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CN109055282B - A new strain of Klebsiella pneumoniae and its isolation method and application - Google Patents

A new strain of Klebsiella pneumoniae and its isolation method and application Download PDF

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CN109055282B
CN109055282B CN201811132568.3A CN201811132568A CN109055282B CN 109055282 B CN109055282 B CN 109055282B CN 201811132568 A CN201811132568 A CN 201811132568A CN 109055282 B CN109055282 B CN 109055282B
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phenol
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klebsiella pneumoniae
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张安龙
赵呈馨
王雪青
王欣奇
罗清
房玉婷
王哲毅
陈朵
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Xi'an Longhua Environmental Protection Technology Co ltd
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Abstract

本发明公开了一株肺炎克雷伯氏菌新菌株及其分离方法和应用，所述新菌株从造纸污水处理厂曝气池活性污泥中分离、筛选得到，保藏在中国普通微生物菌种保藏管理中心，保藏名称为，ZS‑01，保藏号是16041，保藏日期为2018年6月30日。所述菌株特性及性能：(1)特性：在苯酚MedA固体培养基上形成的菌落为较短粗的杆菌，大小0.5～0.8×1～2μm,单独或成双排列，形成淡黄色菌落，质地均匀且不透明；在MPYE固体培养基上形成较大的灰白色粘性菌落；无鞭毛，有荚膜，菌株属肠杆菌科，为革兰氏染色阴性的粗短杆菌。(2)性能：具有一定的苯酚降解能力；对苯酚具有较高的耐受性。其优点是：能够降解较高浓度的苯酚，为有效处理较高浓度的含酚废水提供了新菌源。

Figure 201811132568

The invention discloses a new strain of Klebsiella pneumoniae, a separation method and application thereof. The new strain is obtained by separation and screening from activated sludge of aeration tanks in a papermaking sewage treatment plant, and is preserved in the Chinese Common Microorganism Culture Collection Management Center, the deposit name is ZS‑01, the deposit number is 16041, and the deposit date is June 30, 2018. The characteristics and performance of the strains: (1) Characteristics: the colonies formed on the phenol MedA solid medium are relatively short and thick bacilli, with a size of 0.5-0.8 × 1-2 μm, singly or in pairs, forming pale yellow colonies with texture. Homogeneous and opaque; large gray-white viscous colonies formed on MPYE solid medium; no flagella, with capsule, the strain belongs to Enterobacteriaceae, and is Gram-negative Brevibacterium tumefaciens. (2) Performance: It has a certain phenol degradation ability; it has a high tolerance to phenol. The advantage is that it can degrade phenol with a higher concentration, and provides a new bacterial source for the effective treatment of wastewater containing a higher concentration of phenol.

Figure 201811132568

Description

Novel Klebsiella pneumoniae strain and separation method and application thereof
Technical Field
The invention belongs to the technical field of bioengineering, and relates to a novel Klebsiella pneumoniae strain, a separation method thereof and application thereof.
Background
Phenol is present in various industrial wastewaters of refineries, petrochemical manufacturing, and the like. Phenol is also a major organic component in coal gasification and liquefaction processes, condensate streams. Industrial waste water from pharmaceutical factories, raw wood processing factories, paper mills, plastic factories, etc. also contains phenol. Untreated phenol-containing industrial wastewater cannot be discharged into open waters, otherwise it can produce off-flavors in drinking water and food processing water. The environmental protection agency sets an emission standard: the surface water has a phenol content of less than one part per billion. The phenol-containing industrial wastewater has wide pollution area and great harm effect, and brings serious harm to resident diet, fish culture and crop irrigation. Phenol concentrations in water above 50ppb are toxic to aquatic organisms, 4.7-130mg/100mL phenol in blood is lethal, phenol damages the nervous system and major organs including spleen, pancreas and liver, and prolonged intake of phenol at concentrations of 10-240mg/L causes sore lips, diarrhea, dark urine and impaired vision.
Phenol is a colorless needle crystal with special odor and is toxic, and a phenol molecule is formed by directly connecting a hydroxyl on a benzene ring. Such a structure is hardly converted into a ketone-type structure due to the stability of the benzene ring. The oxygen atom of the phenolic hydroxyl group is hybridized by sp2 to provide a lone pair of electrons to form a delocalized bond with 6 carbon atoms of the benzene ring. The large pi bond strengthens the acidity of the enol, and the electron-pushing effect of the hydroxyl group strengthens the polarity of the O-H bond, so that the hydrogen of the hydroxyl group in the phenol can be ionized.
At present, the treatment methods of phenol are mainly divided into 3 types, namely a chemical method, a physical method and a biological method, phenol is degraded by a microbiological method, the method has mild conditions and strong specificity, the problems of secondary pollution and the like are not involved, and the method is an environment-friendly treatment method. Therefore, the idea of degrading phenol by a microbiological method attracts the wide attention of scholars at home and abroad.
Disclosure of Invention
The invention aims to provide a novel Klebsiella pneumoniae strain as well as a separation method and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:
the invention discloses a Klebsiella Pneumoniae strain which is named Klebsiella Pneumoniae sp and is preserved in the China general microbiological culture collection center, the preservation number of the strain is CGMCC No.16041, and the preservation date is 7 months and 2 days in 2018.
Preferably, the Klebsiella pneumoniae strain forms a thick and short yellow bacillus colony when cultured on a phenol MedA solid medium, the size of the thick and short yellow bacillus colony is 0.5-0.8 multiplied by 1-2 mu m, and the thick and short yellow bacillus colony is singly or doubly arranged and has uniform and opaque texture.
Preferably, the klebsiella pneumoniae strain forms larger off-white sticky colonies when cultured on MPYE solid medium; flagellate, capsulated, gram-negative brevibacterium crassicolum.
Preferably, the Klebsiella pneumoniae strain grows well at 30-35 ℃ and pH 7.
The invention also discloses a separation method of the Klebsiella pneumoniae strain, which comprises the following steps:
1) sampling: injecting sterilized culture solution with phenol as the only carbon source into activated sludge in an aeration tank of a papermaking sewage treatment plant to prepare bacterial solution;
2) enrichment: inoculating the strain prepared in the step 1) into 100mL of MPYE culture solution according to the inoculation amount of 10%, performing shake cultivation at 35 ℃ at 200r/min, observing the turbidity condition of the solution during the cultivation, and taking supernatant to prepare enriched bacterial solution;
3) primary screening: adding 15mL of the enriched bacterial liquid obtained in the step 2) into a conical flask filled with 135mL of MedA culture medium, placing 3 parallel samples in each group into a constant-temperature shaking table at 35 ℃, culturing at 200r/min, gradually inoculating the turbid solution into the MedA culture medium with high phenol content, gradually screening and domesticating strains, and primarily screening to obtain bacteria;
4) re-screening: carrying out flat plate coating on the bacteria obtained by the primary screening in the step 3) on a MedA solid culture medium, and then placing the bacteria in a constant temperature incubator at 35 ℃ for culture; and (3) carrying out plate streaking for multiple times in an ultra-clean workbench to obtain separated pure bacteria, namely the Klebsiella pneumoniae strain.
Preferably, the formulation of the MPYE medium is as follows, with 1000mL of MPYE medium containing: 3g of peptone, 3g of yeast extract, 1M Mgcl 21.6 mL, 1M CaCl 21.6 mL and the balance of pure water.
Preferably, the preparation method of the MedA medium is as follows: preparing 1000mL MedA culture medium, and taking (NH)4)2SO40.4g, 2mL of 2% aspartic acid, 10mL of 10% NaCl, 0.2g of nitrilotriacetic acid, MgSO4·7H2O0.59 g, anhydrous CaCl2 0.05036g、FeSO4·7H2O 251.98mg、(NH4)6Mo24·4H2O 0.186mg、ZnSO4·7H2O 0.5475g、EDTA 125mg、H3BO4 5.7mg、MnSO4·H2O 77mg、CuSO4·5H2O 19.6mg、Co(NO3)2·6H212.4mg of O and the balance of distilled water; loading into 1000mL blue-cap glass bottle, sealing, capping, and autoclaving at 121 deg.C for 30min in Yamato autoclave; taking out, placing into an ultra-clean bench, ultraviolet sterilizing for 30min, cooling to room temperature, adding thiamine hydrochloride 0.5mg, nicotinic acid 1mg, and biotin 0.25 μ L, K2HPO4 1.7418g、KH2PO41.361g, and finally adding 800mg-2500mg of phenol with different gradients to prepare MedA culture media with different phenol gradients.
The invention also discloses application of the Klebsiella pneumoniae strain in degrading phenol-containing wastewater.
Preferably, the Klebsiella pneumoniae strain can degrade phenol-containing wastewater with the concentration of below 2500 mg/L.
Compared with the prior art, the invention has the following beneficial effects:
the novel strain is obtained by separating and screening activated sludge in an aeration tank of a papermaking sewage treatment plant, is preserved in China general microbiological culture collection management center with the preservation name of ZS-01 and the preservation number of 16041, and has the preservation date of 2018, 6 months and 30 days. The characteristics and performances of the strain are as follows: the single or double arrangement forms a light yellow colony which is uniform and opaque; larger off-white sticky colonies were formed on MPYE solid medium; nonflagellated, capsulated, strains belonging to the family Enterobacteriaceae, gram-negative Brevibacterium crassimum. The strain of the invention has certain phenol degradation capability; has higher tolerance to phenol, and provides a new bacterial source for effectively treating phenol-containing wastewater with higher concentration.
Drawings
FIG. 1 shows the form of Klebsiella pneumoniae ZS01 on MedA solid medium using phenol as carbon source;
FIG. 2 is a morphological diagram of Klebsiella pneumoniae ZS01 on MPYE solid medium according to the present invention;
FIG. 3 is a phylogenetic tree of Klebsiella pneumoniae ZS01 of the present invention.
Strain preservation
The new Klebsiella Pneumoniae strain is named ZS-01 and is preserved in China general microbiological culture Collection center, the microbial research institute of China academy of sciences No. 3 of West Lu No.1 of North Chen West of the south facing Yang, Beijing is with the preservation name of ZS-01, the classification name of the strain is Klebsiella Pneumoniae sp, the preservation number is 16041, and the preservation date is 7 months and 2 days in 2018.
Detailed Description
In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
The invention is described in further detail below with reference to the accompanying drawings:
example 1: isolation and characterization of the strains of the invention
(1) Sampling: directly selecting activated sludge in an aeration tank of a papermaking sewage treatment plant, bringing the activated sludge back to a laboratory by using a barrel, taking a small amount of sludge-water mixture of the activated sludge, injecting the sterilized culture solution with phenol as a unique carbon source, and putting the culture solution into a conical flask to prepare a bacterial liquid;
(2) enrichment: prepare 1000mL of MPYE medium: peptone 3g, yeast extract 3g, 1M Mgcl21.6mL、1M Cacl21.6mL and the balance of pure water; subpackaging 500mL of MPYE culture medium into 500mL blue-covered glass bottles, sealing and capping, and autoclaving at 121 deg.C for 30min in a Yamato autoclave; taking out, placing into an ultra-clean workbench, performing ultraviolet sterilization for 30min, inoculating the strain obtained in the step 1 into a 250mL conical flask containing 100mL of MPYE culture solution according to the inoculation amount of 10%, placing into a shaking table at 35 ℃ at 200r/min for culture, observing the turbidity condition of the solution during culture, and taking the supernatant to prepare enriched bacterial liquid;
(3) primary screening: 1000mL MedA medium was prepared: (NH)4)2SO40.4g, 2mL of 2% aspartic acid, 10mL of 10% NaCl, 0.2g of nitrilotriacetic acid, MgSO4·7H2O0.59 g, anhydrous CaCl2 0.05036g、FeSO4·7H2O 251.98mg、(NH4)6Mo24·4H2O 0.186mg、ZnSO4·7H2O 0.5475g、EDTA 125mg、H3BO4 5.7mg、MnSO4·H2O 77mg、CuSO4·5H2O 19.6mg、Co(NO3)2·6H212.4mg of O and the balance of distilled water; loading into 1000mL blue-cap glass bottle, sealing, capping, and autoclaving at 121 deg.C for 30min in Yamato autoclave; taking out, placing into an ultra-clean bench, ultraviolet sterilizing for 30min, cooling to room temperature, adding thiamine hydrochloride 0.5mg, nicotinic acid 1mg, and biotin 0.25 μ L, K2HPO4 1.7418g、KH2PO41.361g, finally adding 800mg-2500mg of phenol with different gradients to prepare MedA culture media with different phenol gradients; will be provided withAnd (3) adding 15mL of the enriched bacterial liquid obtained in the step (2) into a conical flask filled with 135mL of MedA culture medium, placing 3 parallel samples in each group in a constant-temperature shaking table at 35 ℃, culturing at 200r/min, gradually inoculating the solution into the MedA culture medium with high phenol content after the solution is turbid, and gradually screening and domesticating the strains.
(4) Re-screening: screening and domesticating bacteria with MedA culture medium with high phenol content in the step 3, carrying out plate coating on the MedA solid culture medium, and culturing in a constant temperature incubator at 35 ℃; and carrying out plate streaking for multiple times in an ultra-clean workbench to obtain separated pure bacteria.
The above strain screening process is carried out under aerobic condition.
The morphology of ZS01 on MedA solid medium with phenol as carbon source is shown in FIG. 1, and the morphology on MPYE solid medium is shown in FIG. 2.
(5) Strain identification
PCR amplification and sequence analysis of new strain ZS-0116S rDNA gene
Using ZS-01 DNA as a template, using a universal primer of a 16S rDNA gene to carry out PCR amplification, carrying out gel electrophoresis detection on a PCR product, recovering and sequencing an amplified fragment, calculating a genetic distance by using a Kimura-2-parameter method in a MEGA 4.0 software package, constructing a phylogenetic tree by using a Neighbor-Joining method, and repeatedly sampling for 1000 times to analyze the confidence coefficient of each branch of the phylogenetic tree. And compared with the sequences in GenBank, the homology with the Klebsiella pneumoniae is the highest and is 99 percent, as shown in figure 3. The length of 16S rDNA of the strain ZS-01 is 1396bp, and the sequence of the strain is detailed in a nucleotide sequence table and is shown in SEQ ID No. 1.
Example 2: new strain ZS-01 performance
(1) Degradation characteristic of phenol of strain
The 4-amino-pyridine method is adopted for measuring the content of phenol and preparing a standard curve.
The phenol degradation rate was calculated from the following formula:
phenol degradation rate (%) - (A)0－As)/A0×100％
Wherein A is0- -corresponding phenol content in the non-inoculated blank reference test
As- -phenol content of the inoculated sample liquid to be tested
(2) Optimal degradation condition of strain ZS-01 for phenol
The experimental method comprises the following steps: inoculating ZS-01 culture solution under different conditions, and placing in a constant temperature shaking table at 35 ℃ and 200r/min to study the phenol degradation performance of the strain. And (3) measuring the phenol degradation rate in different time periods by using a 4-aminoantipyrine method.
The experimental results are as follows: the optimal phenol degradation condition of the strain is that the inoculation amount is OD600When the concentration of L-glutamic acid was 4.5mmol/L, 30% of the culture was inoculated at 0.8, and the pH of the culture was 7.5; the strain ZS-01 can reach 100% in the degradation rate of phenol of 1000mg/L in 30 h.
The above examples show that the new strain ZS-01 has a higher phenol tolerance threshold and higher phenol degradation performance, and provides a new way for effectively treating phenol-containing wastewater with higher concentration.
The above-mentioned contents are only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited thereby, and any modification made on the basis of the technical idea of the present invention falls within the protection scope of the claims of the present invention.
Sequence listing
<120> novel Klebsiella pneumoniae strain and separation method and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1396
<212> DNA
<213> Klebsiella pneumoniae strain (Klebsiella pneumoniae)
<400> 1
gcaagtcgag cggtagcaca gagagcttgc tctcgggtga cgagcggcgg acgggtgagt 60
aatgtctggg aaactgcctg atggaggggg ataactactg gaaacggtag ctaataccgc 120
ataacgtcgc aagaccaaag tgggggacct tcgggcctca tgccatcaga tgtgcccaga 180
tgggattagc tagtaggtgg ggtaacggct cacctaggcg acgatcccta gctggtctga 240
gaggatgacc agccacactg gaactgagac acggtccaga ctcctacggg aggcagcagt 300
ggggaatatt gcacaatggg cgcaagcctg atgcagccat gccgcgtgtg tgaagaaggc 360
cttcgggttg taaagcactt tcagcgggga ggaaggcgtt aaggttaata accttggcga 420
ttgacgttac ccgcagaaga agcaccggct aactccgtgc cagcagccgc ggtaatacgg 480
agggtgcaag cgttaatcgg aattactggg cgtaaagcgc acgcaggcgg tctgtcaagt 540
cggatgtgaa atccccgggc tcaacctggg aactgcattc gaaactggca ggctagagtc 600
ttgtagaggg gggtagaatt ccaggtgtag cggtgaaatg cgtagagatc tggaggaata 660
ccggtggcga aggcggcccc ctggacaaag actgacgctc aggtgcgaaa gcgtggggag 720
caaacaggat tagataccct ggtagtccac gccgtaaacg atgtcgattt ggaggttgtg 780
cccttgaggc gtggcttccg gagctaacgc gttaaatcga ccgcctgggg agtacggccg 840
caaggttaaa actcaaatga attgacgggg gcccgcacaa gcggtggagc atgtggttta 900
attcgatgca acgcgaagaa ccttacctgg tcttgacatc cacagaactt agcagagatg 960
gattggtgcc ttcgggaact gtgagacagg tgctgcatgg ctgtcgtcag ctcgtgttgt 1020
gaaatgttgg gttaagtccc gcaacgagcg caacccttat cctttgttgc cagcggtccg 1080
gccgggaact caaaggagac tgccagtgat aaactggagg aaggtgggga tgacgtcaag 1140
tcatcatggc ccttacgacc agggctacac acgtgctaca atggcatata caaagagaag 1200
cgacctcgcg agagcaagcg gacctcataa agtatgtcgt agtccggatt ggagtctgca 1260
actcgactcc atgaagtcgg aatcgctagt aatcgtagat cagaatgcta cggtgaatac 1320
gttcccgggc cttgtacaca ccgcccgtca caccatggga gtgggttgca aaagaagtag 1380
gtagcttaac cttcgg 1396

Claims (2)

1.一种能够降解苯酚的肺炎克雷伯氏菌菌株，其特征在于，该肺炎克雷伯氏菌菌株命名为Klebsiella Pneumoniae，保藏于中国普通微生物菌种保藏管理中心，菌种保藏号为CGMCC No.16041，保藏日期为2018年7月2日，该肺炎克雷伯氏菌菌株在30℃~35℃，pH为7时生长状态良好。1. a Klebsiella pneumoniae bacterial strain capable of degrading phenol, is characterized in that, this Klebsiella pneumoniae bacterial strain is named Klebsiella Pneumoniae , is preserved in China General Microorganisms Culture Collection and Management Center, and bacterial classification preservation number is CGMCC No.16041, dated July 2, 2018, the Klebsiella pneumoniae strain grows well at 30°C~35°C and pH 7. 2.权利要求1所述的肺炎克雷伯氏菌菌株在降解含苯酚废水中的应用。2. the application of the described Klebsiella pneumoniae strain of claim 1 in degrading phenol-containing wastewater.
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CN114561323B (en) * 2022-02-23 2024-04-09 浙江台州秀川科技有限公司 Klebsiella pneumoniae capable of resisting salt and efficiently degrading gallic acid and application thereof
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