. 8. The preservative is 1,2-benzisothiazolin-3-one, 5-chloro-2-methyl-4-isothiazolin-3-one, 2-methyl-4-isothiazolin-3-one, quarter Nium-15, phenol, o
-Sodium phenylphenolate, 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 ester, ascorbic acid, citric acid, or sorbic acid, imidazolidinyl urea, diazolidinyl urea,
Dimethylol dimethyl hydantoin, methylene bisthiocyanate, 2-bromo-2-nitropropane-1,3-
Diol, 1,2-benzisothiazolin-3-one,
7. The composition of claim 6 selected from the group consisting of methyl anthranilate, and mixtures thereof. 9. The surfactant is trideceth-3, an ethylene oxide 3 of a linear primary C12-14 alcohol.
Molar adduct, linear primary C12-14 alcohol ethylene oxide 7 molar adduct, sodium lauryl sulfate,
Ammonium lauryl sulfate, dodecylbenzene sulfonic acid, ammonium lauryl sulfate, sodium xylene sulfonate, sodium lauryl sulfate, cocamide diethanolamine, lauramin oxide, α-sulfomethyl C1
2-18 sodium ester and α-sulfo C12-18
Fatty acid disodium salt, sodium dodecylbenzenesulfonate, alkyl polyglycoside, nonylphenoxypoly (ethyleneoxy) ethanol, branched, nonylphenoxypoly (ethyleneoxy) ethanol, branched, alkoxylated linear alcohol, linear primary Mixtures of ethoxylated C12-14 alcohols, linear alkyl sulphates, alkanolamides, octylphenoxypolyethoxyethanol adsorbed on magnesium carbonate, sodium dodecylbenzene sulfonate and isopropyl alcohol, poe (6) tridecyl alcohol, poly ( Oxy-1,2-ethanediyl), α (nonylphenyl)
7. The composition of claim 6 selected from the group consisting of -omega hydroxy, branching, and mixtures thereof. 10. The composition of claim 2, further comprising the following components for the dry formulation: (i) SB3112 (ii) in the range of about 1 × 10 ⁵ to about 5 × 10 ¹¹ CFU / g. 0 to about 10% glycerol (iii) 0 to 10% surfactant (iv) 0 to 1.0% enzyme (v) 0 to 1.0% preservative ( vi) Nutrients in the range of about 0 to 20% (vii) Fillers in the range of 0 to 95%, but all percentages are w / w. 11. The composition according to claim 2, further comprising the following components for the liquid formulation: (i) SB3112 (ii) in the range of about 1 × 10 ⁵ to about 5 × 10 ¹¹ CFU / ml. Glycerol (iii) in the range of about 0 to about 10% Surfactant (iv) in the range of 0 to 10% Preservative in the range of 1 ppm to 1.0% (v) Colorant in the range of 0 to 1% ( vi) Fragrance in the range of 0 to 1.0% (vii) Nutrient in the range of 0 to 20% (viii) Enzyme in the range of 0 to 1% (vii) Thickener in the range of 0 to 5% but percentage Are all w / v. 12. A fatty acid, a fat or oil, or a fatty acid and a fat or oil,
A method for degrading fatty acids or oils comprising contacting a composition comprising a Bacillus subtilis SB-3112 strain having the characteristics of ATCC Deposit No. PTA-3142. 13. The method of claim 12, wherein a biodegradable activator is added to the composition. 14. The method of claim 13, wherein the activator is glycerol in the range of about 0.001% to about 10% (v / v). 15. The method according to claim 12, wherein a surfactant is added to the mixture to enhance biodegradability of fatty acids and oils. 16. A method for enhancing the biodegradation activity of a microorganism, which comprises using glycerol in an effective amount to enhance the biodegradation activity of the microorganism used for biodegradation. 17. The method of claim 16, wherein the amount of glycerol ranges from about 5 ppm to about 1% (w / v). 18. The method of claim 17, wherein the microorganism belongs to Bacillus sp.