CN110607244B

CN110607244B - Heterotrophic nitrification aerobic denitrification candida rugosa strain and application thereof

Info

Publication number: CN110607244B
Application number: CN201911052273.XA
Authority: CN
Inventors: 兰时乐; 杜全能; 胡超; 朱文娟; 陈思宇; 肖思远
Original assignee: Hunan Agricultural University
Current assignee: Hunan Agricultural University
Priority date: 2019-10-31
Filing date: 2019-10-31
Publication date: 2021-04-27
Anticipated expiration: 2039-10-31
Also published as: CN110607244A

Abstract

The invention provides a heterotrophic nitrification aerobic denitrification Candida rugosa strain and application thereof, belonging to the technical field of functional microorganisms. Heterotrophic nitrification-aerobic denitrification Candida rugosa (Diutina rugosa) strain DW-1 with the preservation number of CCTCC NO: m2019166. The invention also provides a microbial preparation containing the strain DW-1 for degrading ammonia nitrogen and nitrite nitrogen in freshwater aquaculture sewage. The DW-1 strain has the functions of heterotrophic nitrification-aerobic denitrification, can directly convert most of ammonia nitrogen in the nitrogen-containing sewage into a gas product through the heterotrophic nitrification-aerobic denitrification, and has less accumulated amounts of nitrite and nitrate nitrogen; the Candida rugosa strain DW-1 or the microbial preparation can be applied to denitrification of freshwater aquaculture water bodies because ammonia nitrogen or nitrite nitrogen is used as a unique nitrogen source for metabolism and high-efficiency denitrification.

Description

Heterotrophic nitrification aerobic denitrification candida rugosa strain and application thereof

Technical Field

The invention belongs to the technical field of functional microorganisms, and particularly relates to a heterotrophic nitrification aerobic denitrification candida rugosa strain and application thereof.

Background

With the increasing severity of the eutrophication problem of the freshwater aquaculture water body, the implementation of policies such as purse net aquaculture, fence aquaculture cancellation, fish returning and lake returning and the like on a large water surface is caused, and the freshwater aquaculture is promoted to play an irreplaceable role in the aquaculture industry of China. However, with the increasing raising of the culture level and the increasing of the fish carrying amount of the unit water body, the culture water quality is seriously polluted due to the fact that a large amount of feed and fish metabolites are put in, and the culture wastewater is directly discharged to a surrounding river channel without being treated, so that the problem of eutrophication of the surrounding water body is increasingly serious.

Nitrogen is an important pollutant in water body pollution, and mainly exists in two forms of organic nitrogen and inorganic nitrogen, wherein the organic nitrogen comprises protein, amino acid, urea and the like; the inorganic nitrogen mainly comprises ammonia nitrogen, nitrate nitrogen and nitrite nitrogen. The method for removing nitrogen in the aquaculture sewage is divided into a physical and chemical denitrification method and a biological denitrification method. The physical and chemical denitrification method has high cost and is difficult to popularize and apply. Biological denitrification is recognized as an economical, effective, environmentally friendly and most promising method for removing nitrogen from sewage. The traditional biological denitrification way comprises two stages of aerobic nitrification and anaerobic denitrification which are respectively completed by nitrifying bacteria and denitrifying bacteria. Because of the different flora and process operating parameters involved, the two processes of nitrification and denitrification need to be carried out in two isolated reactors or in the same reactor which causes alternate anoxic and aerobic environments in time or space. In recent years, some heterotrophic nitrifying bacteria, aerobic denitrifying bacteria and heterotrophic nitrifying aerobic denitrifying bacteria are discovered, and the whole denitrification process can be completed in the same reaction vessel. Compared with the traditional process, the heterotrophic Nitrification-aerobic Denitrification (SND) process can save the volume of a reaction container, shorten the reaction time and is convenient to operate and manage, so that the microbial Denitrification becomes one of the hot problems of the research in the field of water pollution control.

In recent decades, a great deal of research has been conducted by both domestic and foreign scholars on heterotrophic nitrification-aerobic denitrification bacteria. The strains to be studied are mainly Paracoccus (paracoccus), Pseudomonas (pseudomonas), Alcaligenes (alcaligenes), Bacillus (Bacillus), Rhodococcus (rhodococcus), Acinetobacter (Acinetobacter), and the like. Taylor Shauna M and the like screen a providencia rettgeri YL from a membrane bioreactor, under the optimal condition (C/N ratio is 10,30 ℃, 120r/min), when the mass concentration of ammonia nitrogen is 84.63mg/L, the removal rate of Total Nitrogen (TN) after inoculating the providencia germ rettgeri YL is 79% (Taylor Shauna M et al, heter-trophic ammonium removal characteristics of an aerobiotic hemiphotic [ J ] for 48h]Journal of Environmental Sciences,2009, 21(10): 1336-1341.); a heterotrophic nitrification-aerobic denitrification strain Klebsiella sp.y6 is separated from submarine sediments by the Wangxing et al, and researches show that the strain has the optimum pH value of 7.0, the optimum C/N of 17 and the optimum carbon source of trisodium citrate. The strain y6 has high denitrification capacityEffectively remove organic matters (Wangxing Jingjing, ocean heterotrophic nitrification-aerobic denitrifying bacteria y6 synchronous denitrification and decarbonization characteristics [ J]Chinese environmental science, 2017,37(2): 686-; chen et al developed a system for the simultaneous removal of high salinity and high nitrogen organic wastewater in a pressurized biofilm reactor. The results show that at an air supply rate of 200L/h, the salinity is 3.0 +/-0.2%, and the organic load is 10kgCOD/m³D, nitrogen load 0.185kg/m³D, rapid start-up and stable after 30 days of operation. At the same time, the simultaneous degradation of COD and nitrogen is realized in a single-stage reactor, wherein COD and NH⁴⁺The degradation efficiencies of N and TN are 97%, 99% and 98%, respectively (Chen J et. Start-up and microbial compositions of a microbial biomass removal system for high saline and high microbial biomass water vitamin microbial degradation [ J.]Bioresource Technology, 2016, 216:196- & 202.); zhang et al determined the NAR and NIR, HAO activities of Microbacterium sp.strain SFA13 at 0.15. mu. mol/min, 0.21. mu. mol/min and 0.44. mu. mol/min (Zhang D. Removal of ammonium in surface water at low temperature by a new technology, 2013,137C (6): isolated Microba-bacterium sp.strain SFA13[ J]Bioresource, 147-. The nitrogen degradation function of Candida rugosa and the application thereof in the treatment of aquaculture sewage are not reported in domestic and foreign documents.

Disclosure of Invention

In view of the above, the present invention aims to provide a candida rugosa strain for heterotrophic nitrification and aerobic denitrification and an application thereof, wherein the candida rugosa strain has the performance of heterotrophic nitrification and aerobic denitrification under aerobic conditions, and can efficiently remove nitrogen in a freshwater aquaculture water body.

The invention provides a heterotrophic nitrification aerobic denitrification Candida rugosa (Diutina rugosa) strain DW-1 with a preservation number of CCTCC NO: m2019166.

The invention provides a microbial preparation for degrading ammonia nitrogen and nitrite nitrogen in freshwater aquaculture sewage, which comprises Candida rugosa (Diutina rugosa) strain DW-1.

Preferably, the Candida rugosa (Diutina rugosa) strain DW-1 is viableThe concentration of the bacteria is 10¹⁰～10¹⁵CFU/mL。

The invention provides an application of the Candida rugosa (Diutina rugosa) strain DW-1 or the microbial preparation in denitrification of a fresh water aquaculture water body.

Preferably, the method for denitrifying the fresh water aquaculture water body comprises the following steps:

inoculating candida rugosa DW-1 strain bacterial liquid into freshwater aquaculture sewage, and performing aerobic culture and denitrification treatment at the same time.

Preferably, the nitrogen in the denitrification of the freshwater aquaculture water body comprises ammonia nitrogen and/or nitrite nitrogen.

Preferably, in the freshwater aquaculture water body, the concentration of total nitrogen, ammonia nitrogen or nitrite nitrogen consisting of ammonia nitrogen and nitrite nitrogen is 40-100 mg/L.

Preferably, the viable bacteria concentration of the bacteria liquid of the Candida rugosa DW-1 strain is 10¹⁰～10¹⁵CFU/mL; the inoculation amount of the candida rugosa DW-1 strain liquid is 0.1-1%.

Preferably, the initial pH value of the freshwater aquaculture sewage is 4.0-8.5.

Preferably, during the aerobic culture period, the dissolved oxygen amount of the freshwater aquaculture sewage is 1.5-3 mg/L;

the culture temperature of the aerobic culture is 20-36 ℃, the time of the aerobic culture is 24-48 h, and the C/N ratio is 10-25.

The heterotrophic nitrification aerobic denitrification Candida rugosa (Diutina rugosa) strain DW-1 provided by the invention has the preservation number of CCTCC NO: m2019166. The DW-1 strain provided by the invention has the functions of heterotrophic nitrification-aerobic denitrification, can directly convert most of ammonia nitrogen in nitrogen-containing sewage into a gas product through the heterotrophic nitrification-aerobic denitrification, and has less accumulated amounts of nitrite and nitrate nitrogen; the strain DW-1 can be metabolized by taking ammonia nitrogen as a unique nitrogen source, the ammonia nitrogen removal rate can reach 90.28%, and the ammonia nitrogen degradation rate can reach 0.99 mg/(L.h); the strain DW-1 can be metabolized by taking nitrite nitrogen as a unique nitrogen source, the nitrite nitrogen removal rate reaches 98%, and the nitrite nitrogen degradation rate reaches 0.83 mg/(L.h); the DW-1 strain realizes biological denitrification of nitrogen-containing sewage by an SND method, and experiments prove that the DW-1 strain is adopted to treat a freshwater aquaculture water body under an aerobic condition, and the degradation rates of ammonia nitrogen or nitrite nitrogen are 48.6 percent and 71.8 percent respectively when the strain is treated for 24 hours; when the treatment is carried out for 48 hours, the degradation rates are respectively 93.2 percent and 91.3 percent. The Candida rugosa strain DW-1 provided by the invention can realize the nitrification-denitrification function at the same time, is convenient to use and simple to operate, reduces the freshwater aquaculture cost and realizes the purpose of freshwater green aquaculture. The strain DW-1 can be used as a good strain of a novel microecological preparation and has good application prospect.

Drawings

FIG. 1 is a photograph of a colony of Candida rugosa DW-1 on a PDA solid medium plate;

FIG. 2 shows the cell morphology of Candida rugosa DW-1 under a normal light microscope;

FIG. 3 is an electrophoretogram of the 26S rDNA gene amplified fragment of Candida rugosa DW-1;

FIG. 4 is a phylogenetic tree constructed from the 26S rDNA gene of Candida rugosa DW-1;

FIG. 5 is a graph showing the results of the removal rates of total nitrogen, ammonia nitrogen and nitrite nitrogen after the culture wastewater is treated by using the strain DW-1 under different C/N ratios;

FIG. 6 shows the removal rate of total nitrogen, ammonia nitrogen and nitrite nitrogen after the culture wastewater is treated by the strain DW-1 at different culture temperatures.

Biological material preservation information

Candida rugosa DW-1(Diutina rugosa DW-1) is preserved in China Center for Type Culture Collection (CCTCC) in 2019, 4 months and 2 days, and the preservation unit addresses are as follows: china, Wuhan university, the preservation number is CCTCC NO: m2019166.

Detailed Description

The invention provides a heterotrophic nitrification-aerobic denitrification Candida rugosa (Diutina rugosa) strain DW-1 with a preservation number of CCTCC NO: m2019166. The strain DW-1 is identified by morphology and molecular biology respectively, and the result is as follows:

(1) morphological characteristics

The colony morphology of Candida rugosa DW-1 is as follows: the bacterial colony is round, milky white, moist, smooth in surface and opaque.

(2) Molecular biological identification

Carrying out PCR amplification by using an amplification primer of a 26S rDNA gene and using the genome DNA of the DW-1 strain as a template to obtain a PCR product with the total length of 504 bp; the sequence was analyzed for sequence homology by the BLAST search program System of the National Center for Biotechnology Information (NCBI) and found to have 100% similarity to the 26S rDNA D1/D2 region gene sequence of Candida rugosa (Diutina rugosa).

According to the morphological characteristics, physiological and biochemical properties and molecular biological identification characteristics of the DW-1 strain, the DW-1 strain is identified to belong to Candida rugosa and is named as Candida rugosa DW-1(Diutina rugosa DW-1).

The DW-1 strain provided by the invention can realize the nitrification-denitrification function at the same time, directly converts ammonia nitrogen or nitrite nitrogen into a gas product, has less accumulation of nitrite and nitrate nitrogen, metabolizes by taking the ammonia nitrogen or nitrite nitrogen as a unique nitrogen source, and has the ammonia nitrogen removal rate of 90.28% and the nitrite nitrogen removal rate of 98% after being treated for 36 hours.

In the present invention, the method for culturing the strain DW-1 preferably comprises the following steps:

inoculating the strain DW-1 into a liquid seed culture medium, and carrying out constant-temperature shaking culture at the temperature of 28-32 ℃ and at the speed of 160-200 r/min to obtain a seed solution; the liquid seed culture medium: 1000mL of 20% potato juice, 20.0g of glucose and natural pH value; sterilizing at 121 deg.C for 25 min.

Based on the fact that the strain DW-1 can simultaneously realize the nitrification-denitrification function, the invention provides a microbial preparation for degrading ammonia nitrogen and nitrite nitrogen in freshwater aquaculture sewage, which comprises the Candida rugosa (Diutina rugosa) strain DW-1. The viable bacteria concentration of the Candida rugosa (Diutina rugosa) strain DW-1 is preferably 10¹⁰～10¹⁵CFU/mL. The method for preparing the microbial preparation is not particularly limited, and a method for preparing a microbial preparation known in the art may be used.

In the present invention, the method for denitrifying the freshwater aquaculture water body preferably comprises the following steps:

inoculating candida rugosa DW-1 strain bacterial liquid into freshwater aquaculture sewage, and performing aerobic culture and denitrification treatment at the same time. The freshwater aquaculture water body preferably comprises a pond, a lake, a river or a reservoir. The nitrogen in the denitrification of the fresh water aquaculture water body preferably comprises ammonia nitrogen and/or nitrite nitrogen. In the fresh water aquaculture water body, the concentration of total nitrogen, ammonia nitrogen or nitrite nitrogen formed by ammonia nitrogen and nitrite nitrogen is preferably 40-100 mg/L, and more preferably 50-80 mg/L.

In the present invention, the viable bacteria concentration of the bacterial liquid of Candida rugosa strain DW-1 is preferably 10¹⁰～10¹⁵CFU/mL, more preferably 10¹¹～10¹⁴CFU/mL, most preferably 10¹³CFU/mL. The inoculation amount of the candida rugosa DW-1 strain liquid is 0.1-1%, more preferably 0.3-0.8%, and most preferably 0.5%.

In the invention, the initial pH value of the freshwater aquaculture sewage is preferably 4.0-8.5, more preferably 6.0-8.0, and most preferably 7.0-7.5. During the aerobic culture period, the dissolved oxygen amount of the freshwater aquaculture sewage is preferably 1.5-3 mg/L, and more preferably 2.0 mg/L.

In the invention, the culture temperature of the aerobic culture is preferably 20-36 ℃, the culture temperature is different according to the types of the removed nitrogen, and when removing ammonia nitrogen, the culture temperature is preferably 28-36 ℃, and most preferably 32 ℃. When removing the nitrite nitrogen, the culture temperature is preferably 20-28 ℃, and most preferably 20 ℃. The aerobic culture time is preferably 24-48 h.

In the invention, during the aerobic culture, the C/N ratio in the freshwater aquaculture sewage is preferably 10-25. The C/N ratio is different according to the types of the removed nitrogen, and when ammonia nitrogen is removed, the C/N ratio is preferably 15-25, and most preferably 25 ℃. When removing the nitrous nitrogen, the C/N ratio is preferably 10-25, and most preferably 10.

In the invention, after the aerobic culture is finished, the content of ammonia nitrogen and/or nitrite nitrogen in the water body is measured. Ammonia nitrogen is determined by adopting a method for determining the ammonia nitrogen in water by adopting a nano-reagent spectrophotometry (HJ 535-2009); nitrite nitrogen in water is determined by spectrophotometry (GB 7493-87).

The heterotrophic nitrification-aerobic denitrification Candida rugosa strain and its use provided by the present invention are described in detail below with reference to the examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Screening method of bacterial strain

1. Preliminary screening

10mL of water sample is collected from a freshwater aquaculture water body with high ammonia nitrogen content, added into 100mL of culture medium containing 52.39mg/L ammonia nitrogen, and subjected to enrichment culture for 3d under the conditions of 30 ℃ and 180r/min to obtain an enrichment solution for later use.

2. Diluting the prepared enrichment solution to 10 times by a 10-fold dilution method^-7Respectively take 10^-5、10^-6、10^-7And coating the three diluents with different dilutions on a nitrifying solid culture medium plate, and culturing for 48h in a 30 ℃ thermostat to obtain bacterial colonies.

The formula and the preparation method of the nitrifying solid medium (g/L) are as follows: (NH)₄)₂SO₄·7H₂O 0.247g、K₂HPO₄7g、KH₂PO₄3g、MgSO₄·7H₂O 0.1g、FeSO₄·7H₂O 0.05g、H₂O1L, agar 20 g. Diluting with distilled water to constant volume, adjusting initial pH to 7.0, sterilizing at 121 deg.C for 20min, and making into nitrified solid culture medium plate.

3. Purification of

And (3) selecting a single colony on the nitrifying solid culture medium plate, streaking the single colony on the nitrifying solid culture medium plate, and culturing for 48 hours in a 30-DEG C incubator until a single colony is obtained.

4. Preservation of

And respectively inoculating the obtained single colonies on a PDA solid slant culture medium, culturing for 48h in a 30 ℃ incubator, and storing in a 4 ℃ refrigerator.

The formula and the preparation method of the PDA solid slant culture medium are as follows: 1000mL of 20% potato juice, 20.0g of glucose and 20.0g of agar powder, and the pH value is natural. Sterilizing at 121 deg.C for 25min, and making into PDA solid culture medium plate.

5. Double sieve

Inoculating the separated strain into a liquid seed culture medium, and carrying out constant temperature shaking culture at 30 ℃ and 180r/min for 24 h. The formula and the preparation method of the liquid seed culture medium comprise the following steps: 1000mL of 20% potato juice, 20.0g of glucose and natural pH value. Sterilizing at 121 deg.C for 25 min.

Respectively inoculating the cultured liquid seed culture medium into an ammonia oxidation culture medium (initial nitrogen concentration of 52.39mg/L) and a nitrite reduction culture medium (initial nitrogen concentration of 40.58mg/L) according to the inoculation amount of 1%, respectively carrying out constant-temperature shaking culture at 30 ℃ and 180r/min, and measuring ammonia nitrogen and nitrite nitrogen after 36 h. The formula and the preparation method of the ammonia oxidation culture medium comprise the following steps: (NH)₄)₂SO₄·7H₂O 0.247g、K₂HPO₄ 7g、KH₂PO₄ 3g、MgSO₄·7H₂O 0.1g、FeSO₄·7H₂O 0.05g、H₂O1L. Sterilizing at 121 deg.C for 25 min. The formula and the preparation method of the nitrite reduction culture medium comprise the following steps: NaNO₂ 0.2g、K₂HPO₄ 7g、KH₂PO₄ 3g、MgSO₄·7H₂O 0.1g、FeSO₄·7H₂O 0.05g、H₂O1L. Sterilizing at 121 deg.C for 25 min. Ammonia nitrogen is determined by adopting a method for determining the ammonia nitrogen in water by adopting a nano-reagent spectrophotometry (HJ 535-2009); nitrite nitrogen in water is determined by spectrophotometry (GB 7493-87).

6. Determination of heterotrophic nitrification-aerobic denitrification strains

And (5) selecting the strain with the highest heterotrophic nitrification-aerobic denitrification performance according to the result of the step (5), wherein the strain can be metabolized by taking ammonia nitrogen as a unique nitrogen source, the ammonia nitrogen removal rate can reach 90.28%, and the ammonia nitrogen degradation rate can reach 0.99 mg/(L.h). Meanwhile, the DW-1 strain can metabolize by taking nitrite nitrogen as a unique nitrogen source, the nitrite nitrogen removal rate reaches 98%, and the nitrite nitrogen degradation rate reaches 0.83 mg/(L.h). Designated as DW-1 strain.

Example 2

Morphological characteristics and physiological and biochemical characteristics identification of strain DW-1

1. Morphological characterization

The colony morphology of the strain DW-1 is characterized as follows:

inoculating strain DW-1 in liquid seed culture medium, shake culturing at 30 deg.C and 180r/min for 24 times, and diluting to 10 times by 10 times dilution method^-8Take 10^-6、10^-7、10^-80.1mL of diluent with three different dilutions is evenly coated on the surface of a PDA solid culture medium plate, and cultured for 24h under the constant temperature condition of 30 ℃, and the observed colony characteristics are as follows: the colonies were round, milky white, moist, smooth on the surface, and opaque (see FIGS. 1 and 2).

Example 3

26S rDNA sequence analysis

1. Extraction of strain DW-1 genome

(1) And (3) culturing thalli: inoculating the strain DW-1 into a PD liquid culture medium, carrying out shake culture at 30 ℃ and 180r/min for 24 times, and centrifuging at 4 ℃ and 10000r/min to collect thalli.

(2) Extracting genome DNA: the 26S rDNA D1/D2 region genomic DNA of the DW-1 strain was extracted using an Ezup column yeast genomic DNA extraction kit produced by Biotechnology engineering (Shanghai) Ltd.

(3) And (3) storage: the extracted genomic DNA of the DW-1 strain is placed at-20 ℃.

(4) And (3) detection: the extracted genomic DNA of the DW-1 strain was electrophoresed through 1% agarose gel, and then observed and photographed in a gel imaging system, and an electrophoretogram was obtained as shown in FIG. 3.

PCR amplification

The PCR amplification primers used were the common primers NL1(5'-GCATATCAATAAGCGGAGGAAAAG-3', SEQ ID No.1) and NL4(5'-GGTCCGTGTTTCAAGACGG-3', SEQ ID No.2) of the 26S rDNA D1/D2 region.

PCR amplification conditions: pre-denaturation at 95 deg.C for 5min, denaturation at 95 deg.C for 1min, annealing at 55 deg.C for 1min, extension at 72 deg.C for 1min, and 30 cycles; finally, extension is carried out for 10min at 72 ℃ to obtain a PCR product.

The PCR product is obtained by the complete sequence determination and analysis recovery of the 3.26S rDNA D1/D2 region, and sent to the company of Biotechnology engineering (Shanghai) to be sequenced, and the total sequence of the 26S rDNA 1/D2 region gene of the strain DW-1 is obtained to have 504 bases (GCGGAGGAAAAGAAACCAACCGGGATTGCCTCAGTAACGGCGAGTGAAGCGGCAACAGCTCAAATTTGAAAGCCCGCGGGCGTTGTAATTTGCAGGCGGATGTTTTGGGGCGGGCGCTGTCTACGTTCCTTGGAACAGGACGCCGCAGAGGGTGAGAGCCCCGTGCGATGGCGCCTCTAACCGCGTAAAACTCCGCCGACGAGTCGAGTTGTTTGGGAATGCAGCTCCAAGTGGGTGGTAAATTCCATCTAAAGCTAAATACTGGCGAGAGACCGATAGCGAACAAGTACAGTGATGGAAAGATGAAAAGCACTTTGAAAAGAGAGTGAAACAGCACGTGAAATTGTTGAAAGGGAAGGGTATGCGATTAGCGGCCAGCAGGAGGTGCCTTCTCGTGAAAAGGCCGTGCACCGTCTTCGGACACCGTGCGCGGAGATGGCGAGGGGGCGCCTGAGGTCTGCGAACTCGAGGTTGCTGGCGTAATGATTGCATACCACCCGTCTT, SEQ ID No. 3). The sequences were analyzed for sequence homology by the BLAST search program system of the National Center for Biotechnology Information (NCBI) to construct phylogenetic trees (see FIG. 4) and found to have 100% similarity to the gene sequence of the 26S rDNA 1/D2 region of Candida rugosa (Diutina rugosa).

According to the morphological characteristics, physiological and biochemical properties and molecular biological identification characteristics of the DW-1 strain of the present invention, the DW-1 strain is identified as Candida rugosa (Diutina rugosa) and named as Candida rugosa DW-1(Diutina rugosa DW-1) according to the content of Bergey's Manual of bacteria identification (eighth edition).

Example 4

Experiment for treating freshwater aquaculture sewage denitrification by using strain DW-1 with different C/N ratios

Inoculating the strain DW-1 in a culture medium: 1000mL of 20% potato juice, 20.0g of glucose and natural pH value. And culturing at 30 deg.c and 180r/min for 24 hr to obtain strain DW-1 seed liquid.

Inoculating the prepared strain DW-1 seed solution into culture wastewater (ammonia nitrogen is 18.24mg/L, and nitrite nitrogen is 0.104mg/L) according to the inoculation amount of 0.1% (V/V), wherein the C/N ratio of the culture wastewater is respectively 10, 15, 20, 25 and 30, determining the ammonia nitrogen, nitrite nitrogen and total nitrogen content in the culture wastewater after aerobic culture (dissolved oxygen is 2.5mg/L) for 24 hours at 30 ℃, and counting the removal rate.

The results are shown in FIG. 5. FIG. 5 shows the removal rate of total nitrogen, ammonia nitrogen and nitrite nitrogen after the culture wastewater is treated by the strain DW-1 under different C/N ratios. As can be seen from FIG. 5, in the ammonia nitrogen treatment, the removal rate is more than 80% when the C/N ratio is 15-25, wherein the removal rate is 95% at most when the C/N ratio is 25, and the removal rate of ammonia nitrogen is only about 60% when the C/N ratio is 10 or 30; in the nitrous nitrogen treatment, the removal rate is slightly reduced along with the increase of the C/N ratio, wherein the removal rate is more than 85% when the C/N ratio is 10, the removal rate is more than 60% when the C/N ratio is 15-25, and the removal rate is the lowest and only reaches 50% when the C/N ratio is 30.

Example 5

Inoculating the prepared strain DW-1 seed solution into culture wastewater (ammonia nitrogen is 18.24mg/L, and nitrite nitrogen is 0.104mg/L) according to the inoculation amount of 0.1% (V/V), wherein the C/N ratio of the culture wastewater is 10:1, the culture temperatures are respectively set to be 20 ℃, 24 ℃, 28 ℃, 32 ℃ and 36 ℃, and after culturing for 24 hours under aerobic conditions (dissolved oxygen is 2.3mg/L), the ammonia nitrogen, nitrite nitrogen and total nitrogen content in the culture wastewater are measured, and the removal rate is counted.

The results are shown in FIG. 6. FIG. 6 shows the removal rate of total nitrogen, ammonia nitrogen and nitrite nitrogen after the culture wastewater is treated by the strain DW-1 at different culture temperatures. As can be seen from FIG. 6, the temperature has a great influence on the ammonia nitrogen removal effect of the strain DW-1 in freshwater aquaculture sewage treatment, specifically, the ammonia nitrogen removal rate gradually increases with the increase of the temperature, the removal rate reaches the best (more than 90%) at 32 ℃, and the removal rate slightly decreases (85%) at 36 ℃, so that the culture temperature can be selected from the range of 28-36 ℃ during the ammonia nitrogen removal process, and the removal rate reaches more than 75%. Compared with the ammonia nitrogen removal effect, the nitrite nitrogen removal effect is slightly low in the same system, the ammonia nitrogen removal effect of the strain DW-1 in freshwater aquaculture sewage treatment is not greatly influenced by the specific culture temperature, the nitrite nitrogen removal effect is higher at the culture temperature of 20 ℃, the nitrite nitrogen removal rate reaches over 60%, and the nitrite nitrogen removal rate is slightly reduced along with the increase of the culture temperature, so that the culture temperature can be selected from the range of 220-28 ℃ in the nitrite nitrogen removal process, and the nitrite nitrogen removal rate reaches over 45%.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Hunan agriculture university

<120> heterotrophic nitrification-aerobic denitrification Candida rugosa strain and application thereof

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

gcatatcaat aagcggagga aaag 24

<210> 2

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

ggtccgtgtt tcaagacgg 19

<210> 3

<211> 504

<212> DNA

<213> Diutina rugosa

<400> 3

gcggaggaaa agaaaccaac cgggattgcc tcagtaacgg cgagtgaagc ggcaacagct 60

caaatttgaa agcccgcggg cgttgtaatt tgcaggcgga tgttttgggg cgggcgctgt 120

ctacgttcct tggaacagga cgccgcagag ggtgagagcc ccgtgcgatg gcgcctctaa 180

ccgcgtaaaa ctccgccgac gagtcgagtt gtttgggaat gcagctccaa gtgggtggta 240

aattccatct aaagctaaat actggcgaga gaccgatagc gaacaagtac agtgatggaa 300

agatgaaaag cactttgaaa agagagtgaa acagcacgtg aaattgttga aagggaaggg 360

tatgcgatta gcggccagca ggaggtgcct tctcgtgaaa aggccgtgca ccgtcttcgg 420

acaccgtgcg cggagatggc gagggggcgc ctgaggtctg cgaactcgag gttgctggcg 480

taatgattgc ataccacccg tctt 504

Claims

1. A heterotrophic nitrification-aerobic denitrification Candida rugosa (Diutina rugosa) strain DW-1 is characterized in that the preservation number is CCTCC NO: m2019166.

2. A microbial agent for degrading ammonia nitrogen and nitrite nitrogen in freshwater aquaculture wastewater, which comprises candida rugosa (Diutina rugosa) strain DW-1 as claimed in claim 1.

3. The microbial preparation of claim 2, wherein the Candida rugosa (Diutina rugosa) strain DW-1 has a viable concentration of 10¹⁰～10¹⁵CFU/mL。

4. Use of the Candida rugosa (Diutina rugosa) strain DW-1 according to claim 1 or the microbial preparation according to claim 2 or 3 for denitrification of freshwater aquaculture waters.

5. The use of claim 4, wherein the method of denitrification of a body of freshwater aquaculture water comprises the steps of:

6. The use of claim 4, wherein the nitrogen in the denitrification of the freshwater aquaculture water comprises ammonia nitrogen and/or nitrite nitrogen.

7. The application of claim 6, wherein the concentration of total nitrogen, ammonia nitrogen or nitrite nitrogen consisting of ammonia nitrogen and nitrite nitrogen in the fresh water aquaculture water body is 40-100 mg/L.

8. The use of claim 5, wherein the viable bacteria concentration of the bacterial solution of Candida rugosa strain DW-1 is 10¹⁰～10¹⁵CFU/mL; the inoculation amount of the candida rugosa DW-1 strain liquid is 0.1-1%.

9. The use according to claim 5, wherein the initial pH of the freshwater aquaculture wastewater is 4.0-8.5.

10. The use of claim 5, wherein during the aerobic culture, the dissolved oxygen amount of the freshwater aquaculture wastewater is 1.5-3 mg/L;