CN111218410B

CN111218410B - Alkaligenes HO-1 and application thereof

Info

Publication number: CN111218410B
Application number: CN201811423813.6A
Authority: CN
Inventors: 刘志培; 刘双江; 吴梦茹; 刘缨; 苗莉莉; 高喜燕; 彭积森
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2018-11-26
Filing date: 2018-11-26
Publication date: 2021-10-01
Anticipated expiration: 2038-11-26
Also published as: CN111218410A

Abstract

The invention provides an Alcaligenes sp strain HO-1 with the preservation number of CGMCC No.16549, which is separated from a semi-short distance nitration reactor for treating pig farm wastewater. The strain is preserved in China general microbiological culture Collection center (CGMCC) in 2018, 9 and 28 months, and the preservation number is CGMCC No. 16549. The alcaligenes provided by the invention can efficiently remove ammonia nitrogen, and can independently remove NH under aerobic conditions₄ ⁺Conversion into gaseous nitrogen product (N)₂O、N₂). When the ammonia nitrogen-containing wastewater is treated, the whole-course denitrification under the single condition of a single strain can be realized, and the process has more advantages in operation and economic benefits than the traditional biological denitrification process of aerobic nitrification-anaerobic denitrification. In addition, the strain has wider C/N ratio, temperature, pH and dissolved oxygen adaptability, and can tolerate and remove high-concentration ammonia nitrogen.

Description

Alkaligenes HO-1 and application thereof

Technical Field

The invention belongs to the technical field of environmental microorganism denitrifying bacteria.

Technical Field

With the progress of society and the rapid development of industry and agriculture, the excessive discharge of nitrogen and organic matters is brought to the environment by human activities such as aquaculture, livestock and poultry breeding, urban life, agricultural fertilization and irrigation and the like, and a series of environmental problems such as water eutrophication and derived water bloom, black and odorous water bodies and the like caused by the excessive discharge are increasingly serious. The effective denitrification treatment of the sewage to ensure the discharge reaching the standard has important significance for controlling the discharge of nitrogen so as to protect and control the water body environment.

Biological denitrification is currently considered to be the most cost effective means of wastewater nitrogen removal. The traditional biological denitrification technology is to complete denitrification by two processes of nitrification and denitrification in a synergistic way, namely, autotrophic nitrifying bacteria perform nitrification under aerobic conditions to convert ammonia nitrogen into nitrate or nitrite, and heterotrophic denitrifying bacteria perform denitrification under anaerobic conditions to reduce the nitrate or nitrite into gaseous nitrogen. This is the most widely used and mature denitrification technique to date, however it has drawbacks and limitations, mainly including: firstly, the environmental conditions of nitrifying and denitrifying bacteria are different, and the nitrifying and denitrifying bacteria are required to be carried out in two isolated reactors or are carried out by using intermittent aerobic and anaerobic conditions, which causes the problems of high cost and complex operation; secondly, the nitrifying bacteria are chemoautotrophic bacteria, on one hand, the nitrifying bacteria grow and breed for a long time, so that the reactor is started slowly, thereby increasing the operation cost, and on the other hand, the nitrifying bacteria have a low tolerance threshold value on organic load, are sensitive to environmental impact and have high operation difficulty; in addition, denitrifying bacteria are heterotrophic bacteria, which require organic matter, and nitrifying bacteria cannot tolerate organic matter, so that the organic matter is usually removed before the nitrification process during sewage treatment, and carbon sources are supplemented in the denitrification stage, so that the process operation cannot be effectively simplified.

In addition to autotrophic nitrifying bacteria, researchers have recently isolated many heterotrophic nitrifying bacteria from the environment, which grow faster and have more environmental suitability than autotrophic nitrifying bacteria. Moreover, heterotrophic nitrifiers need organic matters to grow and metabolize, and COD can be synchronously removed. In addition, research shows that a plurality of heterotrophic nitrifying bacteria simultaneously have aerobic denitrification capability, so that the denitrification process can be realized in one reactor. Heterotrophic nitrification microorganism can overcome the problems existing in the traditional denitrification process, and has huge development potential in the field of biological denitrification.

Disclosure of Invention

The invention aims to provide an Alcaligenes sp strain HO-1 CGMCC No.16549, which is a heterotrophic bacterium, can denitrify under aerobic condition and can independently convert ammonia nitrogen into gaseous nitrogen products (N)₂O、N₂) Namely, one strain can complete the whole denitrification under the aerobic condition.

The Alcaligenes sp strain HO-1 provided by the invention is preserved in the common microorganism center of China general microbiological culture Collection center (CGMCC) in 2018, 9 and 28, and the preservation number is CGMCC No.16549, and the preservation address is No. 3 of Xilu 1 Beichen of the sunward area in Beijing.

The strain HO-1 is a gram-negative bacterium separated and purified from a small-scale semi-short distance nitration reactor for treating wastewater of a pig farm in a laboratory.

The strain HO-1 is used for biological denitrification in sewage/wastewater under aerobic conditions. Further, the sewage/wastewater may have a carbon-nitrogen (C/N) mass ratio of 2-20:1, preferably 5-20: 1; the pH can be from 5 to 10, preferably from 7 to 10; the temperature can be 15-45 ℃, preferably 25-37 ℃; the dissolved oxygen may be 2-8mg/L, preferably 4-7 mg/L.

Further, the carbon source in the sewage/wastewater is succinic acid, acetic acid, citric acid, malic acid and butyric acid, or the organic matter (COD) is converted into the above-mentioned small molecular organic acid by anaerobic digestion of the sewage/wastewater.

The invention has the beneficial effects that:

1. the strain HO-1 has high-efficiency ammonia nitrogen removal capability and can independently remove NH under aerobic conditions₄ ⁺Conversion into gaseous nitrogen product (N)₂O、N₂) The strain is a novel denitrifying bacterium with the characteristic of independently finishing whole-course denitrification. The ammonia nitrogen is directly inoculated into the sewage/wastewater containing ammonia nitrogen, and the effective removal of the ammonia nitrogen can be realized only by one aerobic condition. The method solves the problem that the two processes of aerobic nitrification and anaerobic denitrification are required to be carried out in stages in the traditional biological denitrification technology, greatly simplifies the denitrification process flow and saves the treatment space and cost.

2. The strain HO-1 has higher ammonia nitrogen and total nitrogen removal efficiency in certain carbon-nitrogen ratio, pH, temperature and dissolved oxygen ranges, is applied to the treatment of nitrogen-containing wastewater under various conditions, and has wide application prospect.

3. The bacterial strain HO-1 is inoculated in sewage/wastewater with initial ammonia nitrogen concentration of about 400mg/L, more than 99% of ammonia nitrogen can be removed in 48 hours, the average ammonia nitrogen removal rate is 189.55mg/L/d, nitrate is not produced in the denitrification process, nitrite is hardly accumulated, and the total nitrogen removal rate including biological nitrogen can reach nearly 47%. Except for synthesizing biological nitrogen, most of the contained ammonia nitrogen is converted into a gaseous nitrogen product, so that the denitrification of sewage/wastewater is realized, and the total nitrogen of effluent is less than 5 mg/L.

4. The strain HO-1 is inoculated into sewage/wastewater with initial ammonia nitrogen concentration of more than 2000mg/L, the ammonia nitrogen removal rate can finally reach more than 80 percent, the total nitrogen removal rate is more than 40 percent, and the total nitrogen of effluent is less than 80mg/L, so that the strain is used for treating high-concentration ammonia nitrogen wastewater, such as wastewater treatment in pig farms.

Drawings

FIG. 1 is a graph showing growth and ammonia nitrogen removal curves of a strain HO-1 by using ammonia nitrogen as a nitrogen source.

FIG. 2 is a diagram showing the detection of nitrogen products during denitrification of the strain HO-1.

Wherein A is a control group; b is the experimental group.

FIG. 3 is a graph showing the growth and ammonia nitrogen removal of the strain HO-1 at different C/N.

FIG. 4 is a graph showing the growth of the strain HO-1 and the removal of ammonia nitrogen at different pH values.

FIG. 5 is a graph showing the growth of the strain HO-1 and the removal of ammonia nitrogen at different temperatures.

FIG. 6 is a graph showing the growth of the strain HO-1 and the removal of ammonia nitrogen in different dissolved oxygen.

FIG. 7 is a graph showing denitrification and intermediate product accumulation of the strain HO-1 in nitrogen-containing sewage/wastewater.

FIG. 8 is a graph showing the growth of the strain HO-1 in high concentration ammonia nitrogen sewage/wastewater and the ammonia nitrogen removal.

Detailed Description

The following detailed description of the present invention will be made in conjunction with the accompanying drawings and examples to better illustrate the aspects of the present invention and its advantages. It should be noted that the following description of specific embodiments and examples is intended for purposes of illustration only and is not intended to limit the invention.

Example 1 identification of Strain HO-1

The Alcaligenes sp strain HO-1 is separated from a short-cut nitrification reactor for wastewater treatment of an autotrophic pig farm.

Inoculating the strain HO-1 in LB solid slant culture medium, culturing at 30 deg.C until obvious lawn appears, selecting a small amount of lawn with sterile inoculating loop, mixing in EP tube filled with 200 μ L sterile water, and vortex shaking to obtain bacterial suspension. The EP tube was then placed in a boiling water bath to boil for 3min, taken out and immediately placed on ice. The somatic cells are easy to break after instantaneous cold and hot alternation, and then release genome DNA. The supernatant is taken out after the bacterial suspension which is cracked by boiling is centrifuged for a short time, and the supernatant can be used as a template for the subsequent 16S rRNA gene PCR amplification.

16S rRNA gene PCR amplification of the strain HO-1 adopts bacterial universal primers 27F/1492R (27F: 5' -AGAGTTTGATCC)TGGCTCAG-3'; 1492R: 5'-GGTTACCTTGTTACGACTT-3') as upstream and downstream primers. The PCR reaction system is as follows: 10 XPCR Buffer 5. mu.L, dNTP mix (2.5mM each of the each) 4. mu.L, upstream and downstream primers (10. mu.M) 2. mu.L each, DNA template 2. mu.L, rTaq DNA polymerase 0.5. mu.L, add ddH₂O34.5. mu.L to a total volume of 50. mu.L.

The PCR reaction program is: at 94 deg.C for 5min, at 94 deg.C for 1min, at 54 deg.C for 1min, at 72 deg.C for 1.5min, and circulating for 30 times at 72 deg.C for 10 min.

The PCR products were detected by agarose gel (1%) electrophoresis, and PCR products with a single electrophoretic band and a band size of about 1500bp were used for subsequent vector ligation and sequencing.

Cloning vector ligation of the qualified PCR product is carried out by using pEASY-T1Cloning Kit according to the Kit instruction, the ligation product is transformed into Trans1-T1 competent cells, then a plate is coated, a positive clone is picked up, PCR identification is carried out on the positive clone by using M13 upstream and downstream primers (M13F: 5'-TGACCGCAGCAAAATG-3'; M13R: 5'-GTCCTTTGTCGATACTG-3') provided by the Kit, and the positive clone with the PCR product length of about 1700bp is screened out. From these, 3 primers were selected for sequencing, with sequencing primers M13F/M13R.

Performing blast comparison on the 16S rRNA gene sequence obtained by sequencing, selecting a strain sequence with the highest similarity, constructing a Neighbor-join phylogenetic evolution tree by using software MEGA 5.0, and analyzing the phylogenetic position of the strain.

The length of the 16S rRNA gene sequence of the strain HO-1 obtained by sequencing is 1525bp, the nucleotide-base sequence of the strain is described in SEQ ID NO.1, and the strain has higher sequence similarity of Alcaligenes faecalis subsp. In the phylogenetic tree, the strain HO-1 also clustered together with the strains of Alcaligenes, and was therefore preliminarily identified as Alcaligenes.

LB culture medium formula (g/L): peptone 10, yeast extract 5, sodium chloride 10, ph 7.0. Solid LB medium on the liquid medium in addition to 15g/L agar powder.

Example 2 growth and Ammonia Nitrogen removal of Strain HO-1 with Ammonia Nitrogen as sole Nitrogen Source

The HO-1 strain frozen by glycerol is inoculated into a test tube filled with LB liquid culture medium, cultured for 24h and activated in a shaker at 30 ℃ and 160rpm, then 1 percent of inoculum size is taken to be transferred into a 250mL triangular flask filled with 100mL LB liquid culture medium, cultured for 20h at 30 ℃ and 160rpm, and the strain is used as seed culture solution.

The seed culture solution is centrifuged at 6000rpm for 5min to collect thalli, the thalli is washed by sterile normal saline for 3 times, and then the thalli is resuspended by the same volume of the sterile normal saline to prepare seed suspension.

The seed suspension was inoculated into a 500mL Erlenmeyer flask containing 200mL of HNM medium at an inoculum size of 1.5%, and cultured at 30 ℃ and 160rpm for 48 hours. Periodically, 3mL of the culture medium was removed, and 1mL was used to determine the growth (OD) of the strain₆₀₀) And the remaining 2mL was centrifuged at 6000rpm for 5min and the supernatant was collected for NH determination₄ ⁺-N concentration.

The LB medium was formulated as described in example 1.

HNM medium formula (g/L): (NH)₄)₂SO₄0.66 parts of sodium succinate hexahydrate 7.88 parts of KH₂PO₄ 0.5，Na₂HPO₄·12H₂O 1.25，MgSO₄·7H₂O0.2, trace element solution 2mL, pH 8.0.

The formula (g/L) of the trace element solution is as follows: EDTA.2 Na 57.1, ZnSO₄·7H₂O 3.9，CaCl₂·2H₂O 7.0，MnCl₂·4H₂O 5.1，FeSO₄·7H₂O 5.0，(NH₄)₆Mo₇O₂₄·4H₂O 1.1，CuSO₄·5H₂O 1.6，CoCl₂·6H₂O 1.6，pH6.0。

The experimental results are shown in the following table and figure 1, the strain HO-1 grows well in an inorganic salt culture medium with ammonia nitrogen as a unique nitrogen source, has higher ammonia nitrogen removal capacity, and is inoculated with OD at the beginning when the initial ammonia nitrogen concentration is about 140mg/L₆₀₀Under the condition of about 0.03, the ammonia nitrogen removal rate can reach 99 percent within 18 hoursThe average removal efficiency was 168.32 mg/L/d.

Growth of strain HO-1 in inorganic salt culture medium with ammonia nitrogen as unique nitrogen source and removal of ammonia nitrogen

Example 3 detection of Nitrogen production during the Denitrification of the Strain HO-1

The strain HO-1 was activated according to the method of example 2, and 4mL of the seed culture was collected by centrifugation at 6000rpm for 5min, and the cells were washed 3 times with sterile physiological saline and then resuspended in 8mL of physiological saline to prepare a 2-fold diluted seed suspension.

1.2mL of the above seed suspension was taken out by a 2mL syringe and inoculated into a 500mL closed flask (not purged) containing 40mL of a nitrogen detection medium, and 1.2mL of physiological saline was added to the control group in the same manner, followed by shaking culture at 160rpm at 30 ℃. After 4 days, the gas in the bottle is taken out, and whether stable isotope labeled nitrogen is generated or not is detected by using a stable isotope ratio mass spectrometry method.

The formula (g/L) of the nitrogen detection culture medium is as follows:¹⁵n-stable isotope labeled NH₄Cl 0.19, sodium succinate hexahydrate 22.51, KH₂PO₄ 0.5，Na₂HPO₄·12H₂O 1.25，MgSO₄·7H₂O0.2, 2mL of trace element solution, pH 8.0. The formulation of the trace element solution was as described in example 2.

The results are shown in FIG. 2, in which the third peak is a nitrogen peak, and the delta of the peak is shown in the control group (FIG. 2A)¹⁵N/¹⁴N is 0.100 and the experimental group (FIG. 2B) is delta for this peak¹⁵N/¹⁴N is 12.122. According to the stable isotope ratio mass spectrum detection method, when delta¹⁵N/¹⁴N>1 hour indicates that the reaction system generates¹⁵N-labelled N₂. Thus, it was demonstrated that the strain HO-1 produced N during denitrification₂That is, the strain can independently convert ammonia into N under single aerobic condition₂. This property provides a great development value for the application of the strain, and can avoid the traditional growthThe denitrification technique has the defects of requiring intermittent aeration conditions or establishing two independent reactors.

Example 4 Ammonia nitrogen removal by Strain HO-1 at different C/N

A seed suspension of strain HO-1 was prepared according to the method of example 2.

HNM media with different C/N mass ratios are prepared, and the content of sodium succinate is changed according to the formula of the medium in the example 2, so that the C/N mass ratios are 2:1, 5:1, 8:1, 10:1, 15:1 and 20: 1.

Inoculating the seed suspension into HNM culture media with different C/N mass ratios according to the inoculation amount of 1.5%, wherein the initial ammonia nitrogen concentration of the culture media is 140mg/L, the initial pH is 8.0, and the seed suspension is cultured for 24 hours at 30 ℃ and 160 rpm. 3mL of the culture broth was taken, and 1mL was used to measure the growth (OD) of the strain₆₀₀) And the remaining 2mL was centrifuged at 6000rpm for 5min and the supernatant was collected for NH determination₄ ⁺-N concentration.

As shown in the following table and FIG. 3, the removal rates of bacterial strain HO-1 for ammonia nitrogen were 60.74%, 96.69%, 97.01%, 96.32%, 98.47% and 98.62% at C/N mass ratios of 2:1, 5:1, 8:1, 10:1, 15:1 and 20:1, respectively. The high carbon-nitrogen ratio is favorable for denitrification of the strain HO-1, but the high carbon-nitrogen ratio still has good removal rate when the C/N is low, and has wider C/N adaptability. When the mass ratio of C to N reaches 5:1, the ammonia nitrogen removal rate can reach more than 95 percent. The most suitable carbon-nitrogen ratio is 5-20: 1.

Growth of strain HO-1 under different C/N mass ratios and table for removing ammonia nitrogen

Example 4 Ammonia nitrogen removal by Strain HO-1 at different pH

HNM media of different pH were prepared, and the pH was changed to 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0 according to the medium formulation of example 2.

Inoculating the seed suspension into HNM culture medium with different pH values at an inoculation amount of 1.5%, and culturingThe initial ammonia nitrogen concentration of the culture medium is 140mg/L, the C/N mass ratio is 8, the culture is carried out for 24 hours at 30 ℃ and 160 rpm. 3mL of the culture broth was taken, and 1mL was used to measure the growth (OD) of the strain₆₀₀) And the remaining 2mL was centrifuged at 6000rpm for 5min and the supernatant was collected for NH determination₄ ⁺-N concentration.

The results are shown in the following table and figure 4, and the strain HO-1 has good growth and ammonia nitrogen removal effects under neutral alkaline conditions and poor growth and ammonia nitrogen removal effects under acidic conditions. When the pH value reaches 7 or above, the ammonia nitrogen removal rate can reach more than 98 percent, and the pH value is preferably 7-10.

Growth of strain HO-1 under different pH values and table for removing ammonia nitrogen

Example 5 Ammonia nitrogen removal by Strain HO-1 at different temperatures

Inoculating the seed suspension into HNM culture medium with 1.5% inoculum size, wherein the initial ammonia nitrogen concentration of the culture medium is 140mg/L, the C/N ratio is 8, the pH is 8.0, and the culture medium is cultured for 24h in a shaking table with the rotation speed of 160rpm and the temperatures of 4 ℃, 15 ℃, 23 ℃, 30 ℃, 37 ℃, 45 ℃ and 55 ℃ respectively. 3mL of the culture broth was taken, and 1mL was used to measure the growth (OD) of the strain₆₀₀) And the remaining 2mL was centrifuged at 6000rpm for 5min and the supernatant was collected for NH determination₄ ⁺-N concentration.

The HNM medium formulation is as described in example 2.

As shown in the following table and FIG. 5, when the culture temperature was low or high, the ammonia nitrogen removal efficiency of the strain HO-1 was poor and the growth was weak, indicating that the growth and denitrification were inhibited by low or high temperature. However, even if the temperature is 15 ℃, the strain has certain ammonia nitrogen removal capacity (the ammonia nitrogen removal rate is about 7%), when the temperature is raised to 23 ℃, the ammonia nitrogen removal capacity of the strain HO-1 is obviously improved, the ammonia nitrogen removal rate for 24 hours can reach 50%, and when the temperature is 30-37 ℃, the denitrification efficiency of the strain can reach more than 98%; if the temperature is further increased, the ammonia nitrogen removal capability of the strain HO-1 is inhibited, and when the temperature is up to 45 ℃, the ammonia nitrogen removal rate for 24h is only about 16 percent. Therefore, the temperature of the ammonia nitrogen containing sewage/wastewater may be 23 to 45 ℃, preferably 30 to 37 ℃.

Growth of strain HO-1 at different temperatures and removal table for ammonia nitrogen

Example 6 Ammonia nitrogen removal by Strain HO-1 at different dissolved oxygen

Inoculating the seed suspension into HNM culture medium with 1.5% inoculation amount, wherein the initial ammonia nitrogen concentration of the culture medium is 140mg/L, the C/N ratio is 8, the pH is 8.0, and the culture medium is cultured for 24h in a shaking table with the temperature of 30 ℃ and the rotating speed of 0(DO about 0.3mg/L), 50rpm (DO about 1.5mg/L), 100rpm (DO about 4mg/L), 150rpm (DO about 6mg/L), 200rpm (DO about 8mg/L) and 250rpm (DO about 8mg/L) respectively. 3mL of the culture broth was taken, and 1mL was used to measure the growth (OD) of the strain₆₀₀) And the remaining 2mL was centrifuged at 6000rpm for 5min and the supernatant was collected for NH determination₄ ⁺-N concentration.

The HNM medium formulation is as described in example 2.

As shown in the following table and FIG. 6, the ammonia nitrogen removal efficiency of the strain HO-1 under the conditions of 0, 50rpm, 100rpm, 150rpm, 200rpm and 250rpm of the strain HO-1 was 46.81%, 57.89%, 98.50%, 98.37%, 98.52% and 98.52%, respectively. The difference of the rotating speeds of the shaking tables can indirectly control the dissolved oxygen level in the reaction solution, and when the rotating speeds of the shaking tables are 0rpm and 50rpm, the dissolved oxygen level in the reaction solution is lower, and the ammonia nitrogen removal rate is relatively lower. When the rotating speed of the shaking table reaches 100rpm, the removal rate of the ammonia nitrogen in 24 hours reaches more than 98 percent. The bacterial strain HO-1 has wider dissolved oxygen adaptability, and the dissolved oxygen of the sewage/wastewater can be 2-8mg/L, preferably 4-8 mg/L.

Growth of strain HO-1 at different table rotation speeds (dissolved oxygen) and table for removing ammonia nitrogen

Example 7 denitrification and intermediate product accumulation of Strain HO-1 in Ammonia containing Sewage/wastewater

Inoculating the seed suspension with 1.5% of inoculum size into a 500mL triangular flask containing 200mL ammonia nitrogen-containing liquid M, and culturing at 30 ℃ and 160rpm for 72 h. Periodically, 3mL of the culture medium was removed, and 1mL was used to determine the growth (OD) of the strain₆₀₀) And the remaining 2mL was centrifuged at 6000rpm for 5min and the supernatant was collected for NH determination₄ ⁺-N and NO₂ ^--N concentration. And the residual total nitrogen (TN, including biological nitrogen) content was determined at 0h and 72 h.

The formula of the nitrogen-containing liquid M comprises: (g/L): (NH)₄)₂SO₄1.89, sodium succinate hexahydrate 22.51, KH₂PO₄ 0.5，Na₂HPO₄·12H₂O 1.25，MgSO₄·7H₂O0.2, trace element solution 2mL, pH 8.0. The formulation of the trace element solution was as described in example 2.

The experimental results are shown in figure 7 and the following table, the bacterial strain HO-1 can remove more than 99% of ammonia nitrogen in the nitrogen-containing liquid with the initial ammonia nitrogen concentration of about 400mg/L within 48 hours, the average ammonia nitrogen removal rate is 189.55mg/L/d, no nitrate is produced in the denitrification process, almost no nitrite is accumulated, and the total nitrogen removal rate including biological nitrogen can reach nearly 47%. The ammonia nitrogen in the nitrogen-containing liquid is mostly converted into gaseous nitrogen products (N) except for being used for synthesizing biological nitrogen₂O、N₂)。

Example 7 growth and Ammonia Nitrogen removal in high concentration Ammonia Nitrogen Sewage/wastewater of Strain HO-1

Inoculating the seed suspension with 1.5% of inoculum size50mL of a high-concentration nitrogen-containing liquid H (initial ammonia nitrogen concentration of about 2240mg/L) was cultured in a 500mL Erlenmeyer flask at 30 ℃ and 160rpm for 9 days. Periodically, 3mL of the culture medium was removed, and 1mL was used to determine the growth (OD) of the strain₆₀₀) And the remaining 2mL was centrifuged at 6000rpm for 5min and the supernatant was collected for NH determination₄ ⁺-N concentration.

The high-concentration nitrogen-containing liquid H comprises the following formula: (g/L): NH (NH)₄Cl 8.56, sodium succinate hexahydrate 109.0, KH₂PO₄1.5，Na₂HPO₄·12H₂O 7.9，MgSO₄·7H₂O0.2, trace element solution 2mL, pH 8.0. The formulation of the trace element solution was as described in example 2.

The experimental results are shown in the following table and figure 8, the growth rate and the ammonia nitrogen removal rate of the strain HO-1 in high-concentration ammonia nitrogen sewage/wastewater are rapidly improved after 1d of adaptation period, the ammonia nitrogen removal rate after 2d reaches 53.0%, and the ammonia nitrogen removal amount in 24h in the next day reaches 1121.53 mg/L. After reacting for 4 days, the ammonia nitrogen is removed to be below 400mg/L, and the removal rate of the ammonia nitrogen is more than 80 percent. Experimental results show that the strain HO-1 can realize the removal rate of ammonia nitrogen to be more than 80% under the condition that the initial ammonia nitrogen concentration is higher than 2000mg/L, so the strain HO-1 has great application potential in the treatment of water containing high-concentration ammonia nitrogen.

While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the principles of the invention.

Growth of strain HO-1 in high-concentration ammonia nitrogen sewage/wastewater and removal table of ammonia nitrogen

Sequence listing

<110> institute of microbiology of Chinese academy of sciences

<120> alkaline producing strain HO-1 and application thereof

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<170> SIPOSequenceListing 1.0

<210> 1

<211> 1525

<212> DNA

<213> Alcaligenes sp

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agagtttgat cctggctcag attgaacgct agcgggatgc tttacacatg caagtcgaac 60

ggcagcacga gagagcttgc tctcttggtg gcgagtggcg gacgggtgag taatatatcg 120

gaacgtgccc agtagcgggg gataactact cgaaagagtg gctaataccg catacgccct 180

acgggggaaa gggggggatc gcaagacctc tcactattgg agcggccgat atcggattag 240

ctagttggtg gggtaaaggc tcaccaaggc aacgatccgt agctggtttg agaggacgac 300

cagccacact gggactgaga cacggcccag actcctacgg gaggcagcag tggggaattt 360

tggacaatgg gggaaaccct gatccagcca tcccgcgtgt atgatgaagg ccttcgggtt 420

gtaaagtact tttggcagag aagaaaaggc atcccctaat acgggatgct gctgacggta 480

tctgcagaat aagcaccggc taactacgtg ccagcagccg cggtaatacg tagggtgcaa 540

gcgttaatcg gaattactgg gcgtaaagcg tgtgtaggcg gttcggaaag aaagatgtga 600

aatcccaggg ctcaaccttg gaactgcatt tttaactgcc gagctagagt atgtcagagg 660

ggggtagaat tccacgtgta gcagtgaaat gcgtagatat gtggaggaat accgatggcg 720

aaggcagccc cctgggataa tactgacgct cagacacgaa agcgtgggga gcaaacagga 780

ttagataccc tggtagtcca cgccctaaac gatgtcaact agctgttggg gccgttaggc 840

cttagtagcg cagctaacgc gtgaagttga ccgcctgggg agtacggtcg caagattaaa 900

actcaaagga attgacgggg acccgcacaa gcggtggatg atgtggatta attcgatgca 960

acgcgaaaaa ccttacctac ccttgacatg tctggaatgc cgaagagatt tggcagtgct 1020

cgcaagagaa ccggaacaca ggtgctgcat ggctgtcgtc agctcgtgtc gtgagatgtt 1080

gggttaagtc ccgcaacgag cgcaaccctt gtcattagtt gctacgcaag agcactctaa 1140

tgagactgcc ggtgacaaac cggaggaagg tggggatgac gtcaagtcct catggccctt 1200

atgggtaggg cttcacacgt catacaatgg tcgggacaga gggtcgccaa cccgcgaggg 1260

ggagccaatc tcagaaaccc gatcgtagtc cggatcgcag tctgcaactc gactgcgtga 1320

agtcggaatc gctagtaatc gcggatcaga atgtcgcggt gaatacgttc ccgggtcttg 1380

tacacaccgc ccgtcacacc atgggagtgg gtttcaccag aagtaggtag cctaaccgca 1440

aggagggcgc ttaccacggt gggattcatg actggggtga agtcgtaaca aggtagccgt 1500

atcggaaggt gcggctggat cacct 1525

Claims

1. Alcaligenes (A) and (B)Alcaligenes sp.) strain, which is characterized in that the name of the alcaligenes strain is HO-1, and the preservation number is CGMCC No. 16549.

2. Use of the strain according to claim 1 for biological denitrification of sewage or wastewater.

3. Use according to claim 2, wherein the C/N mass ratio in the wastewater or waste water is 2-20: 1.

4. Use according to claim 2, wherein the C/N mass ratio in the wastewater or waste water is 5-20: 1.

5. The use according to claim 2, wherein the pH of the wastewater or waste water is in the range of 5 to 10.

6. The use according to claim 2, wherein the pH of the wastewater or waste water is in the range of 7-10.

7. Use according to claim 2, wherein the temperature of the waste water or wastewater is in the range of 15-45 ℃.

8. Use according to claim 2, wherein the temperature of the waste water or wastewater is in the range of 25-37 ℃.

9. The use according to claim 2, wherein said wastewater or waste water has dissolved oxygen in the range of 2-8 mg/L.

10. The use according to claim 2, wherein said wastewater or waste water has dissolved oxygen in the range of 4-8 mg/L.