CN113913336A

CN113913336A - Salt-tolerant aerobic denitrification composite bacterium and application

Info

Publication number: CN113913336A
Application number: CN202111222951.XA
Authority: CN
Inventors: 韩蕊; 刘鹰; 吴英海; 马洪婧; 慕欣廷; 苏鑫
Original assignee: Dalian Ocean University
Current assignee: Dalian Ocean University
Priority date: 2021-10-20
Filing date: 2021-10-20
Publication date: 2022-01-11

Abstract

The invention discloses a salt-tolerant aerobic denitrifying composite bacterium and application thereof, belonging to the technical field of microorganisms. A salt-tolerant aerobic denitrifying composite bacterium comprises aerobic denitrifying strains MAD-44, HRL-9 and AD 2; the preservation number of the MAD-44 strain is GDMCC.No 61854; HRL-9 strain with accession number GDMCC No. 61855; AD2 strain is deposited as GDMCC No. 61856. The inoculation amount of the aerobic denitrification composite bacterial agent is 1-2% (v/v), the C/N is 10-15, the temperature is 30-40 ℃, and the shaking speed is 120-150 rpmUnder the condition of the condition that the concentration of the compound strain to the substrate is 100 mg.L within 24h^‑1NO₃ ^‑The removal rate of-N was 100%, NO NO₂ ^‑-N accumulation.

Description

Salt-tolerant aerobic denitrification composite bacterium and application

Technical Field

The invention relates to a salt-tolerant aerobic denitrification composite bacterium and application thereof, belonging to the technical field of microorganisms.

Background

In the aquaculture process, part of feed is converted into ammonia nitrogen in the metabolic processes of fish digestion, absorption and the like and is discharged into the aquaculture environment, and the produced residual feed and excrement can be mineralized into ammonia under the action of microorganisms and further converted into nitrite nitrogen and nitrate nitrogen. The biological filtration link in the circulating water culture process can convert ammonia nitrogen and nitrite nitrogen with high toxicity in the water body into nitrate nitrogen with low toxicity to cause the accumulation of nitrate in the water body, and the mass concentration of the nitrate can reach 100 mg.L^-1Even 500 mg.L^-1The above (Freetag A R, Thayer L, Leonetti C, et al. effects of elongated nitrate on endocrine function in the anatomical salmon, Salmo salar [ J]Aquaculture,2015,436(1): 8-12.). Studies have shown that nitrate nitrogen has lower toxic effects on cultured subjects than ammonia nitrogen and nitrite nitrogen at the same mass concentration, but nitrate nitrogen at high mass concentration still causes slow growth, decreased survival rate, and decreased immunity in fish (Steinberg K, Zimmermann J, Stiller K T, et al. improved nitrate levels after the fish growth method of lake research in RAS [ J1)]Aquaculture,2018,497: 405-. The discharge of the culture wastewater containing high-quality nitrate nitrogen is one of the important reasons for the eutrophication of the water body in the natural environment, so that the method has the advantages of high efficiency, low cost and high efficiencyThe mass concentration of nitrate nitrogen in the recirculating aquaculture system should be controlled within a reasonable range.

The traditional biological denitrification theory considers that denitrification can only be completed under anaerobic conditions (house snow, screening of efficient aerobic denitrification strains and research on denitrification performance [ D ]. Harbin: university of northeast forestry, 2014.), the aerobic denitrification bacteria can realize synchronous nitrification and denitrification, the understanding of people on the traditional denitrification theory is broken, the ammoniation, nitrification and denitrification reactions are enabled to simultaneously occur in the aerobic environment of mariculture wastewater, the cost of denitrification of the mariculture wastewater can be obviously reduced, and the method has important significance for constructing a synchronous denitrification body in mariculture wastewater treatment.

Researches show that compared with a single strain, the denitrogenation composite flora has stronger adaptability and higher load capacity and has stronger advantages for strengthening biological denitrogenation and actually treating nitrogenous wastewater (Huangzheng Zheng, Caogang, Chenhai Sheng, and the like. the heterotrophic nitrification-aerobic denitrification bacteria synergistically compete for influencing the denitrogenation property [ J ] environmental science research, 2017,30(05): 765-. The compound microbial preparation is concerned due to the advantages of low cost, no toxicity, no side effect, no residual pollution and the like (Li Laiyang, Xie Wei soldier, seawater culture wastewater treatment technology research progress, Hebei fishery, 2009,185(5): 46-50.). Various microecologics are also developed in China successively, but the application effect is difficult to satisfy mainly because the bacterial strains have complex components, lack pertinence and are difficult to adapt to the high-salt oligotrophic seawater environment (poplar, Huweng, and the like. research on ammonia nitrogen wastewater treatment by nitrosobacteria. university of Wuhan theory of technology, 2007,29(3): 63-66; Monui, what is born, BeiDou, and the like. experimental research on purification of aquaculture wastewater by bacillus and nitrobacteria. environmental science and technology, 2009,32(11): 28-31.).

Disclosure of Invention

The invention mainly overcomes the shortage and the deficiency of the aerobic denitrification microbial inoculum of the mariculture wastewater at present, provides an aerobic denitrification composite microbial inoculum capable of effectively removing the nitrate accumulation in the mariculture wastewater, and also provides the application of the aerobic denitrification composite microbial inoculum for the mariculture wastewater denitrification, which is easy to implement, wide in application range, low in cost and high in efficiency. The invention screens 1 strain with high-efficiency denitrification function from the bottom mud of the coastal wetland, combines with 2 strains screened from a seawater circulating water system (RAS) biofilter, develops high-efficiency salt-tolerant (> 30) aerobic denitrification composite bacteria, uses the composite bacteria for treating the mariculture wastewater, explores the influence of single factors (temperature, shaking speed, C/N) on the aerobic denitrification performance of the mariculture wastewater, and provides reference for the application of the aerobic denitrification composite bacteria in the mariculture wastewater treatment. In order to solve the technical problems, the invention adopts the following technical scheme:

a salt-tolerant aerobic denitrifying composite bacterium comprises aerobic denitrifying bacterium strains of Zobellella sp.MAD-44, Halomonas sp.HRL-9 and Vibrio sp.AD2;

MAD-44 in 2021, 8/9 days, deposited in Guangdong province center for culture collection of microorganisms with strain number GDMCC no 61854;

halomonas sp.HRL-9 was deposited in Guangdong province center for culture Collection of microorganisms at 9/8/2021 with the strain number GDMCC No. 61855;

vibrio sp.AD2 was deposited at the Guangdong province Collection of microorganisms at 2021, 8/30/month, with the strain accession number GDMCC No. 61856.

Further, in the technical scheme, the compounding ratio of Vibrio sp.AD2, Halomonas sp.HRL-9 and Zobellella sp.MAD-44 is 1-3: 1-3: 1 to 3.

Further, in the technical scheme, the compounding ratio of Vibrio sp.AD2, Halomonas sp.HRL-9 and Zobellella sp.MAD-44 is 2:2: 3.

The application of salt-tolerant aerobic denitrifying composite bacteria in denitrification of high-salt nitrogen-containing wastewater is characterized in that the salinity of high-salt is more than 30, and the nitrogen contains nitrate nitrogen.

Further, in the above technical scheme, the high-salt nitrogen-containing wastewater comprises mariculture wastewater.

Further, in the technical scheme, the compounding ratio is 1-3: 1-3: 1-3 of Vibrio sp.AD2, Halomonas sp.HRL-9 and Zobellella sp.MAD-44 composite bacteria are inoculated into high-salt nitrogen-containing wastewater to be treated, and sodium acetate is added into KNO₃In a culture medium which is a unique nitrogen source, the C/N is controlled to be 10-15, and the composite bacteria grow in the wastewater under a certain condition, so that the denitrification treatment of the high-salt nitrogen-containing wastewater can be completed.

Further, in the above technical solution, the inoculation amount of the complex bacteria is 1% to 2% (v/v), and the certain conditions are: the temperature is 30-40 ℃, and the shaking speed is 120-150 rpm.

Advantageous effects of the invention

The inoculation amount of the aerobic denitrification composite bacterial agent is 1-2% (v/v), the C/N is 10-15, the temperature is 30-40 ℃, the shaking speed is 120-150 rpm, and the concentration of the composite bacterial strain to a substrate is 100 mg.L within 24h^-1NO₃ ^-The removal rate of-N was 100%, NO NO₂ ^--N accumulation. These results indicate that the complex strains are used for removing NO in high salinity (> 30) wastewater and mariculture wastewater₃ ^-The N aspect has potential good application prospect.

Drawings

FIG. 1 is a graph showing the effect of temperature on aerobic denitrification performance and growth of a composite strain.

FIG. 2 is a graph showing the effect of inoculum size on aerobic denitrification performance of a composite strain and a growth curve.

FIG. 3 is a curve showing the effect of shaking speed on aerobic denitrification performance and growth of a composite strain.

FIG. 4 is a graph showing the effect of C/N on aerobic denitrification performance of a composite strain and the growth curve of the strain.

Detailed Description

The following non-limiting examples will allow one of ordinary skill in the art to more fully understand the present invention, but are not intended to limit the invention in any way.

Example 1

1. Test strains

The strain is separated from bottom mud of coastal wetlands on red beach of Panjin and a biological filter of seawater circulating water systems (RASs) of a culture plant.

2. Culture medium

Denitrification Medium (DM) (L)^-1)：KH₂PO₄ 1.0g，MgSO₄·7H₂O 0.2g，KNO₃0.722g, 3.418g of sodium acetate, 30g of NaCl, 1000mL of pure water and pH 7.0 +/-0.05; 10mL of trace elements, and 20g of agar was added to the solid medium.

BTB Medium (L)^-1)：KH₂PO₄ 1.0g，MgSO₄·7H₂O 0.2g，KNO₃0.722g, 3.418g of sodium acetate, 1mL of BTB (0.1 g of bromothymol blue is dissolved in 10mL of ethanol), 30g of NaCl and 1000mL of pure water; pH 7.0 + -0.05; 10mL of trace elements, and 20g of agar was added to the solid medium.

Trace elements (L)^-1)：EDTA 50.0g，ZnSO₄ 2.2g，CaCl₂ 5.5g，MnCl₂·4H₂O 5.06g，FeSO₄·7H₂O 5.0g，(NH₄)6Mo₇O₂·4H₂O 1.1g，CuSO₄·5H₂O 1.57g，CoCl₂·6H₂O1.61 g, 1000mL of pure water.

The DM culture medium is used for enrichment culture and aerobic denitrification performance test of aerobic denitrifying bacteria, and the BTB culture medium is used for screening the aerobic denitrifying bacteria.

LB Medium (L)^-1): 10g of peptone, 5g of yeast extract powder, 30g of NaCl and 20g of agar added into a solid culture medium. The method is used for enrichment culture of the strain.

3 test method

3.1 optimized selection of combination ratio of functional bacteria in microbial inoculum

The separated high-efficiency aerobic denitrifying strains Vibrio sp.AD2, Halomonas sp.HRL-9 and Zobellella sp.MAD-44 are compounded to prepare the high-efficiency denitrification composite microbial inoculum, and the three experimental strains are all stored in Guangdong province microbial strain preservation center (GDMCC for short, address: No. 59 building 5 of Michelia Tokyo No. 100). The strain preservation number of the Zobellella sp (Zobellella sp.) MAD-44 is GDMCC No. 61854, the preservation date is 2021, 8 months and 9 days, the strain is classified and named as the Zobellella sp (Zobellella sp.), the strain name is MAD-44, and the sequence is shown as SEQ ID NO. 1. The strain of Halomonas salina (Halomonas sp.) HRL-9 has a preservation number of GDMCC No. 61855, a preservation date of 2021, 8 months and 9 days, is classified and named as Halomonas salina (Halomonas sp.), and has a strain name of HRL-9, and the sequence of the strain is shown as SEQ ID NO. 2. The strain accession number of Vibrio (Vibrio sp.) AD2 is GDMCC No. 61856, the preservation date is 2021, 8 months and 30 days, the strain is classified and named as Vibrio (Vibrio sp.), the strain name is AD2, and the sequence is shown in SEQ ID NO. 3.

The addition proportion of three bacteria in the microbial inoculum is determined through an orthogonal test, an L9(33) orthogonal table is adopted, AD2, HRL-9 and MAD-44 are used as A, B, C3 investigation factors, and 3 addition levels (v: v ratio) are selected to prepare the microbial inoculum (table 1). Adding the optimized mixed microbial inoculum into DM culture medium according to the amount shown in Table 1 for denitrification test, culturing at 30 deg.C and 120rpm for 24h, and measuring NH in the culture medium after 24h₄ ⁺-N、NO₃ ^--N、NO₂ ^-N and total nitrogen content, taking the removal rate of the microbial inoculum to the total nitrogen in the culture medium at 24h as an index to investigate the denitrification effect of the microbial inoculum, and setting 3 parallels for each group of experiments.

TABLE 1 orthogonal experimental design

Note: the horizontal value in the table is the addition ratio of 3 microbial inoculum.

The orthogonal test was designed according to the orthogonal factor level of table 1, and the results are shown in table 2. As can be seen from Table 2, the range of (R) A is very poor>B>C, namely the influence of 3 strains on the total nitrogen removal is AD2, HRL-9 and MAD-44 in sequence from large to small. In the range of experimental design, the optimal formula of the aerobic denitrifying bacteria agent is optimized and obtained as A₂B₂C₃The results were the same as those obtained by the direct observation method. Therefore, the bacteria agent has the highest removal rate of the total nitrogen in the culture medium when the adding ratio of AD2, HRL-9 and MAD-44 is 2:2:3, and the removal rate of the total nitrogen in the culture medium is 76.99 percent at the moment.

TABLE 2 results of orthogonal experiments

3.2 growth conditions and aerobic denitrification Properties of Single Strain and Complex Strain

The growth conditions and NO of the obtained 3 strains and a compound bacterium mixed by AD2, HRL-9 and MAD-44 in a ratio of 2:2:3 for 24h₂ ^-Accumulation of-N and TN, NO₃ ^-The removal efficiency of-N was examined, and the results are shown in Table 3. As can be seen from Table 3, in which AD2 and HRL-9 are present for NO₃ ^-the-N has strong removal capacity, the removal rates are respectively 96.55 percent and 98.98 percent, but NO exists₂ ^--N accumulation. The removal efficiency of the two strains to TN under the condition that the C/N is 10 can reach 59.91% and 44.05% respectively. MAD-44 vs. NO₃ ^-The removal rates of-N, TN were high and found to be 99.78% and 72.05%, respectively, and the accumulation of nitrous acid was low and found to be 4.50 mg.L^-1. Composite bacteria to NO₃ ^-The removal rate of-N, TN reaches 100% and 79.95%, respectively, and NO NO is generated₂ ^--N accumulation.

TABLE 3 growth and Nitrogen conversion of the individual strains and of the composite strains

Note: NO in the table₂ ^-N in concentration, expressed in mg.L^-1；NO₃ ^-N and TN are expressed in terms of removal rate.

3.3 microbial inoculum compounding condition optimization

Whether there was antagonism between strains was also investigated. As shown in Table 4, no inhibition zone was generated among 3 strains, and no significant antagonism occurred.

TABLE 4 antagonism between the three strains

Table 3 Antagonism among three strains

3.4 Effect of Single factor on aerobic Denitrification Performance of Complex Strain

The influence of different factors (temperature, inoculation amount, shaking speed and C/N) on the aerobic denitrification performance of the strain composite strain is measured. Temperature experiment: inoculating the composite strain into DM culture medium at 20 deg.C, 30 deg.C and 40 deg.C respectively. And (3) inoculation amount experiment: the strain inoculation amounts (v/v) are set to 1%, 2% and 4%, respectively. Shaking speed experiment: the strain was cultured under conditions of 90rpm, 120rpm and 150rpm shaking, respectively. C/N experiment: by adding different masses of sodium acetate to KNO₃(NO₃ ^-Initial concentration of-N is 100 mg.L^-1) In the culture medium which is a unique nitrogen source, the C/N is controlled to be 5, 10 and 15. In a one-factor experiment, all strains were inoculated in 100mL vials containing 50mL DM medium. Except for the change of the required experimental conditions, the other experiments are cultured for 24 hours at 30 ℃ and 120rpm, and then the OD of the bacterial liquid is tested₆₀₀、NO₂ ^--N、NO₃ ^--N、NH₄ ⁺-N、TN、TOC。

OD₆₀₀Measured by UV spectrophotometer (10s uv-vis, Thermo Fisher Genesys). NH (NH)₄ ⁺Colorimetric method of Nas reagent for determination of-N, NO₃ ^-UV spectrophotometry for determination of-N, NO₂ ^-The N measurement was performed by N- (1-naphthyl) -ethylenediamine photometry using 752 UV-visible spectrophotometer (Shanghai spectrometer Co., Ltd.); TN and TOC were measured by a TOC measuring instrument (TOC-L, Shimadzu).

3.4.1 Effect of temperature on aerobic denitrification Performance of Complex strains

The experimental results show that: the strain grows slowly at 20 ℃ (fig. 1A), the TOC utilization rate is not high (fig. 1B and G); in the denitrification capacity, NO₃ ^-The N24 h removal was only 21.66% (FIGS. 1C and G), and the TN 24h removal was only 19.78% (FIGS. 1F and G). At 40 ℃ NO₃ ^-The removal rate of-N and TN is basically similar to that at the temperature of 30 ℃, and NO is obtained at 24h₃ ^-The N removal was 100% (FIGS. 1C and G). The removal rates of TN are respectively 78.17% and 79.95% (FIGS. 1F and G), and NO NO is generated at 24h₂ ^--N accumulation. Therefore, it is presumed that the optimum conditions for growth of the complex strain and aerobic denitrification capacity range from 30 ℃ to 40 ℃.

3.4.2 influence of inoculation amount on aerobic denitrification performance of composite strain

In order to explore the influence of the inoculation amount on the aerobic denitrification capability of the composite strain, the strain is inoculated in NO₃ ^-N (initial concentration 100.25 mg. L)^-1) In DM liquid medium as the sole nitrogen source, the inoculum size (v/v) was set to 1%, 2%, 4%, respectively. The experimental results are shown in fig. 2: NO when the inoculum size (v/v) is 1%, 2%, 4%₃ ^-The removal rate of-N24 h can reach more than 99.54 percent (shown in figures 2C and G), but when the inoculation amount is 4 percent, the removal rate of TN is very low and is only 31.67 percent (shown in figures 2F and G), and 12h of NO in the reaction system₂ ^-Accumulation of-N was severe (FIG. 2D). The TOC concentration measurement shows that the TOC concentration in the 4% system is only 193.96 mg.L at 24h^-1(FIG. 2B). Thus, at an inoculum size of 4%, NO₃ ^-The removal rate of-N is very high, and the removal rate of TN is lower finally, probably because the concentration of TOC is too low in the later reaction period, the denitrification rate of bacteria is low, and NO in the system is caused₂ ^-The accumulation of-N is severe. When the inoculation amount is 1 percent and 2 percent, the removal rate of 24h TN is 78.52 percent and 79.95 percent respectively (shown in figures 2F and G), and NO NO is generated in the reaction system₂ ^--N accumulation (fig. 2D). The experimental result shows that the inoculation amount can obviously influence the denitrification efficiency of the compound bacteria. The range of the inoculation amount is 1-2% (v/v) is optimal.

3.4.3 Effect of shaking speed on aerobic denitrification Performance of Complex strains

The experimental results show that: at a shaking speed of 90rpm (initial dissolved oxygen concentration of 5.59. + -. 0.1 mg. L)^-1) The strain grew slowly (FIG. 3A) and the TOC utilization was low (FIGS. 3B and G), while most of the NO was present₃ ^-N was almost completely removed at 24h, but the final removal of TN was only 69.41% (FIGS. 3F and G), since under these conditions, NO was present₂ ^-A certain accumulation of-N, 12h NO₂ ^-The concentration of-N is still 2.26 mg.L^-1(FIG. 3D). With the increase of the shaking speed, the strain grows faster and the TOC utilization rate is higher.At 120rpm (initial dissolved oxygen concentration of 6.97. + -. 0.18 mg. L)^-1) And 150rpm (initial dissolved oxygen concentration of 7.48. + -. 0.39 mg. L)^-1) Under conditions of NO₃ ^-N was almost completely removed at 24h (FIGS. 3C and G), TN was removed at 24h with 79.95% and 56.13% respectively (FIGS. 3F and G), and NO NO was present in the reaction system at 24h₂ ^-Accumulation of-N (FIG. 3D), but NO at 120rpm₃ ^-The removal rates of-N and TN are faster. The denitrification capability of the compound strain is best when the shaking speed is 120 rpm-150 rpm, which shows that the compound strain has higher dissolved oxygen tolerance limit.

3.4.4C/N Effect on aerobic denitrification Performance of Complex strains

In order to explore the influence of C/N on the aerobic denitrification capability of the composite strain, the strain is inoculated in NO₃ ^-N (initial concentration 100.25 mg. L)^-1) In DM liquid culture medium as the only nitrogen source, sodium acetate is used as a carbon source, and C/N is respectively set to be 5, 10 and 15. The results of the experiment are shown in FIG. 4: when C/N is 5, NO₃ ^-The removal rate of-N24 h was 66.54% (FIGS. 4C and G), TN was very low and was only 37.37% (FIGS. 4F and G), and NO was present in the reaction system₂ ^-Accumulation of-N was severe (FIG. 4D). The TOC concentration measurement shows that the TOC concentration is only 78.30 mg.L in a system with C/N of 5 at 24h^-1(FIG. 4B). When C/N is 10 to 15, NO₃ ^-the-N removal rate reaches 100%, so that the C/N is 10-15, which is the optimal range for denitrification of the composite strain.

SEQUENCE LISTING

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atctgactta acaaaccacc tgcatgcgct ttacgcccag taattccgat taacgctcgc 900

accctccgta ttaccgcggc tgctggcacg gagttagccg gtgcttcttc tgtcgctaac 960

gtcaaataat gcatctatta aacacactac cttcctcacg actgaaagtg ctttacaacc 1020

cgaaggcctt cttcacacac gcggcatggc tgcatcaggc ttgcgcccat tgtgcaatat 1080

tccccactgc tgcctcccgt aggagtctgg accgtgtctc agttccagtg tggctgatca 1140

tcctctcaga ccagctaggg atcgtcgcct tggtgagccc ttacctcacc aactagctaa 1200

tcccacctag gcatatcctg acgcgagagg cccgaaggtc cccctctttg gcccgtaggc 1260

atcatgcggt attagccatc gtttccaatg gttatccccc acatcagggc aatttcctag 1320

gcattactca cccgtccgcc gctcgacgcc gttaacgttc cccgaaggtt cag 1373

Claims

1. A salt-tolerant aerobic denitrifying composite bacterium is characterized by comprising Zobenella sp.MAD-44, Halomonas sp.HRL-9 and Vibrio sp.AD2;

2. The salt-tolerant aerobic denitrifying complex bacterium according to claim 1, wherein the complex proportion of Vibrio sp.AD2, Halomonas sp.HRL-9, Zobellella sp.MAD-44 is 1-3: 1-3: 1 to 3.

3. The salt-tolerant aerobic denitrifying complex bacterium according to claim 1, wherein the complex ratio of Vibrio sp.AD2, Halomonas sp.HRL-9, Zobellella sp.MAD-44 is 2:2: 3.

4. The application of the salt-tolerant aerobic denitrification composite bacteria in denitrification of the high-salt nitrogen-containing wastewater is characterized in that the salinity of the high-salt is more than 30, and the nitrogen contains nitrate nitrogen.

5. Use according to claim 4, wherein the high-salt nitrogen-containing wastewater comprises a mariculture wastewater.

6. The application of the composition according to claim 4, wherein the composition ratio is 1-3: 1-3: 1-3 of Vibrio sp.AD2, Halomonas sp.HRL-9 and Zobellella sp.MAD-44 composite bacteria are inoculated into high-salt nitrogen-containing wastewater to be treated, and sodium acetate is added into KNO₃In a culture medium which is a unique nitrogen source, the C/N is controlled to be 10-15, and the composite bacteria grow in the wastewater under a certain condition, so that the denitrification treatment of the high-salt nitrogen-containing wastewater can be completed.

7. The use according to claim 6, wherein the amount of the complex bacteria is 1% to 2% (v/v), and the certain condition is: the temperature is 30-40 ℃, and the shaking speed is 120-150 rpm.