CN109593673B

CN109593673B - Flavobacterium JX-1 and application thereof in sewage treatment

Info

Publication number: CN109593673B
Application number: CN201811533538.3A
Authority: CN
Inventors: 李娅; 郑国华; 张莉莉; 邱小忠; 涂祖新
Original assignee: INSTITUTE OF MICROBIOLOGY JIANGXI ACADEMY OF SCIENCES
Current assignee: INSTITUTE OF MICROBIOLOGY JIANGXI ACADEMY OF SCIENCES
Priority date: 2018-12-14
Filing date: 2018-12-14
Publication date: 2021-05-07
Anticipated expiration: 2038-12-14
Also published as: CN109593673A

Abstract

The invention provides a new strain flavobacterium (flavobacterium)Flavobacterium sp.) JX-1 and application thereof in sewage treatment. The invention screens out a flavobacterium strain (f) (in the Poyang lake dry period water body) by separationFlavobacterium sp.) JX-1GDMCC No.60424, the separated strain can degrade organic nitrogen, and the ammoniation capability of the strain is used for purifying nitrogen-containing wastewater, so that the strain has wide application value in the technical field of microbial strain application.

Description

Flavobacterium JX-1 and application thereof in sewage treatment

Technical Field

The invention relates to the technical field of microbial strains and application thereof, in particular to novel flavobacterium JX-1 and the technical field of application thereof in sewage treatment.

Background

Under the influence of human activities, a large amount of nutrient substances such as nitrogen, phosphorus and the like required by organisms enter a lake slow-flow water body to cause the phenomenon of water body eutrophication, and the eutrophication causes disastrous consequences such as water body function reduction, water quality deterioration, massive plankton death and the like. Nitrogen is a basic nutrient element required by biological life activities and is one of key elements for causing water eutrophication, the nitrogen circulation plays a very important role in the water nutrient circulation, the water nitrogen pollution in China is very serious, the availability of water resources is restricted, and the good development of human life health and social economy is directly influenced.

Ammoniation bacteria, which are microorganisms that release ammoniacal nitrogen from macromolecular organic nitrogen-containing substances through oxidation, hydrolysis and reduction, belong to heterotrophic bacteria and are ties connecting organic nitrogen and inorganic nitrogen in nitrogen circulation. The organic nitrogen is an important component of the water body, and the ammoniation bacteria decompose organic nitrogen compounds to generate ammonia nitrogen through the ammoniation effect in the water body, which plays an important role in a water body ecological system. Therefore, the development of the screening of new ammoniated bacteria strains and the research of the biological characteristics of the ammoniated bacteria strains have important theoretical value and practical significance.

Disclosure of Invention

The invention provides a novel strain Flavobacterium sp JX-1 and application thereof in sewage treatment. According to the invention, a Flavobacterium (Flavobacterium sp) JX-1GDMCC No.60424 is separated and screened out from the water body in the Poyang lake dry water period, the separated strain can be used for degrading organic nitrogen, the ammoniation capability of the strain is used for purifying nitrogen-containing wastewater, and the method has a wide application value in the technical field of microbial strain application.

The invention adopts the main technical scheme that:

according to the particularity of the geographical environment of the Poyang lake, the ammoniation bacteria and the denitrifying bacteria are cultured, separated and screened from the water body of the Poyang lake to obtain a batch of bacteria, a Flavobacterium sp (Flavobacterium sp.) with the number of JX-1 is separated and screened from the bacteria, and the JX-1 strain belongs to the Flavobacterium sp (Flavobacterium sp.) as a result of 16S rDNA sequence analysis, phylogenetic analysis, microbiological characteristic analysis and fatty acid component analysis, wherein the strain has the optimal growth conditions as follows: the culture medium can grow at the temperature of 10-37 ℃, the optimal growth temperature is 28 ℃, the pH value is 5.0-11.0, the optimal pH is 7.0, the culture medium can grow under the condition that the NaCl concentration is 0-2.0%, the optimal NaCl concentration is 1%, the culture time is 24-48h, and the culture medium is R2A culture medium. The JX-1 strain is determined to be a member of the genus Flavobacterium by referring to Bojie's system bacteriology appraisal Manual (ninth edition) and the like to carry out morphological, physiological and biochemical tests on the strain, but has the characteristics different from the common member strains of the genus Flavobacterium and the characteristics of some new strains.

Furthermore, the invention carries out gene sequencing on the strain with the number of JX-1, and the sequence is shown in SEQ ID NO. 1 provided in the attachedThe obtained sequences are compared at NCBI website, and the comparison analysis shows that the 16S rRNA gene sequence of the strain JX-1 and Flavobacterium hibernum DSM12611^THas the highest homology of 98.6% with Flavobacterium pertussis DSM 6368^TThe homology of (A) is 98%, strain JX-1 is homologous to Flavobacterium hibernum DSM12611^TAnd Flavobacterium pectinovorum DSM 6368^TThe relationship of (a) is closest. Clustering analysis is carried out by using MEGA 5.0 software through a Neighbor-Joining method, and the results of comparison analysis show that the strain JX-1 and Flavobacterium pectinovorum DSM 6368^TThe credibility of the strain from the same branch is 100%, which shows that the strain has extremely high support rate as a new strain and excellent stability in an evolutionary tree, and the strain JX-1 is determined to be a new species of Flavobacterium (Flavobacterium sp) through serial strain identification, has the characteristic of typicality of the new strain, and is tentatively named as the Flavobacterium (Flavobacterium sp) JX-1 from the taxonomic angle.

This strain was deposited in the international collection of microorganisms under the budapest treaty on the filing date: guangdong province culture Collection of microorganisms (GDMCC). Address: building 5 of the first 100 th college 59 of the first furnance, zhou, china, guangdong province, microbiological research institute, postcode: 510075. the preservation date is 2018, 7 months and 30 days, and the strain preservation number is GDMCC No. 60424. The microorganism is identified and temporarily named as Flavobacterium sp JX-1.

The bacterial colony of a strain Flavobacterium sp (JX-1) on an R2A culture medium is yellow, round or irregular, which shows smooth and translucent and irregular edges; gram staining is negative, the thallus is rod-shaped, the length of the thallus is about 0.9-1.2 μm, the width of the thallus is about 0.3-0.5 μm, the thallus does not move, does not have cilia around the body, does not have capsules, and does not form spores.

Furthermore, the Flavobacterium (Flavobacterium sp.) JX-1GDMCC No.60424 provided by the invention has ammoniation capability.

Further, the Flavobacterium sp JX-1GDMCC No.60424 is applied to degrading organic nitrogen.

Furthermore, the Flavobacterium sp JX-1GDMCC No.60424 has the capability of degrading organic nitrogen, so that the Flavobacterium sp JX-1GDMCC has important value for the utilization of ammonifying bacteria and the research and the utilization of purifying water bodies by degrading organic nitrogen.

By implementing the specific technical scheme provided by the invention, the following beneficial effects can be achieved by implementing the content of the invention:

(1) the Flavobacterium sp JX-1GDMCC No.60424 provided by the invention is a typical new strain which can grow on an R2A culture medium, the bacterial colony is golden yellow and transparent, the surface is smooth, the length of the bacterial colony is about 0.9-1.2 μm, and the width is about 0.3-0.5 μm; the strain JX-1 can grow at the temperature of 10-37 ℃, the optimal growth temperature is 28 ℃, the pH value is 5.0-11.0, the optimal pH is 7.0, the strain can grow under the condition that the NaCl concentration is 0-2.0%, the optimal NaCl concentration is 1%, the culture time is 24-48h, the culture medium is R2A culture medium, the strain has high water purification property, and simultaneously has the characteristics of simple culture condition and rapid propagation, the strain is preliminarily determined to be a new strain of Flavobacterium sp, has the characteristic of typicality of a new strain, and is temporarily named as Flavobacterium sp JX-1 from the taxonomic angle.

(2) According to the invention, a Flavobacterium strain JX-1GDMCC No.60424 is separated and screened out in the Poyang lake dry period water body, the Flavobacterium strain JX-1GDMCC No.60424 has certain ammoniation capability, the separated strain can degrade organic nitrogen, and the JX-1 strain has ammoniation capability and high organic nitrogen removal rate under the conditions that the pH value is 5, the culture temperature is 25 ℃ and the inoculation amount is 3%. The ammoniation capability of the strain is utilized to purify the nitrogen-containing wastewater, and the strain has wide application value in the technical field of microbial strain application.

Drawings

FIG. 1 shows a Flavobacterium (Flavobacterium sp.) JX-1 phylogenetic tree.

FIG. 2 shows the morphology of Flavobacterium (Flavobacterium sp.) JX-1 cells.

FIG. 3 is a graph showing the effect of pH on the performance of Flavobacterium (Flavobacterium sp.) JX-1 ammoniation.

FIG. 4 is a graph showing the effect of temperature on the performance of Flavobacterium (Flavobacterium sp.) JX-1 ammoniation.

FIG. 5 is a graph showing the effect of inoculum size on the performance of Flavobacterium (Flavobacterium sp.) JX-1 ammoniation.

Detailed Description

The present invention will be described below by way of examples, but the present invention is not limited to the following examples. All raw and auxiliary materials selected for use in the present invention, as well as methods for culturing the selected bacterial species, are well known and used in the art, and all percentages referred to herein are by weight unless otherwise indicated.

The samples used in the present invention: water body in dry stage of Yanghu.

The following basic culture media are adopted in the embodiment of the invention: the R2A culture medium comprises yeast extract powder 0.5g, peptone 0.5g, casein hydrolysate 0.5g, glucose 0.5g, soluble starch 0.5g, potassium dihydrogen phosphate 0.3g, anhydrous magnesium sulfate 0.024g, sodium pyruvate 0.3g, agar 15.0g, distilled water 1.0L, and pH 7.2; ammonification ability test medium comprises 5g of peptone, 0.25g of sodium chloride, 0.01g of ferrous sulfate heptahydrate, 0.5g of dipotassium hydrogen phosphate, 0.5g of magnesium sulfate heptahydrate, and distilled water to 1.0L, and the pH value is 7.2.

The following kit is adopted in the embodiment of the invention: DNA extraction kit (Bio-engineering works, Ltd.).

The first embodiment is as follows: separation, screening and identification of flavobacterium JX-1

Separation and purification:

adding 10ml of water sample into 90ml of sterile water from a Poyang lake dry water stage water body sample, uniformly mixing, then carrying out gradient dilution by 10 times to obtain a diluent, taking 1ml of the diluent, inoculating into a test tube containing 5ml of an ammonification bacteria culture medium, culturing at 28 ℃ for 10-14 days, taking out 250ul of the culture solution, reacting with 100ul of a Nashin reagent, wherein ammonia exists if the culture solution is brown, and the culture solution is positive; absorbing the culture solution in the ammonifying bacteria culture medium test tube with positive reaction, and diluting the culture solution to 10 degrees in a gradient manner^-4Uniformly coating 100 mu L of diluent on an ammonifying bacteria culture medium plate, culturing for 2-3 days at 28 ℃, and repeating for 3-4 times to purify the flavobacterium JX-1. The purified Flavobacterium (Flavobacterium sp.) JX-1 is inoculated on an R2A slant, cultured in an incubator at 28 ℃ for 2-3 days at constant temperature, and stored at 4 ℃ for later use.

(II) classification and identification:

1. flavobacterium (Flavobacterium sp.) JX-116S rDNA sequencing and analysis:

(1) extraction of PCR template DNA

Inoculating Flavobacterium (Flavobacterium sp.) JX-1 into an R2A liquid culture medium, culturing at 28 ℃ for 18h to obtain thallus cells, and extracting total genomic DNA by using a magnetic bead method bacterial genomic DNA extraction kit.

(2) PCR amplification

Primer:

PrimerA:5’-AGAGTTTGATCCTGGCTCAG-3’；

PrimerB:5’-TACGGCTACCTTGTTACGACTT-3’。

the total volume of the PCR reaction system is 25 mu L, and the PCR amplification condition is 94 ℃ for 5 min; 94 ℃ for 45s, 56 ℃ for 45s, 72 ℃ for 45s, 30 cycles; 10min at 72 ℃.

(3) Sequence determination

The PCR amplification product is detected by electrophoresis, purified and sequenced, and the sequence is shown as SEQ ID NO. 1. The obtained sequence is compared and analyzed on an NCBI website, and the comparison and analysis show that the 16S rRNA gene sequence of the strain JX-1 and Flavobacterium hibernum DSM12611^THas the highest homology of 98.6% with Flavobacterium pectinovorum DSM 6368^TThe homology of (A) is 98%, strain JX-1 is homologous to Flavobacterium hibernum DSM12611^TAnd Flavobacterium pectinovorum DSM 6368^TThe relationship of (a) is closest. Clustering analysis is carried out by using MEGA 5.0 software through a Neighbor-Joining method, and the results of comparison analysis show that the strain JX-1 and Flavobacterium pectinovorum DSM 6368^TThe credibility of the strain from the same branch is 100%, which shows that the strain has extremely high support rate as a new strain and excellent stability in an evolutionary tree, and the strain JX-1 is determined to be a new species of Flavobacterium (Flavobacterium sp) through serial strain identification, has the characteristic of typicality of the new strain, and is tentatively named as the Flavobacterium (Flavobacterium sp) JX-1 from the taxonomic angle.

2. Physiological and biochemical assays

Flavobacterium JX-1 strain (F.hibernum DSM 12611) with the highest homology with the strain^T、F.pectinovorum DSM 6368^T) The microbiological characteristics were analyzed and the results are shown in Table 1.

Table 1: comparison of physiological and biochemical experimental results of Flavobacterium JX-1 and Flavobacterium strains with high homology

Note: positive; -, negative; w, weak positive.

As can be seen from Table 1, Flavobacterium of the present invention has the highest homology with the strain F.hibernum DSM12611^TPectnovorum DSM 6368^TThe difference exists in physiological and biochemical characteristics, G + C mol% and the like. The strain JX-1 has trypsin activity, nitrate is reduced to be negative, and D-arabinose, L-arabinose, D-xylose, D-galactose, D-fructose, L-rhamnose, arbutin, D-lactose, D-sucrose, inulin and D-raffinose are not utilized. Two strains with the highest homology F.hibernum DSM12611^TPectnovorum DSM 6368^TThere are differences in physiological and biochemical characteristics, G + C mol%, and the kind and content of fatty acids in cell walls. Hibernum DSM12611^TPectnovorum DSM 6368^THas no trypsin activity, and can utilize D-arabinose, L-arabinose, D-xylose, D-galactose, D-fructose, L-rhamnose, arbutin, D-lactose, D-sucrose, inulin and D-raffinose. Combining the above data, it was possible to demonstrate the strains JX-1 and F.hibernum DSM12611^TPectnovorum DSM 6368^TAre not of the same species.

3. Analysis of fatty acid composition

The strain was cultured at 25 ℃ on R2A agar and cells in exponential growth phase were used. The fatty acids of strain JX-1 and its reference strain were extracted by reference to Miller (1982), measured by gas chromatography, and the data obtained were analyzed by MIDI database (Hewlett-Packard Co., Palo Alto, California, USA) to obtain the fatty acid content and content of the strain as shown in Table 2. The content of each component of fatty acid of the strain JX-1 is obviously different from that of a reference strain.

Table 2: comparison of cell wall fatty acid types and contents of Flavobacterium and reference strains thereof

Note: TR, minor amount.

The results of 16S rDNA sequence analysis, phylogenetic analysis, microbiological characteristic analysis and fatty acid component analysis are integrated, which shows that the strain JX-1 is a new bacterial strain of the genus Flavobacterium, and is tentatively named as (Flavobacterium sp.) JX-1. The strain is preserved in Guangdong province microorganism strain preservation center in 2018, 7 and 30 months, and the address is as follows: the strain preservation number is GDMCC No.60424 in No. 59 building 5 of Zhou Lu Dazhou No. 100, the institute of microbiology, Guangdong province.

The morphology of Flavobacterium (Flavobacterium sp.) JX-1GDMCC No.60424 under an electron microscope is shown in figure 2, and the colony of the Flavobacterium cultured on a plate for 36 hours is yellow, round or irregular, which shows smooth and translucent and irregular edge; gram staining is negative, the thallus is rod-shaped, the length of the thallus is about 0.9-1.2 μm, the width of the thallus is about 0.3-0.5 μm, the thallus does not move, does not have cilia around the body, does not have capsules, and does not form spores.

Example two: flavobacterium (Flavobacterium sp.) JX-1 ammoniation performance assay

Flavobacterium (Flavobacterium sp.) JX-1 strain is cultured in R2A culture medium for 18h, then respectively inoculated into 100mL ammoniation capability test culture medium, cultured in a constant temperature shaking table at 150R/min for 40h under different test conditions, culture solution is centrifuged at 6000R/min to take supernatant, NH in the supernatant is measured by adopting a Nashin reagent photometry₄ ⁺-N concentration. The pH values (5.0, 6.0, 7.0, 8.0 and 9.0), the culture temperatures (25 ℃, 30 ℃, 35 ℃ and 40 ℃) and the inoculum sizes (1%, 3%, 5%, 7% and 9%) were investigated for ammonia in the strainInfluence of chemical properties, 3 parallel groups were set for each test, and medium not inoculated with Ammonibacterium was used as a blank control. Except for different temperature experiments, all the other samples were cultured at 25 ℃.

(1) Influence of pH value on ammoniation performance of strain

As shown in the attached figure 3, the experimental result shows that the pH value has a remarkable influence on the ammoniation capacity of the flavobacterium JX-1 strain, the ammonia nitrogen concentration in the culture solution reaches 43.58mg/L when the pH value is 5, and the ammonia nitrogen concentration in the culture solution gradually decreases along with the increase of the pH value, so that the flavobacterium JX-1 strain has a better effect of removing organic nitrogen in an environment with a low pH value.

(2) Influence of temperature on Ammonification Properties of the Strain

The test result of the ammoniation capability of the flavobacterium JX-1 strain at the temperature is shown in figure 4, the ammoniation effect of the flavobacterium JX-1 strain at the temperature is obviously influenced, the capability of removing organic nitrogen is better within the range of 25-30 ℃, and the ammoniation capability of the flavobacterium JX-1 strain is lower when the temperature is lower than 35 ℃.

(3) Influence of inoculum size on ammoniation performance of strain

As shown in figure 5, the test result shows that the inoculation amount has obvious influence on the ammoniation capability of the flavobacterium JX-1 strain, the organic nitrogen removing capability is better within the range of 1% -3%, the ammonia nitrogen concentration in the culture solution is not lower than 43.78mg/L, the organic nitrogen removing effect is better, when the inoculation amount is more than 5%, the ammoniation capability of the flavobacterium JX-1 strain is reduced, but the ammonia nitrogen concentration in the culture solution within the range of 1% -9%, the organic nitrogen removing capability is stronger.

The experiments show that the Flavobacterium sp JX-1GDMCC No.60424 is separated and screened from the water body in the Poyang lake dry period, the Flavobacterium sp JX-1GDMCC has certain organic nitrogen removal capability, the separated strain can degrade the organic nitrogen, and the JX-1 strain has ammoniation capability and high organic nitrogen removal rate under the conditions that the pH value is 5, the culture temperature is 25 ℃ and the inoculation amount is 3%.

Example three: flavobacterium (Flavobacterium sp.) JX-1 ammoniation performance determination in sewage

Carrying out enrichment culture on Flavobacterium (Flavobacterium sp.) JX-1 strain in R2A culture medium for 18h, inoculating 3% of strain into 100mL of polluted lake water, carrying out culture on the polluted lake water at 25 ℃ in a constant temperature shaking table at 150R/min under the condition that the pH value is 5, centrifuging the sewage at 6000R/min, taking supernatant, and measuring NH in the supernatant by adopting a Nassner reagent photometry₄ ⁺-N concentration. The ammonia nitrogen concentration in the sewage is measured to reach 41.69mg/L, and the flavobacterium JX-1 strain has ammoniation capacity and higher organic nitrogen removal rate. The strain is used for purifying the nitrogen-containing wastewater by utilizing the capability of degrading organic nitrogen, and has wide application value in the technical field of microbial strain application.

The above examples are merely illustrative for clearly illustrating the present invention and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications can be made while remaining within the scope of the present invention.

Sequence listing

<110> institute of microbiology of academy of sciences of Jiangxi province

Li Ya

<120> Flavobacterium JX-1 and application thereof in sewage treatment

<141> 2018-12-14

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1436

<212> DNA

<213> Flavobacterium (Flavobacterium sp.)

<400> 1

gatgaacgct agcggcaggc ttaacacatg caagtcgagg ggtatatgtc ttcggatata 60

gagaccggcg cacgggtgcg taacgcgtat gcaatctacc ttttacagag ggatagccca 120

gagaaatttg gattaatacc tcatagcata gcagcttcgc atgaagccac tattaaagtc 180

acaacggtaa aagatgagca tgcgtcccat tagctagttg gtaaggtaac ggcttaccaa 240

ggctacgatg ggtaggggtc ctgagaggga gatcccccac actggtactg agacacggac 300

cagactccta cgggaggcag cagtgaggaa tattggacaa tgggcgcaag cctgatccag 360

ccatgccgcg tgcaggatga cggtcctatg gattgtaaac tgcttttgta cgagaagaaa 420

cactacttcg tgaagtagct tgacggtatc gtaagaataa ggatcggcta actccgtgcc 480

agcagccgcg gtaatacgga ggatccaagc gttatccgga atcattgggt ttaaagggtc 540

cgtaggcggt ttagtaagtc agtggtgaaa gcccatcgct caacggtgga acggccattg 600

atactgctag acttgaatta ttaggaagta actagaatat gtagtgtagc ggtgaaatgc 660

ttagagatta catggaatac caattgcgaa ggcaggttac tactaatgga ttgacgctga 720

tggacgaaag cgtgggtagc gaacaggatt agataccctg gtagtccacg ccgtaaacga 780

tggatactag ctgttgggag caatttcagt ggctaagcgg aagtgataag tatcccacct 840

ggggagtacg ttcgcaagaa tgaaactcaa aggaattgac gggggcccgc acaagcggtg 900

gagcatgtgg tttaattcga tgatacgcga ggaaccttac caaggcttaa atgtagattg 960

accgatttgg aaacagatct ttcgcaagac aatttacaag gtgctgcatg gttgtcgtca 1020

gctcgtgccg tgaggtgtca ggttaagtcc tataacgagc gcaacccctg ttgttagttg 1080

ccatcgagtg atgtcgggaa ctctaacaag actgccagtg caaactgtga ggaaggtggg 1140

gatgacgtca aatcatcacg gcccttacgc cttgggctac acacgtgcta caatggccgg 1200

tacagagagc agccactggg tgaccaggag cgaatctata aagccggtca cagttcggat 1260

cggagtctgc aactcgactc cgtgaagctg gaatcgctag taatcggata tcagccatga 1320

tccggtgaat acgttcccgg gccttgtaca caccgcccgt caagccatgg aagctggggg 1380

tgcctgaagt tggtgaccgc aaggagctgc ctagggtaaa actggtaact agggct 1436

Claims

1. Flavobacterium (A)Flavobacterium sp.) JX-1, characterized in that, said Flavobacterium (F.), (Flavobacterium sp.) The strain accession number of JX-1 is GDMCC No. 60424.

2. The Flavobacterium (F) of claim 1Flavobacterium sp.) Application of JX-1 in sewage treatment.

3. A Flavobacterium (F) of claim 2Flavobacterium sp.) The application of JX-1 in sewage treatment is characterized in that after a flavobacterium JX-1 strain is cultured in an R2A culture medium for 18 hours, 1 to 3 weight percent of flavobacterium JX-1 is inoculated into 100mL of an ammoniation capability test culture medium, and when the temperature is 25 ℃, the pH value is 5.0 to 7.0, and the constant temperature shaking culture is carried out for 40 hours at 150R/min.

4. A Flavobacterium (F) of claim 2Flavobacterium sp.) The application of JX-1 in sewage treatment is characterized in that flavobacterium (F.) (B.)Flavobacterium sp.) After the JX-1 strain is subjected to enrichment culture in an R2A culture medium for 18h, 1-3% of flavobacterium JX-1 is inoculated into 100mL of polluted lake water according to the weight percentage, and the strain is cultured for 40h at 25 ℃ by a constant temperature shaking table at 150R/min under the condition that the pH value is 5.