KR20170019134A - Novel strain pichia caribbica ys03 and uses thereof - Google Patents

Novel strain pichia caribbica ys03 and uses thereof Download PDF

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KR20170019134A
KR20170019134A KR1020150113123A KR20150113123A KR20170019134A KR 20170019134 A KR20170019134 A KR 20170019134A KR 1020150113123 A KR1020150113123 A KR 1020150113123A KR 20150113123 A KR20150113123 A KR 20150113123A KR 20170019134 A KR20170019134 A KR 20170019134A
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김종필
이승훈
장혜란
채한승
박희준
신동윤
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롯데케미칼 주식회사
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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
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    • 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
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
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Abstract

Pichia caribbica YS03 (Pichia caribbica), which has a rapid consumption rate of carbon source such as alkane, fatty acid and fatty acid derivative separated from a wastewater treatment facility of a petrochemical plant and has a characteristic of rapid cell growth using the carbon source as a single carbon source, YS03) (KCTC 12821BP) can be industrially used for industrial wastewater containing carbon sources such as alkane, fatty acid and fatty acid derivatives, and for treatment of harmful environmental substances and decomposition of petroleum.

Description

NOVEL STRAIN PICHIA CARIBBICA YS03 AND USES THEREOF < RTI ID = 0.0 >

The present invention relates to a novel Pichia caricaca YS03 microorganism having a rapid carbon source consumption rate such as an alkane, a fatty acid and a fatty acid derivative, and having a carbon source as a single carbon source and exhibiting rapid cell growth, and a use thereof.

Wastewater treatment is divided into physical, chemical and biological treatment. Physical treatment mainly consists of removal of solids, chemical treatment, pH adjustment through injection of chemicals, removal of heavy metals, and biological treatment, for the primary purpose of removing organic matter by microorganisms. The wastewater treatment process is classified into primary, secondary, and tertiary treatment according to the progression stage. The primary treatment is a physical treatment process using sedimentation and floatation to remove suspended solids, and the secondary treatment It is a biological treatment process to remove biodegradable dissolved organic matter by using microbial activity. The third treatment is a process to remove nitrogen, phosphorus, residual organic matter, solid matter, salt and the like which have not been removed in the first and second treatment. Biological treatment, which is a secondary treatment process, utilizes the ability of microorganisms to ingest, decompose and stabilize wastewater water pollutants as nutrients.

Recent global trends in wastewater treatment processes tend to be biological treatment with a relatively low treatment unit cost, secondary occurrence of secondary pollutants, or biological treatment and chemical treatment. Studies on decomposition microorganisms for specific compounds, refractory substances, heavy metals and the like have been actively conducted at home and abroad.

The wastewater from the petrochemical plant contains various alkanes of high concentration. In the wastewater treatment facility of the conventional petrochemical plant, the wastewater from the oil separator (CPI, coagulated plate interceptor) After treatment, wastewater treatment proceeds through an aeration tank, a settling tank, and the like.

For biodegradation of wastewater in petrochemical plants, new microorganisms are needed for biodegradation and utilization of wastewater from petrochemical plants containing various alkanes as described above.

In order to solve the above problems, the present invention aims to provide a novel strain for decomposing wastewater into a biological process.

In order to achieve the above object,

To provide a novel strain of Pichia caribbica YS03 (KCTC 12821BP).

The novel strain of Pichia caribbica YS03 (KCTC 12821BP) of the present invention may be one using at least one carbon source selected from alkanes, fatty acids and fatty acid derivatives.

The carbon source may be selected from the group consisting of dodecane, methyl laulate lauric acid, and derivatives thereof, or a combination thereof.

The novel Pichia caribbica YS03 (KCTC 12821BP) strain may be characterized in that it shows the reactivity as shown in the following table in terms of carbon magnetization ability.

[table]

Figure pat00001

The present invention also provides a method for treating wastewater using the strain as described above.

The present invention also provides a method for producing dodecanedioic acid (DDDA) from one or more carbon sources selected from alkanes, fatty acids and fatty acid derivatives using the above-mentioned strains.

Pichia caribbica YS03 (Pichia caribbica), which has a rapid consumption rate of carbon source such as alkane, fatty acid and fatty acid derivative separated from a wastewater treatment facility of a petrochemical plant and has a characteristic of rapid cell growth using the carbon source as a single carbon source, YS03) (KCTC 12821BP), industrial wastewater containing at least one carbon source selected from alkane, fatty acid, and fatty acid derivative, and industrial waste water and degradation of petroleum.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram schematically showing a competitive induction continuous type integrated culture apparatus. FIG.
FIG. 2 is a graph showing the change of the OD value and the concentration of the dodecane with time of the microorganism in the competitive induction continuous integrated culture. FIG.
Fig. 3 shows the 18s rRNA base sequence of the isolated strain (picaccaribica YS03).
4 is a graph showing the growth rate of the isolated strain (Pichia caribrica YS03) according to pH.
FIG. 5 is a graph showing the rate of dodecane consumption and the amount of produced bacterium over time in Pichia caricaca YS03 (KCTC 12821BP) and the same strain Pichia carabica (KCTC 17289).
FIG. 6 is a graph showing the rate of dodecane consumption and the amount of produced bacterium over time in Pichia caricaca YS03 (KCTC 12821BP) and Candida tropicallis (ATCC 20336) as a standard strain.

BRIEF DESCRIPTION OF THE DRAWINGS The present invention is capable of various modifications and various embodiments, and specific embodiments are illustrated in the drawings and described in detail in the detailed description. It is to be understood, however, that the invention is not to be limited to the specific embodiments, but includes all modifications, equivalents, and alternatives falling within the spirit and scope of the invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.

Hereinafter, the present invention will be described in detail.

The novel strain of the present invention is characterized by being Pichia caribbica YS03 (KCTC 12821BP).

The novel Pichia caribbica YS03 (KCTC 12821BP) strain of the present invention may use one or more carbon sources selected from alkanes, fatty acids and fatty acid derivatives.

In the present invention, 'alkane' refers to a saturated hydrocarbon which may be represented by 'alkane' or 'alkane' and is a chemical compound consisting exclusively of carbon atoms and hydrogen atoms and exclusively containing a single bond.

Preferably an alkane having 6 to 30 carbon atoms, and more preferably 8 to 20 carbon atoms.

Fatty acids also refer to saturated or unsaturated monocarboxylic acids in the form of chains. May be a fatty acid having 6 to 30 carbon atoms, and preferably a fatty acid having 8 to 20 carbon atoms. And more preferably a saturated fatty acid having 8 to 20 carbon atoms.

Examples of the fatty acid derivatives include, but are not limited to, esters of the above-mentioned fatty acids.

According to a preferred embodiment, the carbon source may be at least one selected from the group consisting of dodecane, methyl laulate, lauric acid and derivatives thereof.

The novel Pichia caribbica YS03 (KCTC 12821BP) strain of the present invention may exhibit reactivity as shown in the following table in terms of carbon atomization ability.

[table]

Figure pat00002

The novel Pichia caribbica YS03 (KCTC 12821BP) strain of the present invention exhibits cell growth at an acidity of pH 4 to pH 7, more preferably at an acidity of pH 6 ± 0.5, It can be an indication.

The present invention also provides a method for treating wastewater by using the strain as described above.

The present invention also provides a method for producing dodecanedioic acid (DDDA) from at least one carbon source selected from alkanes, fatty acids and fatty acid derivatives using the above-mentioned strains.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to be illustrative of the present invention and the scope of the invention as defined by the appended claims. And it is clear that such modifications and variations are included in the scope of the appended claims.

< Example  1> Isolation of strain

In order to treat wastewater from a petrochemical plant with various concentrations of various carbon sources, it is necessary to treat the wastewater by firstly treating it in an oil separator (CPI, Coagulated Plate Interceptor) The samples were collected from the oil separator (CPI), the aeration tank and the settling tank of the wastewater treatment plant in the process.

The sample is prepared by collecting a wastewater sample in a 1 L sterilized water sample pack in an oil separator inflow water, an oil separator effluent water, an equilibrium effluent water, an aeration tank inflow water, a aeration tank effluent water, a sedimentation tank influent water and a sedimentation tank effluent water. And transferred to the laboratory. A portion of the collected samples was spread on a solid agar plate made of the composition of the primary culture medium shown in Table 1 below and incubated for 1 week in a constant temperature incubator at 30 캜.

After culturing, the colonies produced in the solid medium were collected from the culture medium containing Dodecane (C 12 alkane) and selected for rapid growth, and the resulting colonies were cultured in a secondary culture medium , 1 v / v / m aerated amount, 400 rpm stirring speed, and pH 5.0 (manufactured by Wako Pure Chemical Industries, Ltd.) were inoculated into a competitive induction continuous culture medium containing Dodecane (C 12 alkane) controlled by 10N NaOH) (Fig. 1). After 20 g / L of dodecane was depleted in the competition-induced continuous-type co-culture, an additional medium of the following Table 2 containing an additional 40 g / L of dodecane was added to adjust the dilution rate to 0 0.4, and finally the strain Pichia caribbica YS03 (KCTC 12821BP), which has the best growth property, was isolated. In this experiment, 25m g / L of the antibiotic kanamycin was used to inhibit the growth of some microorganisms. The experimental results are shown in Fig.

Medium composition Primary culture medium
(Solid medium) Competitive Induction Continuous Integrated Culture Medium Secondary culture medium Additional badges YNB
(Yeast nitrogen base without amino acid) 6.7 g / L 20 g / L 20 g / L Dodecane 10 g / L 20 g / L 40 g / L Surfactants Gum Arabic 0.5% Tween 80 3 mL / L Tween 80 3 mL / L

< Experimental Example  1> Isolate  18s rRNA  Genetic analysis

The isolated strains isolated in Example 1 were analyzed by 18s rRNA sequencing. The genomic DNA of the isolated strain of Example 1 was extracted using Yeast gDNA prep kit (PureHelix ™, NANOHELIX), and the extracted genomic DNA was amplified by PCR using the 18s ITS 1/4 primer shown in Table 2 below After TA vector cloning, 18s rRNA nucleotide sequence was obtained through DNA sequencing reaction, and the nucleotide sequence was shown in FIG. 3 and SEQ ID NO: 1.

The base sequence (5 '- &gt; 3') SEQ ID NO: Forward primer TCC GTA GGT GAA CCT GCG G SEQ ID NO: 2 Reverse primer TCC TCC GCT TAT TGA TAT GC SEQ ID NO: 3

The nucleotide sequence of the isolated strain shown in SEQ ID NO: 1 was examined using a BLAST (Basic Local Alignment Search Tool) of National Center for Biotechnology Information (NCBI). The results of the investigation are shown in Table 3 below.

Isolated strain Length of sequence Relative species Homology Pichia caribbica YS03 631bp Pichia caribbica strain XTWJX (GU248264) 99%

As shown in Table 3, it was confirmed that the isolate was a close variant having high homology with Pichia capiciva XTWJX.

< Experimental Example  2> Pikia Caribica YS03 ( Pichia caribbica YS03 ) ( KCTC  12821BP) carbon source Magnetizing ability ( Assimilability ) analysis

To investigate the carbonization ability of the above strain (Pichia caribbica YS03 (KCTC 12821BP)), API 20c AUX (Biomerieux) was used to analyze the activity. The API 20c AUX (Biomerieux) One experimental result was compared with the conventional Pichia caribbica KC977491 (Pichia caribbica KC977491) and the synonym thereof, Meyerozyma caribbica UL5-1, and the results are shown in Table 4 Respectively.

Carbon source Example 1
(Pichia caribbica YS03; KCTC 12821BP) Comparative Example 1
(Pichia caribbica KC977491) Comparative Example 2
(Meyerozyma caribbica UL5-1) Glucose + + + Glycerol - - + 2-keto-D-gluconic acid
(2-keto-D-gluconate) + - + L-arabinose (L-arabinose) - + + D-Xylose &lt; / RTI &gt; + + - Adonitol + + Xylitol - + D-galactose &lt; / RTI &gt; + + + Inositol - - - D-sorbitol (D-sorbitol) + - + A-methyl-D-glucoside
(A-Methyl-D-glucoside) + - + N-acetyl-D-glucosamine
(N-Acetyl-D-glucosamine) + - + D-Cellobiose + + + D-lactose (D-lactose) - - D-Maltose + + + D-Saccharose (Sucrose) + + +

As shown in Table 4, when Comparative Example 1 (Pichia caribbica KC977491) and Comparative Example 2 (Meyerozyma caribbica UL5-1) and Pichia Carabica YS03 (KCTC 12821BP) (Example 1) were compared, In Comparative Example 2, glycerol, L-arabinose, and xylitol can be used as carbon sources, while Pichia Carabica YS03 (KCTC 12821BP) (Example 1) can not use glycerol, L-arabinose, or xylitol as a carbon source And it was found. In addition, there was a difference in the availability of D-xylose. As a result of the above experiment, it was confirmed that the novel Pichia carvica YS03 (KCTC 12821BP) of the present invention shows a large difference in the carbon atomization ability as compared with the existing strains.

< Experimental Example  3> Pikia Caribica YS03 ( Pichia caribbica YS03 ) ( KCTC  12821BP) optimum growth pH

In order to examine the optimal growth pH of Pichia caribbica YS03 (KCTC 12821BP), the strain was cultured in an initial pH of 4 to 7 (without amino acid) in a yeast nutrient base (without amino acid) And cultured. The results of the experiment are shown in FIG.

As shown in Fig. 4, it was confirmed that the optimum growth pH of the Pichia caribbica YS03 (KCTC 12821BP) was pH 6.

< Experimental Example  4> Pikia Caribica YS03 ( Pichia caribbica YS03 ) ( KCTC  &Lt; / RTI &gt; 12821BP) C 12 ) Comparison of substrate uptake rate

To investigate the alkane (C 12 ) consumption rate and the amount of produced bacterium in the alkane (C 12 ) substrate culture of Pichia caribbica YS03 (KCTC 12821BP), 20 g / L of alkane to C 12) that are not included in this amino acid was added as a carbon source YNB (Yeast Nitrogen base (without amino acid)) medium Pichia Kariya vicar YS03 (Pichia caribbica YS03; KCTC 12821BP ) ( example 1) and as compared to the strain of the same type Pichia caribbica (KCTC17289) (Comparative Example 3) and the standard strain Candida tropicalis (ATCC 20336) (Comparative Example 4) were cultured. The experimental results are shown in FIGS. 5 and 6. FIG.

As shown in FIG. 5, the alkane (C 12 ) consumption rate of Pichia caribbica YS03 (KCTC 12821BP) (Example 1) was 4.3 g / L per day, (Pichia caribbica; KCTC17289) (Comparative example 3) alkane (C 12) were consumed for consumption Kane (C 12), seen in about 30% faster than the rate of a day 3.3 g / L of cell production also compared to strain ( Which is about 13% higher than that of Comparative Example 3).

In addition, as shown in Figure 6, it was found that at a rate of about 42% faster than the alkane (C 12 ) consumption rate of 3.10 g / L per day of the standard strain Candida tropicalis (ATCC 20336) (Comparative Example 4) Alcaine (C 12 ) was consumed and the amount of the cells produced was about 55% higher than that of the standard strain (Comparative Example 4).

<110> LOTTE CHEMICAL CORPORATION <120> NOVEL STRAIN NOVEL STRAIN PICHIA CARIBBICA YS03 AND USES THEREOF <130> DPA-0674 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 1089 <212> DNA <213> Pichia caribbica YS03 <400> 1 gactcactat agggcgagct cggtacccgg gcgaattcca agctttccgt aggtgaacct 60 gcggaaggat cattacagta ttcttttgcc agcgcttaac tgcgcggcga aaaaccttac 120 acacagtgtc tttttgatac agaactcttg ctttggtttg gcctagagat aggttgggcc 180 agaggtttaa caaaacacaa tttaattatt tttattgata gtcaaatttt gaattaatct 240 tcaaaacttt caacaacgga tctcttggtt ctcgcatcga tgaagaacgc agcgaaatgc 300 gataagtaat atgaattgca gattttcgtg aatcatcgaa tctttgaacg cacattgcgc 360 cctctggtat tccagagggc atgcctgttt gagcgtcatt tctctctcaa acccccgggt 420 ttggtattga gtgatactct tagtcgaact aggcgtttgc ttgaaaagta ttggcatggg 480 tagtactgga tagtgctgtc gacctctcaa tgtattaggt ttatccaact cgttgaatgg 540 tgtggcggga tatttctggt attgttggcc cggccttaca acaaccaaac aagtttgacc 600 tcaaatcagg taggaatacc cgctgaactt aagcatatca ataagcggag gaagatctgg 660 atcccctcta gagtcgacct gcaggcatgc aagcttggcg taatcatggt catagctgtt 720 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa 780 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact 840 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg ggccaacgcg 900 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc 960 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta tacggttatc 1020 cacagaatca ggggattacg cangnngacc atgtgagcna aagggccagc aaagncaggn 1080 anccgtaaa 1089 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 18s ITS quatrer primer Forward <400> 2 tccgtaggtg aacctgcgg 19 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 18S ITS quarter primer Reverse <400> 3 tcctccgctt attgatatgc 20

Claims (7)

Pichia caribbica YS03 (KCTC 12821BP) strain. The method according to claim 1,
The strain is a strain of Pichia caribbica YS03 (KCTC 12821BP), wherein at least one selected from alkanes, fatty acids and fatty acid derivatives is used as a carbon source. 3. The method of claim 2,
Wherein the carbon source is selected from the group consisting of dodecane, methyl laulate, lauric acid, and derivatives thereof, or a combination thereof. The Pichia caribbica YS03) (KCTC 12821BP) strain. The method according to claim 1,
Wherein said strain exhibits reactivity as shown in the following table in terms of carbon atomization ability: Pichia caribbica YS03 (KCTC 12821BP) strain.
[table]
Figure pat00003
 The method according to claim 1,
Wherein said strain exhibits cell growth at an acidity of from pH 4 to pH 7. Pichia caribbica YS03 (KCTC 12821BP) strain. A method for treating wastewater using the strain of any one of claims 1 to 5.  A method for producing dodecanedioic acid (DDDA) using one or more carbon sources selected from alkanes, fatty acids and fatty acid derivatives using the strain of any one of claims 1 to 5.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111334441A (en) * 2020-02-24 2020-06-26 华中农业大学 Acid-reducing yeast strain and application thereof
CN114504000A (en) * 2022-02-17 2022-05-17 浙江省林业科学研究院 Environment-friendly enzyme and rapid preparation method and application thereof
CN115161252A (en) * 2022-06-24 2022-10-11 西南大学 Application of phenylalanine in preparation of reagent for improving antagonistic yeast biocontrol effect and preventing and treating postharvest diseases of jujube fruits

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111334441A (en) * 2020-02-24 2020-06-26 华中农业大学 Acid-reducing yeast strain and application thereof
CN111334441B (en) * 2020-02-24 2021-06-08 华中农业大学 Acid-reducing yeast strain and application thereof
CN114504000A (en) * 2022-02-17 2022-05-17 浙江省林业科学研究院 Environment-friendly enzyme and rapid preparation method and application thereof
CN114504000B (en) * 2022-02-17 2023-06-27 浙江省林业科学研究院 Environment-friendly ferment as well as rapid preparation method and application thereof
CN115161252A (en) * 2022-06-24 2022-10-11 西南大学 Application of phenylalanine in preparation of reagent for improving antagonistic yeast biocontrol effect and preventing and treating postharvest diseases of jujube fruits
CN115161252B (en) * 2022-06-24 2024-03-15 西南大学 Application of phenylalanine in preparation of reagent for improving antagonistic yeast biocontrol efficacy and preventing and treating jujube fruit postharvest diseases

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