CN1603248A - Biological denitrification method for ammonium-nitrogen containing waste water and microbes thereof - Google Patents

Abstract

The invention public a new biotechnology treatment method of containing ammonium and nitrogen wastewater.In this method,adding nitrating activity heterotrophic bacteria or heterotrophic bacteria family into the ammonium wastewater,then removing the ammonium nitrogen of ammonium wastewater directly by the microorganism fuction at the suitable microorganism growth conditions.Also,this invention public one kind of nitrite-oxidizing bacteria which can remove the ammoniacal nitrogen effectively and protect the environment.

Description

Biological denitrification method for ammonium-nitrogen wastewater and microorganisms thereof

The present invention claims priority from patent CN03118600.9 filed in china. The application date of patent application No. CN03118600.9 is 2/14/2003.

The invention belongs to the field of:

the present invention belongs to the field of wastewater treatment, and in particular, the present invention relates to a method for treating ammonium-nitrogen-containing wastewater by a microorganism having nitrification activity, wherein ammonium is mainly converted into dinitrogen gas and other nitrogen-containing gases; the invention also relates to heterotrophic bacteria for use in the method of wastewater treatment.

Background art:

ammonium nitrogen is one of the major nitrogen contaminants responsible for eutrophication of water bodies. And high concentration of NH₄ ⁺The existence and treatment difficulties of-N wastewater in industrial production such as fermentation industry are an important issue to be solved at present.

The nitrification-denitrification mode throughout the brains of most researchers at that moment is NH₄ ⁺Nitrification-denitrification relay mode. Namely:

wherein NH₄ ⁺——→NO₃ ^-The process of (2) is nitrification; and with NO₂ ^-Or NO₃ ^-——→N₂O or N₂Is used for denitrification.

To date, the understanding of the initiators of nitrification in nature is still bound by the theory of chemoautotrophic, i.e., essentially accomplished by the autotrophic nitrifying bacteria. The most important basis of this authoritative theory is: the nitrifying bacteria can not utilize organic matters, and the organic matters are toxic to nitrification. Therefore, nitrifying bacteria cannot be isolated on koch plates, i.e., nutrient gelatin or nutrient agar plates.

At NH₄ ⁺The theory and technology applied in the biological denitrification process of N waste water is still according to the autotrophic nitrification theory, although the problem exists, a plurality of researchers are trying to solve and still search a new answer in the autotrophic concept, such as the treatment of high-concentration ammonia nitrogen waste water by using a combined membrane bioreactor (Lihong rock, Gaomangchun et al, environmental science 2003, 23 (5): 62-66) in the Lihong rock and the like. The recent kluyveromyces institute of Delft, the netherlands, also utilizes a newly discovered concept. (Jetten M S M, Horn S J, et al, Watt. Sci. Tech., 1997, 35 (9): 171-. It is noted that patent CN99808998.2, ｃA specification of ｃA process for treating ammoniｃA-containing waste water, refers to ｃA European patent EP-A-562466 "in which process heterotrophic bacteriｃA are usedIn particular mixtures of microorganisms, e.g. pseudomonadsBacteria, acinetobacter, moraxella, corynebacterium, micrococcus, flavobacterium and bacillus, are grown in separate reactors, so-called breeders, and are continuously or discontinuously dosed from these reactors into the wastewater treatment plant ". The heterotrophic bacteria mentioned in this patent are used to remove organic Carbon (COD) from wastewater before nitrification occurs, to ensure that nitrification by the autotrophic bacteria occurs as well as possible, and are not used as nitrifying bacteria.

Denitrification, the concept of biological denitrification, has long been the removal of NH by nitrifying bacteria₄ ⁺Oxidation of-N to NO₃ ^-And a large amount of-N products are accumulated to be used as a nitrogen source for anaerobic denitrification, so that the aim of denitrification is fulfilled. The SHARON process and the Anammox process are improved by only adding NH₄ ⁺Oxidation of-N to NO₂ ^-N, and the accumulation thereof, should be an improvement over the conventional methods, particularly in terms of the advantages of reduced reaction time, reduced space time with a single reactor, etc.,

nitrification and denitrification, which are mostly considered to occur:

A) nitrification: aerobic condition, no organic matter, substrate NH₄ ⁺Completing self-culture;

B) denitrification: anaerobic condition, organic substance, substrate NO₂ ^-Or NO₃ ^-And finishing the heterotrophic bacteria.

Under the guidance of this theoretical model, most industrial technical applications of biological denitrification of ammonium nitrogen wastewater are carried out according to this model. Technical practice has found considerable problems over the years. Such as: the proliferation speed is slow, the higher biological concentration is difficult to maintain, the concentration of organic matters needs to be reduced by aeration treatment, the strain can grow and express biological activity, and the impact resistance is weak; high-concentration ammonia nitrogen and nitrite can inhibit the growth of nitrifying bacteria, so that the nitrification is incomplete, and the total nitrogen removal rate is low.

At present, researchers in foreign countries have studied the biological denitrification process under aerobic conditions, and certain microbial populations have denitrification under aerobic conditionsThe characteristics of the nitrification and the denitrification are synchronized. The research result shows that microorganisms such as Thiococcus pantoea (Thiospora pantotoph), Alcaligenes faecalis (Alcaligenes faecalis), Pseudomonas (Psadonmonas sp), Comamonas (Comamonas sp.) can utilize NO under aerobic conditions_xN is subjected to denitrification. The nitrifying bacteria and the denitrifying bacteria are placed in the same reactor, such as an aeration tank, and mixed and cultured, although the synchronous nitrification and denitrification of a single reactor can be achieved. However, the denitrification result is not satisfactory, a large amount of nitrogen is converted into oxide nitrogen, and the oxide nitrogen is the main factor of the atmospheric greenhouse gas effect and the formation of ozone cavities and is easy to cause secondary pollution.

Some related research works are also carried out in China: the aerobic denitrifying bacteria and the autotrophic nitrifying bacteria are used for combined denitrification (Gunn chrysanthemum, Liu Dong, etc., applied in the fields of environmental biology, 2002, 8 (1): 78-82). Although the ammonia nitrogen removal capacity is good, the impact resistance is weak, the growth of thalli can be inhibited by high-concentration ammonia nitrogen with the ammonia nitrogen concentration higher than 0.3 g/L, and the residual amount of ammonia nitrogen after denitrification is large when the ammonia nitrogen concentration is higher than 0.2 g/L; meanwhile, the high organic carbon concentration is not endured, and the organic carbon concentration of 0.5 g/L inhibits the growth of thalli and reduces the denitrification effect. Moreover, the culture and growth conditions of various bacteria in the combined flora are inconsistent, one side is in a suppressed state when the other side plays a role, so that the conditions are inconsistent, the biological denitrification process is prolonged, the cost is increased, more importantly, the denitrification efficiency is general, and the denitrified water body has a distance from the environmental protection requirement.

The invention has the technical contents that:

it is an object of the present invention to provide a new process for treating ammonium-nitrogen-containing wastewater, which process only requires one step to convert the ammonia in the wastewater into non-polluting dinitrogen gas and other nitrogen-containing gases.

It is another object of the present invention to provide a group of heterotrophic bacteria or bacterial populations thereof having nitrification activity for use in the present wastewater treatment process.

A method for treating waste water containing ammonium nitrogen is characterized in that organic carbon source for physiologically producing alkali is added into the waste water containing ammonium, and proper amount of heterotrophic bacteria or heterotrophic bacteria group with nitrification activity is added, the bacteria are cultured for 15-35 days under the aerobic condition of pH 6-8 and the temperature under the static or micro-stirring state at 20-35 ℃, and ammonium nitrogen in the waste water containing ammonium is directly removed;

the heterotrophic bacteria with nitrification activity can grow or separate on a PM plate, a Grice reagent is positive when being directly dripped, and the strain has total nitrogen deficiency when being cultured in an aerobic atmosphere by adding inorganic ammonium salt into a carbon source for physiologically producing alkali.

In the present invention, the organic carbon source added to the ammonium-containing wastewater refers to a physiologically alkali-producing organic compound which can cause the pH of the culture substrate to rise by carbon metabolism of the microorganism. Specifically, the physiologically alkali-producing organic carbon source refers to an organic acid having a carboxyl group or a salt thereof, and is preferably pyruvic acid, sodium pyruvate, sodium acetate, citric acid, sodium citrate, sodium formate or the like.

Different chemical forms of N element in chemical reaction (gas, liquid, oxidation-reduction state) in the presence of non-life, and the inorganic chemical diagram indicates that:

the form is as follows: NH (NH)₄ ⁺→N₂H₅ ⁺→NH₂OH→N₂→N₂O→NO→HNO₂

The valence state: -3-2-10 +1 +2 +3

The form is as follows: n is a radical of₂O₄→NO₃ ^-

The valence state: +4 +5

The physicochemical processes as oxidation-reduction are progressive in potential from the highest reduced state of NH₄ ⁺N to NO in the highest oxidation state₃ ^-The oxidation of N may occur in a variety of oxidation products, in particular which products are formed, depending on the other substrate involved in the reaction and the conditions of occurrence, such as temperature, pressure, pH, catalyst, etc. For example, HNO₃As an oxidizing agent, the following substances are mainly reduced:

HNO₃→NO₂→O₂→NO→N₂O→N₂→NH₄ ⁺

+5 +4 +3 +2 +1 0 -3

in chemical reactions, a mixture of several products is usually obtained. At least one of the products is more dependent on the oxidizing agent (HNO)₃) And the activity of the reducing agent.

The inorganic energy nutrition biochemical theory of the microorganism ammonium oxidation and nitrous acid oxidation indicates an extremely isolated biological phenomenon, namely-3 valence N → +5 valence maximum oxidation under organic-free and aerobic conditionsThe nitration theory of the state N is contrary to the diversity of the products of the N oxidation process indicated by the chemical theory on the one hand, and on the other hand, the denitrification organic carbon is added, and the highest oxidation state N can be reduced to zero-valent N under the anaerobic condition₂Or N₂And O. The diversity of the isolated products of the former versus the N metabolites of the latter! The presence of a certain organic species, which constitutes the focal organism of this difference, may lead to "numerous aerobic bacteria capable of utilizing the organic species in NH₄ ⁺Assimilation of N-A multiplicity of N-oxidation products may occur in the decomposition. "in the present invention, such metabolic pathways may exist:

thereby reacting NH₄ ⁺The bacteria transformed into the double-nitrogen gas through the process are heterotrophic bacteria with nitrification activity or a mixture thereof, the heterotrophic bacteria with nitrification activity can grow or separate on a PM plate, a Grice reagent is directly positive when dropped, and the strain with total nitrogen deficiency is cultured in good gas by adding inorganic ammonium salt into a carbon source for physiologically producing alkali.

In the present invention, the temperature for culturing heterotrophic bacteria can be maintained at about 30 ℃, such as 28 ℃, 29 ℃, 30 ℃, 35 ℃ and the like, and the heterotrophic bacteria with nitrification activity can be cultured in ammonia-containing wastewater for about 25 days, such as 18 days, or 20 days, or 25 days, or 28 days, to efficiently convert ammonium nitrogen in the wastewater to dinitrogen gas to different degrees.

On the other hand, the addition of heterotrophic bacteria having nitrifying activity to ammonium-nitrogen-containing wastewater should be controlled to a certain amount, and the amount of cells added to the wastewater may be generally 10⁴-10⁷One/ml. The addition is generally 10⁵-10⁶Preferably one/ml.

In the present invention, heterotrophic bacteria or mixtures thereof having nitrifying activity can be isolated from soil or screened from strains deposited in the culture collection, the process of isolation or screening also known as PM plate method is:

1 coating a sample to be detected on a PM flat plate;

2, picking single bacterial colony or mixed bacteria to a PM flat plate for streaking and purifying;

3 dropping Grignard reagent on the plate colony;

4 within a certain developing time, the red developing person of the Grignard reagent is the positive bacterial colony to be checked

And 5, selecting the positive colony to be checked, repeating the steps 2, 3 and 4, and taking the colony which is red-colored by the Grignard reagent as the positive colony, namely the heterotrophic bacteria or bacteria group with the nitrification activity.

The microorganisms added into the ammonium-containing wastewater can grow or separate on a PM plate, and the Grice reagent is directly dripped to be positive, so that the strains have total nitrogen deficiency when cultured under aerobic conditions by using a carbon source for physiologically producing alkali and adding inorganic ammonium salt.

In the specific example provided by the invention, a bacillus with nitrification activity is obtained by taking alkaline calcareous soil, namely northern China moisture soil, which is sealed in hills in Henan province as a test soil sample, through PM plate separation, purification, activity test, Grice reagent identification and repeated screening. The individual morphological characteristics of the strains are shown in table 1:

individual morphological characteristics of the strains of TABLE 114

Strain number

Colonies on NBB422 PM plates were round, pale yellow, translucent, moist. Gram positive staining; cells are arranged in pairs in an oval, rod-like shape; has spores

Colonies on NBB324 PM plates were round, yellowish, flat, transparent, thin. Gram positive staining; the cell rods are slightly fusiform and arrayed in a fence shape; with spores in the shape of spheres

Colonies on NBB319 PM plates were round, yellowish, flat, transparent, thin. Gram positive staining; the two rod-shaped ends of the cells are slightly sharp and are arranged in a fence shape; with spore and enlarged sporangia

Colonies on NBB295 PM plates were round, irregular-edged, pale, opaque, and dry-surfaced. Gram positive staining; the cell rod is straight or slightly bent, and the two ends are round; with spore and enlarged sporangia

Colonies on NBB247 PM plates were small, round, grey-white, opaque, convex, surface-dried. Weak positive gram staining; the cells are in rod-like, growing chain-like arrangement; with spore and enlarged sporangia

Colonies on NBB204 PM plates were pale yellow, flat, slightly moist, and irregular in edges. Gram positive staining; the cell rods are straight or slightly bent and arranged in a chain shape; has spores

The colonies on the NBB135 PM plate were large, round, white, opaque, wet, and irregular-edged. Gram positive staining; the cells are in rod-shaped, round at two ends, long chain-shaped or splayed arrangement; has spores

Colonies on NBB112 PM plates were round, grey, flat, with irregular edges, dry surfaces, and wrinkled. Gram positive staining; the cells are arranged in a rod shape and a splayed shape; the spore is contained in the culture medium,

colonies on NBB72 PM plates were round, with irregular edges, pale, opaque, and dry on the surface. Gram positive staining; the two ends of the cell rod are round, the staining is uneven, and the cells are arranged in a long chain shape; with spore and enlarged sporangia

Colonies on NBB46 PM plates were round, flat, irregular-edged, pale, opaque, and dry on the surface. Gram positive staining; the two ends of the cell rods are round and are arranged in pairs; spherical spore shape and enlarged sporangia

Colonies on NBB15 PM plates were round, pale yellow, low-protuberant, and surface wet. Gram positive staining; the cell rods are straight or slightly bent and arranged in a long chain shape; spherical spore shape and enlarged sporangia

Colonies on NBB58-3 PM plates were yellowish, flat, large, and diffuse at the edges. Gram positive staining; the two ends of the cell rod are round; has spores

The colony on the NBB609 PM plate is round, gray yellow, flat, dry and has characteristic wrinkles; gram positive staining; the two ends of the cell rod are blunt and round, and are arranged in a fence shape and a splayed shape; has spores

The bacterial colony on the NBB19 PM plate is in an interlaced root shape, is light gray yellow and has a wet surface; gram positive staining; the two ends of the cell rod are blunt and round, and are arranged in a fence shape and a splayed shape; has spores

Furthermore, the inventor performs experiments and identification on 14 strains of bacillus with heterotrophic nitrification capability on physiological and biochemical characteristics, and the results are shown in table 2.

Physiological and biochemical characteristics of the strains of Table 214

Strain number contact Meilan anaerobic VP VP culture starch glucose 7% NaCl indole nitrate xylose mannose

Reduction utilization of enzyme dyeing growth pH hydrolysis fermentation growth experiment

NBB422 + + - - ＜6 + + + - +

NBB324 + - - ＞7 - + - - + + +

NBB319 + - - ＞7 - + - - + + +

NBB295 + + + ＜6 + + - - + + +

NBB247 + - - - ＜6 + + - - + + -

NBB204 + - - - ＞7 + + + - - + -

NBB135 + + + + ＜6 + + + - +

NBB112 + - - + ＜6 + + + - - + +

NBB72 + + + ＜6 + + + - + + +

NBB46 + - - ＞7 + + - - + +

NBB15 + - - ＞7 - - - - -

NBB58-3 + + + + ＜6 + + + - +

NBB609 + - - + ＜6 + + + - +

NBB19 + - + + ＜6 + + + - + -

Note: + indicates a positive reactionor available; negative reaction or unavailability.

It can be seen that there are differences among 14 strains in the indices of catalase, methylene blue staining, anaerobic growth, VP culture pH, starch hydrolysis, glucose fermentation, 7% NaCl growth, indole test, nitrate reduction, xylose utilization, and mannose utilization. Combined with the morphological and reference to the classification identification of the ninth edition of the bergey bacteria identification manual, respectively named of bacillus:

bacillus megaterium NBB-422, CGMCC NO.0554

Bacillus firmus NBB-324 CGMCC NO.0555

Bacillus brevis NBB-319 CGMCC NO.0556

Bacillus circulans NBB-295 CGMCC NO.0557

Bacillus coagulans Bacillus coaggulans NBB-247 CGMCC NO.0558

Bacillus lentus NBB-204 CGMCC NO.0559

Bacillus cereus NBB-135 CGMCC NO.0560

Bacillus pumilus NBB-112 CGMCC NO.0561

Bacillus licheniformis NBB-72 CGMCC NO.0562

Bacillus globisporus NBB-46 CGMCC NO.0563

Bacillus sphaericus strain NBB-15 CGMCC NO.0564

Bacillus badius NBB-58-3 CGMCC NO.0565 of Bacillus badius

Bacillus subtilis NBB-609 CGMCC NO.0565 and

bacillus mycoides NBB-19 CGMCC NO. 0586.

These bacteria were delivered to the China general microbiological culture Collection center, China patent office assigned depositary Committee for culture Collection, on days 4 and 9 and 5 and 31, 2001, respectively.

In the invention, the inventor uses alkaline calcareous soil, namely north China moisture soilfor sealing hills in Henan province as a test soil sample, and obtains S-12 and S-27 strains through PM plate separation, purification, activity test, Grice reagent identification and repeated screening, wherein the individual morphological characteristics of the S-12 and S-27 strains are shown in Table 3. In the present invention, the microorganisms isolated from the evergreen soil of Jiangsu province and the yellow soil of Jiangsu Hongyou respectively also have nitrification property, such as strains NH-2, NH-14, etc., and individual morphological characteristics of NH-2 and NH-14 strains are shown in Table 5:

TABLE 3 Individual morphological characteristics of strains S-12 and S-27

Strain number

The colony on the S-12 PM plate is round, the lawn is thick, the surface is dry, not smooth and slightly rough, the surface is milky white, and no water-soluble pigment is secreted. Gram-positive; the cells are rod-shaped, sometimes with different lengths and obvious bifurcation

The colony on the S-27 PM plate is round, the lawn is thick, the surface is smooth and moist, the color is orange, and the secretion of water-soluble pigment is not seen. Gram-positive; the cells are rod-shaped, sometimes with different lengths, and the thalli are slightly thinner than S-12, so that obvious bifurcation phenomenon is caused.

Furthermore, the inventors conducted experiments and characterization on the physiological and biochemical characteristics of S-12, S-27, NH-2 and NH-14 having heterotrophic nitrification ability, and the results are shown in tables 4-1, 4-2 and 4-3.

TABLE 4-1 physiological and biochemical characteristics of strains S-12, S-27, NH-2 and NH-14 (1)

	Item	NH-2	NH-14	S-12	S-27
						A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12	Control α Cyclodextrin β Cyclodextrin Dextrin Glycogen Inulin powder Mannan Tween 40 Tween 80 N-acetyl-D-galactosamine N-acetyl-D-glucosamine Amygdalin	- - +/- +/- +/- - - + + - - -	- - - - +/- - - + + - - -	+ + + + + + - - - + + +	- +/- + + + - - + + - - -
B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 B12	L-arabinose D-arabitol Hydroquinone glucoside D-Cellobiose D-fructose Fucus vesiculosus sugar D-galactose D-galacturonic acid Gentiobiose D-gluconic acid α -D-glucose m-inositol	- - - - + - - - - +/- +/- -	+ - + - + - - - - +/- +/- -	+ + - + + - + - +/- + + -	- + - - + - - - - - + -

TABLE 4-2 physiological and biochemical characteristics of strains S-12, S-27, NH-2 and NH-14 (2)

	Item	NH-2	NH-14	S-12	S-27
						C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12	α -D-lactose Lactulose (lactulose) Maltose Maltotriose D-mannitol D-mannose D-melezitose D-melibiose α methyl-D-galactoside β methyl-D-galactoside 3-methyl-glucose α -methyl-D-glucoside	+ - - - + +/- - - - - - -	- - - - - - - - - - - -	+ +/- + + - - + - +/- +/- + +	- - - - + + - - - - +/- +
D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 D11 D12	β -methyl-D-glucoside α -methyl-D-mannoside palatinose D-psicose D-raffinose L-rhamnose D-ribose Salicin Sedum heptaglycan D-sorbose Stachyose (four) candy Sucrose	- - - +/- - - - - +/- + - +	+/- - - + - - + + +/- - - -	+ - - - + - - - - + - +	- - + + - - + - + + - +
						E1 E2 E3 E4 E5 E6 E7 E8 E9 E10 E11 E12	D-tagatose D-trehalose Turanose Shu sugar alcohol D-xylose Acetic acid α -hydroxy-butyric acid β -hydroxy-butyric acid Gamma-hydroxy-butyric acid p-hydroxyphenylacetic acid α ketoglutaric acid β Ketolvaleric acid	- - - - - + - - - - - -	+/- +/- - - + + + + - - + +	- + - - + + + + + - + +	- + + + + + + + + - + +

TABLE 4-3 physiological and biochemical characteristics of strains S-12, S-27, NH-2 and NH-14 (3)

	Item	NH-2	NH-14	S-12	S-27
						F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12	Lactamide D-lactic acid methyl ester L-lactic acid D-malic acid L-malic acid Pyruvic acid methyl group Succinic acid methyl group Propionic acid Pyruvic acid Succinamic acid Succinic acid N-acetic acid-L-lactamic acid glutamic acid	- - - - - - - - - - - -	- + + - + + + - + + + -	+ + + - + + + +/- + + + -	+ + + - + + + + + + + +
G1 G2 G3 G4 G5 G6 G7 G8 G9 G10 G11 G12	L-propionamide D-alanine L-alanine L-alanylglycine L-asparagine L-glutamic acid Glycine-L-glutamic acid L-pyroglutamic acid L-serine Butanediamine 2, 3-butanediol Glycerol	- - - - - +/- +/- +/- - - - -	+/- +/- - - - - +/- +/- - - - +	- + + +/- + + +/- +/- - - - +	+/- - - - +/- +/- + - + - + +
						H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12	Adenosine (I) 2-deoxyadenosine Inosine derivative Thymidine Uridine (uridine) 5' -monophosphate adenosine 5' -Thymidine monophosphate 5' -monophosphate guanosine 6-phospho-D-fructose 1-phosphoric acid- α -D-glucose 6-phosphoric acid D-glucose D-L- α -Glycerol phosphate	- - - - - +/- +/- - - - - -	- - +/- - - - - - + - +/- +/-	+ + + +/- + - - - + + + +/-	- - - + - - - - + + + -

TABLE 5 Individual morphological characteristics of strains NH-2 and NH-14

Strain number

The colony on the NH-2 PM plate is round, the lawn is thick, the surface is smooth and moist, the color is lemon, and no water-soluble pigment is secreted. Gram-positive; the cells are in a long rod shape; endospore formation can be seen, the spores are terminal, the cysts are not expanded, and the fallen spores are oval.

The bacterial colony on the NH-14 PM plate is round, the bacterial lawn is thick, the surface is slightly dry, non-wet and milk white, and no water-soluble pigment is secreted. Gram-positive; the cells are short rod-shaped and coarse; endospore formation can be seen, the spores are terminal, the cysts are not expanded, and the spores are oval.

Referring to the ninth edition of the Bergey' S Manual of bacteriological identification, the S-12 strain is Mycobacterium (Arthrobacter ramosus), and thus it is named as Mycobacterium S-12; the mycobacterium S-12 has been preserved in China Center for Type Culture Collection (CCTCC), the preservation number of which is CCTCC M203103, in the preservation unit designated by the China patent office on 3.1.2004. The S-27 strain is Chromobacterium sulphureus (Arthrobacter sulphureus), so the strain is named as Chromobacterium sulphureus S-27, the Chromobacterium sulphureus S-27 is preserved in China Center for Type Culture Collection (CCTCC) which is a preservation unit designated by China patent office on 1/3 in 2004, and the preservation numbers are CCTCC M203104 respectively.

With reference to the ninth edition of the Bergey's Manual of bacteriological identification, the NH-2 strain is Bacillus firmus (Bacillus pseudobacteriumus), and is therefore named Bacillus firmus NH-2; the bacillus firmus NH-2 is preserved in China Center for Type Culture Collection (CCTCC), a preservation unit designated by China patent office, on 3.1.2004, with the preservation number of CCTCC M203101. The NH-14 strain is Bacillus candelilla (Paenibacillus campinaensis), so the strain is named as Bacillus candelilla NH-14, and the Bacillus candelilla NH-14 is preserved in China Center for Type Culture Collection (CCTCC), a preservation number of CCTCC M203102, on 3 days 1 month in 2004, in thepreservation unit designated by the China patent office.

The 18 strains of bacteria provided by the invention can independently perform a nitrification function, can also perform the nitrification function by combining at least one and a plurality of bacteria, and can complete denitrification on a water body together with nitrifying bacteria and denitrifying bacteria to eliminate the pollution of nitrogen on the environment. The nitrifying bacteria and denitrifying bacteria acceptable in the water body denitrification process refer to the commonly used autotrophic or heterotrophic nitrifying and denitrifying bacteria, such as CCTCC-M202043, CCTCC-M202044 and the like.

In the present invention, a group of heterotrophic bacteria having nitrification activity, such as Alcaligenes faecalis (CGMCC No.1.1799), Sarcina lutea (CGMCC No.1.880), etc., is also selected from the bacteria that have been preserved in the depository. That is, the bacteria separated or screened by the PM plate method can be added into the ammonium nitrogen-containing wastewater, and after the bacteria are cultured for 15-35 days at a static or micro-stirring state at 20-35 ℃, the ammonium nitrogen is converted into pollution-free dinitrogen gas, and the ammonium nitrogen-containing wastewater becomes clean water.

In the invention, the invention also provides soil or activated sludge with nitrification activity, and the method can be used for preparing the ammonium-nitrogen-containing wastewater into clean water. Such soil or activated sludge having nitrification activity can be obtained by the PM plate method as follows:

1 coating a sample to be detected such as soil or activated sludge on a PM flat plate;

2, picking single bacterial colony or mixed bacteria to a PM flat plate for streaking and purifying;

3 dropping Grignard reagent on the plate colony;

4 within a certain developing time, the red developing person of the Grignard reagent is the positive bacterial colony to be checked

And 5, selecting the positive bacterial colony to be checked, repeating the steps 2, 3 and 4, and taking the red-appearing colony of the Grignard reagent as the positive bacterial colony, namely the soil or the activated sludge of the bacteria with the nitrification activity.

80-95% of the ammonium nitrogen in the water body can be removed by applying the bacteria or heterotrophic bacteria group or soil or activated sludge containing nitrification activity provided by the invention. The invention overcomes the recognition and research miszone that the nitrification of ammonia is finished by autotrophic nitrifying bacteria, and overcomes the difficulties that the eutrophic water body needs aeration treatment, the number of autotrophic nitrifying bacteria is insufficient, the biological reaction tank is large, the process is complex and the like in the prior autotrophic nitrification and denitrification biological denitrification process.

The method provided by the invention has the advantages that:

the energy and time are saved, the generated reaction can be carried out in one step in the same reactor, and the COD is not required to be removed firstly; no obvious accumulation of nitrite is generated in the process; the high-concentration ammonium nitrogen wastewater with the ammonium nitrogen concentration higher than 0.5 g/L cannot inhibit the growth of thalli, the ammonium nitrogen removal rate is more than 80%, the denitrification effect is good, and the residual ammonium nitrogen concentration is low.

In the present invention, heterotrophic bacteria having nitrification activity are those which can be obtained by subjecting natural soil, sludge, wastewater, or these natural products to NH₄ ⁺Can be grown or separated on a PM plate in a culture system; and shows positive by direct drip with Grice reagent and shows NO₂ ^-The result is obtained; and the strain with total nitrogen deficiency is cultured in aerobic inorganic ammonium salt with carbon source for physiologically producing alkali, such as formic acid, acetic acid, pyruvic acid and other organic acid salt. Such bacteria include various bacteria of the genera Bacillus, Corynebacterium, Arthrobacter, and Pseudomonas.

The following embodiments are incorporated herein. The present invention is further illustrated with the understanding that these examples are provided for illustrative purposes only and are not intended to limit the scope of the invention as claimed.

The various units used in the present invention have national standards, which are uniformly adopted, and no national standards, which are adopted, and the nitrite concentration is 60.0mgNO₂-NL^-1It is expressed as 60 mg nitrite nitrogen per liter of solution, and the nitrate content is 0.18mgNL^-1Indicating 0.18mg of nitrate nitrogen per liter of solution.

The following embodiments are incorporated herein. The present invention is further illustrated, it being understood that these examples are intended to be illustrative only and are not intended to limit the scope of the claimed invention, and that specific experimental conditions and procedures are not set forth in the following examples, generally in accordance with conventional conditions such as: the soil microorganism research society, eds. (Ri), leaf Vinine, science publishers, 1983, soil microorganism experiments; shuanghou, ed by Schlegelia, et al, Beijing agricultural Press, 1986, handbook of soil microbiological analysismethods; and the conditions described in the institute of soil and microorganism, edited by the institute of soil and institute of soil, science publishers, 1985, soil microbial research act, etc., of the national academy of sciences, or following the conditions recommended by the manufacturers.

Examples

EXAMPLE 1 preparation of culture Medium, simulated ammonium-containing wastewater and reagents

1.1 preparation of PM liquid Medium (beef extract peptone Medium)

3 g of beef extract and 5 g of peptone are weighed and dissolved in 1000 ml of distilled water to form a PM liquid medium, and 20 g of agar is added to the unsterilized liquid PM medium to form a PM solid medium.

The pH of the medium was adjusted to 7.1 with 1N sodium hydroxide. Subpackaging in triangular flask, sterilizing, cooling the culture medium to 56 deg.C, and pouring into flat plate. The high pressure liquid chromatography analysis of the PM liquid culture medium shows that the contents of nitrite and nitrate are trace: nitrite was not detected, and the nitrate content was 0.18mgNL^-1。

1.2 preparation of Grignard reagent

1.2.1 sulfanilic acid reagent (solution A): 0.5 g of Sulfanilic acid (sulfamilic acid) is dissolved in 150 ml of 20% dilute acetic acid solution, stored in a brown bottle and refrigerated for standby.

1.2.2 α -naphthylamine reagent (solution B) 0.5 g α -naphthylamine (α -naphthylamine) was added to 20 ml distilled water and 150 ml 20% diluted acetic acid solution, and stored in a brown bottle and refrigerated for use.

1.3 preparation of NB liquid Medium

1.3.1 preparation of inorganic salt solutions

Weighing (NH)₄)₂SO₄2.1 g, NaH₂PO₄0.25 g, K₂HPO₄0.75 g of the total weight of the powder,

MgSO₄·7H₂o0.03 g, MnSO₄·H₂0.01 g of O, and the like,

FeSO₄·7H₂0.01 g of O, dissolved in 1000 ml of distilled water,

adjusting pH of the culture medium to 7.1 with 1M NaOH, packaging in triangular flask, and sterilizing.

1.3.2 preparation of organic carbon solution

1.3.2.1 preparation of glucose solution: 36 g of glucose was weighed and dissolved in 1000 ml of distilled water to form a 0.20M glucose solution, which was sterilized.

1.3.2.2 preparation of sodium acetate solution: 27.2 g of sodium acetate trihydrate are weighed out and dissolved in 1000 ml of distilled water to form a 0.20M sodium acetate solution which is sterilized.

1.3.2.3 preparation of sodium formate solution: 20.8 g of sodium formate dihydrate are weighed out and dissolved in 1000 ml of distilled water to form a 0.20M sodium formate solution, which is sterilized.

1.3.2.4 preparation of pyruvic acid solution: sucking 14.0ml pyruvic acid and dissolving in 1000 ml distilled water to form 0.20M pyruvic acid solution, and sterilizing.

When in use, 90 parts (volume ratio) of inorganic salt solution is mixed with 10 parts of organic carbon solution to form the NB medium.

1.4 preparation of simulated ammonium-containing wastewater

1.4.1 preparation of inorganic salt solutions

Weighing (NH)₄)₂SO₄2.1 g, NaH₂PO₄0.25 g, K₂HPO₄0.75 g of the total weight of the powder,

MgSO₄·7H₂o0.03 g, MnSO₄·H₂0.01 g of O, and the like,

FeSO₄·7H₂0.01 g of O, dissolved in 1000 ml of distilled water,

adjusting pH of the culture medium to 7.1 with 1M NaOH, packaging in triangular flask, and sterilizing.

1.4.2 preparation of organic carbon solution

1.4.2.1 preparation of sodium acetate solution: 27.2 g of sodium acetate trihydrate are weighed out and dissolved in 1000 ml of distilled water to form a 0.20M sodium acetate solution which is sterilized.

1.4.2.2 preparation of pyruvic acid solution: sucking 14.0ml pyruvic acid and dissolving in 1000 ml distilled water to form 0.20M pyruvic acid solution, and sterilizing.

1.4.2.3 preparation of sodium formate solution: 20.8 g of sodium formate dihydrate are weighed out and dissolved in 1000 ml of distilled water to form a 0.20M sodium formate solution, which is sterilized.

When in use, 90 parts (volume ratio) of inorganic salt solution is mixed with 10 parts of organic carbon solution to form simulated ammonium-containing wastewater.

Example (b): 2. separation and identification of heterotrophic bacterial strains with nitrifying activity

2.1 soil samples, see Table 6.

TABLE 6 sources of various soil samples

Sample number place classification name Source pH (Water extract)

1 south of Henan, sealing North China, dry land and 8.48 of plough layer soil

2 soil in paddy field and plough layer of evergreen soil of Jiangsu province 7.41

3 Sulianyuankangyellow soil 6.32 f of plough layer soil

2.2 weigh 1.0 g of air-dried soil into a 250ml Erlenmeyer flask containing 50ml of sterile distilled water and shake for 4 hours on a shaker at 90 r/min.

2.3 soil suspension was diluted 10-fold and spread on PM plates, three replicates per dilution. After 7 days of incubation at 28 ℃ single colonies were picked onto PM plates and streaked for purification. Microscopic examination proves the purity.

2.4 preliminary screening (identification of active bacteria)

Inoculating the obtained heterotrophic strain after separation and purification on a PM plate, culturing at 28 ℃ for 10 days, directly dripping a Grignard reagent on the PM plate, confirming the nitration activity, and taking the plate without inoculation as a blank control. Within 1 minute, the Grignard reagent developed a red color indicating the production of nitrite. And (5) repeatedly verifying that the chromogenic reaction is still positive, and preliminarily determining the chromogenic reaction as the nitrifying active bacteria. (see Table 7). The high pressure liquid chromatography analysis of the PM culture medium shows that the contents of nitrite and nitrate are trace amount: wherein nitrite is not detected, nitreThe acid salt content is less than 0.2mgNL^-1And the result is not positively interfered.

TABLE 7 Classification of partially active strains

Strain represented by species (genus) name

Bacillus (Bacillus)

Bacillus megaterium NBB422, NBB217

Bacillus pumilus (Bacillus brevis) NBB319, WY-2

Bacillus licheniformis (Bacillus licheniformis) NBB072, WX-2

Bacillus circulans NBB295, WR-8

Bacillus firmus NBB324, WR-9

Bacillus coagulans NBB247, NBB300

Bacillus lentus (Bacillus lentus) NBB204, NBB118

Bacillus cereus NBB135, NBB310

Bacillus pumilus NBB112, NBB557

Bacillus globisporus NBB46, NBB534

Bacillus sphaericus (Bacillus sphaericus) NBB15, NBB614

Bacillus badius NBB58-3, NBB101

Bacillus subtilis NBB609 and NBB617

Bacillus mycoides NBB19, NBB59

Bacillus firmus (Bacillus pseudobacteri) NH-2, NH-19

Paenibacillus canpi (Paenibacillus campinaensis) NH-14, NH-22

Mycobacterium (Arthrobacter ramosus) S-12, S-4

S-27, S-11 of Chromobacterium sulphureus (Arthrobacter sulfureus)

2.5 double sifter

Selecting representative strains with stronger activity in preliminary screening. Scraping pure bacterial lawn growing on PM plate, and adding 30 ml sterile water to make it fully and uniformly dispersed to obtain bacterial suspension. 1ml of each bacterial suspension was inoculated into a 250ml Erlenmeyer flask containing 50ml of NB medium with different organic carbon sources, and each set was repeated three times. And the non-inoculated medium was used as a blank control. After static culture at 28 ℃ for 21 days, the culture solution is centrifuged for 15min at 5000g at 4 ℃, and the nitrite concentration in the supernatant is measured by Grignard reagent colorimetric method. The difference between the colorimetric value of the culture supernatant of the strain sample and the colorimetric value of the blank control supernatant is more than 0.3mgNL^-1It wasjudged as heterotrophic nitrifying active bacteria (see Table 8).

2.6 physiological identification of active strains reference is made to Bergey's Manual of bacteria identification, ninth edition. The bacterial characteristics are shown in tables 1 and 2.

TABLE 8 colorimetric values (expressed as nitrite, NO) for the individual strains₂ ^--NmgL^-1)

Strain number carbon source sodium citrate sodium acetate

NBB422 0.7 3.0

NBB217 0.5 4.7

NBB319 1.7 3.3

NBB72 0.0 5.4

NBB295 0.7 2.8

NBB324 0.6 7.2

NBB247 0.3 2.9

NBB204 0.0 3.4

NBB135 0.4 1.9

NBB112 0.9 1.7

NBB46 0.7 5.3

NBB15 0.0 3.4

NBB58-3 0.5 3.8

NBB609 0.6 5.2

NBB19 0.3 4.9

WY-2 0.0 1.7

NH-2 0.1 5.8

NH-14 0.1 1.8

S-12 0.0 1.1

S-27 0.0 1.4

CK 0.0 0.1

Example (b): 3 identification of Bacillus megaterium NBB-422

1. And (3) colony morphology characteristics: after aerobic culture for 4 days at constant temperature of 28 ℃ on nutrient agar PM plates, the colonies are round, pale yellow, translucent and moist.

2. Morphological characteristics of the thallus: gram positive staining; cells are arranged in pairs in an oval, rod-like shape; has spores

3. The main physiological and biochemical characteristics are shown in Table 2, the optimum culture temperature is 28-32 deg.C, pH7.0-7.2, and the culture is aerobic.

4. Ammoxidation activity: the nitrite concentration accumulated in the shaking culture at 90-100r/min for 14 days is higher than 15.0mgNO₂-NL^-1The individual plant can reach 23mgNO₂-NL^-1The above.

The strain is Bacillus megaterium (Bacillus megaterium) according to the ninth edition of the Bergey's Manual of identification of bacteria. Therefore, it was named Bacillus megaterium NBB-422. The bacillus megaterium NBB-422 has been preserved in China general microbiological culture Collection center (CGMCC), a preservation unit specified by China patent office at 9.4.2001, with the preservation number of CGMCC NO. 0554.

Example (b): identification of Bacillus firmus NBB-324

1. And (3) colony morphology characteristics: after aerobic culture for 4 days at constant temperature of 28 ℃ on nutrient agar PM plates, the colonies are round, yellowish, flat, transparent and thin.

2. Morphological characteristics of the thallus: gram positive staining; the cell rods are slightly fusiform and are arranged in a fence shape; the spore is spherical.

3. The main physiological and biochemicalcharacteristics are shown in Table 2, the optimum culture temperature is 28-32 deg.C, pH7.0-7.2, and the culture is aerobic.

4. Ammoxidation activity: the nitrite concentration accumulated in the shaking culture at 90-100r/min for 14 days is higher than 25.0mgNO₂-NL^-1The individual plant can reach 36mgNO₂-NL^-1The above.

The strain is Bacillus firmus (Bacillus firmus) according to the ninth edition of Bergey's Manual of identification of bacteria. Therefore, it was named Bacillus firmus NBB-324. Bacillus firmus NBB-324 has been preserved in China general microbiological culture Collection center (CGMCC) which is a preservation unit specified by China patent office at 9.4.2001, and the preservation number is CGMCC NO. 0555.

Example (b): identification of Bacillus brevis NBB-319

1. And (3) colony morphology characteristics: after aerobic culture for 4 days at constant temperature of 28 ℃ on nutrient agar PM plates, the colonies are round, yellowish, flat, transparent and thin.

2. Morphological characteristics of the thallus: gram positive staining; the two rod-shaped ends of the cells are slightly sharp and are arranged in a fence shape; with spores, the sporangia dilate.

3. The main physiological and biochemical characteristics are shown in Table 2, the optimum culture temperature is 28-32 deg.C, pH7.0-7.2, and the culture is aerobic.

4. Ammoxidation activity: the nitrite concentration accumulated in the shaking culture at 90-100r/min for 14 days is higher than 15.0mgNO₂-NL^-1The individual plant can reach 26mgNO₂-NL^-1The above.

The strain is Bacillus brevis (Bacillus brevis) according to the ninth edition of Bergey's Manual of identification of bacteria. Therefore, it was named Bacillus brevis NBB-319. The Brevibacillus brevis NBB-319 has been preserved in China general microbiological culture Collection center (CGMCC), a preservation unit specified by China patent office at 9.4.2001, and the preservation number is CGMCC NO. 0556.

Example (b): identification of Bacillus circulans NBB-295

1. And (3) colony morphology characteristics: after aerobic culture on nutrient agar plates (PM plates) at a constant temperature of 28 ℃ for 4 days, the colonies are round, irregular in edge, grey-white, opaque and dry on the surface.

2. Morphological characteristics of the thallus: gram positive staining; the cell is rod-shaped or slightly bent, and the two ends of the cell are round; with spores, the sporangia dilate.

3. The main physiological and biochemical characteristics are shown in Table 2, the optimum culture temperature is 28-32 deg.C, pH7.0-7.2, and the culture is aerobic.

4. Ammoxidation activity: the nitrite concentration accumulated in the shaking culture at 90-100r/min for 14 days is higher than 15.0mgNO₂-NL^-1The individual plant can reach 22mgNO₂-NL^-1The above.

The strain is Bacillus circulans (Bacillus circulans) according to the ninth edition of Bergey's Manual of identification of bacteria. Therefore, it was named Bacillus circulans NBB-295. The Bacillus circulans NBB-295 has been preserved in China general microbiological culture Collection center (CGMCC) which is a preservation unit specified by China patent office at 4 month and 9 th 2001, and the preservation number is CGMCC NO. 0557.

Example (b): 7 identification of Bacillus coagulans NBB-247

1. And (3) colony morphology characteristics: after aerobic culturefor 4 days on nutrient agar PM plates at constant temperature of 28 ℃, the bacterial colony is small, round, grey white, opaque, convex and dry on the surface.

2. Morphological characteristics of the thallus: gram positive staining; the cells are arranged in a rod shape and a long chain shape; with spores, the sporangia dilate.

3. The main physiological and biochemical characteristics are shown in Table 2, the optimum culture temperature is 28-32 deg.C, pH7.0-7.2, and the culture is aerobic.

4. Ammoxidation activity: the nitrite concentration accumulated in the shaking culture at 90-100r/min for 14 days is higher than 15.0mgNO₂-NL^-1The individual plant can reach 20mgNO₂-NL^-1The above.

The strain is Bacillus coagulans (Bacillus coagulans) according to the ninth edition of Bergey's Manual of identification of bacteria. Therefore, it was named Bacillus coagulans NBB-247. Bacillus coagulans NBB-247 has been deposited in China general microbiological culture Collection center (CGMCC) which is a preservation unit specified by China patent office at 9.4.2001, and the preservation number is CGMCC NO. 0558.

Example (b): identification of Bacillus lentus NBB-204

1. And (3) colony morphology characteristics: after aerobic culture for 4 days at constant temperature of 28 ℃ on nutrient agar PM plates, colonies are pale yellow, flat, slightly moist and irregular in edges.

2. Morphological characteristics of the thallus: gram positive staining; the cell rods are straight or slightly bent and arranged in a chain shape; has spores.

3. The main physiological and biochemical characteristics are shown in Table 2, the optimum culture temperature is 28-32 deg.C, pH7.0-7.2, and the culture is aerobic.

4. Ammoxidation activity: the nitrite concentration accumulated in the shaking culture at 90-100r/min for 14 days is higher than 20.0mgNO₂-NL^-1The individual plant can reach 27mgNO₂-NL^-1The above.

The strain is Bacillus lentus (Bacillus lentus) according to the ninth edition of Bergey's Manual of identification of bacteria. Therefore, it was named Bacillus lentus NBB-204. The Bacillus lentus NBB-204 is preserved in China general microbiological culture Collection center (CGMCC) which is a preservation unit specified by China patent office at 9.4.2001, and the preservation number is CGMCC NO. 0559.

Example (b): 9 identification of Bacillus cereus NBB-135

1. And (3) colony morphology characteristics: after aerobic culture for 4 days on nutrient agar PM plates at constant temperature of 28 ℃, the colonies are large, round, white, opaque, moist and irregular in edges.

2. Morphological characteristics of the thallus: gram positive staining; the cells are in rod-shaped, round at two ends, long chain-shaped or splayed arrangement; has spores.

3. The main physiological and biochemical characteristics are shown in Table 2, the optimum culture temperature is 28-32 deg.C, pH7.0-7.2, and the culture is aerobic.

4. Ammoxidation activity: the nitrite concentration accumulated in the shaking culture at 90-100r/min for 14 days is higher than 10.0mgNO₂-NL^-1The individual plant can reach 17mgNO₂-NL^-1The above.

The strain is Bacillus cereus (Bacillus cereus) according to the ninth edition of Bergey's Manual of identification of bacteria. Therefore, it was named Bacillus cereus NBB-135. Bacillus cereus NBB-135 has been deposited in China general microbiological culture Collection center (CGMCC), a preservation unit specified by China patent office, on 9.4.2001 with the preservation number of CGMCC NO. 0560.

Example (b): identification of Bacillus pumilus NBB-112

1. And (3) colony morphology characteristics: after aerobic culture for 4 days at constant temperature of 28 ℃ on nutrient agar PM plates, the colonies are round, grey-white, flat, irregular in edge, dry in surface and wrinkled.

2. Morphological characteristics of the thallus: gram positive staining; the cells are arranged in a rod shape and a splayed shape; has spores.

3. The main physiological and biochemical characteristics are shown in Table 2, the optimum culture temperature is 28-32 deg.C, pH7.0-7.2, and the culture is aerobic.

4. Ammoxidation activity: the nitrite concentration accumulated in the shaking culture at 90-100r/min for 14 days is higher than 10mgNO₂-NL^-1The individual plant can reach 18mgNO₂-NL^-1The above.

The strain is Bacillus pumilus (Bacillus pumilus) according to the ninth edition of the Bergey's Manual of identification of bacteria. Therefore, it was named Bacillus pumilus NBB-112. Bacillus pumilus NBB-112 has been deposited in China general microbiological culture Collection center (CGMCC), a preservation unit specified by China patent office at 9.4.2001 with the preservation number of CGMCC NO. 0561.

Example (b): identification of 11 Bacillus licheniformis NBB-72

1. And (3) colony morphology characteristics: after aerobic culture for 4 days at constant temperature of 28 ℃ on nutrient agar PM plates, the colonies are round, grey white, irregular in edge, opaque and dry in surface.

2. Morphological characteristics ofthe thallus: gram positive staining; the two ends of the cell rod are round, the staining is uneven, and the cells are arranged in a long chain shape; with spores, the sporangia dilate.

3. The main physiological and biochemical characteristics are shown in Table 2, the optimum culture temperature is 28-32 deg.C, pH7.0-7.2, and the culture is aerobic.

4. Ammoxidation activity: the nitrite concentration accumulated in the shaking culture at 90-100r/min for 14 days is higher than 25.0mgNO₂-NL^-1The individual plant can reach 37mgNO₂-NL^-1The above.

The strain is Bacillus licheniformis (Bacillus licheniformis) by referring to the ninth edition of the Bergey's Manual of identification of bacteria. Therefore, it was named Bacillus licheniformis NBB-072. Bacillus licheniformis NBB-072 has been preserved in China general microbiological culture Collection center (CGMCC), a preservation number of CGMCC, which is a preservation unit specified by China patent office at 4, 9 and 2001, and the preservation number is CGMCC NO. 0562.

Example (b): identification of Bacillus circulans NBB-46

1. And (3) colony morphology characteristics: after aerobic culture for 4 days at constant temperature of 28 ℃ on nutrient agar PM plates, the colonies are round, grey white, irregular in edge, opaque and dry in surface.

2. Morphological characteristics of the thallus: gram positive staining; the two ends of the cell rods are round and are arranged in pairs; spherical spore shape, and expanded sporangia.

3. The main physiological and biochemical characteristics are shown in Table 2, the optimum culture temperature is 28-32 deg.C, pH7.0-7.2, and the culture is aerobic.

4. Ammoxidation activity: the nitrite concentration accumulated in the shaking culture at 90-100r/min for 14 days is higher than 25.0mgNO₂-NL^-1The individual plant can reach 34mgNO₂-NL^-1The above。

The strain is Bacillus globisporus (Bacillus globisporus) according to the ninth edition of Bergey's Manual of identification of bacteria. Therefore, it was named Bacillus circulans NBB-046. Bacillus circulans NBB-046 has been deposited in China general microbiological culture Collection center (CGMCC), a preservation unit specified by China patent office, in 2001, 4, 9 and the preservation number is CGMCC NO. 0563.

Example (b): identification of Bacillus sphaericus NBB-15

1. And (3) colony morphology characteristics: after aerobic culture on nutrient agar plates (PM plates) at a constant temperature of 28 ℃ for 4 days, the colonies are round, pale yellow, low in projections and moist in surface.

2. Morphological characteristics of the thallus: gram positive staining; the cell rods are straight or slightly bent and arranged in a long chain shape; spherical spore shape, and expanded sporangia.

3. The main physiological and biochemical characteristics are shown in Table 2, the optimum culture temperature is 28-32 deg.C, pH7.0-7.2, and the culture is aerobic.

4. Ammoxidation activity: the nitrite concentration accumulated in the shaking culture at 90-100r/min for 14 days is higher than 20.0mgNO₂-NL^-1The individual plant can be as high as 28mgNO₂-NL^-1The above.

The strain is Bacillus sphaericus (Bacillus sphaericus) according to the ninth edition of Bergey's Manual of identification of bacteria. Therefore, it was named Bacillus sphaericus NBB-015. Bacillus sphaericus NBB-015 has been preserved in China general microbiologicalculture Collection center (CGMCC), a preservation unit specified by China patent office at 9.4.2001, and the preservation number is CGMCC NO. 0564.

Example (b): identification of 14-Bacillus badius NBB-58-3

1. And (3) colony morphology characteristics: after aerobic culture for 4 days at constant temperature of 28 ℃ on nutrient agar PM plates, the colonies are round, gray yellow, flat and dry.

2. Morphological characteristics of the thallus: gram positive staining; the two ends of the cell rod are round; has spores.

3. The main physiological and biochemical characteristics are shown in Table 2, the optimum culture temperature is 28-32 deg.C, pH7.0-7.2, and the culture is aerobic.

4. Ammoxidation activity: the nitrite concentration accumulated in the shaking culture at 90-100r/min for 14 days is higher than 25.0mgNO₂-NL^-1The individual plant can be as high as 32mgNO₂-NL^-1The above.

The strain is Bacillus badius (Bacillus badius) according to the ninth edition of Bergey's Manual of identification of bacteria. Therefore, it was named Bacillus badius NBB-58-3. The Bacillus badius NBB-58-3 is preserved in China general microbiological culture Collection center (CGMCC) which is a preservation unit appointed by China patent office in 2001, 4 and 9, and the preservation number is CGMCC NO. 0565. Example (b): identification of 15 Bacillus subtilis NBB-609

1. And (3) colony morphology characteristics: after aerobic culture on nutrient agar PM plates at a constant temperature of 28 ℃ for 4 days, the colonies are round, grey yellow, flat, dry and have characteristic wrinkles.

2. Morphological characteristics of the thallus: gram positive staining; the two ends of the cell rod are blunt and round, and are arranged in a fence shape and a splayed shape; has spores.

3. The main physiological and biochemical characteristics are shown in Table 2, the optimum culture temperature is 28-32 deg.C, pH7.0-7.2, and the culture is aerobic. 4. Ammoxidation activity: the nitrite concentration accumulated in the shaking culture at 90-100r/min for 14 days is higher than 25.0mgNO₂-NL^-1The individual plant can be as high as 38mgNO₂-NL^-1The above.

The strain is Bacillus subtilis (Bacillus subtilis) according to the ninth edition of Bergey's Manual of identification of bacteria. Therefore, it was named Bacillus subtilis NBB-609. The bacillus subtilis NBB-609 has been preserved in China general microbiological culture Collection center (CGMCC) which is a preservation unit appointed by China patent office in 2001, 4, 9 and has a preservation number of CGMCC NO. 0566.

Example (b): identification of Bacillus mycoides NBB-19

1. And (3) colony morphology characteristics: after aerobic culture on nutrient agar plates (PM plates) at a constant temperature of 28 ℃ for 4 days, the bacteria fell into an interlaced root shape, which was pale yellow and had a moist surface.

2. Morphological characteristics of the thallus: gram positive staining; the two ends of the cell rod are blunt and round, and are arranged in a fence shape and a splayed shape; has spores.

3. The main physiological and biochemical characteristics are shown in Table 2, the optimum culture temperature is 28-32 deg.C, pH7.0-7.2, and the culture is aerobic.

4. Ammoxidation activity: the nitrite concentration accumulated in the shaking culture at 90-100r/min for 14 days is higher than 25.0mgNO₂-NL^-1The individual plant can be as high as 32mgNO₂-NL^-1The above.

The strain is Bacillus mycoides (Bacillus species) according to the ninth edition of Bergey's Manual of identification of bacteria. Therefore, it was named Bacillus mycoides NBB-19. Bacillus mycoides NBB-19 has been preserved in China general microbiological culture Collection center (CGMCC), a preservation unit specified by China patent office in 31.5.2001, with the preservation number of CGMCC NO. 0586.

Example 17 identification of Mycobacterium S-12

1. And (3) colony morphology characteristics: after aerobic culture for 4 days on nutrient agar plates (PM plates) at a constant temperature of 28 ℃, the bacterial colony is round, the bacterial lawn is thick, the surface is dry, not smooth and slightly rough, the surface is milky white, and no water-soluble pigment is secreted.

2. Morphological characteristics of the thallus: gram-positive; the cells are rod-shaped, sometimes with different lengths, with obvious bifurcation phenomenon.

3. The main physiological and biochemical characteristics are shown in tables 4-1, 4-2 and 4-3, the optimum culture temperature is 28-32 ℃, pH is 7.0-7.2, and the culture is aerobic.

4. Ammoxidation activity: the nitrite concentration accumulated in the shaking culture at 90-100r/min for 14 days is higher than 11.0mgNO₂-NL^-1The individual plant can be up to 20mgNO₂-NL^-1The above.

Referring to the ninth edition of the Bergey's Manual of bacteriological identification, this strain is Mycobacterium (Arthrobacter ramosus). Therefore, it was named as Mycobacterium S-12. The mycobacterium S-12 has been preserved in China Center for Type Culture Collection (CCTCC), a preservation unit designated by the Chinese patent office,on 3.1.2004 with the preservation number of CCTCC M203103.

Example 18 identification of Chromenobacter sulphureus S-27

1. And (3) colony morphology characteristics: after aerobic culture for 4 days on nutrient agar plates (PM plates) at constant temperature of 28 ℃, the bacterial colony is round, the bacterial lawn is thick, the surface is smooth and moist, the bacterial colony is orange, and no water-soluble pigment is secreted.

2. Morphological characteristics of the thallus: gram-positive; the cells are rod-shaped, sometimes with different lengths, and the thalli are slightly thinner than S-12, so that obvious bifurcation phenomenon is caused.

3. The main physiological and biochemical characteristics are shown in tables 4-1, 4-2 and 4-3, the optimum culture temperature is 28-32 ℃, pH is 7.0-7.2, and the culture is aerobic.

4. Ammoxidation activity: the nitrite concentration accumulated in the shaking culture at 90-100r/min for 14 days is higher than 5.0mgNO₂-NL^-1The individual plant can be up to 14mgNO₂-NL^-1The above.

Referring to the ninth edition of Bergey's Manual of identification of bacteria, the strain is Chromobacterium sulphureus (Arthrobacter sulfurous). Therefore, it was named as Chromobacterium sulphureus S-27. The Chroobacter sulphureus S-27 has been preserved in China Center for Type Culture Collection (CCTCC), a preservation unit designated by the China patent office, on 3.1.2004 with the preservation number of CCTCC M203104.

Example 19 identification of Paenibacillus camphenius NH-14

1. And (3) colony morphology characteristics: after aerobic culture for 4 days on nutrient agar plates (PM plates) at a constant temperature of 28 ℃, the bacterial colony is round, the bacterial lawn is thick, the surface is slightly dry, non-moist and milk white, and no water-soluble pigment is secreted.

2. Morphological characteristics of the thallus: gram-positive; the cells are short rod-shaped and coarse; endospore formation can be seen, the spores are terminal, the cysts are not expanded, and the spores are oval.

3. The main physiological and biochemical characteristics are shown in tables 4-1, 4-2 and 4-3, the optimum culture temperature is 28-32 ℃, pH is 7.0-7.2, and the culture is aerobic.

4. Ammoxidation activity: the nitrite concentration accumulated in the shaking table culture at 90-100r/min for 14 days is highAt 7.0mgNO₂-NL^-1Individual strains can be as high as 12mg NO₂-NL^-1The above.

The strain is Paenibacillus camppi (Paenibacillus campinaensis) according to the ninth edition of Bergey's Manual of identification of bacteria. Therefore, it was named Paenibacillus candelilla NH-14. The Paenibacillus camphensis NH-14 is preserved in China Center for Type Culture Collection (CCTCC), a preservation unit designated by China patent office, on 3.1.2004, with the preservation number of CCTCC M203102.

EXAMPLE 20 identification of Bacillus firmus NH-2

1. And (3) colony morphology characteristics: after 4-day aerobic culture at constant temperature of 28 ℃ on a nutrient agar plate (PM plate), the bacterial colony is round, the bacterial lawn is thick, the surface is smooth and moist, the bacterial lawn is lemon, and no water-soluble pigment is secreted.

2. Morphological characteristics of the thallus: gram-positive; the cells are in a long rod shape; endospore formation can be seen, the spores are terminal, the cysts are not expanded, and the fallen spores are oval.

3. The main physiological and biochemical characteristics are shown in tables 4-1, 4-2 and 4-3, the optimum culture temperature is 28-32 ℃, pH is 7.0-7.2, and the culture is aerobic.

4. Ammoxidation activity: the nitrite concentration accumulated in the shaking culture at 90-100r/min for 14 days is higher than 27.0mgNO₂-NL^-1The individual plant can be as high as 35mgNO₂-NL^-1The above.

The strain is Bacillus firmus (Bacillus pseudobacteriumus) according to the ninth edition of the Bergey's Manual of identification of bacteria. Therefore, it was named Bacillus firmus NH-2. The bacillus firmus NH-2 is preserved in China Center for Type Culture Collection (CCTCC), a preservation unit designated by China patent office, on 3.1.2004, with the preservation number of CCTCC M203101.

Example 21 Denitrification of Bacillus megaterium NBB-422

21.1 the culture medium is 0.015mol L^-1The preparation method of the NB medium with sodium acetate as a carbon source is the same as 1.3.

21.2 preculture:

21.2.1 bacterial lawn attached to a ring NBB-422 from PM slant surface and filled with 0.015mol L^-1Shaking and culturing the strain liquid in a 250ml conical flask of 50ml NB medium with sodium acetate as a carbon source for 24 hours at 30 ℃ and 120-₆₀₀＝0.45。

21.2.2 inoculating 2% of the pre-cultured bacterial liquid into a culture medium containing 0.060mol L^-1Model of pyruvic acidAmmonium-containing wastewater (preparation method is the same as 1.4) is cultured for 28 days at 30 ℃ in a static way. The indophenol blue colorimetric method is used for measuring ammoniacal nitrogen, the Grignard reagent colorimetric method is used for measuring nitritenitrogen, and the Kelvin method is used for measuring total nitrogen.

21.3 results: after the mixed culture is cultured for 28 days, 74 percent of total nitrogen in the system is removed, the ammonium nitrogen removal rate is 93.5 percent, and the nitrite concentration is only 0.15mgNL^-1。

Example 22 Denitrification of Bacillus firmus NBB-324

22.1 the culture medium is 0.015mol L^-1The preparation method of the NB medium with sodium acetate as a carbon source is the same as 1.3.

22.2 preculture:

22.2.1 lawn of NBB-324 from PM slope^-1Shaking and culturing the strain liquid in a 250ml conical flask of 50ml NB medium with sodium acetate as a carbon source for 24 hours at 30 ℃ and 120-₆₀₀＝0.45。

22.2.2 inoculating 2% of the pre-cultured bacterial liquid into a culture medium containing 0.060mol L^-1The simulated ammonium-containing wastewater of pyruvic acid (preparation method is the same as 1.4) is cultured for 28 days at 30 ℃ in a static way. The indophenol blue colorimetric method is used for measuring ammoniacal nitrogen, the Grignard reagent colorimetric method is used for measuring nitrite nitrogen, and the Kelvin method is used for measuring total nitrogen.

22.3 results: after the mixed culture is cultured for 28 days, 67.4 percent of total nitrogen in the system is removed, the ammonium nitrogen removal rate is 83.7 percent, and the nitrite concentration is only 0.18mgNL^-1。

Example 23 Denitrification of Brevibacillus brevis NBB-319

23.1 the culture medium is 0.015mol L^-1The preparation method of the NB medium with sodium acetate as a carbon source is the same as 1.3.

23.2 preculture:

23.2.1 lawn containing 0.015mol L of NBB-319 from PM slant^-1Shaking and culturing the strain liquid in a 250ml conical flask of 50ml NB medium with sodium acetate as a carbon source for 24 hours at 30 ℃ and 120-₆₀₀＝0.45。

23.2.2 inoculating 2% of the pre-cultured bacterial liquid into a culture medium containing 0.060mol L^-1The simulated ammonium-containing wastewater of pyruvic acid (preparation method is the same as 1.4) is cultured for 28 days at 30 ℃ in a static way. The indophenol blue colorimetric method is used for measuring ammoniacal nitrogen, the Grignard reagent colorimetric method is used for measuring nitrite nitrogen, and the Kelvin method is used for measuring total nitrogen.

23.3 results: after the mixed culture is cultured for 28 days, 71.2 percent of total nitrogen in the system is removed, the ammonium nitrogen removal rate is 85.1 percent, and the nitrite concentration is only 0.22mgNL^-1。

Example 24 Denitrification of Bacillus circulans NBB-295

24.1 the culture medium is 0.015mol L^-1The preparation method of the NB medium with sodium acetate as a carbon source is the same as 1.3.

24.2 preculture:

24.2.1 bacterial lawn attached to a ring NBB-295 from PM slope into a bag containing 0.015mol L^-150ml of NB medium with sodium acetate as carbon source in a 250ml conical flask, shaking the flask at 30 ℃ and 120-Shaking for 24 hr, and adjusting OD value of the inoculum solution to OD₆₀₀＝0.45。

24.2.2 inoculating 2% of the pre-cultured bacterial liquid into a culture medium containing 0.060mol L^-1The simulated ammonium-containing wastewater of pyruvic acid (preparation method is the same as 1.4) is cultured for 28 days at 30 ℃ in a static way. The indophenol blue colorimetric method is used for measuring ammoniacal nitrogen, the Grignard reagent colorimetric method is used for measuring nitrite nitrogen, and the Kelvin method is used for measuring total nitrogen.

24.3 results: after the mixed culture is cultured for 21 days, 62.8 percent of total nitrogen in the system is removed, the ammonium nitrogen removal rate is 74.3 percent, and the nitrite concentration is only 0.18mgNL^-1。

Example 25 Denitrification of Bacillus coagulans NBB-247

25.1 the medium was 0.015mol L^-1NB medium with sodium acetate as carbon sourceThe preparation method is the same as 1.3.

25.2 preculture:

25.2.1 bacterial lawn with 0.015mol L attached from PM slant to NBB-247^-1Shaking and culturing the strain liquid in a 250ml conical flask of 50ml NB medium with sodium acetate as a carbon source for 24 hours at 30 ℃ and 120-₆₀₀＝0.45。

25.2.2 inoculating 2% of the pre-cultured bacterial liquid into a culture medium containing 0.060mol L^-1The simulated ammonium-containing wastewater of pyruvic acid (preparation method is the same as 1.4) is cultured for 28 days at 30 ℃ in a static way. The indophenol blue colorimetric method is used for measuring ammoniacal nitrogen, the Grignard reagent colorimetric method is used for measuring nitrite nitrogen, and the Kelvin method is used for measuring total nitrogen.

25.3 results: after the mixed culture is cultured for 21 days, 57.2 percent of total nitrogen in the system is removed, the removal rate of ammonium nitrogen is 73.5 percent, and the concentration of nitrite is only 0.15mgNL^-1。

Example 26 Denitrification of Bacillus lentus NBB-204

26.1 the culture medium is 0.015mol L^-1The preparation method of the NB medium with sodium acetate as a carbon source is the same as 1.3.

26.2 preculture:

26.2.1 bacterial lawn with 0.015mol L attached from PM slant to NBB-204^-1Shaking and culturing the strain liquid in a 250ml conical flask of 50ml NB medium with sodium acetate as a carbon source for 24 hours at 30 ℃ and 120-₆₀₀＝0.45。

26.2.2 inoculating 2% of the pre-cultured bacterial liquid into a culture medium containing 0.060mol L^-1The simulated ammonium-containing wastewater of pyruvic acid (preparation method is the same as 1.4) is cultured for 28 days at 30 ℃ in a static way. The indophenol blue colorimetric method is used for measuring ammoniacal nitrogen, the Grignard reagent colorimetric method is used for measuring nitrite nitrogen, and the Kelvin method is used for measuring total nitrogen.

26.3 results: after the mixed culture is cultured for 28 days, 69.7 percent of total nitrogen in the system is removed, the ammonium nitrogen removal rate is 84.1 percent, and the nitrite concentration is only 0.16mgNL^-1。

Example 27 Denitrification of Bacillus cereus NBB-135

27.1 the medium was 0.015mol L^-1The preparation method of the NB medium with sodium acetate as a carbon source is the same as 1.3.

27.2 preculture:

27.2.1 bacterial lawn with 0.015mol L attached from PM slant to NBB-135^-1Shaking and culturing the strain liquid in a 250ml conical flask of 50ml NB medium with sodium acetate as a carbon source for 24 hours at 30 ℃ and 120-₆₀₀＝0.45。

27.2.2 inoculating 2% of the pre-cultured bacterial liquid into a culture medium containing 0.060mol L^-1The simulated ammonium-containing wastewater of pyruvic acid (preparation method is the same as 1.4) is cultured for 28 days at 30 ℃ in a static way. The indophenol blue colorimetric method is used for measuring ammoniacal nitrogen, the Grignard reagent colorimetric method is used for measuring nitrite nitrogen, and the Kelvin method is used for measuring total nitrogen.

27.3 results: after the mixed culture is cultured for 28 days, 60.5 percent of total nitrogen in the system is removed, the ammonium nitrogen removal rate is 72.7 percent, and the nitrite concentration is only 0.45mgNL^-1。

Example 28 Denitrification of Bacillus pumilus NBB-112

28.1 the culture medium is 0.015mol L^-1The preparation method of the NB medium with sodium acetate as a carbon source is the same as 1.3.

28.2 preculture:

28.2.1 bacterial lawn with 0.015mol L attached from PM slant to NBB-112^-1Shaking and culturing the strain liquid in a 250ml conical flask of 50ml NB medium with sodium acetate as a carbon source for 24 hours at 30 ℃ and 120-₆₀₀＝0.45。

28.2.2 inoculating 2% of the pre-cultured bacterial liquid into a culture medium containing 0.060mol L^-1The simulated ammonium-containing wastewater of pyruvic acid (preparation method is the same as 1.4) is cultured for 28 days at 30 ℃ in a static way. The indophenol blue colorimetric method is used for measuring ammoniacal nitrogen, the Grignard reagent colorimetric method is used for measuring nitrite nitrogen, and the Kelvin method is used for measuring total nitrogen.

28.3 results: after the mixed culture is cultured for 28 days, 72.5 percent of total nitrogen in the system is removed, the removal rate of ammoniumnitrogen is 87.7 percent, and the concentration of nitrite is only 0.20mgNL^-1。

Example 29 Denitrification of Bacillus licheniformis NBB-72

29.1 the medium was 0.015mol L^-1The preparation method of the NB medium with sodium acetate as a carbon source is the same as 1.3.

29.2 preculture:

29.2.1 bacterial lawn with 0.015mol L attached from PM slant to NBB-72^-1Shaking and culturing the strain liquid in a 250ml conical flask of 50ml NB medium with sodium acetate as a carbon source for 24 hours at 30 ℃ and 120-₆₀₀＝0.45。

29.2.2 inoculating 2% of the pre-cultured bacterial liquid into a culture medium containing 0.060mol L^-1The simulated ammonium-containing wastewater of pyruvic acid (preparation method is the same as 1.4) is cultured for 14 days at 30 ℃ in a static way. The indophenol blue colorimetric method is used for measuring ammoniacal nitrogen, the Grignard reagent colorimetric method is used for measuring nitrite nitrogen, and the Kelvin method is used for measuring total nitrogen.

29.3 results: after the mixed culture is cultured for 14 days, 60.3 percent of total nitrogen in the system is removed, the ammonium nitrogen removal rate is 73.5 percent, and the nitrite concentration is only 0.15mgNL^-1。。

Example 30 Denitrification of Bacillus circulans NBB-46 Strain

30.1 the culture medium is 0.015mol L^-1The preparation method of the NB medium with sodium acetate as a carbon source is the same as 1.3.

30.2 preculture:

30.2.1 bacterial lawn with 0.015mol L attached from PM slant to NBB-46^-1Shaking and culturing the strain liquid in a 250ml conical flask of 50ml NB medium with sodium acetate as a carbon source for 24 hours at 30 ℃ and 120-₆₀₀＝0.45。

30.2.2 inoculating 2% of the pre-cultured bacterial liquid into a culture medium containing 0.060mol L^-1The simulated ammonium-containing wastewater of pyruvic acid (preparation method is the same as 1.4) is cultured for 28 days at 30 ℃ in a static way. The indophenol blue colorimetric method is used for measuring ammoniacal nitrogen, the Grignard reagent colorimetric method is used for measuring nitrite nitrogen, and the Kelvin method is used for measuring total nitrogen.

30.3 results: after the mixed culture is cultured for 28 days, 73.3 percent of total nitrogen in the system is removed, the ammonium nitrogen removal rate is 92.7 percent, and the nitrite concentration is only 0.15mgNL^-1。

Example 31 Denitrification of Bacillus sphaericus NBB-15

31.1 the culture medium is 0.015mol L^-1The preparation method of the NB medium with sodium acetate as a carbon source is the same as 1.3.

31.2 preculture:

31.2.1 bacterial lawn with 0.015mol L attached from PM slant to a ring NBB-15^-1Shaking and culturing the strain liquid in a 250ml conical flask of 50ml NB medium with sodium acetate as a carbon source for 24 hours at 30 ℃ and 120-₆₀₀＝0.45。

31.2.2 inoculating 2% of the pre-cultured bacterial liquid into a culture medium containing 0.060mol L^-1The simulated ammonium-containing wastewater of pyruvic acid (preparation method is the same as 1.4) is cultured for 28 days at 30 ℃ in a static way. The indophenol blue colorimetric method is used for measuring ammoniacal nitrogen, the Grignard reagent colorimetric method is used for measuring nitrite nitrogen, and the Kelvin method is used for measuring total nitrogen.

31.3 results: after the mixed culture is cultured for 28 days, 55.7 percent of total nitrogen in the system is removed, the ammonium nitrogen removal rate is 73.2 percent, and the nitrite concentration is only 0.18mgNL^-1。

Example Denitrification of 32 Bacillus badius NBB-58-3

32.1 the culture medium is 0.015mol L^-1The preparation method of the NB medium with sodium acetate as a carbon source is the same as 1.3.

32.2 preculture:

32.2.1 bacterial lawn with 0.015mol L attached from PM slant to NBB-58-3^-1Shaking and culturing the strain liquid in a 250ml conical flask of 50ml NB medium with sodium acetate as a carbon source for 24 hours at 30 ℃ and 120-₆₀₀＝0.45。

32.2.2 inoculating 2% of the pre-cultured bacterial liquid into a culture medium containing 0.060mol L^-1The simulated ammonium-containing wastewater of pyruvic acid (preparation method is the same as 1.4) is cultured for 28 days at 30 ℃ in a static way. The indophenol blue colorimetric method is used for measuring ammoniacal nitrogen, the Grignard reagent colorimetric method is used for measuring nitrite nitrogen, and the Kelvin method is used for measuring total nitrogen.

32.3 results: after the mixed culture is cultured for 28 days, 64.2 percent of total nitrogen in the system is removed, the removal rate of ammonium nitrogen is 85.7 percent, and nitrite is concentratedDegree of only 0.15mgNL^-1。

Example 33 Denitrification of Bacillus subtilis NBB-609

33.1 the medium was 0.015mol L^-1The preparation method of the NB medium with sodium acetate as a carbon source is the same as 1.3.

33.2 preculture:

33.2.1 lawn of NBB-609 was placed on PM slant and filled with 0.015mol L^-1Shaking and culturing the strain liquid in a 250ml conical flask of 50ml NB medium with sodium acetate as a carbon source for 24 hours at 30 ℃ and 120-₆₀₀＝0.45。

33.2.2 inoculating the pre-cultured bacterial liquid 1ml to a culture medium containing 0.060mol L^-1The simulated ammonium-containing wastewater of pyruvic acid (preparation method is the same as 1.4) is cultured for 28 days at 30 ℃ in a static way. The indophenol blue colorimetric method is used for measuring ammoniacal nitrogen, the Grignard reagent colorimetric method is used for measuring nitrite nitrogen, and the Kelvin method is used for measuring total nitrogen.

33.3 results: after the mixed culture is cultured for 28 days, 68.4 percent of total nitrogen in the system is removed, the removal rate of ammonium nitrogen is 87.1 percent, and the concentration of nitrite is only 0.18mgNL^-1。

Example 34 Denitrification of Bacillus mycoides NBB-19

34.1 the culture medium is 0.015mol L^-1The preparation method of the NB medium with sodium acetate as a carbon source is the same as 1.3.

34.2 preculture:

34.2.1 bacterial lawn with 0.015mol L attached from PM slant to NBB-19^-1Shaking and culturing the strain liquid in a 250ml conical flask of 50ml NB medium with sodium acetate as a carbon source for 24 hours at 30 ℃ and 120-₆₀₀＝0.45。

34.2.2 inoculating 2% of the pre-cultured bacterial liquid into a culture medium containing 0.060mol L^-1The simulated ammonium-containing wastewater of pyruvic acid (preparation method is the same as 1.4) is cultured for 28 days at 30 ℃ in a static way. The indophenol blue colorimetric method is used for measuring ammoniacal nitrogen, the Grignard reagent colorimetric method is used for measuring nitrite nitrogen, and the Kelvin method is used for measuring total nitrogen.

34.3 results: the mixed culture was removed after 28 days of culture62.7 percent of total nitrogen of the system, 76.3 percent of ammonium nitrogen removal rate and 0.18mgNL of nitrite concentration^-1。

Example 35 Denitrification of Mycobacterium S-12

35.1 the medium was 0.015mol L^-1The preparation method of the NB medium with sodium acetate as a carbon source is the same as 1.3.

35.2 preculture:

35.2.1 bacterial lawn with 0.015mol L attached to the surface of PM^-1Shaking and culturing the strain liquid in a 250ml conical flask of 50ml NB medium with sodium acetate as a carbon source for 24 hours at 30 ℃ and 120-₆₀₀＝0.45。

35.2.2 inoculating 2% of the pre-cultured bacterial liquid into a culture medium containing 0.060mol L^-1The simulated ammonium-containing wastewater of pyruvic acid (preparation method is the same as 1.4) is cultured for 28 days at 30 ℃ in a static way. The indophenol blue colorimetric method is used for measuring ammoniacal nitrogen, the Grignard reagent colorimetric method is used for measuring nitrite nitrogen, and the Kelvin method is used for measuring total nitrogen.

35.3 results: after the mixed culture is cultured for 28 days, 80 percent of total nitrogen in the system is removed, the removal rate of ammonium nitrogen is 96.5 percent, and the concentration of nitrite is only 0.15mgNL^-1。

Example 36 Denitrification of Chromobacterium Sulforum S-27

36.1 the culture medium is 0.015mol L^-1The preparation method of the NB medium with sodium acetate as a carbon source is the same as 1.3.

36.2 preculture:

36.2.1 bacterial lawn with 0.015mol L attached to the surface of PM^-1Shaking and culturing the strain liquid in a 250ml conical flask of 50ml NB medium with sodium acetate as a carbon source for 24 hours at 30 ℃ and 120-₆₀₀＝0.45。

36.2.2 inoculating 2% of the pre-cultured bacterial liquid into a culture medium containing 0.060mol L^-1The simulated ammonium-containing wastewater of pyruvic acid (preparation method is the same as 1.4) is cultured for 28 days at 30 ℃ in a static way. The indophenol blue colorimetric method is used for measuring ammoniacal nitrogen, the Grignard reagent colorimetric method is used for measuring nitrite nitrogen, and the Kelvin method is used for measuring total nitrogen.

36.3 results: after the mixed culture is cultured for 28 days, 45 percent of total nitrogen in the system is removed, the removal rate of ammonium nitrogen is 56.5 percent, and the concentration of nitrite is only 0.15mgNL^-1。

Example 37 Denitrification of Paenibacillus candelilla NH-14

37.1 the culture medium is 0.015mol L^-1The preparation method of the NB medium with sodium acetate as a carbon source is the same as 1.3.

37.2 preculture:

37.2.1 bacterial lawn with a ring NH-14 attached to the PM slant and filled with 0.015mol L^-1Shaking and culturing the strain liquid in a 250ml conical flask of 50ml NB medium with sodium acetate as a carbon source for 24 hours at 30 ℃ and 120-₆₀₀＝0.45。

37.2.2 inoculating 2% of the pre-cultured bacterial liquid into aculture medium containing 0.060mol L^-1The simulated ammonium-containing wastewater of pyruvic acid (preparation method is the same as 1.4) is cultured for 21 days at 30 ℃ in a static way. The indophenol blue colorimetric method is used for measuring ammoniacal nitrogen, the Grignard reagent colorimetric method is used for measuring nitrite nitrogen, and the Kelvin method is used for measuring total nitrogen.

37.3 results: after the mixed culture is cultured for 21 days, 77 percent of total nitrogen in the system is removed, and the removal rate of ammonium nitrogen is

88.2%, nitrite concentration of only 0.15mgNL^-1。

Example 38 Denitrification of Bacillus firmus NH-2

38.1 the medium was 0.015mol L^-1The preparation method of the NB medium with sodium acetate as a carbon source is the same as 1.3.

38.2 preculture:

38.2.1 bacterial lawn with a ring NH-2 attached to the PM slant and filled with 0.015mol L^-1Shaking and culturing the strain liquid in a 250ml conical flask of 50ml NB medium with sodium acetate as a carbon source for 24 hours at 30 ℃ and 120-₆₀₀＝0.45。

38.2.2 inoculating the pre-cultured bacterial liquid 1ml to a culture medium containing 0.060mol L^-1The simulated ammonium-containing wastewater of pyruvic acid (preparation method is the same as 1.4) is cultured for 14 days at 30 ℃ in a static way. Determination of ammonia by indophenol blue colorimetric methodNitrogen, griissThe reagent colorimetry is used for measuring nitrite nitrogen, and the Kelvin method is used for measuring total nitrogen.

38.3 results: after the mixed culture is cultured for 14 days, 73 percent of total nitrogen in the system is removed, the ammonium nitrogen removal rate is 88.5 percent, and the nitrite concentration is only 0.15mgNL^-1。

Example (b): 39 the combined denitrification 39.1 culture medium of the bacillus circulans NBB-46 and the bacillus megaterium NBB-422 is 0.015mol L^-1The preparation method of the NB medium with sodium acetate as a carbon source is the same as 1.3.

39.2 preculture:

39.2.1A bacterial lawn of Bacillus circulans NBB-46 was inoculated from PM slant into a bag filled with 0.015mol L^-1Shaking and culturing the strain liquid in a 250ml conical flask of 50ml NB medium with sodium acetate as a carbon source for 24 hours at 30 ℃ and 120-₆₀₀＝0.45。

37.2.2 inoculating Bacillus megaterium NBB-422 lawn from PM slant into 50ml NB medium (0.015mol L)^-1Sodium acetate as carbon source) in a 250ml conical flask, shaking and culturing for 24 hours at 30 ℃ and 130r/min by using a shaking table, and adjusting the OD value of the bacterial solution to OD₆₀₀＝0.45。

39.3 culturing: NBB-422(37.2.2 culture) and NBB-46(37.2.1 culture) were mixed at a volume ratio of 1: 1, and 1ml of the inoculated mixed bacterial suspension (2%) was inoculated into 0.060mol L^-1The simulated ammonium nitrogen wastewater with acetate as a carbon source (the preparation method is the same as 1.4) is statically cultured for 28 days at the temperature of 30 ℃. The indophenol blue colorimetric method is used for measuring ammoniacal nitrogen, the Grignard reagent colorimetric method is used for measuring nitrite nitrogen, and the Kelvin method is used for measuring total nitrogen. 39.4 results: after the mixed culture is cultured for 28 days, 77.5 percent of total nitrogen in the system is removed, the ammonium nitrogen removal rate is 94.7 percent, and the nitrite concentration is only 0.25mgNL^-1。

Example (b): the combined denitrification 40.1 culture medium of 40 bacillus cereus NBB-135 and bacillus circulans NBB-295 is 0.015mol L^-1The preparation method of the NB medium with sodium acetate as a carbon source is the same as 1.3.

40.2 preculture:

40.2.1A bacterial lawn of Bacillus cereus NBB-135 was inoculated from PM slant to a cell containing 0.015mol L^-1Shaking and culturing the strain liquid in a 250ml conical flask of 50ml NB medium with sodium acetate as a carbon source for 24 hours at 30 ℃ and 120-₆₀₀＝0.45。

40.2.2 inoculating the bacterial lawn of Bacillus circulans NBB-295 from PM slant into 50ml NB medium (0.015mol L)^-1Sodium acetate as carbon source) in a 250ml conical flask, shaking and culturing for 24 hours at 30 ℃ and 130r/min by using a shaking table, and adjusting the OD value of the bacterial solution to OD₆₀₀＝0.45。

40.3, culturing: NBB-295(38.2.2 culture) and NBB-135(38.2.1 culture) were mixed at a ratio of 3: 7 (by volume), and 1ml of the inoculated mixed bacterial suspension (2%) was inoculated into 0.060mol L^-1The simulated ammonium nitrogen wastewater (formula 1.4) with acetate as carbon source is cultured for 21 days at 30 ℃ in a static way. The indophenol blue colorimetric method is used for measuring ammoniacal nitrogen, the Grignard reagent colorimetric method is used for measuring nitrite nitrogen, and the Kelvin method is used for measuring total nitrogen.37.4 results: after the mixed culture is cultured for 21 days, 61.3 percent of total nitrogen in the system is removed, the ammonium nitrogen removal rate is 75.1 percent, and the nitrite concentration is only 0.32mg NL^-1。

Example (b): 41 Bacillus (Bacillus cereus NBB-135, Bacillus pumilus NBB-112, Bacillus circulansNBB-295, Bacillus licheniformis NBB-72) and other nitrifying bacteria: joint denitrification effect of arthrobacter globiformis WR-2(CCTCC M202043)

41.1 the culture medium is 0.015mol L^-1The preparation method of the NB medium with sodium acetate as a carbon source is the same as 1.3.

39.2 preculture:

41.2.1 from PM slant each loop Bacillus cereus NBB-135, Bacillus pumilus NBB-112, Bacillus circulans NBB-295, Bacillus licheniformis NBB-072 lawn into 50ml NB medium (0.015mol L)^-1Sodium acetate as carbon source) in a 250ml conical flask, shaking and culturing for 24 hours at 30 ℃ and 130r/min by using a shaking table, and adjusting the OD value of the bacterial solution to OD₆₀₀＝0.45。

41.2.2 inoculating thallus Porphyrae of Arthrobacter globiformis WR-2 from PM slant, placing into 250ml conical flask containing 70ml PM liquid culture medium, shake culturing at 30 deg.C and 130r/min for 28 hr, and adjusting OD value of the bacterial liquidIs a thallus OD₆₀₀＝0.45。

41.3 culturing: a culture of Arthrobacter globiformis WR-2 (39.2.2 culture) and a mixed culture of nitrifying bacteria such as Bacillus cereus NBB-135 (39.2.1 culture) were mixed at a volume ratio of 1: 1, and 1ml of the mixed culture was added to a mixture of 0.075mol L^-1The simulated ammonium nitrogen wastewater (formula 1.4) with acetate as carbon source is cultured for 21 days at 30 ℃ in a static way. The indophenol blue colorimetric method is used for measuring ammoniacal nitrogen, the Grignard reagent colorimetric method is used for measuring nitrite nitrogen, and the Kelvin method is used for measuring total nitrogen.

41.4 results: after the mixed culture is cultured for 21 days, 76.3 percent of total nitrogen in the system is removed, the removal rate of ammonium nitrogen is 87.9 percent, and the concentration of nitrite is only 0.10mgNL^-1。

Example 42 isolation and enumeration of heterotrophic nitrification-active bacterial strains from different types of soil samples in different regions 42.1 a large number of representative heterotrophic nitrification-active bacterial strains were isolated and identified from 9 different types of soil samples in 4 regions of our country (table 9) by the same procedures and methods of example 2 (table 10).

TABLE 9 sources of various soil samples

Source pH of classification name of numbered sampling site

1 Shanxi Yangling red oil dry land, 8.04 of plough layer soil

2 The south China Jianbei south China moisture soil dry land, 8.48 plough layer soil

3 The soil of the irrigated land and plough layer 8.24 of the south China hillock

4 West of the Yangtze river Queen river fourth red clay dry land, 5.46 arable layer soil

5 Jiangxi Yingtan quaternary red clay paddy field, 6.13 arable layer soil

6 Jiangxi Yingtan quaternary red clay barren slope, surface soil 4.88

7.41 pieces of plough layer soil of paddy field of UK-grid soil of Jiangsu province

8 soil in dry land and plough layer 6.41

9 Su Lian Yun gang yellow soil paddy field, 6.32 of topsoil

TABLE 10 Nitrification Activity bacteria count statistics for each soil sample

Numbered soil sample fresh soil (Cell/g dry soil) acclimatization (Cell/g dry soil)

1 red oil soil, 5.55X 10 dry land⁶4.03×10¹²

2 North China moisture soil, 2.71X 10 dry land⁶5.95×10¹⁰

3 North China moisture soil, 1.99X 10 in paddy field⁶2.23×10¹²

4 quaternary red clay, 1.16X 10 dry land⁷1.72×10¹¹

5 quaternary red clay, 1.88X 10 in paddy field⁶4.02×10¹⁰

6 quaternary red clay, 3.23X 10 on barren slopes⁵7.95×10⁷

7 soil with black grid, 3.11X 10 for paddy field⁷3.05×10¹⁰

8 soil with black grid, 7.06X 10 dry land⁷1.72×10¹¹

9 yellow soil, 1.06X 10 in paddy field⁸

42.2 with reference to Bergey's Manual of identification of bacteria, ninth edition, the active strains were identified as belonging to various genera such as Arthrobacter, Erwinia, Corynebacterium, Micromonospora, Bacillus, Acinetobacter, Rhodococcus, Alcaligenes, etc.

EXAMPLE 43 Denitrification of different soil samples

43.1 sources of respective soil samples, see Table 11.

TABLE 11 sources of respective soil samples

Sample number place classification name Source pH (Water extract)

1 south of Henan, sealing North China, dry land and 8.48 of plough layer soil

2 Jiangxi Yingtan quaternary red clay dry land, 5.43% plough layer soil

3 soil of fourth red clay old paddy field and plough layer 4.81

4 Jiangxi Yingtan quaternary red clay barren slope, surface soil 5.69

5 soil in paddy field and plough layer of evergreen Uygur soil of Jiangsu province 7.41

6.32 of topsoil in 6 Sulianyuankang yellow soil paddy field

43.2 weigh 10 g of air-dried soil into a 500 ml Erlenmeyer flask containing 100 ml of sterile distilled water and shake for 4 hours on a shaker at 90 r/min.

43.3 inoculating 10 ml of soil suspension cultured by shaking to a solution containing 0.060mol L^-1Simulated ammonium-containing wastewater of pyruvic acid (preparation)The same procedure as in 1.4) was followed, and the culture was allowed to stand at 30 ℃ for 21 days. The indophenol blue colorimetric method is used for measuring ammoniacal nitrogen, the Grignard reagent colorimetric method is used for measuring nitrite nitrogen, and the Kelvin method is used for measuring total nitrogen.

43.4 results: after the soil suspension culture is cultured for 21 days, 76.5-89.8 percent of total nitrogen in the system is removed, the ammonium nitrogen removal rate is 97.3-98.9 percent, and the nitrite concentration is only 0.02-0.73mgNL^-1。

TABLE 12 denitrification Effect of various soil samples

Method for treating red soil, peanut land, red soil, paddy field and dry land yellow soil paddy field black grid soil paddy field in red soil forest land

The ammonia nitrogen removal rate is 98.1 percent, 97.3 percent, 96.8 percent, 98.9 percent, 98.8 percent and 97.4 percent

The total nitrogen removal rate is 76.5 percent, 84.7 percent, 79.1 percent, 89.8 percent, 85.8 percent and 84.0 percent

Nitrite 0.090.070.040.730.090.02

(mgNL^-1)

Example (b): denitrogenation of 44 immobilized Bacillus megaterium NBB422 strain

44.1 preparation of immobilized Membrane of Bacillus megaterium NBB422

44.1.1 preparation of Bacillus megaterium NBB422 concentrated cell

Inoculating a ring of lawn from PM slant with a solution of 0.015mol L^-150ml of NB medium with sodium acetate as carbon source was shake-cultured at 30 ℃ for 24 hours at 120-. Inoculating the strain to a culture medium containing 0.015mol L of bacteria at a concentration of 1%^-1150 ml of NB medium with sodium acetate as carbon source was shake-cultured at 30 ℃ for 72 hours at 120-. Centrifuging at 4 deg.C for 15min at 5000r/min, washing with physiological saline twice, centrifuging, and suspending in 15ml physiological salineBrine.

44.1.2 Bacillus megaterium NBB422WR-2 fixation

Adding the concentrated thallus into 20% PVA and 1.0mol L^-1In (C) is₂The mixed solution is evenly stirred and then is spread on an organic glass plate, the organic glass plate is placed in a refrigerator, frozen overnight at the temperature of minus 20 ℃, and then unfrozen at room temperature. And repeating the freezing and thawing for 3-4 times, and fully washing with distilled water to obtain the flat plate-shaped immobilized cell membrane.

44.1.3 immobilized membrane reactor and denitrification experiment

And fixing the obtained immobilized cell membrane by using a flange and assembling the biological denitrification reactor. The amount of liquid contained in the reactor was 1600 ml at 0.015mol L^-1Simulated ammonium nitrogen wastewater with acetate as carbon source (preparation method is the same as 1.4), wherein the concentration of ammonium nitrogen is reduced to 100mgNL^-1And (3) placing the cell in a constant-temperature incubator at 28 ℃ for activation, and performing a denitrification experiment after the cell activity is stable. During the experiment, the concentration of dissolved oxygen is controlled to be 5-8 mg/L. A small amount of samples are taken at regular intervals to analyze the concentrations of ammonium nitrogen, nitrite nitrogen and nitrate nitrogen in the samples. The results showed that after 16 days of culture, the ammonium nitrogen removal was 87.7%, and the nitrite concentration was only 0.22mgNL^-1. Example 45 Combined Denitrification of Bacillus pumilus NBB-112 and Bacillus pumilus WO-8(CCTCC M202044)

45.1 the culture medium is 0.015mol L^-1The preparation method of the NB medium with sodium acetate as a carbon source is the same as 1.3.

45.2 preculture:

45.2.1A bacterial lawn containing 0.015mol L of Bacillus pumilus NBB-112 was inoculated from PM slant^-1Shaking and culturing the strain liquid in a 250ml conical flask of 50ml NB medium with sodium acetate as a carbon source for 24 hours at 30 ℃ and 120-₆₀₀＝0.45。

45.2.2 inoculating the bacterial lawn of the Bacillus pumilus WO-8 from PM slant into a medium containing 50ml NB medium (0.015mol L)^-1Sodium acetate as carbon source) in a 250ml conical flask, shaking and culturing for 24 hours at 30 ℃ and 130r/min by using a shaking table, and adjusting the OD value of the bacterial solution to OD₆₀₀＝0.45。

45.3, culturing: WO-8 (culture of 45.2.2) and NBB-112 (culture of 45.2.1) were mixed at a volume ratio of 1: 1 and inoculated at a rate of 2%1ml of mixed bacterial liquid is inoculated with 0.075mol L^-1The simulated ammonium nitrogen wastewater with acetate as a carbon source (the preparation method is the same as 1.4) is statically cultured for 21 days at the temperature of 30 ℃. The indophenol blue colorimetric method is used for measuring ammoniacal nitrogen, the Grignard reagent colorimetric method is used for measuring nitrite nitrogen, and the Kelvin method is used for measuring total nitrogen.

45.4 results: after the mixed culture is cultured for 21 days, 57.5 percent of total nitrogen in the system is removed, the removal rate of ammonium nitrogen is 72.7 percent, and the concentration of nitrite is only 0.45mgNL^-1。

Example 46 Combined Denitrification of Bacillus cereus NBB-135 and Bacillus pumilus plant WO-8(CCTCCM202044)

46.1 the culture medium is 0.015mol L^-1The preparation method of the NB medium with sodium acetate as a carbon source is the same as 1.3.

46.2 preculture:46.2.1A bacterial lawn of Bacillus cereus NBB-135 was inoculated from PM slant to a cell containing 0.015mol L^-1Shaking and culturing the strain liquid in a 250ml conical flask of 50ml NB medium with sodium acetate as a carbon source for 24 hours at 30 ℃ and 120-₆₀₀＝0.45。

46.2.2 inoculating the bacterial lawn of the Bacillus pumilus WO-8 from PM slant into a medium containing 50ml NB medium (0.015mol L)^-1Sodium acetate as carbon source) in a 250ml conical flask, shaking and culturing for 24 hours at 30 ℃ and 130r/min by using a shaking table, and adjusting the OD value of the bacterial solution to OD₆₀₀＝0.45。

46.3 culturing: WO-8(46.2.2 culture) and NBB-135(46.2.1 culture) were mixed at a ratio of 3: 7 (by volume), and 1ml of the inoculated mixed inoculum of 2% was inoculated into 0.075mol L^-1The simulated ammonium nitrogen wastewater with acetate as a carbon source (the preparation method is the same as 1.4) is statically cultured for 21 days at the temperature of 30 ℃. Theindophenol blue colorimetric method is used for measuring ammoniacal nitrogen, the Grignard reagent colorimetric method is used for measuring nitrite nitrogen, and the Kelvin method is used for measuring total nitrogen. 46.4 results: after the mixed culture is cultured for 21 days, 61.3 percent of total nitrogen in the system is removed, the removal rate of ammonium nitrogen is 75.1 percent, and the concentration of nitrite is only 0.32mgNL^-1。

Example 47 Bacillus (Bacillus cereus NBB-135, Bacillus pumilus NBB-112, Bacillus circulans NBB-295, Bacillus licheniformis NBB-072) and other nitrifying bacteria: combined denitrification effect of mixed culture of mycobacterium S-12 and chromycobacteria sulphureus S-27 and denitrifying bacteria plant bacillus pumilus WO-8(CCTCCM202044)

47.1 the culture medium is 0.015mol L^-1The preparation method of the NB medium with sodium acetate as a carbon source is the same as 1.3.

47.2 preculture:

47.2.1 cells of Bacillus cereus NBB-135, Bacillus pumilus NBB-112, Bacillus circulans NBB-295, Bacillus licheniformis NBB-072, Mycobacterium S-12 and Chromobacterium sulphureus were inoculated onto PM slant with 50ml NB medium (0.015mol L)^-1Sodium acetate as carbon source) in a 250ml conical flask, shaking and culturing for 24 hours at 30 ℃ and 130r/min by using a shaking table, and adjusting the OD value of the bacterial solution to OD₆₀₀＝0.45。

47.2.2 inoculating Bacillus pumilus WO-8(CCTCC M202044) from PM slant to the bag

Shaking and culturing 70ml PM liquid culture medium in a 250ml conical flask at 30 ℃ and 120-₆₀₀＝0.45。

47.3 culturing: a culture of Bacillus pumilus WO-8 (47.2.2 culture) and a mixed culture of nitrifying bacteria such as Bacillus cereus NBB-135 (47.2.1 culture) were mixed at a volume ratio of 1: 1, and 1ml of the mixed culture was inoculated into 0.075mol L of the mixed culture^-1The simulated ammonium nitrogen wastewater (formula 1.4) with acetate as carbon source is cultured for 21 days at 30 ℃ in a static way. The indophenol blue colorimetric method is used for measuring ammoniacal nitrogen, the Grignard reagent colorimetric method is used for measuring nitrite nitrogen, and the Kelvin method is used for measuring total nitrogen.

47.4 results: after the mixed culture is cultured for 21 days, 76.3 percent of total nitrogen in the system is removed, the removal rate of ammonium nitrogen is 87.9 percent, and the concentration of nitrite is only 0.10mgNL^-1。

List of bacteria protection

Strain number

WO-8 CCTCC M202044 Curtobacterium plantarum WO-8 plant Bacillus pumilus

WR-2 CCTCC M202043 Arthrobacter globiformis WR-2 Arthrobacter globiformis

NBB422 CGMCC NO.0554 Bacillus megaterium NBB-422 Bacillus megaterium

NBB324 CGMCC NO.0555 Bacillus firmus NBB-324 Bacillus firmus

NBB319 CGMCC NO.0556 Bacillus brevis NBB-319 Brevibacillus

NBB295 CGMCC NO.0557 Bacillus circulans NBB-295 Bacillus circulans

NBB247 CGMCC NO.0558 Bacillus coaggulans NBB-247 Bacillus coagulans

NBB204 CGMCC NO.0559 Bacillus lentus NBB-204 Bacillus lentus

NBB135 CGMCC NO.0560 Bacillus cereus NBB-135 Bacillus cereus

NBB112 CGMCC NO.0561 Bacillus pumilus NBB-112 Bacillus pumilus

NBB72 CGMCC NO.0562 Bacillus licheniformis NBB-072 Bacillus licheniformis

NBB46 CGMCC NO.0563 Bacillus globisporus NBB-046 Bacillus circulans

NBB15 CGMCC NO.0564 Bacillus sphaericus NBB-015 Bacillus sphaericus

NBB58-3 CGMCC NO.0565 Bacillus badius NBB-58-3 Bacillus badius

NBB609 CGMCC NO.0566 Bacillus subtilis NBB-609 Bacillus subtilis

NBB19 CGMCC NO.0586 Bacillus mycoides NBB-019 Bacillus mycoides

NH-2 CCTCC M203101 Bacillus pseudofiirmus NH-2 Bacillus firmus

NH-14 CCTCC M203102 Paenibacillus campinaensis NH-14 campinaensis Paenibacillus

S-12 CCTCC M203103 Arthrobacter ramosus S-12 Mycobacterium

S-27 CCTCC M203104 Arthrobacter subsufus S-27 Chromobacterium sulphureus

Claims (24)

1. A method for treating waste water containing ammonium nitrogen is characterized in that organic carbon source for physiologically producing alkali is added into the waste water containing ammonium, and proper amount of heterotrophic bacteria or heterotrophic bacteria group with nitrification activity is added, the bacteria are cultured for 15-35 days under the aerobic condition of pH 6-8 and the temperature under the static or micro-stirring state at 20-35 ℃, and ammonium nitrogen in the waste water containing ammonium is directly removed;

the heterotrophic bacteria with nitrification activity can grow or separate on a PM plate, a Grice reagent is positive when being directly dripped, and the strain has total nitrogen deficiency when being cultured in an aerobic atmosphere by adding inorganic ammonium salt into a carbon source for physiologically producing alkali.

2. The method according to claim 1, wherein theheterotrophic bacteria having nitrification activity are statically cultured on the ammonium nitrogen-containing wastewater for 28 days at a temperature of 30 ℃.

3. The method according to claim 1, wherein the heterotrophic bacteria that have nitrification activity in the ammonium-nitrogen containing wastewater are present at 10⁵-10⁶One/ml.

4. The method according to claim 1, wherein the physiologically alkali-producing organic carbon source is an organic acid having a carboxyl group or a salt thereof.

5. The method according to claim 1, wherein the heterotrophic bacterium having nitrifying activity is Bacillus megaterium NBB-422, CGMCC NO. 0554.

6. The process as claimed in claim 1, wherein the heterotrophic bacterium having nitrifying activity is Bacillus firmus NBB-324 CGMCC NO. 0555.

7. The method according to claim 1, wherein the heterotrophic bacterium having nitrifying activity is Bacillus brevis NBB-319 CGMCC NO. 0556.

8. The method according to claim 1, wherein the heterotrophic bacterium having nitrification activity is Bacillus circulans NBB-295 CGMCC NO. 0557.

9. The method according to claim 1, wherein the heterotrophic bacterium having nitrifying activity is Bacillus coagulans NBB-247 CGMCC NO. 0558.

10. The method according to claim 1, wherein the heterotrophic bacterium having nitrifying activity is Bacillus lentus NBB-204 CGMCC NO. 0559.

11. The process as claimed in claim 1, wherein the heterotrophic bacterium having nitrifying activity is Bacillus cereus NBB-135 CGMCC NO. 0560.

12. The method according to claim 1, wherein the heterotrophic bacterium having nitrifying activity is Bacillus pumilus NBB-112 CGMCC NO. 0561.

13. The process as claimed in claim 1, wherein the heterotrophic bacterium having nitrifying activity is Bacillus licheniformis NBB-72 CGMCC NO. 0562.

14. The method according to claim 1, wherein the heterotrophic bacterium having nitrifying activity is Bacillus globisporus NBB-46 CGMCC NO. 0563.

15. The method according to claim 1, wherein the heterotrophic bacterium having nitrifying activity is Bacillus sphaericus NBB-15 CGMCC No. 0564.

16. The method according to claim 1, wherein the heterotrophic bacterium having nitrifying activity is Bacillus badius NBB-58-3 CGMCC NO. 0565.

17. The process as claimed in claim 1, wherein the heterotrophic bacterium having nitrifying activity is Bacillus subtilis NBB-609 CGMCC NO. 0566.

18. The method according to claim 1, wherein the heterotrophic bacterium having nitrifying activity is Bacillus mycoides NBB-19 CGMCC NO. 0586.

19. The process according to claim 1, wherein the heterotrophic bacterium having nitrifying activity is Bacillus firmus Bacillus pseudobacteriumus NH-2 CCTCC M203101.

20. The process according to claim 1, wherein the heterotrophic bacterium having nitrifying activity is Paenibacillus canpi Paenibacillus Campinascens NH-14 CCTCC M203102.

21. The method as claimed in claim 1, wherein the heterotrophic bacterium having nitrifying activity is Arthrobacter ramosus S-12 CCTCC M203103.

22. The method according to claim 1, wherein the heterotrophic bacterium which has nitrifying activity is Arthrobacter sulphureus S-27 CCTCC M203104.

23. The method according to claim 1, wherein the heterotrophic bacteria having nitrifying activity are a mixed population of any two or more of claims 5 to 22.

24. The method according to claim 1, wherein the heterotrophic bacteria having nitrification activity are soil samples or activated sludge containing heterotrophic bacteria or mixed flora having nitrification activity that grow on PM plates, are positive for direct spot-feeding of Grignard reagent, and are deficient in total nitrogen when cultured aerobically with a physiologically alkali-producing carbon source plus an inorganic ammonium salt.