CN114231448B - Burkholderia contaminated with indole degradation capability and application thereof - Google Patents

Burkholderia contaminated with indole degradation capability and application thereof Download PDF

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CN114231448B
CN114231448B CN202111491187.6A CN202111491187A CN114231448B CN 114231448 B CN114231448 B CN 114231448B CN 202111491187 A CN202111491187 A CN 202111491187A CN 114231448 B CN114231448 B CN 114231448B
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indole
burkholderia
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degrading
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CN114231448A (en
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王颖思
周刚
谢小保
彭红
施庆珊
李彩玲
李素娟
彭如群
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Institute of Microbiology of Guangdong Academy of Sciences
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    • 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
    • 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
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2101/00—Nature of the contaminant
    • C02F2101/30—Organic compounds
    • C02F2101/38—Organic compounds containing nitrogen

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Abstract

The invention discloses a Burkholderia contaminated with indole degradation capability and application thereof. The strain is Burkholderia plantarii (Burkholderia contaminans) DM32, and is deposited in the Guangdong province microorganism strain collection center, the deposition number is GDMCC No. 62032, and the deposition date is 2021, 11 months and 4 days. Experiments prove that the strain DM32 can completely remove 1mmol/L indole within 24 hours, the optimal temperature for degrading the indole is 30 ℃, the optimal pH is 5-7, and simultaneously, the degradation speed of the indole can be effectively accelerated by increasing the initial inoculation amount of the strain DM32. The efficiency of degrading indole by the Burkholderia DM32 is good, and the method has a certain application prospect.

Description

Burkholderia contaminated with indole degradation capability and application thereof
Technical Field
The invention relates to the technical field of microorganisms and environmental engineering, in particular to Burkholderia contaminated with indole degradation capability and application thereof.
Background
Indole is an azaaromatic compound, is one of common pollutants in industrial and agricultural wastewater, has stable chemical properties and strong foul smell, is mainly secreted by algae which are propagated in large scale due to eutrophication in water bodies, contains high concentration of indole in excreta of animals such as mice, dogs, pigs and the like, and can produce a large amount of wastewater containing indole in industrial production such as petroleum processing, pharmaceutical production and the like, thereby polluting soil, groundwater, surface water and even indoor environment. Indoles are also known as mutagens and carcinogens, and present a long-lasting hazard to human health and the ecological environment.
The traditional physical and chemical methods degrade or eliminate indole, and have the problems of large reagent usage amount, high cost, chemical residue and the like. The microorganism has the characteristics of high growth and propagation speed, large biomass, high environmental adaptability and the like, so that the microorganism is utilized to degrade indole, and the microorganism-utilized indole has the advantages of high efficiency, green and the like, and intermediate metabolites such as indigo, isatin, indirubin and the like obtained by utilizing the microorganism to degrade indole can be developed, and the indole-degraded intermediate products have high application value in the fields of printing and dyeing, preparation of special high polymer materials, food pigments and the like, so that the microorganism is utilized to degrade indole to obtain high added value products, and certain economic benefit is generated.
The study of the aerobic degradation of indole by microorganisms starts in the last 20 th century, and indole degrading strains mainly comprise pseudomonas, alcaligenes, copper rod, comamonas, streptomyces and the like. Meanwhile, some fungi such as Aspergillus niger, thermomyces lanuginosus, endophytic fungi, white rot fungi and the like can also complete indole degradation under specific conditions.
Disclosure of Invention
Aiming at the difficult problem that indole is difficult to remove in polluted environment in the prior art, and effectively utilizing the characteristic that indole can be used as a bacterial carbon source, the invention provides a bacterial strain for degrading indole and an application optimal condition, thereby improving the biological removal efficiency of indole.
The first object of the present invention is to provide a burkholderia contaminated (Burkholderia contaminans) DM32, wherein the burkholderia contaminated DM32 is isolated from production water, the gene sequence of the 16s RNA from 1 st to 1434 th is shown in SEQ ID No.1, and the strain is deposited in the cantonese province microorganism strain collection (GDMCC), address: first, the middle road 100 # building 59 building 5 in the View district of Guangzhou city, guangdong province, post code: 510070 the deposit number is GDMCC No. 62032 and the deposit date is 2021, 11 and 4.
The second object of the invention is to provide the application of the Burkholderia contaminated (Burkholderia contaminans) DM32 in indole degradation. Further, the application of the Burkholderia (Burkholderia contaminans) DM32 in bioremediation of indole polluted environments is provided.
A third object of the present invention is to provide the use of said Burkholderia contaminated (Burkholderia contaminans) DM32 for culturing in a medium having indole as sole carbon source.
The fourth object of the invention is to provide the application of the Burkholderia contaminated DM32 in preparing indole degrading microbial agents.
The fifth object of the invention is to provide an indole degrading microbial agent, which contains the Burkholderia contaminated (Burkholderia contaminans) DM32 as a main active ingredient.
The sixth object of the invention is to provide the application of the microbial agent in bioremediation of indole polluted environment.
The seventh object of the present invention is to provide a method for degrading indole, specifically, inoculating the Burkholderia contaminated (Burkholderia contaminans) DM32 into an indole-containing system for culturing, so as to degrade indole.
Preferably, the culture conditions of the culture are 30 ℃ and the pH is 5-7.
According to the invention, the efficiency of degrading indole by DM32 is improved by adjusting the initial consumption of the contaminated Burkholderia DM32 bacterial liquid and the culture conditions (such as temperature and pH value), so that the efficient degradation of indole is realized.
The invention has the beneficial effects that:
(1) The Burkholderia contaminated DM32 obtained by the invention has the capacity of degrading indole, and the bacterial strain, namely the Burkholderia contaminated DM32, is found for the first time to have the capacity of degrading indole.
(2) The invention is proved by experiments: indole is used as the only carbon source, the efficiency of removing indole of Burkholderia DM32 at 30 ℃ and pH 5-7 is highest, and 1mmol/L indole can be degraded within 24 hours.
(3) The invention is proved by experiments: the indole degradation speed of the Burkholderia DM32 is accelerated with the increase of the initial added bacterial liquid concentration.
(4) The optimal use condition of the Burkholderia DM32 and the indole degradation provided by the invention lays a research foundation for preparing biological products to remove indole pollutants in the environment.
Burkholderia contaminated according to the invention (Burkholderia contaminans) was deposited at the Cantonese microbiological culture Collection center (GDMCC) at 11/4/2021, address: building 5, building 59, guangzhou City, guangdong, first, china, qinghai, china: 510070 and the deposit number is GDMCC No. 62032.
Drawings
FIG. 1 is a graph showing the growth and indole degradation curves of Burkholderia contamination DM32
FIG. 2 is the effect of temperature on the degradation of indole by Burkholderia DM32.
FIG. 3 is the effect of pH on indole degradation by Burkholderia DM32.
FIG. 4 is the effect of varying initial concentrations of Burkholderia contamination DM32 broth on indole degradation.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
M9 medium: each liter contains Na 2 HPO 4 6.8g，KH 2 PO 4 3.0g，NaCl 0.5g，NH 4 Cl 1.0g，MgSO 4 2.0mmol，CaCl 2 0.1mmol, the solvent is water.
Indole extraction and content determination in a culture medium: centrifuging the culture medium at 10000rpm for 1min, collecting supernatant, adding equal volume of ethyl acetate, extracting, shaking thoroughly, standing for 30min, and collecting supernatant. The supernatant was filtered through a 0.22 μm filter and stored in a sample bottle. High performance liquid chromatography (Thermo U3000) was run on a C18 column (Thermo, 5 μm, 250X 4.6 mm) flow rate: 0.5mL/min, mobile phase: 80% methanol-water, column temperature: indole peak appearance was detected at 265nm ultraviolet wavelength at 30℃and each sample was kept for 30min. Standard curves were made using 1mM, 0.8mM, 0.6mM, 0.4mM, 0.2mM, 0mM indole standards as controls.
Example 1 isolation, purification and identification of indole degrading bacteria
1. Separation of indole degrading bacteria
1mL of the sample is taken and added into nutrient agar culture medium (10.0 g of peptone, 3.0g of beef extract powder, 5.0g of sodium chloride, 15.0g of agar and water as solvent) for 2 days at 37 ℃ for collecting production water, and single colony is picked into M9 culture medium with 0.5mmol/L indole as a unique carbon source for 2 days at 37 ℃. Observing turbidity of the culture medium, picking out turbid colony, inoculating into M9 culture medium of 1mmol/L indole, standing at 37deg.C, culturing, sampling every 4 hr, and measuring OD within 48 hr 600 And indole content in the medium, 3 replicates per set of experiments. Selecting the strain with highest indole degradation rate as an experimental strain, and naming the strain DM32.
As shown in FIG. 1, the indole content in the medium decreased as the bacteria grew, indicating that the bacteria grew using indole as a carbon source, and that indole was fully utilized for about 28 hours, but no indole was detected in the medium, but bacteria were still able to continue to grow.
2. Identification of indole degrading bacteria
The monoclonal strain DM32 was picked up from the separation plate, inoculated into LB medium (10.0 g/liter of peptone, 5.0g of yeast extract, 10.0g of NaCl, water as solvent) and cultured overnight at 37 ℃. And (3) taking overnight culture bacterial liquid, centrifuging at 10000rpm for 1min, collecting bacterial bodies, extracting DM32 genome as a template, amplifying 16s RNA fragments, and obtaining a PCR product for sequencing. The sequence of the gene from 1 st to 1434 st is shown in SEQ ID NO. 1. The similarity between the strain DM32 and Burkholderia contaminated (B.contaminans) is found to be 99.86 percent at most. Thus, strain DM32 was identified as Burkholderia contaminated (B.contaminans).
Example 2 investigation of degradation efficiency of indole degrading bacterium by environmental factors
1. Effect of different temperatures on degradation of indole by Strain DM32
Strain DM32 was grown overnight in LB medium, the next day the cells were washed 3 times with M9 medium, and the bacteria were resuspended in M9 medium to adjust the bacterial concentration to OD 600 =1.0 ready for use. The M9 culture medium with 1mmol/L indole is added into the bacterial suspension with 10% v/v, and the bacterial suspension is placed into an incubator with the temperature of 20 ℃,25 ℃, 30 ℃,37 ℃ and 40 ℃ respectively for static culture for 24 hours. Samples were sampled at 0h and 24h. The amount of indole remaining in the medium was measured by the method described above, and each experiment was repeated 3 times.
As a result, the culture temperature was 20℃and 25℃and the degradation rate was 24.3%, 43.3%, 100%, 94.7% and 53.7% at 37℃and 40℃respectively, as shown in FIG. 2. The strain DM32 has the best effect of degrading indole at the culture temperature of 30 ℃ and completely degrades 1mmol/L indole within 24 hours.
2. Effect of different pH on degradation of indole by strain DM32
The M9 culture medium was adjusted to pH=4, 5, 6, 7, 8, 9 with 1mol/L HCl and 1mol/L NaOH, respectively, sterilized at high temperature and high pressure, 1mmol/L indole and 10% v/v bacterial suspension treated in step 1 were added, and the mixture was placed in a 30℃incubator for stationary culture for 24 hours. Samples were sampled at 0h and 24h. Indole content in the medium was measured by the method described above and each set of experiments was repeated 3 times.
As shown in FIG. 3, strain DM32 degraded the indole at the fastest rate at medium pH values of 5, 6 and 7, with an initial concentration of 1mmol/L indole for 24h. At pH values of 4, 8 and 9, strain DM32 was degraded by 44.8%, 46.1% and 51.7% within 24 hours, respectively.
3. Influence of different bacterial liquid concentrations on degradation of indole by strain DM32
1mmol/L M9 culture medium of indole is prepared, 10% v/v, 20% v/v and 40% v/v of bacterial suspension treated in step 1 are respectively added into different volumes, and the bacterial suspension is placed into a 30 ℃ incubator for static culture for 24 hours. Samples were sampled at 0h, 16h and 24h. Indole content in the medium was measured by the method described above and each set of experiments was repeated 3 times.
As shown in FIG. 4, the indole degrading speed of the strain DM32 is accelerated along with the increase of the initial bacterial liquid concentration, and the indole degrading efficiency of the bacterial liquid added with 20% v/v and 40% v/v is respectively improved by 14.0% and 23.9% in 16h compared with that of the bacterial liquid added with 10% v/v, but at 24h, the indole in the culture medium can be completely degraded no matter the initial inoculation amount of the bacterial liquid.
The above results indicate that the indole contaminants in the environment can be degraded by contaminating burkholderia (b.contacts) DM32, the optimal degradation condition is 30 ℃ and ph=5-7, and the amount of the bacterial cells can be appropriately increased to increase the indole degradation rate, so that the contamination burkholderia (b.contacts) DM32 has practical application potential and prospect under specific conditions.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that the above-mentioned preferred embodiment should not be construed as limiting the invention, and the scope of the invention should be defined by the appended claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and such modifications and adaptations are intended to be comprehended within the scope of the invention.
Sequence listing
<110> the institute of microbiology of the academy of sciences of Guangdong province (microbiological analysis and detection center of Guangdong province)
<120> Burkholderia contaminated with indole degrading ability and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1434
<212> DNA
<213> Burkholderia DM32 (Burkholderia contaminans DM)
<400> 1
gcggtggcgc gcttacctgc agtcgaacgg cagcacgggt gcttgcacct ggtggcgagt 60
ggcgaacggg tgagtaatac atcggaacat gtcctgtagt gggggatagc ccggcgaaag 120
ccggattaat accgcatacg atctacggat gaaagcgggg gaccttcggg cctcgcgcta 180
tagggttggc cgatggctga ttagctagtt ggtggggtaa aggcctacca aggcgacgat 240
cagtagctgg tctgagagga cgaccagcca cactgggact gagacacggc ccagactcct 300
acgggaggca gcagtgggga attttggaca atgggcgaaa gcctgatcca gcaatgccgc 360
gtgtgtgaag aaggccttcg ggttgtaaag cacttttgtc cggaaagaaa tccttggctc 420
taatacagtc gggggatgac ggtaccggaa gaataagcac cggctaacta cgtgccagca 480
gccgcggtaa tacgtagggt gcaagcgtta atcggaatta ctgggcgtaa agcgtgcgca 540
ggcggtttgc taagaccgat gtgaaatccc cgggctcaac ctgggaactg cattggtgac 600
tggcaggcta gagtatggca gaggggggta gaattccacg tgtagcagtg aaatgcgtag 660
agatgtggag gaataccgat ggcgaaggca gccccctggg ccaatactga cgctcatgca 720
cgaaagcgtg gggagcaaac aggattagat accctggtag tccacgccct aaacgatgtc 780
aactagttgt tggggattca tttccttagt aacgtagcta acgcgtgaag ttgaccgcct 840
ggggagtacg gtcgcaagat taaaactcaa aggaattgac ggggacccgc acaagcggtg 900
gatgatgtgg attaatycra tgcaacgcga aaaaccttac ctaccctttg acatggtcgg 960
aatcccgctg agaggtggga gtgctcgaaa gagaaccggc gcacaggtgc tgcatggctg 1020
tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa cccttgtcct 1080
tagttgctac gcaagagcac tctaaggaga ctgccggtga caaaccggag gaaggtgggg 1140
atgacgtcaa gtcctcatgg cccttatggg tagggcttca cacgtcatac aatggtcgga 1200
acagagggtt gccaacccgc gagggggagc taatcccaga aaaccgatcg tagtccggat 1260
tgcactctgc aactcgagtg catgaagctg gaatcgctag taatcgcgga tcagcatgcc 1320
gcggtgaata cgttcccggg tcttgtacac accgcccgtc acaccatggg agtgggtttt 1380
accagaagtg gctagtctaa ccgcaaggag gacggtcacc acgtgatacc ctgg 1434

Claims (10)

1. Burkholderia contaminated (Burkholderia contaminans) DM32, deposited with the accession number: GDMCC No. 62032.
2. Use of burkholderia contaminated according to claim 1 (Burkholderia contaminans) DM32 for degrading indole.
3. The use according to claim 2, characterized in that it is in the bioremediation of indole-contaminated environments.
4. Use of burkholderia contaminated according to claim 1 (Burkholderia contaminans) DM32 for cultivation in medium with indole as sole carbon source.
5. Use of burkholderia contaminated according to claim 1 (Burkholderia contaminans) DM32 for the preparation of an indole degrading microbial agent.
6. An indole-degrading microbial agent, which is characterized by comprising Burkholderia contaminated (Burkholderia contaminans) DM32 according to claim 1 as a main active ingredient.
7. The use of the microbial agent of claim 6 in bioremediation of indole-contaminated environments.
8. A method for degrading indole, wherein the method comprises inoculating the burkholderia contaminated (Burkholderia contaminans) DM32 of claim 1 into an indole-containing system for culturing to degrade indole.
9. The method for degrading indole according to claim 8, wherein the cultivation temperature of the cultivation is 30 ℃.
10. The method for degrading indole according to claim 8, wherein the culture pH of the culture is 5 to 7.
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