CN110029072B - Agrobacterium and application thereof in degradation of 3-hydroxypyridine - Google Patents
Agrobacterium and application thereof in degradation of 3-hydroxypyridine Download PDFInfo
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Abstract
The invention belongs to the technical field of microorganisms and biodegradation thereof, and discloses an Agrobacterium tumefaciens 3-PB belonging to Agrobacterium (Agrobacterium sp.) aiming at the problem of lack of a strain for efficiently and specifically degrading 3-hydroxypyridine, wherein the strain has a preservation number of: CCTCC M2018821. The invention also discloses application of the strain in the aspect of 3-hydroxypyridine. The strain can grow by using 3-hydroxypyridine as the only carbon nitrogen source and energy, can completely degrade the 3-hydroxypyridine with the concentration of less than or equal to 1500mg/L within 66 hours, can tolerate 2000mg/L of 3-hydroxypyridine, has high substrate specificity and good degradation effect on high-concentration pyridine-polluted wastewater, can be used for biological treatment processes of petroleum wastewater and the like, and has good application prospect.
Description
Technical Field
The invention belongs to the technical field of microorganisms and biodegradation thereof, and particularly relates to agrobacterium tumefaciens and application thereof in degrading 3-hydroxypyridine.
Background
Pyridine and its derivatives are important aromatic nitrogen heterocyclic compounds. The pyridine ring is an important composition structure of plant alkaloid, coenzyme and the like, and along with the death and the putrefaction of a host, the pyridine compounds enter the soil environment and are quickly degraded without polluting the environment. However, a large amount of pyridine and its derivatives are used in the industrial synthesis of pesticides, herbicides and petroleum products, and are inevitably released into the environment beyond the self-cleaning capability of the environment. Pyridine and its derivatives have high biological toxicity to humans and other organisms and therefore it is desirable to reduce these contaminants to a suitable concentration range before they enter the environment.
One effective way to remove pyridine is to completely convert it into carbon dioxide, water, ammonium ions, etc. by means of biodegradation. Since 1910, only several strains of Achromobacter and Nocardia which can degrade 3-hydroxypyridine have been reported, but all strains which can degrade 3-hydroxypyridine are not specific, so that the degradation efficiency is low, and no specific process for degrading 3-hydroxypyridine is reported. Therefore, the screening of 3-hydroxypyridine degrading strains with strong substrate specificity, high degradation efficiency, saline-alkali tolerance and stable properties has important practical significance and research value for removing pyridine compounds in the environment and clarifying the metabolic mechanism of 3-hydroxypyridine.
Disclosure of Invention
Aiming at the problems of weak specificity, low 3-hydroxypyridine degradation efficiency and lack of efficient and specific 3-hydroxypyridine degradation strains of the existing pyridine degradation strains, the invention provides an efficient and specific 3-hydroxypyridine degradation bacterium Agrobacterium (Agrobacterium sp) 3-PB, and the Agrobacterium is applied to removal of 3-hydroxypyridine pollutants in environmental petroleum polluted water.
The inventor obtains an Agrobacterium sp 3-PB from the oil polluted soil of Liaohekou oil field through enrichment, separation, purification and screening, the bacterial strain is preserved in China center for type culture collection with the preservation number of CCTCC M2018821. This strain constitutes a first aspect of the invention.
The strain can efficiently and specifically degrade 3-hydroxypyridine. This constitutes a second aspect of the invention. The water body polluted by petroleum contains 3-hydroxypyridine, so the strain can be used for removing the 3-hydroxypyridine pollutant in the water body polluted by petroleum. Preferably, the agrobacterium is added into the wastewater containing 3-hydroxypyridine, the reaction temperature is 25-40 ℃, and the reaction pH is 7.0-9.0.
An Agrobacterium (Agrobacterium sp.)3-PB, which is preserved in China center for type culture collection with the preservation number of CCTCC M2018821.
The microbial agent is characterized by containing the agrobacterium.
The agrobacterium or the microbial inoculum is applied to degradation of 3-hydroxypyridine.
The application is characterized in that the agrobacterium is added into a water body containing 3-hydroxypyridine.
Further, the application is characterized in that the reaction temperature of the degradation process is 25-40 ℃, preferably 25-37 ℃, and more preferably 30 ℃.
Further, the use is characterized in that the pH of the degradation process is 7.0-9.0, preferably 8-9, more preferably 8.
Furthermore, the application is characterized in that the concentration of the 3-hydroxypyridine is 200mg/L-2000 mg/L.
Further, the application is characterized in that the water body is a petroleum polluted water body.
Has the advantages that:
(1) the agrobacterium 3-PB of the invention can utilize 3-hydroxypyridine as the only carbon nitrogen source and energy source, does not need to be induced, has stable performance, can grow in LB solid/liquid culture medium, and can also grow in basic inorganic salt liquid culture medium with 3-hydroxypyridine concentration of 0.2 mg/mL-2.0 mg/mL, the growth temperature is 25-42 ℃, the pH value is 7.0-10.0, the salt concentration is 0-3%, the optimum growth temperature is 30 ℃, and the optimum pH value is 8.0.
(2) The strain has high substrate specificity, can only degrade 3-hydroxypyridine, but not pyridine, 3-methylpyridine, 2-methylpyridine, 4-hydroxypyridine, 2-hydroxypyridine and other substrates, and can be used for specifically removing 3-hydroxypyridine pollutants in the environment.
(3) The strain has high degradation efficiency, can completely degrade 3-hydroxypyridine with the concentration of less than or equal to 1500mg/L within 66 hours, can tolerate 2000mg/L of 3-hydroxypyridine, has good degradation effect on high-concentration pyridine-polluted wastewater, can be used for biological treatment processes of petroleum wastewater and the like, and has good application prospect.
(4) The agrobacterium is used as plant symbiotic bacteria, has the advantage of environmental friendliness, is prepared into a microbial inoculum and is added into the environment to remove the 3-hydroxypyridine pollutants in the environment, and has the advantages of mild treatment conditions, high efficiency, no secondary pollution and the like.
(5) The genetic operation system of the agrobacterium is complete, and provides a convenient condition for researching the metabolic mechanism of the agrobacterium.
Drawings
FIG. 1 is a photograph of a colony of Agrobacterium 3-PB isolated in the present invention on LB solid medium;
FIG. 2 is a scanning electron micrograph of Agrobacterium 3-PB isolated according to the present invention;
FIG. 3 is a tree of the evolution of the strain Agrobacterium 3-PB;
FIG. 4 is a graph showing the growth and degradation curves of the strain Agrobacterium 3-PB;
FIG. 5 is a graph showing the effect of degradation process of Agrobacterium 3-PB at different substrate concentrations;
FIG. 6 shows the effect of different culture temperatures on the degradation of 3-hydroxypyridine by Agrobacterium 3-PB;
FIG. 7 effect of initial pH on 3-hydroxypyridine degradation by Agrobacterium strain 3-PB;
FIG. 8 enhanced treatment effect of Agrobacterium 3-PB on high concentration 3-hydroxypyridine-added petroleum wastewater
The preservation date of the strain is 11 months and 23 days in 2018, and the preservation number is as follows: CCTCC M2018821, classified name of Agrobacterium sp 3-PB, preservation unit name of China center for type culture Collection, address of eight Lopa Jia mountain in Wuhan City, Wuhan university Collection center, Taisui code: 430072.
Detailed Description
The present invention will be described in further detail with reference to the following embodiments and the accompanying drawings.
Example 1: isolation and identification of the Strain
1. Isolation of Agrobacterium 3-PB
(1) The strain is derived from the surface soil (0-8cm) polluted by oil in Liaohe oil field.
(2) Weighing about 5g of petroleum-contaminated soil, and placing in 50mL of MSN culture medium containing 500 mg/L3-hydroxypyridine (MSN culture medium formula: each 1L contains 12.6g K)2HPO4·3H2O，3.4g KH2PO4，1.0g Na2SO4，0.2g MgSO4·7H2O and 0.5mL of a metal ion solution. ) In the medium, the cells were cultured for 7d at 30 ℃ and 170rpm on a constant temperature shaking table. Taking 5mL of the upper layer culture solution, transferring the upper layer culture solution into a fresh 3-hydroxypyridine + MSN culture medium, continuing to culture, and repeating the process for 4-5 times to obtain a relatively clear culture solution. The culture solution was mixed according to the following formula 10-3，10-4，10-5，10-6The mixture is diluted according to the proportion, coated on a 3-hydroxypyridine + MSN solid culture medium plate and placed in a 30 ℃ constant temperature incubator for 48 hours. And (3) selecting a single colony on the plate to a fresh 3-hydroxypyridine + MSN liquid culture medium, verifying the growth condition of the single colony, preserving the strain with higher degradation efficiency, and performing subsequent experiments.
2. Identification of Agrobacterium 3-PB
(1) Morphological characteristics of strain and physiological and biochemical characteristics thereof
Bacterial colonies of the strain on an LB solid plate are white and semitransparent, are round, have neat edges, are smooth and moist in surface, and have white round dots in the center (figure 1).
The scanning electron microscope picture of the strain is shown in figure 2, the length is 0.8-1.4 μm, the diameter is about 0.2-0.4 μm, the strain is short rod-shaped, and the surface is smooth.
The gram staining result of the strain is negative, and the strain is a gram-negative bacterium.
(2) 16S rRNA identification of Agrobacterium 3-PB
The 16S rRNA sequence of the strain Agrobacterium 3-PB is amplified by bacterial 16S rRNA universal primers 27F (5'-GAG AGT TTG ATC CTG GCT CAG-3') and 1492R (5'-ACG GAT ACC TTG TTA CGA CTT-3'), sequenced and the sequencing result is shown in SEQ ID NO. 1. BLAST alignment showed 99% sequence similarity of the strain to Agrobacterium, and the clade is shown in fig. 3, classifying the strain to Agrobacterium (Agrobacterium sp.).
Example 2: degradation performance of 3-hydroxypyridine by agrobacterium tumefaciens 3-PB
Method for measuring 3-hydroxypyridine by monitoring the change in the concentration of 3-hydroxypyridine with High Performance Liquid Chromatography (HPLC) from Waters, column 4.6 × 250mm, 5 μm Welch Xtimate C18,the mobile phase is as follows: 20% methanol and 80% 1mM H2SO4The sample injection amount is 2 mu L, the column temperature is 30 ℃, the flow rate is 1mL/min, the detector is a DAD detector, and the detection wavelength range is 200nm-400 nm. The sample processing method comprises the following steps: adding 2 times volume of methanol into the sample, standing at 4 deg.C for 10min, centrifuging at 10000r/min for 2min, collecting supernatant, filtering with 0.22 μm filter membrane, and detecting.
(1) Inoculating the activated agrobacterium 3-PB strain into a fresh MSN liquid culture medium containing 1000 mg/L3-hydroxypyridine according to the inoculation amount of 1%, placing the MSN liquid culture medium in a constant-temperature shaking table at 30 ℃ and 170rpm for culture, sampling every 12h, and measuring an absorption value (representing the thallus concentration) of OD600nm and an ultraviolet absorption value (representing the substrate concentration) of 200nm-350 nm. The results are shown in FIGS. 4 and 5: with the continuous increase of the concentration of the bacteria, the concentration of the 3-hydroxypyridine is gradually reduced, and the growth trend is basically consistent with the degradation process, which shows that the strain can grow by using the 3-hydroxypyridine as a unique carbon nitrogen source and an energy source, and has higher degradation effect on the 3-hydroxypyridine.
(2) Influence of initial substrate concentration on the strain degradation process:
the activated agrobacterium 3-PB strain is inoculated into MSN liquid culture media with different initial concentrations (200, 500, 1000 and 1500mg/L) of 3-hydroxypyridine according to the inoculation amount of 1 percent, the MSN liquid culture media are placed in a constant-temperature shaking table at 30 ℃ and 170rpm for culture, the residual amount of the 3-hydroxypyridine in the culture solution is measured by sampling every 12 hours, and as shown in figure 5, the delay period required by the strain degradation is longer along with the increase of the concentration of the 3-hydroxypyridine in the culture medium, and the time required for completely degrading the strain is longer.
(3) Influence of different culture temperatures on degradation of 3-hydroxypyridine by Agrobacterium 3-PB strain:
the activated agrobacterium 3-PB strain is inoculated into MSN liquid culture medium of 3-hydroxypyridine of 1000mg/L according to the inoculation amount of 1%, the MSN liquid culture medium is respectively placed in constant-temperature shaking tables of 170rpm at different temperatures (25 ℃, 30 ℃ and 37 ℃) for culture, samples are taken every 12 hours to determine the residual amount of the 3-hydroxypyridine in the culture solution, and the degradation effect is best at the temperature of 30 ℃ as shown in figure 6.
(4) Effect of initial pH on 3-hydroxypyridine degradation by Agrobacterium 3-PB strains:
inoculating the activated agrobacterium tumefaciens 3-PB strain into a fresh MSN liquid culture medium containing 1000 mg/L3-hydroxypyridine according to the inoculation amount of 1%, adjusting the initial pH of the culture medium to 7.0, 8.0 and 9.0 respectively, culturing in a constant-temperature shaking table at 30 ℃ and 170rpm, sampling every 12h to determine the residual amount of the 3-hydroxypyridine in the culture solution, as shown in FIG. 7, the degradation effect is best under the condition that the initial pH is 8.0, and the degradation effect of the initial pH of 9.0 is better than that of the pH7.0, which indicates that the strain is suitable for an alkaline environment.
Example 3: enhanced treatment of petroleum wastewater added with high-concentration 3-hydroxypyridine by using bacterial strain
Inoculating the activated agrobacterium 3-PB strain into 50mL LB liquid culture medium according to the inoculation amount of 1%, culturing for 48h in a constant temperature shaking table at 30 ℃ and 170rpm, and collecting the strain. The collected bacteria were added to a flask containing 50mL of petroleum wastewater (from Liaohe oil field, pH 8.1) +1000 mg/L3-hydroxypyridine, the control group was petroleum wastewater without 3-PB degrading bacteria plus 1000 mg/L3-hydroxypyridine, and the flask was placed in a 30 ℃ and 170rpm constant temperature shaking table to detect the residual amount of 3-hydroxypyridine in the system, as shown in FIG. 8, it can be seen that the concentration of 3-hydroxypyridine in the control group was slightly decreased, while the concentration of 3-hydroxypyridine in the wastewater with 3-PB degrading bacteria was gradually decreased and completely degraded within 60h, indicating that the strain had a good effect of degrading 3-hydroxypyridine in petroleum wastewater.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Sequence listing
<110> Qingdao agricultural university
<120> agrobacterium tumefaciens and application thereof in degradation of 3-hydroxypyridine
<160>1
<170>SIPOSequenceListing 1.0
<210>1
<211>1409
<212>DNA
<213> Agrobacterium sp.)
<400>1
agagcgggta ggcggaggct aacacatgca agtcgagcgc cccgcaaggg gagcggcaga 60
cgggtgagta acgcgtggga atctaccgtg ccctacggaa tagctccggg aaactggaat 120
taataccgta tacgccctac gggggaaaga tttatcgggg tatgatgagc ccgcgttgga 180
ttagctagtt ggtggggtaa aggcctacca aggcgacgat ccatagctgg tctgagagga 240
tgatcagcca cattgggact gagacacggc ccaaactcct acgggaggca gcagtgggga 300
atattggaca atgggcgcaa gcctgatcca gccatgccgc gtgagtgatg aaggtcttag 360
gattgtaaag ctctttcacc ggtgaagata atgacggtaa ccggagaaga agccccggct 420
aacttcggtg ccaagcagcc gcgggtaata cgaagggggc tagcgttgtt cggaattact 480
gggcgtaaag cgccacgtag gcggatattt aagtcagggg tgaaatccca gagctcaact 540
ctggaaactg cctttgatac tgggtatctt gagtatggaa agaggtgagt ggaaattccg 600
agtgtagagg tgaaattcgt agatattcgg aggaacacca gtggcgaagg cggctcactg 660
gtccattact gacgctgagg tgcgaaagcg tggggagcaa acaggattag ataccctggt 720
agtccacgcc gtaaacgatg aatgttagcc gtcgggcagt atactgttcg gtggcgcagc 780
taacgcatta aacattccgc ctggggagta cggtcgcaag attaaaactc aaaggaattg 840
acgggggccc gcacaagcgg tggagcatgt ggtttaattc gaagcaacgc gcagaacctt 900
accagctctt gacatccggg tcgcggacag tggagacatt gtccttcagt taggctggac 960
ccaggacagg tgctgcatgg ctgtcgtcag ctcgtgtcgt gagatgttgg ggttaagtcc 1020
cgcaacgagc gcaaccctcg cccttagttg ccagcattca gttgggcact ctaaggggac 1080
tgcccgtgat aagcccaaag gaaggtgggg atgacgtcaa gtcctcatgg cccttacggg 1140
ctgggctaca cacgtgctac aatggtggkg acagtgggca gcgagacagc gatgtcgagc 1200
taatctccaa aagccatctc agttcggatt gcactctgca actcgaagtg catgaagttg 1260
gaatcgctag taatcgcgga atcagcatgc cgcggtgaat acgttcccgg gccttgtaca 1320
caccgcccgt ccacaccatg gggagttggt tttacccgaa ggtagtgcgc taacccgcaa 1380
gggaggcagc taaccacggt gtcaggcgg 1409
Claims (10)
1. An Agrobacterium (Agrobacterium sp.)3-PB, which is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO: m2018821.
2. A microbial agent, characterized in that it contains the Agrobacterium of claim 1.
3. Use of the agrobacterium of claim 1 or the microbial inoculum of claim 2 for degrading 3-hydroxypyridine.
4. The use according to claim 3, wherein the Agrobacterium is added to a body of water containing 3-hydroxypyridine.
5. Use according to claim 4, wherein the water body is a petroleum contaminated water body.
6. The use according to any one of claims 3 to 5, wherein the 3-hydroxypyridine is present in a concentration of from 200mg/L to 2000 mg/L.
7. Use according to claim 3, wherein the reaction temperature of the degradation process is between 25 ℃ and 40 ℃.
8. Use according to claim 3, wherein the reaction temperature of the degradation process is between 30 ℃ and 37 ℃.
9. Use according to claim 3, characterized in that the pH of the degradation process is 7.0-9.0.
10. Use according to claim 3, characterized in that the pH of the degradation process is 8.0.
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