NL2032098B1 - Aerobic denitrifying bacteria for denitrifying tail water of mariculture and application thereof - Google Patents

Abstract

Disclosed is an aerobic denitrifying bacteria for denitrifying tail water of mariculture and application thereof, belongs to the technical field of microorganisms and environmental science, the aerobic denitrifying bacterium CF1 - 6 is deposited in the General Microbiological Culture Collection Center of China Microbe Preservation Management Committee, on 2 August, 2021, with a deposit number of CGCMCC NO.23103, a deposit address of No.3, yard 1, Beichen West Road, Chaoyang District, Beijing, and a taxonomic designation of Bruce/la anthropi. The strain exhibits strong aerobic denitrification capability at 1% - 3% salinity, can effectively remove ammonia nitrogen, nitrite nitrogen and nitrate nitrogen, and only accumulates a small amount of N20 gas, which shows great application potential in the denitrifying treatment of seawater aquaculture tail water.

Description

AEROBIC DENITRIFYING BACTERIA FOR DENITRIFYING TAIL WATER OF MARICULTURE AND APPLICATION THEREOF

TECHNICAL FIELD The application belongs to the technical field of microorganisms and environmental science, and in particular to an aerobic denitrifying bacteria for denitrifying tail water of mariculture and application thereof.

BACKGROUND In recent years, the rapid development of mariculture has created great social and economic value. However, in order to improve the production speed and reduce costs, small aquaculture farmers adopt the methods of multi - input, multi - output and artificial addition of various disinfectants, growth promoters and other drugs for culturing. This extensive aquaculture method has a low feed utilization rate, thus resulting in relatively high total value of organic matter, nitrogen and phosphorus in the aquaculture wastewater. If the wastewater is directly discharged into the sea without treatment, the environment in the offshore waters will be seriously polluted and the coastal ecological system will be imbalanced. The environmental problems of mariculture wastewater in China in the last century have not attracted enough attention, and the country has not formulated relevant laws and regulations to restrict the discharge of mariculture wastewater. Therefore, most of the mariculture wastewater is directly discharged without treatment. In 2007, the Ministry of Agriculture of the PRC formulated and promulgated the “Marine Aquaculture Water Emission Regulations” (SC/T9103 - 2007) and began its implementation on September 1 of that year. According to the function of the discharging area and the characteristics of the mariculture water, the standard divides the mariculture water emission requirements into grade | and grade Il. The primary emission grade for permanganate index (CODMn), inorganic nitrogen and active phosphate is not higher than 10, 0.50 and 0.05 mg/l, and the secondary grade is not higher than 20, 1.00 and 0.10 mg/l. Compared with industrial wastewater and domestic wastewater, the quality and quantity of mariculture wastewater have different characteristics. Firstly, the discharging amount is large but the pollutant content is low. For embodiment, the inorganic nitrogen content is generally only 3 - 5 mg/l, and the COD content is 20 - 40 mg/l, which is lower than those of industrial wastewater and domestic wastewater. At the same time, its dissolved oxygen content is very high, even close to saturation state. In addition, the particularity of pollutant structure in mariculture wastewater and the salinity effect and ionic strength effect of seawater greatly increase the processing difficulty and complexity. The biological nitrogen removal technology based on nitrification and denitrification is an economical and efficient sewage treatment technology. However, due to the high dissolved oxygen and salinity in seawater, the common denitrifying bacteria are not competent. The complete denitrification process is a process in which nitrous nitrogen, nitric oxide and nitrous oxide (N20) are finally reduced to nitrogen through nitrous nitrogen. N20 is one of the world's three largest greenhouse gases. Therefore, an excellent and efficient aerobic denitrifying strain should not only have the ability to rapidly reduce nitrate, but also have the characteristics of small accumulation or strong reduction of N20. In this way, it can not only strengthen the degradation of nitrogen pollution in the wastewater, but also reduce the emission of greenhouse gases and play the real characteristics of high efficiency, environmental protection and economic efficiency.

SUMMARY One object of the present application is to provide an aerobic denitrifying bacterium, the aerobic denitrifying bacterium CF1 - 6 being deposited in the General Microbiological Culture Collection Center of China Microbe Preservation Management Committee, on August 2, 2021 with a deposit number of CGCMCC NO.23103, with deposit address of No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing, having the taxonomic designation of Brucella anthropi.

Through comparative analysis of the 16S rRNA sequence (SEQ ID NO .1), the strain CF1 - 6 has 99.92% homology with the 16S rRNA sequence of Brucella anthropi X7, and further determining that the strain CF1 - 6 of the application belongs to the Brucella anthropi strain by combining the strain morphological characteristics and growth conditions.

The second object of the present application is to provide an application of the aerobic denitrifying bacteria in denitrification under the condition of containing salt.

Preferably, the salt - containing condition is that the sodium chloride concentration is < 35.0 g/l.

Preferably, the pH of the denitrification reaction condition is 6 - 10, and the total nitrogen concentration is < 180 mg/l.

Preferably, the inoculation amount of the aerobic denitrifying bacteria is 1 - 8% (v/v).

Compared with the prior art, the application has the following beneficial effects: In the application, a bacterial strain CF1 - 6 with good denitrification capability is screened out from the activated sludge through the processes of enrichment, separation, purification, performance identification and the like by adopting a microbial culture method, and the denitrification performance of the bacterial strain is studied under different carbon sources and different salinity. The experimental results show that the strain exhibits strong aerobic denitrification capability at 1% ~ 3% salinity, can effectively remove ammonia nitrogen, nitrite nitrogen and nitrate nitrogen, and only accumulates a small amount of N20 gas, which shows great application potential in the denitrifying treatment of seawater aquaculture tail water.

Preservation institution: General Microbiological Culture Collection Center of China Microbe Preservation Management Committee; Collection Number: CGMCC No.23103; Date of preservation: 2 August, 2021;

Deposit Address: No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Taxonomic designation: Brucella anthropi.

BRIEF DESCRIPTION OF THE FIGURES FIG. 1 shows the phylogenetic tree in embodiment 2. FIG. 2a is a graph showing the concentration change of nitrate nitrogen in embodiment 3. FIG. 2b is a numerical change diagram of OD600 in embodiment 3. FIG. 2c is a graph showing the concentration change of nitrite nitrogen in embodiment 3. FIG. 2d is a graph showing the concentration change of N20 in embodiment 3. FIG. 3a is a numerical change diagram of OD600 in embodiment 4. FIG. 3b is a graph showing the concentration change of nitrate nitrogen in embodiment 4. FIG. 4a is a numerical change diagram of OD600 in embodiment 5. FIG. 4b is a graph showing the concentration change of N20 in embodiment 5.

DESCRIPTION OF THE APPLICATION Embodiment 1 (1) Enrichment of denitrifying bacteria: Placing 1ml of sludge from seawater farm in a 300 ml conical flask, adding into 100 ml of LB medium, adding 30g/l NaCl, and placing the mixture in a shaker at 30°C at 120r/min, for constant temperature oscillation culture for 24 hours. Where, the peptone liquid medium (LB): 10g tryptone, 5 g NaCl, 5 g yeast extract, 1000 ml distilled water, pH at 7.2 - 7.4, the temperature is 121°C, sterilizing for 30 min.

(2) Separation of denitrifying bacteria: Preparing the enriched bacterial solution into 1073, 10-4 10% and 10" bacterial suspensions according to a gradient dilution method, coating 1ml of each bacterial suspension on a BTB solid culture medium supplemented with 30g/INaCl by the spread plate method, culturing the bacterial suspension in a constant - temperature incubator at 30°C until colonies generating blue halos grow out. Where BTB (bromothymol blue) medium: 20 g agar, 1.0 g KNO3, 1.0 g KH, PO,, 0.5 g FeCl; 6H0, 0.2 g CaCl; 7H,0, 1.0g MgS0,7H,O, 8.5 g sodium succinate, Iml BTB (1% dissolved in alcohol), 1000 ml distilled water, pH at 7.2 value, the temperature is 121°C, sterilizing for 30 min.

(3) Purification of denitrifying bacteria: Streaking, separating and purifying the strain on a solid culture medium for a plurality of times until no miscellaneous bacteria can be seen under a microscope, obtaining a single colony. Transferring the isolated single colony to a slant culture medium for storage.

(4) Screening of denitrifying bacteria:

Inoculating the single colony preserved in the slant culture medium into 100 ml of high - salt denitrification medium with an inoculation ring. Culturing the single colony in a shaking table at 30°C for 120r/min at constant temperature. Detecting the contents of nitrate nitrogen, nitrite nitrogen and ammonia nitrogen in the culture solution every 6 - 10h. Then selecting the strain CF1 - 6 with the highest denitrification efficiency. Embodiment 2 Submitting the 16SrRNA sequence of strain CF1 - 6 to the NCBI database for comparison, downloading the representative sequences with high similarity for comparison, constructing the phylogenetic tree, (the results are shown in Fig. 1), to determine the phylogenetic status and species of the strain. As shown in Fig. 1, strain CF1 - 6 belongs to Brucella anthropi. Embodiment 3 Test of denitrification ability of strain CF1 - 6: (1) Strain activation Preparing the strain activation medium with the following formula: 3.42g/l anhydrous sodium acetate, 0.38g/l NH4Cl, 1.6g/l KoHPO4, 0.1g/1 MgSO4-7H,O, 0.02g/1 CaCl;, 0. 005 g/l FeSO47H920 and 0.1ml trace element mother solution. The trace element mother liquor is formulated with 3.5 g EDTA, 2.0g ZnSO4-7H»0, 1.0g CuSO4 5H90, 2.0 g MnSO47H90, 0.9g Co(NO3)26H50, 1.0g H3BO3 and 1.0g Na2MoO4 per litre of water. (2) High - salt denitrification test medium The high - salt denitrification test medium formula: 3.42g/l anhydrous sodium acetate, 0.6g/ NaNO3, 1.6g/l K5HPO,4, 0.1 g/l MnSQO4-7H20, 0.02g/l CaCls, 0.005 g/l FeSO4 7H90, 30g/l NaCl, 0.1ml trace element mother solution, pH at 7.2 value; the formula of trace element mother liquor is: 3.5 g EDTA, 2.0g ZnSO04 7H20, 1.09 CuSO::5H20, 2.09 MnSO47H50,

0.99 Co{NO3)2'6H20, 1.0g H3BO3 and 1.0g Na2MoO4 per liter of water. Culturing the bacterial strains to the late logarithmic growth phase, and collecting 10 ml of bacterial suspension. After being washed and re - suspended with sterile water, the bacterial suspension is inoculated into a 300 ml closed anaerobic flask fitted with 50 ml of high - salt denitrification test medium. (3) Test of denitrification performance Sealing the anaerobic bottle with an airtight rubber stopper, and then culturing under the condition of 30°C and 150rpm. Extracting 5 ml of bacterial suspension by a syringe every 6 - 12 hours to determine OD600. After centrifugation, taking the supernatant to determine the concentration of ammonia nitrogen, nitrite nitrogen and nitrate nitrogen. At the same time, extracting 100uL of upper space gas to determine the concentration of N20, and determining its aerobic denitrification capacity and N>O production.

The experimental results: the denitrification of simulated aquaculture wastewater by strain CF1 - 6 is shown in Fig. 2a - 2d.

It could be seen from Fig. 2a - 2d that the strain grew well and plays an good aerobic denitrification role.

Under the condition of initial nitric nitrogen 5 concentration of 176.45 mg/l, 92.70% nitric nitrogen is removed within 70h. nitric nitrogen is accumulated along with the reduction of nitric nitrogen, and gradually decreased when the highest concentration of 162mg/l is reached.

N20 increases all the time during the process, and reached the highest when the nitrate and nitrite nitrogen are almost completely consumed, but the maximum amount is only 150ppm, which may be ignored in the proportion of the initial nitrate nitrogen.

The raw data corresponding to Fig. 2a is shown in Table 1: Table 1 Sampling 10 22 35 46 59 70 time(h) Nitrate nitrogen 176.45 [176.26 150.11 66.36 |26.27 20.34 12.88 concentration (mg/l)

The raw data corresponding to Fig. 2b is shown in Table 2: Table 2 OD600 0.087 0.135 0.162 0.244 0.279 0.315 0.313 The raw data corresponding to Fig. 2c is shown in Table 3: Table 3 Nitrate nitrogen 0.002 |0.006 [0.172 [72.200 |162.000 |58.200 |5.200 concentration(mg/l)

The raw data corresponding to Figure 2d is shown in Table 4:

Table 4 rn] 0] ol Ee] EE N20 1.25 19.39 54.48 93.00 117.82 158.80 13.18

ETT (ppm) Embodiment 4 Test of salt tolerance of strain CF1 —6 Preparing 50 ml of high - salt denitrification test medium with salinity gradient of 0%, 1%, 2%, 3%, and 4% (i.e., 0, 10, 20, 30, and 40g/l) of sodium chloride(the other components are the same as the formulation of the high - salt denitrification test medium in embodiment 3, except the concentration of sodium chloride), and placing in a 300 ml closed anaerobic flask sealed with an airtight rubber stopper. Then collecting 10 ml of bacterial strain culture medium of the bacterial CF1 - 6 cultured to the late logarithmic growth stage. After being washed and re - suspended with sterile water, the medium is inoculated into an anaerobic flask and cultured under the shaking condition of 30°C and 150rpm. Then extracting 5 ml of bacterial suspension by a syringe every 6 - 12 hours to determine OD600. After centrifugation, taking the supernatant to determine the concentration of ammonia nitrogen, nitrite nitrogen and nitrate nitrogen.

The experimental results: The growth and denitrification of strain CF1 - 6 at different salinity are shown in Fig. 3a - Fig. 3b. It could be seen that the strain grew well at 0%, 1%, 2%, and 3% salinity, and OD600 reaches 0.21 - 0.27 after 20h, respectively. The removal rate of nitrate and nitrogen is above 89.35%. The bacterial growth rate is the fastest at 1% salt concentration, and the denitrification effect is the best. The growth of strain is inhibited at 4% salinity. Considering that the salinity of the mariculture tail water is generally around 3%, it can be speculated that the strain CF1 - 6 can exert good denitrification performance in the mariculture tail water treatment.

The raw data corresponding to Fig. 3a is shown in Table 5: Table 5 Tse ooo ews es | | eo a [om [om | om | ow | wo a [om em ow Tew [0

The raw data corresponding to Fig. 3b is shown in Table 6: Table 6 | me 2 | Gs | 1% | Fy | Wa | $9 | Embodiment 5 Test of reduction performance of strain CF1 - 6 on N20; Preparing a 50 ml high - salt denitrification medium using N20 as a nitrogen source (compared with the formulation of the high - salt denitrification test medium in Embodiment 3,

0.6g/INaNO:3 is removed, and the other components are the same), and placing in a 300 ml closed anaerobic bottle sealed with an airtight rubber stopper, and collecting 10 ml of strain culture solution of the bacterial CF1 - 6 cultured to the later logarithmic growth stage , followed by washing and re - suspending with sterile water, and inoculating to an anaerobic flask, under the condition of 30°C and 150rpm, extracting 5 ml of bacterial suspension by a syringe every 6 - 12 hours to determine OD600. At the same time, extracting 100uL of upper space gas to determine the concentration of N20. The experimental results: The experimental results show that the initial concentration of N20 reaches 1312.69ppm by introducing N20 gas. The reduction effect of strain CF16 on N>O under aerobic condition is tested. The results are shown in Fig. 4a - Fig. 4b. lt can be seen from Fig. 4a and Fig. 4b that the strain CF16 can remove 99.13% NzO within 72h time, showing a good N20 reduction ability. It is of great application significance for greenhouse gas emission reduction in the seawater aquaculture wastewater treatment process. The raw data corresponding to Fig. 4a is shown in Table 7: Table 7 To | DW] 2] ww] w] Ww] ®

The raw data corresponding to Fig. 4b is shown in Table 8: Table 8 N20 1312.70 1225.80| 1166.10 | 886.16 | 590.41 | 154.59 | 29.09 | 11.48 concentration (ppm)

The above - mentioned embodiments only describe the preferred mode of the present application, and do not limit the scope of the present application.

Without departing from the design spirit of the present application, those of ordinary skill in the art have made various contributions to the technical solutions of the present application.

Variations and improvements should fall within the protection scope determined by the claims of the present application.

Sequence Listing

<110> TIANJIN RESEARCH INSTITUTE FOR WATER TRANSPORT ENGINEERING, M.O.T. <120> Aerobic denitrifying bacteria for denitrifying tail water of mariculture and application thereof

<130> SHX-Denitrification NL

<150> 2021-09-27

<151> CN202111136889.2

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1234

<212> DNA

<213> Human Brucella strain CF1-6

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Claims (5)

CONCLUSIONS 1. An aerobic denitrifying bacterium with the taxonomic designation Brucella anthropi, deposited on August 2, 2021 with deposit number CGCMCC NO.23103 at the General Microbiological Culture Collection Center of China Microbe Preservation Management Committee, with deposit address No.3, yard 1, Beichen West Road, Chaoyang District, Beijing, A use of the aerobic denitrifying bacteria according to claim 1 in denitrification under conditions where salt is present. The use according to claim 2, wherein conditions where salt is present is obtained by a sodium chloride concentration of < 35.0g/l. The use according to claim 2, wherein the pH of the denitrification reaction conditions is 6-10, and the total nitrogen concentration is < 180 mg/l. The use according to claim 2, wherein the inoculation amount of the aerobic denitrifying bacteria is 1 - 8% (v/v).