WO2010059028A1 - Bactéries pour la dégradation et la modification de matières grasses, huiles et graisse - Google Patents

Bactéries pour la dégradation et la modification de matières grasses, huiles et graisse Download PDF

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Publication number
WO2010059028A1
WO2010059028A1 PCT/MY2008/000150 MY2008000150W WO2010059028A1 WO 2010059028 A1 WO2010059028 A1 WO 2010059028A1 MY 2008000150 W MY2008000150 W MY 2008000150W WO 2010059028 A1 WO2010059028 A1 WO 2010059028A1
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WIPO (PCT)
Prior art keywords
bacillus
grease
composition
strain
ranging
Prior art date
Application number
PCT/MY2008/000150
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English (en)
Inventor
Nor Aripin Shamaan
Mohd Yunus Abd Shukor
Fakhru'l Razi Ahmadun
Norhafizah Abdullah
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University Putra Malaysia
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Application filed by University Putra Malaysia filed Critical University Putra Malaysia
Priority to PCT/MY2008/000150 priority Critical patent/WO2010059028A1/fr
Publication of WO2010059028A1 publication Critical patent/WO2010059028A1/fr

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Classifications

    • 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
    • C02F3/341—Consortia of bacteria
    • 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
    • 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
    • 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
    • C12N1/205—Bacterial isolates
    • 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
    • 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
    • C12R2001/085—Bacillus cereus

Definitions

  • the invention discloses several Gram-positive microorganisms that effectively and efficiently degrade fats, oils and greases.
  • a composition comprising said microorganism and a method for degrading fatty acids and grease are also disclosed. 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 formulation comprising said Gram-positive organism.
  • Fats, oils and greases (FOGs) in wastewater create problems including the production of foul odours, the blockage of sewer lines and may interfere with the proper operation of sewage treatment works. Removal of FOG from wastewater is thus critically important to ensure that wastewater is disposed of efficiently and economically.
  • One of the major mechanisms of sewer line blockage due to oil is the conversion of oil to a greasy-like substance. When oil was incubated with the wastewater bacteria, an opaque, semi-solid sticky material was formed. Some of this material stuck to the sides of the culture vessels. The composition of the fatty acids was found to be different from the original oil.
  • 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 malodours and requiring the establishment to close until the problem is corrected.
  • grease traps can function to biologically mediate 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 on the size of the grease trap and wastewater flow. Other factors that 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 effluents 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 malodours. In addition to grease traps, bioaugmentation has also been used to help remove grease from lift stations, drain lines, septic tanks and other situations where grease accumulation can cause flow problems and malodours. Bioaugmentation products can be either liquid or dry. Because of ease of handling, liquid products are generally preferred and added by a liquid metering pump drawing on a container that is replenished on a periodic basis.
  • Gram-negative microorganisms 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 formulations. Therefore, products containing Gram-negative organisms cannot contain biocides and surfactants. Then, unpreserved liquid products may develop severe malodours from microbial contaminants growing in the product. Some of these contaminants may be undesirable in a food service environment. Furthermore, unpreserved products may also suffer from decreased shelf life and efficacy. Clearly, while Gram-negative microorganisms have an advantage in fatty acid degradation, their use in residential and food service products have serious drawbacks.
  • Dry Gram-negative products do have a slight advantage of improved shelf life over liquid Gram-negative formulations. However, this advantage is only marginal and varies significantly from strain to strain. Disadvantages of dry products include contaminating dust and difficulty in handling dry materials. 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.
  • WO200294181-A by Tisinger J L, Paone D A, Leder J, Drahos DJ, Pacne DA, Brahos DJ, Tisinger J, Paone D, Drahos D, Tisinger L, Paone A and Drahos J (2003)- use a novel Bacillus megaterium SB3112 (A) having the ATCC deposit number PTA-3142 for degrading fatty acids and grease flowing into a sewer or to an on-site waste disposal system.
  • JP2004009044-A by Ekomatekku Kenkyusho, Kaji Y, Takahashi M (2004) - using an oil absorbing-decomposing composition comprises cellulosic fibrous material; Bacillus subtilis, B.brevis, B.licheniformis, B.thuringiensis, B.cereus and/or B.tumilus and Pseudomonas aeruginosa, P.fluorescens, P.putida, P.syringae, P.mallei, P.diminuta, P.vesicularis and/or P.pickettii.
  • Figure 1 the comparison of palm oil-degrading efficiency between the commercial product and individual strains of Bacillus spp. locally isolated
  • Figure 2 is 16sRNA gene sequence for Bacillus cereus Strain Dr.Y135 with accession number of EF121823
  • Figure 3 is 16sRNA gene sequence for Bacillus sp. strain SeAG 1 Y135 with accession number of EU828795
  • Figure 4 is 16sRNA gene sequence for Uncultured Bacillus sp. clone A.rzi Y135 with accession number of EU835195
  • Figure 5 is 16sRNA gene sequence for Bacillus amyloliquefaciens strain RXZ Y135 with accession number of EU835195
  • 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.
  • An additional object of the present invention is to provide a method for degrading fatty acid and grease using a Gram-positive strain of Bacillus spp.
  • Yet another object of the present invention is to enhance the biodegrading activity of Gram-positive strain of Bacillus species.
  • the present invention relates to Gram positive microorganisms, Bacillus spp.
  • the disclosed Bacillus species is effective for degradation of fats, oils and greases.
  • the above and various other objects and advantages of the present invention are achieved by biologically pure cultures Gram-positive microorganism, Bacillus spp.
  • the said microorganisms with their 16srRNA gene sequence submitted to Genbank with the accession numbers of EF121823, EU828795, EU835195 and EU835195.
  • biodegradation means that the substrate is broken down, oxidized or degraded by the microorganism.
  • activity enhancement 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”.
  • the preserved, spore-based Gram-positive containing product can contain preservatives and surfactants to aid in the biodegradation of fats, oils and greases (FOGs), because the spores are relatively resistant to biocides and surfactants. Furthermore, these products may also contain micronutrients promoting the growth of the microorganisms.
  • 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.
  • Liquid 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 and the like.
  • Acinetobacter Aspergillus, Azospirillum, Burkholderia, Bacillus, Ceriporiopsis,
  • Rhodococcus Syphingomonas, Streptococcus, Thiobacillus, Trichoderma and
  • the microorganism may be selected from the group consisting of Bacillus subtilis, Bacillus cereus, Bacillus pumilus, and Bacillus thuringiensis and a combination thereof.
  • the 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-nitropropyl
  • the 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; sodium dodecylbenzene sulfonate; alkyl polyglycoside; nonylphenoxypoly (ethyleneoxy) ethanol, branched; nonylphenoxypoly (ethyleneoxy) ethanol, branched; alkoxylated linear alcohol; blend of ethoxylate
  • preservative ranging from 1 ppm to 1.0%
  • color ranging from 0.02% to 1 %
  • fragrance ranging from 0.02% to 1.0%
  • a commercial oil-degrading supplement, Microblaze, in the form of block was initially chipped using sterile spatula to give approximately 1 cm3 block having an approximate spore count of 1 x 10 8 CFU bacteria and were directly added into the oil growth medium.
  • Bacillus spp. strains were initially grown overnight at 3O 0 C on an orbital shaker (250 rpm) and were added into the flask to a final concentration of 1 x 10 8 CFU/ml.
  • Controls were prepared in the same manner but without the addition of the supplement. The flasks were incubated for 21 or 28 days at 30 0 C with shaking at 130 rev/min before the remaining lipids were extracted. All treatments and controls were prepared in triplicate.
  • Lipids were extracted by transferring the contents of the flasks into separating funnels and adding 40 ml of dichloromethane. The funnels were shaken vigorously, allowed to settle and the organic phase transferred to a florentine flask. If emulsification had occurred, separation of the two phases was achieved by centrifugation at 4500 g for 2-10 min assisted by the addition of a few drops of saturated sodium chloride solution. The remaining aqueous phase was re-extracted a further 3 times and the solvent phases pooled together and dried using a rotary evaporator.
  • the dried oil was redissolved in a few ml of dichloromethane, transferred to a vial containing anhydrous sodium sulphate and then filtered through Whatman number 6 filter paper into a pre-weighed vial.
  • the solvent was evaporated under oxygen free nitrogen at 40°C and the weight of oil determined.
  • Bacillus amyloliquefaciens strain RXZ (EU835195), Bacillus pumilus Strain Lubn2 (EU851976), Bacillus cereus Strain DRY135 (DQ 851857), Bacillus sp. strain SeAG 1 (EU828795) were obtained from the Dept. of Biochemistry Culture Collection, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia. Values in parentheses indicate Genbank Accession number. Benchmarking product, BioAccess, was obtained from and contained several undiclosed Bacillus strains;
  • BioAccess product properties are as follow:
  • Figure 1 showed the comparison of palm oil-degrading efficiency between the commercial product (BioAccess) product and individual strains of Bacillus spp. locally isolated. The degradation was carried out at 30 0 C since this is the normal environmental temperatures of Malaysian climate. Only Bacillus sp. strain SeAG 1 gave significantly higher reduction in palm oil than the commercial Bioaccess consortium (p ⁇ 0.05). The rest of the strains gave no significant difference in terms of palm oil degradation capacity compared to the commercial BioAccess consortium (p>0.05). However, the combination of all of the local strains gave significantly higher degradation of palm oil compared to the commercial BioAccess consortium (p ⁇ 0.05).

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Water Supply & Treatment (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Virology (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Environmental & Geological Engineering (AREA)
  • Hydrology & Water Resources (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)

Abstract

L'invention porte sur plusieurs microorganismes à Gram-positif qui dégradent de façon effective et efficace les matières grasses, les huiles et la graisse. L'invention porte sur une composition comprenant ledit microorganisme et sur un procédé pour dégrader des acides gras et la graisse.
PCT/MY2008/000150 2008-11-21 2008-11-21 Bactéries pour la dégradation et la modification de matières grasses, huiles et graisse WO2010059028A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/MY2008/000150 WO2010059028A1 (fr) 2008-11-21 2008-11-21 Bactéries pour la dégradation et la modification de matières grasses, huiles et graisse

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/MY2008/000150 WO2010059028A1 (fr) 2008-11-21 2008-11-21 Bactéries pour la dégradation et la modification de matières grasses, huiles et graisse

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WO2010059028A1 true WO2010059028A1 (fr) 2010-05-27

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3187579A4 (fr) * 2014-08-25 2017-07-26 Japan Environmental Science Company Composition, support, système de traitement des eaux usées, procédé de traitement des eaux usées, procédé de désodorisation, et procédé de traitement des eaux usées par lot
FR3068688A1 (fr) * 2017-07-05 2019-01-11 Exochems Environnement Composition bacterienne et utilisation pour le traitement des eaux usees et le traitement des dechets
FR3115293A1 (fr) * 2020-10-16 2022-04-22 Open Medical System Composition liquide de bactéries et procédé de conservation de bactéries

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6171847B1 (en) * 1999-04-14 2001-01-09 Roebic Laboratories, Inc. Enzyme-producing strain of Bacillus bacteria
WO2002094181A2 (fr) * 2001-05-18 2002-11-28 Novozymes A/S Agent gram-positif de degradation des acides gras

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6171847B1 (en) * 1999-04-14 2001-01-09 Roebic Laboratories, Inc. Enzyme-producing strain of Bacillus bacteria
WO2002094181A2 (fr) * 2001-05-18 2002-11-28 Novozymes A/S Agent gram-positif de degradation des acides gras

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE BIOSIS [online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; 2001, ARCHAMBAULT J G ET AL: "Development of a Bacillus consortium for digestion of food waste in grease trap system", XP002537501, Database accession no. PREV200200251470 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3187579A4 (fr) * 2014-08-25 2017-07-26 Japan Environmental Science Company Composition, support, système de traitement des eaux usées, procédé de traitement des eaux usées, procédé de désodorisation, et procédé de traitement des eaux usées par lot
US10052399B2 (en) 2014-08-25 2018-08-21 Japan Environmental Science Company Composition, support, wastewater treatment system, wastewater treating method, deodorization method, and batch wastewater treating method
FR3068688A1 (fr) * 2017-07-05 2019-01-11 Exochems Environnement Composition bacterienne et utilisation pour le traitement des eaux usees et le traitement des dechets
FR3115293A1 (fr) * 2020-10-16 2022-04-22 Open Medical System Composition liquide de bactéries et procédé de conservation de bactéries

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