CN113005063B

CN113005063B - Pseudomonas putida GY13 and application thereof in sewage treatment

Info

Publication number: CN113005063B
Application number: CN202110322121.8A
Authority: CN
Inventors: 李少杰; 曹先贺; 孙宪昀; 张振颖; 胡成成
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2021-03-25
Filing date: 2021-03-25
Publication date: 2022-08-23
Anticipated expiration: 2041-03-25
Also published as: CN113005063A

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to the technical field of microbial wastewater treatment. The invention discloses a strain of Pseudomonas pseudomonads (Pseudomonas aeruginosa ivorans) GY13 with a preservation number of CGMCC No. 21281. The strain is preserved in China general microbiological culture Collection center (CGMCC) in 12 months and 2 days in 2020, is named as CGMCC No. 3 of Xilu No.1 of Beijing, facing Yang district, and has a preservation number of CGMCC No. 21281. The strain can realize the synchronous removal of ammonia nitrogen, total nitrogen and COD under aerobic conditions.

Description

Pseudomonas putida GY13 and application thereof in sewage treatment

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to the technical field of microbial wastewater treatment.

Background

Main pollution index substances with overproof detection in fresh water resources (including surface water, underground water and the like of rivers, lakes, various large hydraulic projects and the like) in China comprise Chemical Oxygen Demand (COD) and ammonia Nitrogen (NH) ₄ -N), Total Nitrogen (TN), and Total Phosphorus (TP), among others. Wherein the nitrogen element has a plurality of existing pollutant forms and is difficult to remove, and when nitrogen-containing pollutants such as ammonia, nitrite and the like with higher concentration are discharged into the environment, on one hand, eutrophication of water bodies can be caused, and on the other hand, biotoxicity can be generated to various organisms, so thatThe denitrification theory and technology are always the research hotspots in the field of sewage treatment.

Biological denitrification is the most economic and effective way for removing nitrogen in sewage at present. The traditional biological denitrification process comprises two processes of aerobic autotrophic nitrification and anaerobic heterotrophic denitrification, but has the following defects: (1) the autotrophic nitrifying bacteria have slow growth, poor environmental adaptability and weak impact load resistance, and are easily inhibited by high-concentration ammonia nitrogen and nitrite nitrogen. (2) The two reactions of nitrification and denitrification can not be unified in time and space, and the investment and operation cost is increased.

Heterotrophic nitrification and aerobic denitrification are a novel denitrification process. The device can realize nitrification and denitrification in one reactor simultaneously, complete synchronous removal of carbon and nitrogen pollutants, shorten reaction period, save space and reduce operation cost. At present, a plurality of heterotrophic nitrification-aerobic denitrification strains are separated and screened, for exampleBacillus subtilis、 Pseudomonas stutzeri、Klebsiella pneumoniae、Alcaligenes faecalisAnd so on. However, the suitable growth conditions of most of the strains separated at present are harsh, and the application of denitrification to some special types of wastewater (such as acid-base wastewater, high-salt wastewater and high-ammonia nitrogen wastewater) is limited.

Disclosure of Invention

The invention aims to provide a pseudomonas putida (a) which can feed on various kinds of resinPseudomonas multiresinivorans) GY13 with the preservation number of CGMCC number 21281. The strain is preserved in China general microbiological culture Collection center (CGMCC) at 12 months and 2 days in 2020, is called CGMCC for short, and has the address of No. 3 of West Lu No.1 of Beijing, Chaoyang district and the preservation number of CGMCC number 21281.

The invention has the beneficial effects that:

1. the pseudomonas putida (A) and (B) provided by the inventionPseudomonas multiresinivorans) GY13 has good effect in the aspect of sewage microbial treatment of types such as domestic sewage, livestock and poultry breeding sewage, and not only has good removal effect to ammonia nitrogen and total nitrogen, but also can get rid of most COD in the water simultaneously to realize the synchronous denitrogenation decarbonization of sewage, greatly simplifyThe denitrification process flow is simplified, and the treatment space and the cost are saved.

2. The strain GY13 has wide pH adaptability, and can effectively remove ammonia nitrogen within the pH range of 4-11.5.

3. The strain GY13 of the invention has salt tolerance, and the ammonia nitrogen removal rate can still reach more than 95% under the salt concentration of 40 g/L.

4. The strain GY13 provided by the invention can realize the rapid denitrification treatment of high-concentration ammonia nitrogen sewage, wherein the ammonia nitrogen removal rate reaches 93.1%, the TN removal rate reaches 63%, and the COD removal rate is 78.4% after 72 h of aerobic treatment in the denitrification process of pig-raising wastewater with the initial ammonia nitrogen concentration of 465 mg/L.

Drawings

FIG. 1 shows Pseudomonas putida (Pseudomonas multiresinivorans) Growth and heterotrophic nitrification profiles of GY 13.

FIG. 2 shows Pseudomonas putida (Pseudomonas multiresinivorans) GY13 growth and denitrification profiles at different pH.

FIG. 3 shows Pseudomonas putida (Pseudomonas multiresinivorans) Growth and denitrification performance plots of GY13 at different salinity.

FIG. 4 shows Pseudomonas putida (A)Pseudomonas multiresinivorans) Graph of denitrification performance of GY13 in unsterilized synthetic wastewater.

FIG. 5 shows Pseudomonas putida (A)Pseudomonas multiresinivorans) The denitrification performance of GY13 in sterilized domestic sewage is shown.

FIG. 6 shows Pseudomonas putida (A)Pseudomonas multiresinivorans) The denitrification performance of GY13 in sterilized pig wastewater.

Detailed Description

The media used in the examples are as follows:

LB culture medium: 5 g of yeast extract, 10 g of tryptone, 10 g of sodium chloride and 1L of distilled water.

Heterotrophic nitrification culture medium: 4 g of sodium chloride, 2.66 g of disodium hydrogen phosphate, 1 g of monopotassium phosphate, 4.41 g of potassium citrate, 0.38 g of ammonium chloride, 3 mL of trace element solution and 1L of distilled water, and the pH value is natural.

Synthesizing wastewater: disodium hydrogen phosphate 4.78 g, sodium dihydrogen phosphate 1.24 g, sodium chloride 4 g, magnesium sulfate heptahydrate 0.08 g, trisodium citrate 4.08 g, ammonium chloride 0.305 g, trace element solution 3 mL, distilled water 1L, natural pH.

Solution of trace elements: 3 g of magnesium sulfate heptahydrate, 3 g of manganese sulfate monohydrate, 3 g of zinc sulfate heptahydrate, 1.12 g of boric acid, 0.3 g of ferrous sulfate heptahydrate, 0.6 g of calcium chloride dihydrate and 1L of distilled water.

Example 1

Separation and identification of strains:

enrichment culture: the sample is aerobic activated sludge collected from a certain sewage treatment plant in Beijing by Cao Shi ahead in 2020 and 4 months, 20 mL of fully mixed activated sludge is taken and placed in 180 mL of 0.2% sodium chloride solution for suspension, 5 mL of suspension is taken and placed in a 250 mL conical flask filled with 100 mL of heterotrophic nitrification culture medium, and domestication enrichment culture is carried out at 30 ℃ and 180 rpm. The acclimation takes 48 h as a period. After each period, 10 mL of the enrichment solution is added into a new 100 mL of heterotrophic nitrification culture medium to continue enrichment culture, and the enrichment culture is continuously carried out for 5 periods. During which the removal of ammonia nitrogen from the culture broth was examined.

Collecting culture solution after 5 generations enrichment, and adding sterile water according to the ratio of 10 ^-3 -10 ^-7 The method comprises the following steps of (1) diluting in different proportions in a gradient manner, taking 100 mu L of diluent from each gradient, coating the diluent on a heterotrophic nitrification solid culture medium, placing the culture medium in a biochemical incubator for 48 hours at 30 ℃, observing the growth condition of bacterial colonies, selecting single bacterial colonies with different forms from the culture medium, respectively scribing on the heterotrophic nitrification solid culture medium by using a plate scribing method, and culturing for 48 hours under the same condition. Then selecting single colony to perform multi-time partition streak purification until obtaining pure strain, placing the purified strain in 20% glycerol, and freezing and preserving at-80 ℃ in a refrigerator for later use.

Strain screening: respectively selecting purified strains with inoculating loop, inoculating into sterilized LB liquid culture medium, culturing at 30 deg.C and 180 rpm for 24 hr, sucking a certain amount of bacterial liquid according to 5% (v/v) inoculum size, centrifuging at 4000 rpm for 5 min, collecting thallus, washing with sterile waterThen inoculating into 150 mL conical flask containing 50 mL heterotrophic nitrification medium, culturing at 30 deg.C and 180 rpm for 72 h in shaking table, and sampling for detecting NH in culture solution at 12 h, 24 h, 36 h, 48 h, 60 h and 72 h respectively ₄ N, TN and COD content. Screening out bacterial strains with heterotrophic nitrification function.

The heterotrophic nitrification-aerobic denitrification bacterium GY13 is obtained through the separation and purification process, and is characterized by being short-rod-shaped in morphology, the size of the bacterium is (0.3-0.5) mu× (0.9-1.3) mum, no spores are produced, the colony is circular, the edge is neat and smooth, and the bacterium is faint yellow on an LB culture medium and is a gram-negative aerobic bacterium.

The obtained strain is identified in molecular biology, and is subjected to sequencing comparison after the 16S rDNA sequence (SEQ ID NO. 1) of the bacteria is amplified by PCR.

The amplification primers were 27F: AGAGTTTGATCMTGGCTCAG, 1492R: TACGGYTACCTTGTTACGACTT, respectively;

the reaction system is as follows: 10 Xbuffer 2 muL, 2.5 mM dNTP 1.5 muL, Primer 11 muL, Primer 21 muL, template 1 muL, enzyme 0.3 muL, water 13.2 muL and total volume 20 muL;

the reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min, denaturation at 95 ℃ for 30 sec at 30 cycles, annealing at 55 ℃ for 30 sec, extension at 72 ℃ for 1.5 min, extension at 72 ℃ for 10 min, and heat preservation at 4 ℃ for forever.

The PCR product was detected by agarose gel electrophoresis and sequenced.

Sequencing in forward and reverse directions, splicing to obtain 16S rDNA sequence of the strain, performing Blast comparison on the sequence in NCBI database, and displaying the comparison result with pseudomonas (pseudomonas) as a result of various kinds of resinPseudomonas multiresinivorans) ATCC 700690 has the highest homology of 99.93%, and the strain is named as Pseudomonas resinovorans (A), (B)Pseudomonas multiresinivorans) GY13, hereinafter abbreviated as strain GY13 in this application.

Pseudomonas putida (A) and (B)Pseudomonas multiresinivorans) GY13 has been deposited in China general microbiological culture Collection center (CGMCC) at 12.2.2020 for short, with the address of No. 3 Xilu-Beijing province of Chaoyang, the deposition number of CGMCC No. 3. 21281。

Example 2

Growth and heterotrophic nitrification characteristics of strain GY 13:

activating and culturing strain GY13 in LB liquid test tube for 24 h, absorbing a certain amount of bacterial liquid according to the inoculum size of 2% (v/v), centrifuging for 5 min at 4000 rpm, collecting thallus, washing with sterile water, inoculating into a 150 mL conical flask containing 50 mL sterilized heterotrophic nitrification medium (initial ammonia nitrogen concentration 100 mg/L, C/N = 20), culturing for 72 h in a shaker at 30 ℃ and 180 rpm, sampling every 4 h during the period, measuring OD600 value, centrifuging the sample at 10000 rpm for 10 min, removing thallus, taking supernatant, and detecting COD and NH in a water sample ₄ -N、NO ₂ -N、NO ₃ -N, TN content.

The results are shown in the following table and in FIG. 1. The strain started to grow rapidly at 4 h after inoculation, when NH was present ₄ N, TN and the COD content decreased rapidly. The growth of the strain was substantially complete by 16 h, at which time the OD600 was 1.358 and the maximum removal rate, NH, was reached ₄ The removal rates of-N, COD and TN are respectively 93.3%, 95.8% and 89%, and the treatment process is free from nitrite nitrogen and nitrate nitrogen.

Example 3

Effect of pH on growth and denitrification of strain GY 13:

adjusting the pH of the heterotrophic nitrification culture medium to be 3, 4, 5, 7, 8.5, 9.5, 10.5 and 11.5 respectively, activating the strain GY13 according to a conventional method, absorbing a certain amount of bacterial liquid according to the inoculum size (v/v) of 2%, centrifuging for 5 min at 4000 rpm, collecting thalli, washing with sterile water, inoculating into the heterotrophic nitrification culture medium after adjusting the pH, culturing for 72 h in a shaker at 30 ℃ and 180 rpm, detecting the OD600 value under the final pH conditions, centrifuging the sample for 10 min at 10000 rpm, removing the thalli, taking the supernatant, and detecting COD and NH in the water sample ₄ -the content of N.

The results are shown in the following table and fig. 2. The strain GY13 can grow well and perform heterotrophic nitrification in the pH range of 4-11.5, when the pH values are 5, 7, 8.5, 9.5 and 10.5, the ammonia nitrogen removal rates are 90%, 88%, 91.7%, 84.7% and 92%, and the COD removal rates are 100%, 100% and 90.5%, respectively. The strain GY13 is shown to have wide application prospect in sewage treatment with lower or higher pH.

pH	OD600	Ammonia nitrogen removal (%)	COD removal Rate (%)
				3	0.266	0.0	0.0
4	0.252	0.0	2.8
				5	1.380	90.0	100.0
7	1.020	88.0	100.0
				8.5	1.175	91.7	100.0
9.5	1.235	84.7	100.0
				10.5	0.880	92.0	90.5
11.5	0.035	0.0	0.0

Example 4

Effect of salinity on growth and denitrification of strain GY 13:

adjusting the salinity of the heterotrophic nitrification culture medium to 5, 20, 40 and 60 g/L by using NaCl, activating a strain GY13 according to a conventional method, absorbing a certain amount of bacterial liquid according to an inoculum size (v/v) of 2%, centrifuging for 5 min at 4000 rpm, collecting thalli, washing with sterile water, inoculating into the heterotrophic nitrification culture medium after adjusting the salinity, culturing for 72 h in a shaking table at 30 ℃ and 180 rpm, detecting the OD600 value under each pH condition finally, centrifuging the sample for 10 min at 10000 rpm, removing the thalli, taking the supernatant to detect NH in a water sample ₄ -N content.

The results are shown in the following table and fig. 3. The strain GY13 can perform better growth and heterotrophic nitrification in the salinity range of 0-40 g/L, and when the salinity is respectively 5, 20 and 40 g/L, the ammonia nitrogen removal rates are respectively 90.3%, 91.8% and 99.1%. When the salinity reaches 60 g/L or more, the growth of the strain and the ammonia nitrogen removal rate are seriously inhibited, so the strain GY13 has better application prospect in the sewage treatment with the salinity of 40 g/L or less.

NaCl (g/L)	OD600	Ammonia nitrogen removal (%)
			5	0.865	90.3
20	1.108	91.8
			40	1.625	99.1
60	0.283	7.5

Example 5

Denitrification performance of the strain GY13 in synthetic wastewater:

the strain GY13 is inoculated into unsterilized synthetic wastewater according to the proportion of 2% (v/v) after being activated conventionally, the initial COD of the synthetic wastewater is 1600 mg/L, and the initial ammonia nitrogen is 80 mg/L. Aerobic treatment is carried out in a shaking table at 30 ℃ and 180 rpm, meanwhile, synthetic wastewater without inoculation is set as a control group, and the contents of ammonia nitrogen, TN and COD in the wastewater are detected every 12 hours, and the results are shown in the following table and figure 4.

Time (h)

OD600- CK

OD600- GY13

COD- CK (mg/L)

COD- GY13 (mg/L)

NH4-N- CK (mg/L)

NH4-N- GY13 (mg/L)

TN- CK (mg/L)

TN- GY13 (mg/L)

0

0±0

0.12±0

1628±0

80.4±0

79.2±0

12

0±0

0.86±0.01

1583.5±72.83

1396±0

80.5±0.71

42±0

80.1±0.14

49.8±8.2

24

0±0

1.26±0.01

1602±25.46

593±72.12

83.85±0.49

18.4±0.57

84.15±0.92

34.35±7.28

36

0±0

1.53±0.01

1591±55.15

61±4.24

86.45±1.91

5.25±0.49

85.95±4.6

9.3±2.97

The results show that after the treatment for 36 h, the removal rates of ammonia nitrogen, TN and COD of the GY13 treatment group are 93.5%, 88.3% and 96.3%, respectively, while the ammonia nitrogen, TN and COD content of the control group are not obviously reduced. The strain GY13 has good application prospect in the denitrification treatment of sewage with medium and low ammonia nitrogen concentration.

Example 6

Denitrification performance of the strain GY13 in sterilized domestic sewage:

the C/N ratio of actual domestic sewage (initial ammonia nitrogen content of about 100 mg/L) is adjusted to about 20 (initial COD content of about 1980 mg/L) by adding citrate, then the sterilization treatment is carried out, the strain GY13 is inoculated into the sterilized domestic sewage according to the proportion of 2% (v/v) after being subjected to conventional activation, aerobic treatment is carried out in a shaking table at 30 ℃ and 180 rpm, and the ammonia nitrogen, TN and COD content in the sewage are detected every 24 hours, and the results are shown in the following table and figure 5.

The result shows that the strain GY13 can remove most of ammonia nitrogen and all COD in domestic sewage within 24 hours after inoculation, the ammonia nitrogen removal rate within 24 hours reaches 89.8%, and the total nitrogen removal rate reaches 88.9%; after 48 hours, the ammonia nitrogen removal rate reaches 91%, the total nitrogen removal rate reaches 90.5%, and the COD removal rate is 100%, which shows that the strain GY13 has good application prospect in domestic sewage denitrification treatment with medium and low ammonia nitrogen concentration.

Example 7

The denitrification performance of the strain GY13 in sterilized pig wastewater:

the pretreatment method of the laboratory pig raising wastewater comprises the steps of uniformly mixing fresh pig urine collected from a certain pig farm of great Xing in Beijing with tap water according to the proportion of 1:4, adding fresh pig manure with the mass fraction of 5%, uniformly stirring, standing for 24 hours, and taking the upper liquid as the laboratory pig raising wastewater for later use.

The C/N ratio of the actual pig raising wastewater (the initial ammonia nitrogen content is about 465 mg/L) is adjusted to about 20 (the initial COD content is about 11700 mg/L) by adding citrate, the sterilization treatment is carried out, the strain GY13 is inoculated into the sterilized pig raising wastewater according to the proportion of 5% (v/v) after being activated conventionally, aerobic treatment is carried out in a shaking table at 30 ℃ and 180 rpm, and the ammonia nitrogen, TN and COD content in the wastewater are detected every 36 h, and the results are shown in the following table and figure 6.

The result shows that the ammonia nitrogen content is rapidly reduced from 465 mg/L to 146 mg/L and the COD is reduced from 11700 mg/L to 5394 mg/L at 36 h after inoculation of the strain GY 13; the ammonia nitrogen content is reduced to 32 mg/L by 72 h, COD is reduced to 2523 mg/L, the ammonia nitrogen removal rate reaches 93.1 percent in 72 h, the COD removal rate is 78.4 percent, and the TN removal rate reaches 63 percent, wherein the ammonia nitrogen content meets the requirements of the discharge standard of pollutants for livestock and poultry breeding (GB 18596-2001) in China. The strain GY13 has good application prospect in denitrification treatment of livestock and poultry breeding sewage with medium and high ammonia nitrogen concentration.

The above description is only an exemplary embodiment of the present invention, and is not intended to limit the scope of the present invention. Any equivalent changes and modifications of the invention without departing from the spirit and principles of the invention should fall within the protection scope of the invention.

Sequence listing

<110> institute of microbiology of Chinese academy of sciences

<120> Pseudomonas putida GY13 and application thereof in sewage treatment

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<212> DNA

<213> Pseudomonas multiresidue (Pseudomonas multiresiniivorans)

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tcaccgtgac attctgattc acgattacta gcgattccga cttcacgcag tcgagttgca 120

gactgcgatc cggactacga tcggttttat gggattagct ccacctcgcg gcttggcaac 180

cctctgtacc gaccattgta gcacgtgtgt agccctggcc gtaagggcca tgatgacttg 240

acgtcatccc caccttcctc cggtttgtca ccggcagtct ccttagagtg cccaccataa 300

cgtgctggta actaaggaca agggttgcgc tcgttacggg acttaaccca acatctcacg 360

acacgagctg acgacagcca tgcagcacct gtgttccgat tcccgaaggc actcccgcat 420

ctctgcagga ttccggacat gtcaaggcca ggtaaggttc ttcgcgttgc ttcgaattaa 480

accacatgct ccaccgcttg tgcgggcccc cgtcaattca tttgagtttt aaccttgcgg 540

ccgtactccc caggcggtcg acttatcgcg ttagctgcgc cactaaaatc tcaaggattc 600

caacggctag tcgacatcgt ttacggcgtg gactaccagg gtatctaatc ctgtttgctc 660

cccacgcttt cgcacctcag tgtcagtatc agtccaggtg gtcgccttcg ccactggtgt 720

tccttcctat atctacgcat ttcaccgcta cacaggaaat tccaccaccc tctaccgtac 780

tctagtcagg cagttatgga tgcagttccc aggttgagcc cggggatttc acatccatct 840

taccaaacca cctacgcgcg ctttacgccc agtaattccg attaacgctt gcacccttcg 900

tattaccgcg gctgctggca cgaagttagc cggtgcttat tctgttggta acgtcaaaac 960

agcaaggtat taacttactg cccttcctcc caacttaaag tgctttacaa tccgaagacc 1020

ttcttcacac acgcggcatg gctggatcag gctttcgccc attgtccaat attccccact 1080

gctgcctccc gtaggagtct ggaccgtgtc tcagttccag tgtgactgat catcctctca 1140

gaccagttac ggatcgtcgc cttggtaggc ctttacccca ccaactagct aatccgacct 1200

aggctcatct gatagcgcaa ggcccgaagg tcccctgctt tctcccgtag gacgtatgcg 1260

gtattagcgt tcctttcgaa acgttgtccc ccactaccag gcagattcct aggcattact 1320

cacccgtccg ccgctgaatc cgggagcaag ctcccatca 1359

Claims

1. Pseudomonas putida (A) and (B)Pseudomonas multiresinivorans) GY13 with preservation number of CGMCCC No. 21281。

2. A microbial preparation comprising, as an active ingredient, the Pseudomonas resinovorans (Pseudomonas aeruginosa) according to claim 1Pseudomonas multiresinivorans）GY13。

3. Pseudomonas resinovorans (Pseudomonas difficile) according to claim 1Pseudomonas multiresinivorans) Application of GY13 in removing COD, ammonia nitrogen or/and total nitrogen in sewage.

4. Use according to claim 3, wherein the sewage is domestic sewage or swine wastewater.

5. Use according to claim 3, wherein the pH of the effluent is in the range of 5 to 10.5.

6. Use according to claim 3, wherein the salinity of the effluent is in the range of 5-40 g/L.