CN106148227B

CN106148227B - Low-temperature nitrifying pseudomonas and application thereof

Info

Publication number: CN106148227B
Application number: CN201610525084.XA
Authority: CN
Inventors: 林学政; 李晓亮; 何培青; 张培玉
Original assignee: First Institute of Oceanography SOA
Current assignee: First Institute of Oceanography SOA
Priority date: 2016-07-05
Filing date: 2016-07-05
Publication date: 2020-01-07
Anticipated expiration: 2036-07-05
Also published as: CN106148227A

Abstract

The invention discloses a low-temperature nitrifying pseudomonas and application thereof, wherein the low-temperature nitrifying pseudomonas is obtained by screening from ocean sediments in the arctic region and is named as pseudomonasPseudomonassp, CC6-YY-74, which is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC 12611 and the preservation date of 2016, 6 and 15 days. The pseudomonas has stronger nitrification and denitrification activity at low temperature and wider salinity adaptation range, and can solve the problem that the effluent quality of a sewage treatment plant in northern areas of China is difficult to reach the standard due to low temperature when the pseudomonas is used for sewage treatment.

Description

Low-temperature nitrifying pseudomonas and application thereof

Technical Field

The invention belongs to the technical field of environmental microorganisms, and particularly relates to a nitrifying-arctic low-temperature pseudomonas strain, a screening method of the nitrifying-arctic low-temperature pseudomonas strain, and application of the nitrifying-arctic low-temperature pseudomonas strain in treatment of nitrogen-containing wastewater.

Background

With the continuous improvement of urbanization in China, the control of medium and small point source pollution is the key to improve the water environment quality and solve the problem of water resource shortage. The biological treatment of domestic sewage in cold areas of China has the problems of low microbial activity, poor biochemical treatment effect, difficult effluent reaching the standard and the like all the time, and the reduction of temperature has obvious influence on the adsorption performance, the sedimentation performance, the growth and development of microorganisms, the population composition, the oxygen transfer efficiency in an aeration tank and the like of activated sludge in the sewage treatment process.

At present, in order to relieve the influence of low temperature on sewage treatment in cold areas in winter in northern China, measures such as prolonging sewage retention time, reducing sludge load, increasing sludge backflow amount, and insulating or heating structures are generally adopted in engineering practice to ensure that effluent of sewage treatment in winter reaches the standard, but the measures can increase engineering investment and operating cost, the treatment effect is poor, and the problem of sludge bulking often occurs. Meanwhile, salinity also has certain influence on microbial activity, and can inhibit microbial growth when salinity is too high. In order to solve the influence of overhigh salinity, the method generally adopts a precipitation biological process of acclimatization of activated sludge, adding some halophilic bacteria into an activated sludge system and strengthening physical and chemical treatment. These measures extend the treatment cycle and increase the engineering costs. Therefore, a low-temperature efficient salt-tolerant degrading bacterium needs to be found, is applied to the treatment of low-temperature domestic sewage, and is an effective method for solving the problem that the quality of the effluent of a sewage plant in winter in northern areas of China is difficult to reach the standard.

The arctic region has unique geographical, environmental and climatic characteristics, a special polar region microbial ecosystem is created, the surrounding sea area is in a low-temperature high-salt environment all the year round, and long-time precipitation and accumulation are realized, so that the polar region marine sediment becomes a microbial habitat with complex and huge components. And these microorganisms often have morphological, physiological specificities adapted to their particular environment. And thus is considered to be a resource pool of new microbial germplasm, genes and products. By screening salt-tolerant and low-temperature-resistant nitrobacteria from the arctic ocean sediments, the problem that the quality of the winter sewage in northern areas of China is difficult to reach the standard can be effectively solved.

Disclosure of Invention

The invention aims to provide a low-temperature pseudomonas nitroreducens screened from arctic ocean sediments so as to solve the problem that the water quality of effluent of a sewage treatment plant is difficult to reach the standard due to low temperature in northern areas of China.

The invention provides low-temperature Pseudomonas which is obtained by screening from ocean sediments in the arctic, is named as Pseudomonas sp.CC6-YY-74 and is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No 12611 and the preservation date of 2016, 6 and 15.

The invention also provides application of the low-temperature pseudomonas, which is suitable for water purification.

Furthermore, the applicable temperature for water purification is 0-30 ℃, and the NaCl concentration is 0-60 per mill.

Compared with the prior art, the invention has the advantages and positive effects that: the strain is obtained by screening from arctic ocean sediments, has strong nitrification and denitrification activity at low temperature, has high sewage treatment efficiency, and omits the measures of heat preservation or temperature rise and the like for structures by prolonging the retention time of sewage; in addition, the salinity of the strain is wide in application range, sewage with different salinity can be treated, and the acclimatization time and the cost of facility construction are saved; thereby reducing the cost of the process on the basis of ensuring the efficiency of the process.

Drawings

FIG. 1 is a phylogenetic tree of strain CC6-YY-74 constructed based on the 16S rRNA gene sequence;

FIG. 2 is a graph showing nitrification at different temperatures of the strain CC6-YY-74 in example 2;

FIG. 3 is the nitrification curve under different NaCl concentrations for the strain CC6-YY-74 in example 2;

FIG. 4 is a graph showing denitrification at different temperatures for the strain CC6-YY-74 in example 3;

FIG. 5 is a graph showing the denitrification effect of the strain CC6-YY-74 in example 3 at different NaCl concentrations;

FIG. 6 is a graph showing COD degradation at different temperatures of the strain CC6-YY-74 in example 4;

FIG. 7 is a graph showing COD degradation curves of the strain CC6-YY-74 in example 4 at different NaCl concentrations.

Detailed Description

The technical solution of the present invention will be described in further detail with reference to the following embodiments. The proportions, parts and proportions referred to herein are by weight unless otherwise specifically indicated.

The embodiment provides a low-temperature nitrifying pseudomonas, which is a low-temperature salt-tolerant nitrifying bacterium and has strong nitrification and denitrification effects in a low-temperature environment; the method is applied to biological treatment of sewage, simultaneously solves the problems of inhibiting the growth of microorganisms due to overhigh salinity and reducing the activity of the microorganisms under low temperature conditions, and effectively solves the problem that the water quality of the effluent of a sewage treatment plant in northern areas of China is difficult to reach the standard in winter.

The low-temperature Pseudomonas is obtained by screening from arctic ocean sediments and is named as Pseudomonas sp.CC6-YY-74, is preserved in China general microbiological culture Collection center (CGMCC), has the preservation number of CGMCC No. 12611, has the preservation date of 2016, 6 and 15 days, has the preservation address of No. 3 of Xilu No. 1 of the sunward area of Beijing, and the institute of microbiology of China academy of sciences. The low-temperature pseudomonas can carry out nitrification and denitrification under the low-temperature condition, and provides new strain resources and basic data for the biological treatment of the low-temperature domestic sewage in winter in northern areas.

Example 1

The low-temperature pseudomonas related to the embodiment is obtained by screening arctic ocean sediments by methods of enrichment, dilution, selective medium culture and the like.

1. The materials used were:

7-9 months 2014, China scientifically explores the marine sediments in the sixth north pole.

2. The components of the culture medium:

heterotrophic nitrification medium (g/L): (NH)₄)₂SO₄1, sodium acetate 2.5, sodium citrate 2.5, K₂HPO₄ 5，MgSO₄2.5, NaCl 20 and 2mL of trace element solution.

Wherein the trace element solution (g/L): ZnSO₄ 2.2，CaCl₂ 5.5，MnCl₂ 5.06，FeSO₄5.0 ammonium molybdate 1, CuSO₄ 1.57，CoCl₂1.61. Seawater Zobell 2216E medium (g/L): peptone 5, yeast powder 1, filtered old seawater: tap water (v/v2: 1).

3. The screening method comprises the following steps:

taking a proper amount of an arctic ocean sediment sample, transferring the arctic ocean sediment sample into 50mL of liquid heterotrophic nitrification culture medium, carrying out shake culture (130r/min) at 10 ℃ for 7d, then transferring the culture solution according to the inoculum size of 2%, transferring the culture solution into a new same liquid heterotrophic nitrification culture medium, carrying out culture under the same conditions for 7d, repeating the steps twice, diluting the enriched culture solution by 10000 times, taking 100 mu L of a coating screening culture medium flat plate, placing the coated screening culture medium flat plate in a 10 ℃ culture box, after the bacterial solution completely permeates into the culture medium, inverting, carrying out culture for 14d, selecting different single bacterial colonies according to morphological characteristics of the bacterial colonies, carrying out streaking separation again until the bacterial colonies are pure, and obtaining the purified bacterial strain CC 6-YY-74. 4. Phylogenetic analysis of the strain CC 6-YY-74:

extracting strain genome DNA by adopting a genome DNA extraction kit of Tiangen biochemical technology (Beijing) Limited company, wherein a primer for PCR amplification of a 16S rRNA gene is 27F: 5'-AGAGTTTGATCCTGGCTCAG-3', 1492R: 5'-GGTTACCTTGTTACGACTT-3', PCR the conditions are: pre-denaturation at 94 deg.C for 5min, denaturation at 1min, annealing at 55 deg.C for 30s, and annealing at 72 deg.C for 90s, circulating for 35 times, extending at 72 deg.C for 10min, and subjecting PCR property to agarose gel electrophoresis detection and sequence determination by Nanjing Kingsry Biotech Co. The effective length of the obtained 16S rRNA gene sequence is 1336bp, and BLAST comparison analysis is carried out on the sequencing result at NCBI. Sequence alignment adopts a BioEdit multiple sequence alignment arrangement (Clustalw multiple alignment), phylogenetic analysis adopts a Mega5.0 adjacency method (Neighbor-Joining method), 16S rRNA gene similarity with a model strain is analyzed by EzTaxon-Database, 16SrRNA gene sequences of the most similar strains are selected, alignment is carried out by software BioEdit, MEGA5 software is applied, and a phylogenetic tree is constructed by the adjacency method (Neighbor-join).

The 16S rRNA gene sequence alignment result shows that the strain CC6-YY-74 has the highest similarity with Pseudomonas taanensis MS-3, and the similarity is 98.73%. As can be seen from the phylogenetic tree of FIG. 1, the strain CC6-YY-74 belongs to the genus Pseudomonas.

5. The study on the physicochemical properties of the low-temperature pseudomonas comprises the following steps:

the strain is gram-negative bacteria, can produce urease, alpha-glucosidase and the like, and can utilize glucose, potassium gluconate, malic acid, sodium citrate, sodium acetate, sodium citrate and other carbon sources. The colonies on 2216E solid medium are light yellow, round, neat in edge, smooth and moist in surface and convex.

6. temperature/NaCl concentration resistance growth suitability determination of Strain CC 6-YY-74:

(1) temperature growth range: inoculating into sea water 2216E culture medium (peptone 5g/L, yeast powder 1g/L, sea water 1L) according to 1% inoculum size, respectively, shake culturing at different temperatures (0 deg.C, 10 deg.C, 20 deg.C and 30 deg.C) (130r/min), sampling after 2d, and determining culture solution concentration (OD)₆₀₀) The temperature adaptation range of the strain was observed, and the results are shown in table 1.

(2) NaCl concentration resistant growth range: inoculating purified strain CC6-YY-74 into culture medium with different NaCl concentrations (peptone 5g/L, yeast powder 1g/L, distilled water 1L; NaCl with different mass concentrations is added to make the final mass concentrations respectively 0 ‰, 15 ‰, 30 ℃, 45 ‰, 60 ℃and90 ℃), shake culturing at 10 deg.C (130r/min), sampling after 2d to determine culture solution concentration (OD)₆₀₀) The adaptation range of the strain to NaCl concentration was thus observed, and the results are shown in Table 2.

(3) And (3) detection results: as shown in Table 1, the strain CC6-YY-74 has activity at 0-30 ℃, wherein the growth condition is best at 20 ℃ under the condition of the same NaCl concentration, and then the strain of the embodiment is adaptive to the low-temperature living environment at 10 ℃, 0 ℃ and 30 ℃ in sequence.

TABLE 1 concentration of the culture broth measured at different temperatures

As shown in Table 2, the strains can grow in the culture medium with NaCl concentration of 0-90 per mill. Wherein, under the same temperature, the growth condition is best under the condition that the NaCl concentration is 15 per thousand, then 30 per thousand, 0 per thousand, 45 per thousand, 60 per thousand and 90 per thousand are carried out in sequence, which shows that the strain of the embodiment can survive in the living environment with a wider salinity range.

TABLE 2 culture solution concentrations measured at different NaCl concentrations

Example 2

Nitration activity detection of strain CC6-YY-74

(1) Inoculating purified strain CC6-YY-74 with 4% inoculum size in 100mL sterilized nitration culture medium with NaCl concentration of 30 ‰, and shake culturing at 0 deg.C, 10 deg.C, 20 deg.C and 30 deg.C in incubator at rotation speed of 130 r/min. Detecting ammonia Nitrogen (NH) in the culture medium every 24h₄N) concentration change, the results are shown in FIG. 2.

(2) Inoculating purified strain CC6-YY-74 with 4% inoculum size in 100mL sterilized nitrification culture medium of NaCl of different concentrations (NaCl concentration is 0 ‰, 15 ‰, 30 ‰, 45 ‰, 60 ‰ and 90 ‰), shake culturing at 10 deg.C and 130r/min, detecting ammonia Nitrogen (NH) in culture medium every 24 hr₄-N) concentration change, the results are shown in FIG. 3.

The above-mentioned nitrification medium component (g/L): NH (NH)₄Cl 0.191，KH₂PO₄ 0.05，MgSO₄0.1, 2.5 parts of sodium acetate, 2.5 parts of sodium citrate and 2mL of trace element solution. NH (NH)₄the-N concentration measurement was performed by means of Nagowski reagent photometry.

(3) And (3) detection results: in the presence of NH₄Cl is the only nitrogen source, sodium acetate and sodium citrate are the carbon sources, the initial pH is 7.2, and the nitrification efficiency is different when the culture is carried out at different temperatures and NaCl concentrations. As can be seen from figure 2, when the NaCl concentration is the same, the treatment effect is the best at 20 ℃, and the ammonia nitrogen content is reduced from 45mg/L to 13mg/L after 24 hours; the treatment effect is better when the temperature is 10 ℃, the ammonia nitrogen content is reduced from 45mg/L to 40mg/L after 24 hours, and is reduced to 20mg/L after 48 hours. As can be seen from FIG. 3, when the temperature is the same, the treatment effect is best when the NaCl concentration is 15 ‰, the ammonia nitrogen content is reduced from 45mg/L to 10mg/L after 48h, and the ammonia nitrogen removal rate is close to 80%.

Example 3

Denitrification activity detection of strain CC6-YY-74

(1) The purified strain CC6-YY-74 is inoculated in 100mL of sterilized denitrification culture medium with NaCl concentration of 30 per mill by the inoculation amount of 4 percent, and is cultured in an incubator with 0 ℃, 10 ℃, 20 ℃ and 30 ℃ in a shaking way, and the rotating speed is 130 r/min. Detecting nitrate Nitrogen (NO) in the culture medium every 24h₃-N), nitrous Nitrogen (NO)₂-N) concentration change, the results are shown in FIG. 4.

(2) Inoculating purified strain CC6-YY-74 with 4% inoculum size in 100mL sterilized denitrification culture medium with NaCl of different concentrations (NaCl concentration is 0 ‰, 15 ‰, 30 ‰, 45 ‰, 60 ‰ and 90 ‰), shake culturing at 10 deg.C and 130r/min, and detecting nitrate Nitrogen (NO) in the culture medium every 24 hr₃-N), nitrous Nitrogen (NO)₂-N) concentration change, the results are shown in FIG. 5.

The above-mentioned denitrifying medium ingredient (g/L): KNO₃ 0.144，KH₂PO₄ 0.05，MgSO₄0.1, 2.5 parts of sodium acetate, 2.5 parts of sodium citrate and 2mL of trace element solution. Measurement of NO₃Measurement of NO by thymol photometry of the-N concentration₂The concentration of-N was determined photometrically using N- (1-naphthyl) -ethylenediamine.

(3) And (3) detection results: in the presence of KNO₃Sodium acetate and sodium citrate are used as carbon sources, the initial pH is 7.2, and the denitrification efficiency is different when the culture is carried out at different temperatures and NaCl concentrations. As can be seen from FIG. 4, when the NaCl concentration is the same, the treatment effect is the best at 20 ℃, and the content of nitrate nitrogen is reduced from 53mg/L to 23mg/L after 24 hours; the treatment effect is better when the temperature is 10 ℃, the nitrate nitrogen is reduced from 53mg/L to 43mg/L after 24 hours, and is reduced to 36mg/L after 48 hours. As can be seen from FIG. 5, when the temperature is the same, the treatment effect is best when the NaCl concentration is 15%, and the content of nitrate nitrogen is reduced from 54mg/L to 27mg/L after 24 h.

Example 4

Detection of treatment effect of strain CC6-YY-74 in treatment of simulated domestic sewage

(1) Inoculating the purified strain CC6-YY-74 into 100mL of sterilized simulated domestic sewage with NaCl concentration of 30 per mill in an inoculation amount of 4%, wherein the formula of the simulated domestic sewage is (g/L): 10 parts of peptone, 5 parts of yeast extract, 3 parts of glucose and 3 parts of dipotassium phosphate, wherein the diluted components are diluted by 80 times for experiment, the COD is about 1300mg/L, and the components are subjected to shake culture in an incubator at 0 ℃, 10 ℃, 20 ℃ and 30 ℃ at the rotating speed of 130 r/min. The COD degradation rate in the culture medium was measured every 24h, and the COD measurement was performed by potassium dichromate method, and the results are shown in FIG. 6.

(2) The purified strain CC6-YY-74 is inoculated into 100mL of sterilized liquid simulation domestic sewage with different NaCl concentrations (the NaCl concentrations are respectively 0 per thousand, 15 per thousand, 30 per thousand, 45 per thousand, 60 per thousand and 90 per thousand) with the inoculation amount of 4 percent, the strain is diluted by 80 times for experiment, the COD is about 1300mg/L, the strain is subjected to shaking culture at 10 ℃ and 130r/min, the COD degradation rate in the culture medium is detected every 24h, the COD determination adopts a potassium dichromate method, and the result is shown in figure 7.

(3) And (3) detection results: the initial pH was 7.2, and the removal rate of COD was different when cultured at different temperatures and NaCl concentrations. As can be seen from FIG. 6, when the NaCl concentration is the same, the treatment effect is the best at 20 ℃, the COD content is reduced from 1337mg/L to 714mg/L after 24h, and is reduced to 205mg/L after 48 h; when the temperature is 10 ℃, the COD content is reduced from 1321mg/L to 823mg/L after 24h, and is reduced to 365mg/L after 48 h; when the temperature is 0 ℃, the COD content is reduced from 1356mg/L to 987mg/L after 24h, and is reduced to 471mg/L after 48h, and the strain can be seen to have better activity at the low temperature of 0-20 ℃.

As can be seen from FIG. 7, when the temperature is the same, the treatment effect is best when the NaCl concentration is 15 ‰, and the COD content is reduced from 1324mg/L to 789mg/L after 24 h.

It is understood from the combination of examples 2, 3 and 4 that the low-temperature pseudomonas can be applied to low-temperature or high-salinity treatment of domestic sewage.

The above examples are only a few of the several preferred embodiments of the present invention, and it should be noted that the present invention is not limited to the above examples; for a person skilled in the art, modifications may be made to the technical solutions described in the foregoing embodiments, or equivalents may be substituted for some of the technical features; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.

Claims

1. The low-temperature Pseudomonas is named as Pseudomonas sp.CC6-YY-74, is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No 12611 and the preservation date of 2016, 6 and 15.

2. The use of pseudomonas aeruginosa according to claim 1 for water purification.