CN114590911B - Denitrifying microbial inoculum with broad-spectrum dissolved oxygen and organic carbon tolerance and application thereof - Google Patents

Denitrifying microbial inoculum with broad-spectrum dissolved oxygen and organic carbon tolerance and application thereof Download PDF

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CN114590911B
CN114590911B CN202210497333.4A CN202210497333A CN114590911B CN 114590911 B CN114590911 B CN 114590911B CN 202210497333 A CN202210497333 A CN 202210497333A CN 114590911 B CN114590911 B CN 114590911B
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yel14
cho6
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许玫英
周少锋
杨旭楠
杨永刚
陈杏娟
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Institute of Microbiology of Guangdong Academy of Sciences
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Abstract

The invention discloses a denitrogenation microbial inoculum with broad-spectrum dissolved oxygen and organic carbon tolerance and application thereof. The microbial inoculum comprises bacillus amyloliquefaciens yel14 and alcaligenes faecalis CHO 6. The strain yel14 has both heterotrophic ammonia oxidation function and aerobic denitrification function, and the strain CHO6 has denitrification function under both anaerobic and aerobic conditions. Compared with the verification result that the strains yel14 and CHO6 are singly inoculated in the landfill leachate, the compound flora CHO6-yel14 has stronger denitrification function. The microbial inoculum is put into wastewater containing nitrate nitrogen, nitrite nitrogen and ammonia nitrogen, and is uniformly mixed, so that the effect of efficiently removing total nitrogen is achieved. The microbial inoculum developed by the strains yel14 and CHO6 realizes NH in the polluted water body under aerobic and anaerobic conditions 4 + —NO 3 ‑ —NO 2 ‑ —N 2 The whole denitrification process has the characteristics of simple operation, low cost and environmental friendliness.

Description

Denitrifying microbial inoculum with broad-spectrum dissolved oxygen and organic carbon tolerance and application thereof
Technical Field
The invention belongs to the technical field of environmental microorganisms, and particularly relates to a bacillus amyloliquefaciens strainBacillus amyloliquefaciens) yel14 and an Alcaligenes faecalisAlcaligenes faecalis) CHO6, and a strain preparation composed of the two microorganisms and application thereof in water denitrification.
Background
The problem of nitrogen pollution of water environment is increasingly severe in the industrialized and urbanization high-speed development process. Because the nitrogen circulation is closely coupled with the circulation process of various substances and is also an important factor for determining the eutrophication degree of the water body, the denitrification efficiency of the polluted water body is concerned. The biological denitrification technology is a common method in the field of water pollution treatment at present because of good economic benefit and environmental benefit. In the traditional biological denitrification process, autotrophic ammonia-oxidizing bacteria and nitrosobacteria are used for oxidizing ammonia nitrogen into nitrate under aerobic conditions, and denitrifying bacteria are used for reducing the nitrate into nitrogen under anoxic conditions, so that denitrification is realized. However, the dissolved oxygen concentration of the polluted water usually fluctuates greatly and often contains a plurality of toxic pollutants for inhibiting the functional activity of microorganisms, so that ammonia nitrogen and nitrate nitrogen in the water are difficult to effectively remove. Denitrification of water has become a key and difficult point of water pollution prevention and control.
With the development of research, various novel microorganisms with denitrification functions are continuously discovered, including heterotrophic ammonia-oxidizing bacteria, anaerobic ammonia-oxidizing bacteria, aerobic denitrifying bacteria and the like. Compared with the common detoxification functional microorganisms, the novel denitrification functional microorganisms have wider applicability and more obvious practicability in the treatment of the polluted water body. Compared with the traditional ammonia oxidation process, the heterotrophic ammonia-oxidizing bacteria can oxidize ammonia nitrogen into nitrite nitrogen under the condition of the existence of an organic carbon source, thereby overcoming the inhibiting effect of the organic carbon source commonly existing in the polluted water body on ammonia oxidation; compared with the traditional denitrification process, the aerobic denitrifying bacteria can play a role of denitrification under the condition of high dissolved oxygen, thereby overcoming the defect that the traditional denitrifying bacteria can reduce nitrate into nitrogen under the condition of oxygen deficiency. The combination of the two functional bacteria is expected to overcome the ubiquitous inhibition of organic carbon sources on ammonia oxidation and the inhibition of dissolved oxygen on denitrification in the polluted water treatment process, so that the total nitrogen can be removed more flexibly and efficiently. However, there are very few heterotrophic ammonia-oxidizing bacteria and aerobic denitrification bacteria available so far, and no report has been made on microorganisms having both heterotrophic ammonia-oxidizing bacteria and aerobic denitrification functions.
Disclosure of Invention
Aiming at the problem of water pollution treatment of inhibiting biological denitrification activity by organic carbon and dissolved oxygen in a polluted water body, the invention provides a denitrification functional microbial inoculum with the tolerance capability of the dissolved oxygen and the organic carbon, and realizes the high-efficiency removal of ammonia nitrogen and total nitrogen in the water body.
The invention firstly separates and obtains bacillus amyloliquefaciens yel14 with heterotrophic ammonia oxidation function and aerobic denitrification function and alcaligenes faecalis CHO6 with denitrification function under anaerobic and aerobic conditions.
The invention also utilizes the two functional bacteria to develop the functional bacteria agent which can effectively remove ammonia nitrogen and total nitrogen in the polluted water body under anaerobic and aerobic conditions.
The invention also verifies the denitrification capability of the developed denitrification bacterial agent on the garbage leachate containing high-concentration ammonia nitrogen wastewater.
The bacillus amyloliquefaciens separated and obtained by the inventionBacillus amyloliquefaciens) yel14 and an Alcaligenes faecalisAlcaligenes faecalis) CHO6 was deposited at the guangdong province collection of microorganisms (CGMCC) at 2021, 5/27, address: building 5 of first furnance, large yard, 100, building 59, Guangdong province, Guangzhou, China, zip code: 510070, the preservation numbers are respectively: GDMCC No. 61699 and GDMCC No. 61700.
The alcaligenes faecalis of the present inventionAlcaligenes faecalis) CHO6 is from water body sediment of Guangzhou city stone well river in Guangdong province, the separation method is that the flora SB8 is separated and screened by a single cell screening instrument in a visualized way, and the obtained single cells are inoculated in a liquid culture medium for 20 days, and the components of the culture medium are as follows: glucose (10 g +)L)、NaHCO 3 (2.52 g/L)、KH 2 PO 4 (0.5 g/L)、NH 4 Cl(1 g/L)、MgCl 2 ⋅6H 2 O (0.06 g/L)、CaCl 2 ⋅6H 2 O(0.06 g/L)、K 2 SO 4 (3.48 g/L) and FeSO 4 ⋅7H 2 O (0.05 g/L) and the solvent is water. The bacterial liquid grown in the liquid culture medium is taken and grows on an agar solid plate of the culture medium, and the bacterial colony is irregular round, opaque and white and can survive in aerobic and anaerobic environments. The strain is discovered by 16S rRNA gene sequencing, andAlcaligenes faecalisNBRC 13111 has 99.41% similarity, so it is namedAlcaligenes faecalis CHO6。
The invention provides bacillus amyloliquefaciens (A), (B), (C)Bacillus amyloliquefaciens) yel14 was isolated from a plastic bag filled with trash of Liupan Water, Guizhou province, and the medium grown was potato medium (PDB, 24 g/L). The morphological and physiological and biochemical characteristics are as follows: after the bacterium is cultured for 48 hours at 30 ℃, the bacterial colony is irregular oval, light yellow and opaque, and water drops are arranged on the surface of the bacterial colony. The strain can survive under aerobic and facultative anaerobic conditions, and has a growth pH of 6.5-8.5. The strain is sequenced by 16S rRNA, and BLAST analysis is carried out to find outBacillus amyloliquefaciensMPA strains have 99.78% similarity, so they are namedBacillus amyloliquefaciens yel14。
The invention provides the verification of the denitrification capability of two strains in a common culture medium:
will be provided withAlcaligenes faecalisCHO6 was inoculated separately in R 2 A(12 g/L)+NO 2 - (17.5 mM)+NH 4 + (3.5 mM) and R 2 A(12 g/L)+NO 3 - (16 mM) and aerobic (180 rpm, 30 ℃) and anaerobic (30 ℃ C.) cultures (99.99% N aeration) 2 Anaerobic tank) to verify the nitrification ability, denitrification ability and denitrification ability. As a result, it was found that 2 A(12 g/L)+NO 2 - (17.5 mM)+NH 4 + After 10 days of culture in (3.5 mM) medium, the aerobic denitrification capacity and the anaerobic denitrification capacity of the strain CHO6 were about 25% (FIG. 2). And thenAt R 2 A(12 g/L)+NO 3 - The denitrification capacity was most pronounced in the (16 mM) medium, which was able to reach 66% and 71% denitrification capacity under aerobic and anaerobic conditions, respectively (FIG. 2). Further analysis of the nitrogen ion-containing compounds revealed that strain CHO6 was able to convert R, both under aerobic and anaerobic conditions 2 A(12 g/L)+NO 2 - (17.5 mM)+NH 4 + (3.5 mM) NO in Medium 2 – Is totally removed by denitrification (in FIG. 3
Figure 27950DEST_PATH_IMAGE001
、
Figure 40031DEST_PATH_IMAGE002
) NH, as compared with control group 4 + No significant change occurred. And in R 2 A(12 g/L)+NO 3 - In (16 mM) culture medium, the strain CHO6 has strong denitrification capability and can utilize denitrification process to remove NO 3 - Almost total conversion removal (FIG. 4
Figure 288610DEST_PATH_IMAGE001
、
Figure 480557DEST_PATH_IMAGE002
)。
Will be provided withBacillus amyloliquefaciensyel14 was inoculated into PDB (24 g/L) + NO 2 - (13.2 mM)+NH 4 + (3.3 mM) and PDB (24 g/L) + NO 3 - (11.8 mM), and aerobic culture (180 rpm, 30 ℃) and anaerobic culture (30 ℃ C., 99.99% N aeration) were performed 2 The anaerobic tank). Strain yel14 in PDB (24 g/L) + NO 2 - (13.2 mM)+NH 4 + (3.3 mM) had no aerobic denitrification capacity, and anaerobic denitrification was only 11.42% (FIG. 2). Similarly, strain yel14 was found to be PDB (24 g/L) + NO 3 - The medium (11.8 mM) also had no aerobic denitrification capacity, but the anaerobic denitrification capacity was only 7.37% (FIG. 2). The denitrification capability of the strain yel14 is very weak, probably because of the systemThe nitrogen ion-containing compounds are not converted to nitrogen gas, but are interconverted in ionic form. Specifically, strain yel14 was found to be PDB (24 g/L) + NO 2 - (13.2 mM)+NH 4 + (3.3 mM) in aerobic culture, NO not present in the culture can be produced 3 - (1.248 mM) (FIG. 3
Figure 888404DEST_PATH_IMAGE003
) And can culture 47% of NH under anaerobic condition 4 + (of FIG. 3
Figure 773184DEST_PATH_IMAGE004
) Conversion to NO 3 - (0.447 mM) (FIG. 3
Figure 509059DEST_PATH_IMAGE005
) And NO 2 - (2.683 mM) (FIG. 3
Figure 65548DEST_PATH_IMAGE006
). In PDB (24 g/L) + NO 3 - In (11.8 mM), the aerobic denitrification capability of the strain yel14 is stronger and can reach 94.8%, but the anaerobic denitrification capability is very weak, and a large amount of NO can be accumulated 2 - (2.98 mM) (FIG. 4
Figure 531165DEST_PATH_IMAGE003
、
Figure 993370DEST_PATH_IMAGE005
) This may be the reason why the Total Nitrogen (TN) in the system cannot be removed.
In summary, under aerobic or anaerobic conditions,Alcaligenes faecalisCHO6 has strong denitrification capability (removing NO) 3 - And NO 2 - ) But no ammonia oxidation capability. WhileBacillus amyloliquefaciensyel14 has certain denitrification capability (removing NO) under aerobic condition 3 - ) However, under anaerobic conditions, the denitrification capacity is very weak, and a large amount of NO is accumulated 2 - . More interestingly, strain yel14 was able to achieve 47% NH under anaerobic conditions 4 + Conversion to NO 2 - And NO 3 - . Therefore, strains CHO6 and yel14 can be mixed and cultured, and NH in landfill leachate is treated by using strain yel14 4 + Conversion to NO 2 - And NO 3 - Then converted into N by denitrification of a bacterial strain CHO6 2 So as to achieve the aim of denitrification.
The invention also provides denitrification condition verification of aerobic, anaerobic and normal-temperature standing culture of the bacillus amyloliquefaciens yel14 and the alcaligenes faecalis CHO6 in the landfill leachate.
Strains CHO6 and yel14 and 1:1 complex flora CHO6-yel14 were inoculated into landfill leachate (Huizhou landfill, mature,>10 years) and no nutrient solution was added. Compared with the situation that the strains CHO6 and yel14 are singly inoculated in the landfill leachate for culture, the compound flora CHO6-yel14 has better denitrification effect under aerobic and anaerobic conditions, and can respectively remove 470.5 mg/L and 724.8 mg/L of total nitrogen (shown in figure 5)
Figure 341175DEST_PATH_IMAGE001
、
Figure 547028DEST_PATH_IMAGE003
). Ion chromatography determination shows that the compounded flora CHO6-yel14 has better ammonia nitrogen removal capacity and adaptability in high ammonia nitrogen landfill leachate, and the ammonia nitrogen removal rates of the compound flora CHO6-yel14 under aerobic, normal-temperature standing and anaerobic conditions can respectively reach 44.4%, 37.53% and 38.95% (shown in figure 6)
Figure 663889DEST_PATH_IMAGE001
、
Figure 562575DEST_PATH_IMAGE002
、
Figure 899140DEST_PATH_IMAGE003
）。
Accordingly, it is a first object of the present invention to provide a denitrifier comprising Bacillus amyloliquefaciens (Bacillus amyloliquefaciens)Bacillus amyloliquefaciens) And Alcaligenes faecalis: (Alcaligenes faecalis）。
Preferably, the bacillus amyloliquefaciens is bacillus amyloliquefaciens yel14 with the preservation number of: the GDMCC No. 61699, wherein the Alcaligenes faecalis is Alcaligenes faecalis CHO6, and the preservation number is as follows: GDMCC No. 61700.
Preferably, the denitrifier further comprises a functional carrier.
The second purpose of the invention is to provide the application of the denitrification microbial inoculum in the preparation of denitrification biological membranes.
The third purpose of the invention is to provide a denitrification biological film which contains the denitrification bacterial agent.
The fourth purpose of the invention is to provide the application of the denitriding agent or the denitriding biological film in water denitrification.
Preferably, the denitrification microbial inoculum or the denitrification biological membrane is provided for removing nitrogen in the water body under aerobic or anaerobic conditions.
Preferably, the nitrogen in the water body comprises ammonia nitrogen NH 4 + -N, nitrate nitrogen NO 3 - -N or nitrous nitrogen NO 2 - -N.
The fifth purpose of the invention is to provide the application of the denitrification microbial inoculum or the denitrification biomembrane in sewage deodorization and/or landfill leachate deodorization.
The sixth purpose of the invention is to provide a water body denitrification method, which is to directly put the denitrification microbial inoculum into the nitrogen-containing wastewater or mix the denitrification microbial inoculum and the nitrogen-containing wastewater in a biochemical reactor for treatment. Preferably, the denitrifier is mixed with a certain amount of functional carrier and then put into a biochemical reactor for treating nitrogen-containing wastewater.
The invention has the following beneficial effects:
the invention develops a functional microbial inoculum which can efficiently remove ammonia nitrogen and total nitrogen in the polluted water body under anaerobic and aerobic conditions by utilizing bacillus amyloliquefaciens yel14 and alcaligenes faecalis CHO6, and effectively overcomes the inhibiting effect of dissolved oxygen and organic carbon in the polluted water body on biological denitrification. The microbial inoculum developed by the invention can effectively remove ammonia nitrogen and total nitrogen in landfill leachate under anaerobic and aerobic conditions, compared with the condition that the strains yel14 and CHO6 are independently inoculated in the landfill leachate for culture, the compound flora CHO6-yel14 has better denitrification effect under aerobic and anaerobic conditions, and the two strains play a synergistic denitrification role, thereby having larger engineering application potential and wide market application prospect.
Bacillus amyloliquefaciensyel14, deposited at 27/5/2021 at the Guangdong province culture Collection (GDMCC), address: building 5 of first furnance, large yard, 100, building 59, Guangdong province, Guangzhou, China, zip code: 510070, accession number: GDMCC No. 61699.
Alcaligenes faecalisCHO6, deposited at 27 days 5/2021 at the guangdong province collection of microorganisms (GDMCC) address: building 5 of first furnance, large yard, 100, building 59, Guangdong province, Guangzhou, China, zip code: 510070, accession number: GDMCC No. 61700.
Drawings
FIG. 1 shows Alcaligenes faecalisAlcaligenes faecalisCHO6 and Bacillus amyloliquefaciensBacillus amyloliquefaciens yel 14A process for denitrification in landfill leachate.
FIG. 2 shows Alcaligenes faecalisAlcaligenes faecalisCHO6 and Bacillus amyloliquefaciensBacillus amyloliquefaciens yel14 in PDB Medium and R, respectively 2 And (B) verifying the denitrification capability in the culture medium A.
FIG. 3 shows Alcaligenes faecalisAlcaligenes faecalisCHO6 and Bacillus amyloliquefaciensBacillus amyloliquefaciens yel14 for nitrite removal and ammonia oxidation.
FIG. 4 shows Alcaligenes faecalisAlcaligenes faecalisCHO6 and Bacillus amyloliquefaciensBacillus amyloliquefaciens yel14 verification of denitrification capability.
FIG. 5 shows Alcaligenes faecalisAlcaligenes faecalisCHO6, Bacillus amyloliquefaciensBacillus amyloliquefaciens yel14 and the verification of the denitrification capability in the landfill leachate after the two bacteria are compounded in a ratio of 1: 1.
FIG. 6 shows Alcaligenes faecalisAlcaligenes faecalisCHO6, Bacillus amyloliquefaciensBacillus amyloliquefaciens yel14 and the capability of removing ammonia nitrogen in the landfill leachate after the two bacteria are compounded in a ratio of 1: 1.
FIG. 7 is a flow cytometer for visually screening Alcaligenes faecalisAlcaligenes faecalis CHO6。
Detailed Description
In order to make the objects, technical solutions and advantageous technical effects of the present invention clearer, the present invention is further described in detail with reference to the following embodiments. It should be understood that the embodiments described in this specification are only for explaining the present invention and do not limit the scope of the present invention. Test methods in which specific conditions are not noted in the following examples are generally performed under conventional conditions or conditions recommended by each manufacturer.
Example 1
Alcaligenes faecalisCHO6 andBacillus amyloliquefaciensyel14 the specific separation and breeding process comprises the following steps:
（1）Alcaligenes faecalisCHO6 isolation culture:
the invention providesAlcaligenes faecalisCHO6 was derived from industrial contaminated water sediments from Guangzhou city Stone well river, Guangdong province by isolating the colony SB8 (a single colony of bacteria capable of growing on a plate, which is available in the prior art and is disclosed in Qian Y. F., Xu M. Y., et al. synthetic interactions ofDesulfovibrio and Petrimonasfor sulfate-reduction coupling polymeric carbohydrate degradation, J. Hazard. mater, 2021, 407) single cells obtained by visually screening rod-shaped microorganisms using a single cell screening apparatus (Changchun Chang Ying bioscience apparatus Co., Ltd.) (FIG. 7), culturing in a liquid medium for 20 days, and observing whether the bacterial solution is turbid (30 ℃, 10% H) or not (30 ℃, 10%) 2 +10%CO 2 +80%N 2 Anaerobic chamber) of the culture medium, streaking the turbid bacterial liquid on an agar solid plate of the culture medium, and identifying.Alcaligenes faecalisThe CHO6 colony is irregular round, opaque and white, and can survive in both aerobic and anaerobic environments. The bacterium was found by 16S rRNA gene sequencing, andAlcaligenes faecalisNBRC 13111 has 99.41% similarity, so the nomenclature isAlcaligenes faecalis CHO6，Alcaligenes faecalisThe 16S rRNA sequence of CHO6 is shown in SEQ ID NO. 1. The components of the liquid culture medium are as follows: glucose (10 g/L), NaHCO 3 (2.52 g/L)、KH 2 PO 4 (0.5 g/L)、NH 4 Cl(1 g/L)、MgCl 2 ⋅6H 2 O (0.06 g/L)、CaCl 2 ⋅6H 2 O(0.06 g/L)、K 2 SO 4 (3.48 g/L)、FeSO 4 ⋅7H 2 O (0.05 g/L), and the solvent is water; the solid plate of the culture medium is prepared by adding agar with the mass fraction of 1.5% into a liquid culture medium. The preparation method of the culture medium comprises adding the above components into water, stirring to dissolve, and sterilizing.
（2）Bacillus amyloliquefaciensyel14 separation and culture:
the invention providesBacillus amyloliquefaciensyel14 was isolated from plastic bags of Liupai water, Guizhou province, and the isolated culture medium was potato culture medium (PDB, 24 g/L, supplied by Guangdong Huaka microbial science, Inc.). Adding the flora on the garbage plastic into sterile water, blowing back and forth for 5 times by using a pipette, taking 50 mu L of the flora on a PDB plate, coating the PDB plate (containing agar with the mass fraction of 1.5%), placing the PDB plate in a constant temperature box at the temperature of 30 ℃ for culture, incubating for 1 week, and selecting single colonies for further purification.Bacillus amyloliquefaciensyel14 the colony is irregular oval, light yellow, opaque, and has water drop on the surface. The bacteria can survive under aerobic and facultative anaerobic conditions, and grow at pH 6.5-8.5. The strain is subjected to 16S rRNA gene sequencing and BLAST analysis to find outBacillus amyloliquefaciensMPA strains have 99.78% similarity and are so namedBacillus amyloliquefaciens yel14，Bacillus amyloliquefaciensyel14 16S rThe RNA sequence is shown as SEQ ID NO. 2. And verifying the ammonia oxidation capacity and the denitrification capacity of the obtained microorganisms. In the validation of numerous microorganisms, it was finally foundBacillus amyloliquefaciensyel14 has good ammonia oxidation function, and is selected as a candidate strain for next experiment.
Bacillus amyloliquefaciens (A), (B), (C), (B) and (C)Bacillus amyloliquefaciens) yel14, deposited at the Guangdong province culture Collection (GDMCC) at 27 th month 5.2021, address: building 5 of first furnance, large yard, 100, building 59, Guangdong province, Guangzhou, China, zip code: 510070, accession number: GDMCC No. 61699.
Alcaligenes faecalis (A), (B), (C), (B), (C), (B), (C), (B), (C), (B), (C), (B), (C), (B), (C)Alcaligenes faecalis) CHO6, deposited at 27 days 5/2021 at the guangdong province collection of microorganisms (GDMCC) address: building 5 of first furnance, large yard, 100, building 59, Guangdong province, Guangzhou, China, zip code: 510070, accession number: GDMCC No. 61700.
Example 2
Alcaligenes faecalisCHO6 andBacillus amyloliquefaciensyel14 confirmation of denitrification capability in common culture medium:
two strains were separated on different media, so two different basal media were selected to verify denitrification capacity, R respectively 2 A and PDB medium. With the inoculation amount of 1 percent by volume fraction, the method comprises the following steps ofAlcaligenes faecalisCHO6 was inoculated in 1 liter of Medium R 2 A(12 g/L)+NO 2 - (17.5 mM)+NH 4 + (3.5 mM) verified its pairing with NH 4 + Oxidation capacity and p-NO 2 - And inoculated in 1 liter of the medium R 2 A(12 g/L)+ NO 3 - In (16 mM), the denitrifying ability of the strain CHO6 was verified. And require aerobic (180 rpm, 30 ℃) and anaerobic (30 ℃ C., 99.99% N charge, respectively 2 Anaerobic chamber of (c) under conditions to verify the denitrification capability of CHO 6. Wherein NO 3 - 、NO 2 - And NH 4 + The ion concentration of (d) was determined by ion chromatography (Thermo Scientific) and the Total Nitrogen (TN) was determined by TOC instrument (shimadzu).
With the inoculation amount of 1 percent by volume fraction, the method comprises the following stepsBacillus amyloliquefaciensyel14 in 1L of PDB medium (24 g/L) + NO 2 - (13.2 mM)+ NH 4 + (3.3 mM) and PDB (24 g/L) + NO 3 - (11.8 mM), aerobic culture (180 rpm, 30 ℃) and anaerobic culture (30 ℃ C., charged with 99.99% N) were performed simultaneously 2 Anaerobic box) to verify yel14 denitrification capability.
And the corresponding medium not inoculated with strain CHO6 or yel14 was set as a blank Control (CK).
The results show that the strain CHO6 is in R 2 A(12 g/L)+NO 2 - (17.5 mM)+NH 4 + After 10 days of culture in (3.5 mM) medium, the strain CHO6 exhibited aerobic denitrification capability equivalent to the anaerobic denitrification capability, and the aerobic denitrification capability and the anaerobic denitrification capability were 25.32% and 26.35%, respectively (FIG. 2). And in R 2 A(12 g/L)+ NO 3 - After 10 days of culture in (16 mM) medium, the denitrification capability of the strain CHO6 is most obvious, and 66% and 71% denitrification capability can be achieved under aerobic and anaerobic conditions respectively (figure 2). Further analysis of the nitrogen ion-containing compounds revealed that strain CHO6 was able to bind R both aerobically and anaerobically 2 A(12 g/L)+NO 2 - (17.5 mM)+NH 4 + (3.5 mM) NO in Medium 2 - Is totally removed by denitrification (in FIG. 3
Figure 439843DEST_PATH_IMAGE001
、
Figure 552156DEST_PATH_IMAGE002
) NH, as compared with control group 4 + No significant change occurred. And in R 2 A(12 g/L)+ NO 3 - In (16 mM) culture medium, the strain CHO6 has strong denitrification capability and can utilize the denitrification process to remove NO 3 - Almost all conversion was removed (99%) (in FIG. 4
Figure 480797DEST_PATH_IMAGE001
、
Figure 678561DEST_PATH_IMAGE002
）。
Strain yel14 in PDB (24 g/L) + NO 2 - (13.2 mM)+ NH 4 + (3.3 mM) after 10 days of culture, the aerobic denitrification capacity was no more than 0, and the anaerobic denitrification efficiency was only 11.42% (FIG. 2). Similarly, strain yel14 was found to be PDB (24 g/L) + NO 3 - After 10 days of culture in the (11.8 mM) medium, the aerobic denitrification capacity was also no more than 0, while the anaerobic denitrification capacity was only 7.37% (FIG. 2). The strain yel14 has very weak denitrification capability, probably because the nitrogen ion-containing compounds in the system are not converted into nitrogen gas, but are interconverted in an ionic form. Specifically, strain yel14 was found to be PDB (24 g/L) + NO 2 - (13.2 mM)+ NH 4 + (3.3 mM) in aerobic culture, NO not contained in the culture can be produced 3 - (1.248 mM) (FIG. 3
Figure 819692DEST_PATH_IMAGE003
) While 47% of NH can be added during anaerobic culture 4 + (of FIG. 3
Figure 114407DEST_PATH_IMAGE004
) Conversion to NO 3 - (0.447 mM) (FIG. 3
Figure 978064DEST_PATH_IMAGE005
) And NO 2 - (2.683 mM) (FIG. 3
Figure 663124DEST_PATH_IMAGE006
). At PDB (24 g/L) + NO 3 - In (11.8 mM), the aerobic denitrification capability of the strain yel14 is stronger, and 94.8 percent of NO can be realized 3 - Conversion, but anaerobic denitrification capacity is very weak and large amounts of NO accumulate 2 - (2.982 mM) (FIG. 4
Figure 342367DEST_PATH_IMAGE003
、
Figure 553905DEST_PATH_IMAGE005
) This may be the reason why the Total Nitrogen (TN) in the system cannot be removed.
In a comprehensive sense, the method has the advantages that,Alcaligenes faecalisstrain CHO6 has strong denitrification function (removing NO) under aerobic or anaerobic condition 3 - And NO 2 - ) But lack ammonia oxidation capability; and thenBacillus amyloliquefaciensstrain yel14 has a certain degree of NO only under aerobic conditions 3 - Denitrification (removal of NO) 3 - ) Although hardly having a denitrifying function under anaerobic conditions, the bacterium has an ability to oxidize ammonia and convert NH 4 + Conversion to NO 2 - Or NO 3 - (FIGS. 2-4).
Example 3
Alcaligenes faecalisstrain CHO6 andBacillus amyloliquefaciensthe application of strain yel14 in landfill leachate is specifically implemented as follows:
from a huizhou landfill leachate treatment plant (the permanent connection in huizhou city, guangdong province, mature type,>for 10 years), the stock solution is taken back and placed in a refrigeration house with the temperature of minus 4 ℃ for storage and use. To verifyAlcaligenes faecalisCHO6 andBacillus amyloliquefaciensyel14, respectively culturing strains CHO6 and yel14 to logarithmic phase, inoculating flora CHO6-yel14 compounded in a volume ratio of 1:1 into 1 liter of landfill leachate stock solution, wherein the inoculation amount is 1 percent of volume. And setting landfill leachate without inoculated strain CHO6 or yel14 as a blank control group (CK), wherein the setting culture method is as follows:
aerobic culture: CK (group 3), CHO6 (group 3), yel14 (group 3), CHO6-yel14 (group 3), with the proviso that 180 rpm is applied at 30 ℃ in a shaker;
and (3) normal-temperature standing culture: CK (group 3), CHO6 (group 3), yel14 (group 3), CHO6-yel14 (group 3);
anaerobic culture: CK (group 3), CHO6 (group 3), yel14 (group 3), CHO6-yel14 (group 3), with the proviso that 30 ℃ contains 99.99% N 2 The anaerobic incubator of (1).
The cultivation time was 10 days.
The process of denitrification in landfill leachate by the strains CHO6 and yel14 is schematically shown in figure 1. The results show that under the three culture conditions, the strains CHO6 and yel14 and the flora CHO6-yel14 have certain denitrification capability and ammonia nitrogen removal capability in the landfill leachate. Compared with the situation that the strains CHO6 and yel14 are singly inoculated in the landfill leachate for culture, the compound flora CHO6-yel14 has better denitrification effect, and can respectively remove 470.5 mg/L, 241.67 mg/L and 724.83 mg/L of total nitrogen (shown in figure 5) in the landfill leachate under aerobic, normal-temperature standing and anaerobic conditions (shown in figure 5)
Figure 293191DEST_PATH_IMAGE001
、
Figure 465546DEST_PATH_IMAGE002
、
Figure 981104DEST_PATH_IMAGE003
). Ion chromatography determination shows that the compound flora CHO6-yel14 has better ammonia nitrogen removal capacity and adaptability in a high ammonia nitrogen landfill leachate environment, and the ammonia nitrogen removal rates of the compound flora CHO6-yel14 under aerobic, normal-temperature standing and anaerobic conditions can reach 44.4%, 37.53% and 38.95% respectively (the ammonia nitrogen removal rate in figure 6 is shown in the specification)
Figure 922515DEST_PATH_IMAGE001
、
Figure 629440DEST_PATH_IMAGE002
、
Figure 351408DEST_PATH_IMAGE003
）。
The above are only preferred embodiments of the present invention, and it should be noted that the above preferred embodiments should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.
Sequence listing
<110> institute of microbiology, academy of sciences of Guangdong province (center for microbiological analysis and detection of Guangdong province)
<120> denitrification bacterial agent with broad-spectrum dissolved oxygen and organic carbon tolerance and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1346
<212> DNA
<213> Alcaligenes faecalis CHO6(Alcaligenes faecalis CHO6)
<400> 1
ctacttctgg tgaaacccac tcccatggtg tgacgggcgg tgtgtacaag acccgggaac 60
gtattcaccg cgacattctg atccgcgatt actagcgatt ccgacttcac gcagtcgagt 120
tgcagactgc gatccggact acgatcgggt ttctgagatt ggctccccct cgcgggttgg 180
cgaccctctg tcccgaccat tgtatgacgt gtgaagccct acccataagg gccatgagga 240
cttgacgtca tccccacctt cctccggttt gtcaccggca gtctcattag agtgctcttg 300
cgtagcaact aatgacaagg gttgcgctcg ttgcgggact taacccaaca tctcacgaca 360
cgagctgacg acagccatgc agcacctgtg ttccggttct cttgcgagca cggccaaatc 420
tcttcggcat tccagacatg tcaagggtag gtaaggtttt tcgcgttgca tcgaattaat 480
ccacatcatc caccgcttgt gcgggtcccc gtcaattcct ttgagtttta atcttgcgac 540
cgtactcccc aggcggtcaa cttcacgcgt tagctgcgct actaaggcct aacggcccca 600
acagctagtt gacatcgttt agggcgtgga ctaccagggt atctaatcct gtttgctccc 660
cacgctttcg tgtctgagcg tcagtattat cccagggggc tgccttcgcc atcggtattc 720
ctccacatat ctacgcattt cactgctaca cgtggaattc tacccccctc tgacatactc 780
tagctcggca gttaaaaatg cagttccaag gttgagccct gggatttcac atctttcttt 840
ccgaaccgcc tacacacgct ttacgcccag taattccgat taacgcttgc accctacgta 900
ttaccgcggc tgctggcacg tagttagccg gtgcttattc tgcagatacc gtcagcagca 960
tcccgtatta ggggatgcct tttcttctct gccaaaagta ctttacaacc cgaaggcctt 1020
catcatacac gcgggatggc tggatcaggg tttcccccat tgtccaaaat tccccactgc 1080
tgcctcccgt aggagtctgg gccgtgtctc agtcccagtg tggctggtcg tcctctcaaa 1140
ccagctacgg atcgttgcct tggtgagcct ttaccccacc aactagctaa tccgatatcg 1200
gccgctccaa tagtgagagg tcttgcgatc cccccctttc ccccgtaggg cgtatgcggt 1260
attagccact ctttcgagta gttatccccc gctactgggc acgttccgat atattactca 1320
cccgtccgcc actcgccacc aagaga 1346
<210> 2
<211> 1391
<212> DNA
<213> Bacillus amyloliquefaciens yel14(Bacillus amyloliquefaciens yel14)
<400> 2
tcgagcggac agatgggagc ttgctccctg atgttagcgg cggacgggtg agtaacacgt 60
gggtaacctg cctgtaagac tgggataact ccgggaaacc ggggctaata ccggatggtt 120
gtctgaaccg catggttcag acataaaagg tggcttcggc taccacttac agatggaccc 180
gcggcgcatt agctagttgg tgaggtaacg gctcaccaag gcgacgatgc gtagccgacc 240
tgagagggtg atcggccaca ctgggactga gacacggccc agactcctac gggaggcagc 300
agtagggaat cttccgcaat ggacgaaagt ctgacggagc aacgccgcgt gagtgatgaa 360
ggttttcgga tcgtaaagct ctgttgttag ggaagaacaa gtgccgttca aatagggcgg 420
caccttgacg gtacctaacc agaaagccac ggctaactac gtgccagcag ccgcggtaat 480
acgtaggtgg caagcgttgt ccggaattat tgggcgtaaa gggctcgcag gcggtttctt 540
aagtctgatg tgaaagcccc cggctcaacc ggggagggtc attggaaact ggggaacttg 600
agtgcagaag aggagagtgg aattccacgt gtagcggtga aatgcgtaga gatgtggagg 660
aacaccagtg gcgaaggcga ctctctggtc tgtaactgac gctgaggagc gaaagcgtgg 720
ggagcgaaca ggattagata ccctggtagt ccacgccgta aacgatgagt gctaagtgtt 780
agggggtttc cgccccttag tgctgcagct aacgcattaa gcactccgcc tggggagtac 840
ggtcgcaaga ctgaaactca aaggaattga cgggggcccg cacaagcggt ggagcatgtg 900
gtttaattcg aagcaacgcg aagaacctta ccaggtcttg acatcctctg acaatcctag 960
agataggacg tccccttcgg gggcagagtg acaggtggtg catggttgtc gtcagctcgt 1020
gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc cttgatctta gttgccagca 1080
ttcagttggg cactctaagg tgactgccgg tgacaaaccg gaggaaggtg gggatgacgt 1140
caaatcatca tgccccttat gacctgggct acacacgtgc tacaatggac agaacaaagg 1200
gcagcgaaac cgcgaggtta agccaatccc acaaatctgt tctcagttcg gatcgcagtc 1260
tgcaactcga ctgcgtgaag ctggaatcgc tagtaatcgc ggatcagcat gccgcggtga 1320
atacgttccc gggccttgta cacaccgccc gtcacaccac gagagtttgt aacacccgaa 1380
gtcggtgagg t 1391

Claims (9)

1. A denitrificaion agent with broad-spectrum dissolved oxygen and organic carbon tolerance is characterized by comprising bacillus amyloliquefaciens (Bacillus amyloliquefaciens)Bacillus amyloliquefaciens) And Alcaligenes faecalis: (Alcaligenes faecalis) (ii) a The bacillus amyloliquefaciensIs bacillus amyloliquefaciens yel14 with the preservation number as follows: the GDMCC No. 61699, wherein the Alcaligenes faecalis is Alcaligenes faecalis CHO6, and the preservation number is: GDMCC No. 61700; the compounding ratio of the bacillus amyloliquefaciens yel14 to the alcaligenes faecalis CHO6 is 1: 1.
2. The denitrifier according to claim 1, further comprising a functional carrier.
3. The use of the denitrification bacterial agent of claim 1 in the preparation of denitrification biofilms.
4. A denitrification biofilm comprising the denitrification bacterial agent according to claim 1.
5. The denitrification microbial inoculum of claim 1 or the denitrification biofilm of claim 4 for denitrification of water.
6. The use of claim 5, wherein the nitrogen is removed from the body of water under aerobic or anaerobic conditions.
7. Use according to claim 6, wherein the nitrogen in the body of water comprises ammonia nitrogen, NH 4 + -N, nitrate nitrogen NO 3 - -N or nitrous nitrogen NO 2 - -N.
8. Use of the denitrification microbial inoculum of claim 1 or the denitrification biofilm of claim 4 in sewage deodorization and/or landfill leachate deodorization.
9. A method for denitrifying a water body, characterized in that the denitrifying bacteria agent of claim 1 is directly put into nitrogen-containing wastewater, or the denitrifying bacteria agent of claim 1 is mixed with a functional carrier and then put into a biochemical reactor for treating the nitrogen-containing wastewater.
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