CN1210395C - Heterotrophic nitrobacteri, culturing method and application thereof - Google Patents

Abstract

The invention discloses a novel bacterium with both nitrification activity and single-plant ammonia nitrogen removal ability and a composition containing the bacterium; the bacterium can effectively remove ammonia nitrogen and protect the environment. The invention also discloses a method for cultivating the bacterium and the application of the bacterium in biological denitrification and screening nitrification inhibitors.

Description

Heterotrophic nitrifying bacteria, culture method and application thereof

Technical field:

The present invention belongs to the field of microbes, in particular, the present invention relates to a new bacterium with denitrification biological activity, a method for cultivating the bacterium, a composition containing the bacterium capable of denitrification in water, and the bacterium Application in biological denitrification and screening of nitrification inhibitors.

Background technique:

Nitrogen is an essential nutrient element for all life and an important part of the material cycle of global ecosystems. The use of nitrogen fertilizer has become the main means of high yield and increase of crops, especially food crops. However, the use of nitrogen fertilizers has brought serious environmental problems while greatly increasing crop yields: such as the continuous growth of nitrate accumulation in drinking water, eutrophication of closed and semi-enclosed waters such as lakes, and the entry of NH ₄ ⁺ -NH ₃ into water bodies Harm to fish and shellfish and other aquatic resources, and the continuous emission of N ₂ O, one of the greenhouse gases, aggravates the atmospheric greenhouse effect and damage to the ozone layer. Improving nitrogen use efficiency, reducing the loss of nitrogen fertilizer and reducing the negative impact of nitrogen on the environment have become a huge challenge for industrial and agricultural production and environmental protection. At the same time, the transformation of nitrogen form is closely related to the improvement of sewage sludge denitrification efficiency and denitrification process, and the solution of water eutrophication. At present, there are physical methods, chemical methods and biological methods to solve the nitrogen eutrophication of water bodies. With the aggravation of nitrogen pollution and people's increasing attention to environmental issues, nitrogen removal technology, especially biological nitrogen removal technology has become an important direction and means to control water pollution.

Ammonia nitrogen is one of the main nitrogen pollutants causing water eutrophication. Recently, it is generally believed that in the process of biological denitrification, ammonia nitrogen in wastewater is first oxidized to NO _x ^- by autotrophic nitrifying bacteria under aerobic conditions, and then NO _x ^- is reduced to gaseous nitrogen by denitrifying bacteria under anoxic conditions. _N2 etc. escape from the water. Nitrification and denitrification can be carried out in both activated sludge reactors and biofilm reactors, but the most practical application is the activated sludge method, where nitrifying bacteria and denitrifying bacteria are in the same activated sludge. Since the aerobic and autotrophic characteristics of the currently discovered and used nitrifying bacteria are significantly different from the anoxic and heterotrophic characteristics of the denitrifying bacteria, the denitrification process usually needs to be carried out independently in two reactors, such as the Bardenpho process, UCT (University of Capetwon) process, double-ditch oxidation ditch process, etc., or sequentially in a reactor such as SBR (Sequencing Batch Reactor, sequencing batch reactor). When the mixed sludge enters the anoxic tank or is in an anoxic state, the denitrifying bacteria work and the nitrifying bacteria are in an inhibited state; Bacteria are suppressed.

However, regardless of the traditional microbial attachment sewage treatment structures, such as biofilters, biological turntables and submerged biofilters, or some newly developed high-efficiency biofilm treatment systems, such as two-phase fluidized beds, three-phase fluidized beds, Anaerobic fluidized bed, electrode-biofilm method, etc. The nitrifying bacteria used in these methods are mostly autotrophic bacteria, the proliferation rate is slow and it is difficult to maintain a high biological concentration. It needs to be aerated to reduce the concentration of organic matter before the strain can Growth and biological activity, weak impact resistance; high concentration of ammonia nitrogen and nitrite will inhibit the growth of nitrifying bacteria, make nitrification incomplete, resulting in low removal rate of total nitrogen.

At present, some foreign researchers have studied the biological denitrification process under aerobic conditions, using the denitrification characteristics of certain microbial populations under aerobic conditions to achieve simultaneous nitrification and denitrification. The research results showed that microorganisms such as Thiosphera pantotroph, Alcaligenea faecalis, Pseadonmonas sp, and Comamonos sp. could utilize _NOx -N for denitrification. Nitrifying bacteria and denitrifying bacteria are placed in the same reactor such as an aeration tank for mixed culture, although simultaneous nitrification and denitrification of a single reactor can be achieved. But the result of denitrification is unsatisfactory and has no practical application value.

Some related research work has also been carried out in China: nitrogen removal by combination of aerobic denitrifying bacteria and autotrophic nitrifying bacteria (Geng Jinju, Liu Dengru, etc., Journal of Applied and Environmental Biology, 2002, 8(1): 78-82). Although it has a good ability to remove ammonia nitrogen, it has weak impact resistance. High concentrations of ammonia nitrogen with an ammonia nitrogen concentration higher than 0.3 g/L can inhibit the growth of bacteria, and when the ammonia nitrogen concentration is higher than 0.2 g/L, denitrification The residual amount of ammonia nitrogen is more; at the same time, it is not resistant to high organic carbon concentration, and the organic carbon concentration of 0.5 g/L inhibits the growth of bacteria and reduces the denitrification effect.

Nitrification inhibitors are specialized chemical preparations that selectively inhibit ammonia oxidizing bacteria. The development of various nitrification inhibitors is one of the effective measures to control nitrification and improve nitrogen utilization efficiency. Although there are many kinds of nitrification inhibitors at present, the actual application and widespread reports at home and abroad are mostly limited to 2-chloro-6 (trichloromethyl) pyridine (N-serve, also known as N-serve) which was screened in the early 1960s and 1970s. called nitrapyrin or cetyl CP), 4-amino-1,2,4-triazole hydrochloride (ATC), dicyandiamide (DCD), thiourea (TU), amidinothiourea (ASU), 2- Amino-4-chloro-6-methylpyrimidine (AM) and a handful of compounds; the new research isn't quite as good either. One of the main reasons for this situation is that the composition of nitrifying microorganisms is complex and the mechanism of action of nitrification inhibitors on target microorganisms is not very clear.

Existing research results show that the inhibitory concentration of nitrification inhibitors to different soils varies greatly, such as N-Serve, the concentration can vary in 0.5-20g N-Serve g ^-1 soil (Norton JM, Handbook of soil science, 2000, 160 -175). In addition to the close relationship between soil texture, pH, water content, organic matter content, temperature, etc. (Keeney DR, Ntrification inhibitors potentialsnandlimitations, 1980, 33-46), the composition of the target nitrifying microorganisms, the difference in the composition of different soil nitrifying microorganisms , especially the existence of heterotrophic nitrifying microorganisms and their activities have an important impact on the application effect of nitrification inhibitors. Experiments have shown that some nitrification inhibitors commonly used at present, such as acetylene, N-Serve, and 1-allyl-2-thiourea, cannot inhibit some heterotrophic nitrifying bacteria tested at concentrations that can inhibit autotrophic nitrifying bacteria. Nitrification of pure cultures, they require much higher inhibitory concentrations (Verstraete W. and Alexander M., J. Bacterial., 1972, 110: 955-961; Hynes RK and Knowles R., Can. J. Microbiol., 1982 , 28: 334-340). Experiments with purified ammonia monooxygenase (AMO) from heterotrophic nitrifying bacteria obtained similar results, and 1mM (equivalent to 2.5%) acetylene did not inhibit the enzyme activity of heterotrophic nitrifying microorganisms - Paracoccus denitrificans AMO , Pseudomonas putida, 5% concentration of acetylene can inhibit its oxidation of ammonia, while Nitrosomonas europeanum, 0.25μM acetylene can strongly inhibit its oxidation of ammonia, 3.8μM acetylene can completely inhibit its oxidation Enzyme activity (HynesR.K.and Knowles R., Can.J.Microbiol., 1982,28:334-340; Moir JWBet al, FEBS Lett., 387:71-74; Daum M.et al, Curr.Microbiol ., 1998, 37: 281-288).

At present, most of the screening research and effect comparison of nitrification inhibitors are carried out under soil culture; so far, autotrophic nitrification bacteria are still considered to be the main executors of soil nitrification, especially agricultural soil. Development and screening are also mainly carried out with autotrophic nitrifying bacteria as targets. In addition, from the perspective of inhibitor screening methodology, except for a few inhibitors such as ATC, sodium or potassium azide, dicyandiamide, and thiourea, a large number of test compounds are mostly organic compounds that are poorly soluble in water. When adding organic solvents or surfactants, researchers often ignore the impact of these organic substances on the composition and activity of nitrifying microorganisms.

Invention content:

One object of the present invention is to provide a new bacterium with denitrification biological activity, which has the ability to remove nitrogen from water.

Another object of the present invention is to provide a combination culture condition, so that the bacteria can exhibit stronger nitrification activity and ammonia nitrogen removal ability.

Another object of the present invention is to provide the application of the new bacteria in the biological denitrification of water body. The new bacteria can be used alone or in combination to effectively remove nitrogen from water bodies.

Another object of the present invention is to provide the application of new bacteria in screening nitrification inhibitors. The new strain can be used as a model strain to screen nitrification inhibitors.

Another object of the present invention is to provide a bacterial composition with denitrification biological activity, and the application of the bacterial composition in biological denitrification of water bodies.

The above objects can be achieved by the present invention.

A bacterium with denitrification biological activity, the bacterium has high nitrification activity and single plant ammonia nitrogen removal ability. These strains are heterotrophic nitrifying bacteria, which can effectively convert NH ₄ ⁺ into NO ₂ ^- or NO ₃ ^- through nitrification in biological denitrification, and can also directly convert NH ₄ ⁺ into gases of various nitrogen elements Such as NO, N ₂ O and N ₂ gases escape. More specifically, such bacteria are bacteria of the genus Arthrobacter sp. Arthrobacter globosa. In a specific example of the present invention, the bacterium that not only has higher nitrification activity but also has the ability to remove ammonia nitrogen in a single strain is Arthrobacter globiformis WR-2, which was released in China on November 5, 2002. The depository designated by the Patent Office—China Center for Type Culture Collection (CCTCC) deposits, and the deposit registration number is CCTCC M202043.

The present invention takes the alkaline calcareous soil of North China fluvo-aquic soil in Fengqiu, Henan Province as the test soil sample, through PM plate separation, purification, activity test, and Gliese reagent identification, a large amount of Gliess reactions are positive Heterotrophic strains, initially confirmed that they have ammonia oxidation ability. These strains belong to Arthrobacter, Erwinia, Corynebacterium, Micromonospora, Bacillus and so on.

Specifically, 8 strains of bacteria belonging to the genus Arthrobacter globosa were screened from the soil. These 8 strains of bacteria all have similar biological functions, and their nitrification activity and single ammonia nitrogen removal ability are higher than those screened from the soil. The other species of strains had high denitrification biological activity, but the 8 strains were different in morphology. The characteristics of each strain of bacteria are as follows:

(1) B173: The colonies on the PM plate are round, light yellow, and slightly raised; Gram staining is positive, without endophytic spores, bacilli of different lengths at 12 hours, and spherical cells with specific arrangements at 7 days;

(2) B256: The colonies on the PM plate are round, yellow, and slightly raised; Gram staining is positive, without endophytic spores, small straight or curved bacilli at 12 hours, some with branches, and club-shaped at 7 days cell;

(3) B459: The colony on the PM plate is round, light yellow, slightly raised; Gram staining is positive, no endophytic spores, bacillus at 12 hours, with two branches, irregular shape, cocci and cocci at 7 days ;

(4) B464: The colonies on the PM plate are round, light yellow, and slightly raised; Gram staining is positive, without endophytic spores, bacilli with branches at 12 hours, and cocci and cocci at 7 days;

(5) B602: The colonies on the PM plate are round, egg yellow, opaque, raised, and smooth; Gram staining is positive, no endophytic spores, bacilli at 12 hours, cocci, spherical or ellipsoid at 7 days;

(6) B605: The colonies on the PM plate are round, egg-yellow, raised, and smooth; Gram staining is positive, without endophytic spores, bacillus, slender and curved at 12 hours, and cocci and coccus at 7 days;

(7) WY-1: The colonies on the PM plate are round, yellow, raised, and smooth; Gram staining is positive, no endophytic spores, bacilli at 12 hours, some with branches, and cocci and cocci at 7 days;

(8) WR-2: It can form very small colonies on PM plates, round, flat, and entire; the surface is not smooth and wrinkled, and the surface of the lawn is khaki; Gram-positive; in the early stage of culture, the cells are uneven Regular rod shape, spherical shape after prolonged culture time, almost all aged cells are spherical; mostly arranged in chain form; no movement, no endospore formation.

The present inventor repeatedly detects and compares further from 8 strains of bacteria, and as a result, separates a strain of bacteria with higher nitrification activity and single ammonia nitrogen removal ability from the North China fluvo-aquic soil in Fengqiu, Henan Province, which is identified as Arthrobacter globosa ( Arthrobacter globiformis) WR-2. The Arthrobacter globosa WR-2 strain has the following characteristics:

1. Morphological characteristics of the colony: On the agar plate cultured on the PM plate, after 7 days of aerobic culture at a constant temperature of 28°C, small colonies are formed, round, flat, and entire; No obvious soluble pigment was produced.

2. Morphological characteristics of the bacteria: Gram-positive; in the early stage of culture, the cells are irregular rod-shaped, and after prolonged culture, they appear spherical, and the aged cells are almost all spherical; most of them are arranged in a chain form; they do not move, do not form Endospores.

3. The main physiological and biochemical characteristics are shown in Table 1: the optimum culture temperature is 28-32°C, pH 7.0-7.2, aerobic.

          Table 1 Physiological and biochemical characteristics of WR-2 strain

Test Item Result Result Test Item Result

Biotin for growth + + liquefied gelatin +

Thiamine - Hydrolyzed Starch +

NO ₃ →NO ₂ + D-xylose -/+

Uric acid + + melezitose +

m-Hydroxybenzoic acid + + D-glucuronate -/+

α-Keto-glutaric acid + + Cyclohexanol -

Raffinose - D-Mannose -

D-Glycine + + Sucrose -/+

Lactose -

Note: + indicates a positive reaction or can be used; - indicates a negative reaction or cannot be used.

4. Ammonia oxidation activity: With pyruvate as carbon source and ammonium sulfate as nitrogen source, the strain can grow well and nitrify at an initial pH of 5.0-9.0 and a culture temperature of 15-35°C; at 90- The concentration of nitrite accumulated in 100r/min shaker for 14 days was higher than 60.0mg NO ₂ -N L ^-1 , and some individual plants could be as high as 95mg NO ₂ -N L ^-1 or more. When the initial pH is lower than 5, the bacteria have no growth and no nitrification activity.

5. Nitrogen removal activity: with the concentration of 0.075mol L ^-1 sodium acetate as the carbon source, when C/N is 5:1, culture for 28 days, the removal rate of ammonia nitrogen is 65%, and the removal rate of total nitrogen is 62%; Pyruvate of 0.050 mol L ^-1 is the carbon source, and when the C/N ratio is 5:1, the removal rate of ammonia nitrogen is 90% and the removal rate of total nitrogen is 68% after 28 days of cultivation. The remaining total nitrogen is almost bacterial cells.

According to the classification and identification of the ninth edition of Bergey's Bacteria Identification Manual, the strain is Arthrobacter globiformis. Therefore, it was named Arthrobacter globosa WR-2. Arthrobacter globosa WR-2 was deposited on November 5, 2002 at the China Center for Type Culture Collection (CCTCC), a depository designated by the China Patent Office, with a deposit number of CCTCC M202043.

Arthrobacter globosa WR-2, which we isolated from the soil, not only has high nitrification activity in the presence of nitrogen such as ammoniacal nitrogen and/or nitrate and/or nitrite, but also can The ability of nitrogen to convert dinitrogen gas _N2 , the environment for this effect is under aerobic conditions, and under the condition of higher carbon/nitrogen ratio, the biological activity is stronger, so, for eutrophic water body , without chemical aeration treatment process.

A method for cultivating Arthrobacter globosa to exhibit stronger nitrification activity and ammonia nitrogen removal ability, the method comprising: a) pH 5.0-9.0; b) temperature 15-35°C; c) iron ion concentration measured as FeSO ₄ · 7H ₂ O is calculated as 0.005-0.02 g/L; d) Bacteria are cultivated under the condition of carbon/nitrogen ratio of 1:10-20:1. A preferred combination of methods includes a) a pH of 6.0-8.0; b) a temperature of 18-30° C.; c) an iron ion concentration of 0.01-0.02 g/L in terms of FeSO ₄ ·7H ₂ O; d) a carbon/nitrogen ratio of Bacteria are cultivated under the conditions of 1:1-10:1, and the optimal method combination includes a) pH is 7.0; b) temperature is 30°C; c) iron ion concentration is 0.01 g/L in terms of FeSO ₄ ·7H ₂ O; d) Cultivate bacteria under the condition of carbon/nitrogen ratio of 5:1.

Arthrobacter globosa WR-2 is a bacterium with both high nitrification activity and the ability to remove ammonia nitrogen in a single strain. It can show nitrification activity under normal growth conditions, and can show ammonia nitrogen removal ability when it exists alone. Under the appropriate combination culture conditions of pH value, temperature, carbon/nitrogen ratio and iron ion concentration, Arthrobacter sphaericus WR-2 can exhibit stronger nitrification activity and ammonia nitrogen removal ability.

The Arthrobacter globosa WR-2 isolated and screened from the soil in the present invention can grow and multiply in nitrogen-polluted water bodies. Through the biological reaction process in the bacteria, part of the nitrogen is converted into dinitrogen gas and discharged, and the other part Converted into bacterial substances, the residual nitrogen is within the reasonable content range of ordinary water, and the sewage treated by Arthrobacter globosa WR-2 meets the environmental protection discharge standard. The invention provides the application of Arthrobacter globosa WR-2 isolated and screened from soil in biological denitrification of water body. In a specific embodiment of the present invention, Arthrobacter globosa WR-2, CCTCC M202043 is applied to the removal of nitrogen pollutants in water bodies.

Arthrobacter globosa WR-2 is a heterotrophic aerobic microorganism, which can carry out nitrogen harmless biological treatment in water bodies, especially eutrophic water bodies, including water bodies with ammonium salts, nitrites, and organic nitrogen. The water body treated by Arthrobacter globosa WR-2 meets the qualified water standard for discharge or drinking. The harmless biological treatment of Arthrobacter globosa WR-2 is carried out under aerobic conditions, without special equipment and additional experimental conditions, and the biological harmless treatment of nitrogen in water is simultaneously nitrified in a reaction tank and denitrification process.

The invention also discloses the application of Arthrobacter globosa WR-2 in screening nitrification inhibitors. This model bacterium of Arthrobacter globiformis WR-2 can be used as the inhibitor of experimental bacteria screening for nitrification. Bacteria, found that vitamin C can effectively inhibit the occurrence of nitrification in soil, which is helpful for rational application of nitrogen fertilizer.

A bacterial composition for denitrification in water body, which contains effective amount of Arthrobacter sphaericus WR-2 and acceptable carrier in water body denitrification process. Acceptable carriers in water body denitrification process include adsorbents, nitrifying and denitrifying bacteria, etc. The adsorbent adopted in the present invention is such as 20% polyvinyl alcohol; Nitrifying bacteria such as Bacillus cereus NBB-135 (CGMCC No.0560); Denitrifying bacteria such as Brevibacterium plantum WO-8 (CCTCCM202044) etc. Bacillus cereus NBB-135, CGMCC No.0560 is preserved in the patent procedure at the General Microbiology Center of China Microbiological Culture Collection Management Committee; Brevibacterium plantarum WO-8 (CCTCC M202044) is preserved in the patent procedure at the China Type Culture Collection Center.

The invention provides the application of the bacterial composition in the biological denitrification of water body. The ammoniacal nitrogen in the water body can be effectively removed by using the bacterial composition provided by the invention.

Arthrobacter sphaericus WR-2 provided by the present invention is a strain of heterotrophic aerobic microorganisms, which not only has high nitrification activity and the ability to remove ammonia nitrogen from a single plant, but also is effective against commonly used nitrification inhibitors such as 4-amino- 1,2,4-triazole hydrochloride (ATC) has no sensitivity; the inventors used Arthrobacter globosa WR-2 as a model strain to screen nitrification inhibitors. In a specific embodiment of the present invention, we used Arthrobacter globosa WR-2 as a model bacterium, and found that vitamin C can significantly inhibit its growth and reproduction, and affect its biological activity.

In the present invention, ammonium nitrogen or ammonium nitrogen refers to soluble ammonium ion NH ₄ ⁺ -NH ₃ ; nitrogen oxide refers to NO and N ₂ O.

In the present invention, nitrification refers to the process in which microorganisms oxidize NH ₄ ⁺ to NO ₂ ^- , and then NO ₂ ^- to NO ₃ ^- , or the process in which oxidized nitrogen increases due to the action of microorganisms. It is an important part of the nitrogen cycle in nature and one of the key ways of nitrogen loss in farmland. Biological nitrification can be divided into three types: chemoautotrophic nitrification, heterotrophic nitrification and methanotrophic nitrification.

In the present invention, heterotrophic nitrification broadly refers to the process of organic and inorganic nitrogen compounds from the reduced state through biological oxidation to various oxidation states. The oxidation of nitrogen or organic nitrogen to hydroxylamine, nitrite and nitrate.

In the present invention, denitrification refers to the process in which microorganisms reduce NO ₃ ^- to NO ₂ ^- , and then NO ₂ ^- to N ₂ O, NO or N ₂ .

In the present invention, the carbon/nitrogen ratio, also known as the C/N ratio, refers to the ratio of the molar concentration of the carbon element to the molar concentration of the nitrogen element, such as C/N=1 is equivalent to ammonium sulfate 0.015mol L ^-1 : sodium acetate 0.015 mol L ^-1 .

In the present invention, denitrification refers to the removal of soluble nitrogen in water, and soluble nitrogen refers to NH ₄ ⁺ , NO ₂ ^- and NO ₃ ^- .

Acceptable carriers in the water body denitrification process in the present invention include adsorbents, nitrifying and denitrifying bacteria, and the like. For various units used in the present invention, those with national standards shall adopt national standards, and those without national standards shall adopt industry standards. A nitrite concentration of 60.0mg NO ₂ -N L ^-1 means that each liter of solution contains 60 mg of nitrite nitrogen, and a nitrate content of 0.18 mg N L ^-1 means that each liter of solution contains 0.18 mg of nitrate nitrogen.

In the present invention, Arthrobacter sphaericus WR-2 is isolated and screened from the soil, and has been preserved under a patent procedure with a preservation number of CCTCC M202043. In the present invention, Arthrobacter sphaericus WR-2 is equivalent to CCTCC M202043.

Description of drawings

Accompanying drawing 1 Morphology of Arthrobacter globosa WR-2 Figure 1: A plate colony diagram; B bacteria microphotograph

Accompanying drawing 2 is the growth curve of Arthrobacter globosa WR-2 cultured at different concentrations of sodium acetate, wherein C1, C3, C5, and C10 respectively indicate that the sodium acetate concentrations are 0.015mol L ^-1 , 0.045mol L ^-1 , and 0.075mol L ^-1 , 0.15mol L ^-1

Accompanying drawing 3 Accumulation of nitrite of Arthrobacter globosa WR-2 cultured in different concentrations of sodium acetate, wherein C1, C3, C5, and C10 respectively indicate that the concentrations of sodium acetate are 0.015mol L ^-1 , 0.045mol L ^-1 , and 0.075mol L ^-1 , 0.15mol L ^-1

Accompanying drawing 4 growth status of Arthrobacter globosa WR-2 in different concentrations of ATC, wherein the carbon source is pyruvate, and the culture temperature is static culture at 28°C

Figure 5 shows the nitrification activity of Arthrobacter globosa WR-2 in different concentrations of ATC, wherein the carbon source is pyruvate, and the culture temperature is static culture at 28°C

Below in conjunction with specific embodiment. To further set forth the present invention, it should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of protection of the present invention. Concrete experimental conditions and methods are not indicated in the following examples, usually according to conventional conditions such as: soil microbial research Edited by Hui〖日〗, translated by Ye Weiqing, etc., Science Press, 1983, Soil Microbial Experimental Method; edited by Xu Guanghui, etc., Beijing Agricultural Press, 1986, Handbook of Soil Microbial Analysis Methods; The conditions described in Science Press, 1985, Soil Microorganism Research Method, etc., or the conditions recommended by the manufacturer.

Detailed ways:

Embodiment 1. Preparation of culture medium and reagents

1.1 Preparation of PM liquid medium, also called beef extract peptone medium

Weigh 3 grams of beef extract, 5 grams of peptone, and dissolve in 1000 milliliters of distilled water to form a PM liquid medium, add 20 grams of agar to the above-mentioned non-sterilized liquid PM medium to form a PM solid medium.

Adjust the pH of the medium to 7.1 with 1 N sodium hydroxide. Divide into Erlenmeyer flasks, sterilize, and pour the plate when the culture medium is cooled to 50°C. The PM liquid medium was analyzed by high-pressure liquid chromatography, and the results showed that the contents of nitrite and nitrate were trace amounts, no nitrite was detected, and the nitrate content was 0.18mg N L ^-1 .

1.2 Preparation of Gliese reagent

1.2.1 Sulfanilic acid reagent (liquid A): Dissolve 0.5 g of sulfanilic acid in 150 ml of 20% dilute acetic acid solution, store in a brown bottle, and keep refrigerated for later use.

1.2.2 α-naphthylamine reagent (solution B): add 0.5 g of α-naphthylamine to 20 ml of distilled water and 150 ml of 20% dilute acetic acid solution, store in a brown bottle, and keep refrigerated for later use.

1.2.3 The solution used for Gliese reagent: Take equal proportions of A solution and B solution and mix it for use.

1.3 Preparation of NB liquid medium

1.3.1 Preparation of inorganic salt solution

Weigh (NH ₄ ) ₂ SO ₄ 2.1 g, NaH ₂ PO ₄ 0.25 g, K ₂ HPO ₄ 0.75 g, MgSO ₄ 7H ₂ O 0.03 g, MnSO ₄ H ₂ O 0.01 g, FeSO ₄ 7H ₂ O 0.01 g, dissolved in 1000 ml of distilled water,

Use 1M NaOH to adjust the pH of the medium to 7.0, divide it into Erlenmeyer flasks, and sterilize.

1.3.2 Preparation of organic carbon solution

1.3.2.1 Preparation of sodium acetate solution: Weigh 27.2 g of sodium acetate trihydrate and dissolve it in 1000 ml of distilled water to form a 0.20 M sodium acetate solution, and then sterilize.

1.3.2.2 Preparation of sodium formate solution: Weigh 20.8 g of sodium formate dihydrate and dissolve it in 1000 ml of distilled water to form a 0.20 M sodium formate solution, and then sterilize.

1.3.2.3 Preparation of pyruvic acid solution: Dissolve 14.0 ml of pyruvic acid in 1000 ml of distilled water to form a 0.20 M pyruvic acid solution, and then sterilize.

When in use, take 90 parts of inorganic salt solution and 10 parts of organic carbon solution according to volume ratio and mix to form NB medium.

1.4 Preparation of 4-amino-1,2,4-triazole hydrochloride (ATC): Weigh 18 grams of ATC and dissolve in 1000 ml of distilled water to form an ATC solution of 1800 mg/L, and sterilize.

1.5 Preparation of vitamin C: Weigh 2.60 grams of vitamin C and dissolve in 100 ml of distilled water to form a 2.60% vitamin C solution, and then sterilize.

1.6 Preparation of polyvinyl alcohol (PVA2000) solution: 20.0 g of PVC2000 was weighed and dissolved in 100 ml of distilled water to form a 20% PVA solution.

1.7 Preparation of calcium chloride (CaCl ₂ ) solution: 11.1 g of CaCl ₂ was weighed and dissolved in 100 ml of distilled water to form a 1.0M CaCl ₂ solution.

1.8 Preparation of polyvinyl alcohol and calcium chloride mixed solution: Take 80 parts of 20% PVA solution and 20 parts of _1.0MCaCl solution by volume ratio, and mix them evenly to obtain a mixed solution of polyvinyl alcohol and calcium chloride.

Embodiment: 2, isolate and identify the heterotrophic bacterial strain with nitrifying activity

2.1 Soil samples

Sampling location: Zhaogang Township, Fengqiu County, Henan Province; classification name: medium loamy yellow fluvo-aquic soil;

Source: plow layer soil, silt soil; Soil sample treatment: air-dried and ground through a 20-mesh sieve;

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

2.3 The soil suspension was diluted 10 times and spread on the PM plate, and each dilution was repeated three times. After culturing at 28°C for 7 days, pick a single colony onto a PM plate and streak for purification. Microscopic examination to prove the purity.

2.4 Initial screening, identification of active bacteria

The isolated and purified heterotrophic strains were inoculated on PM plates and cultured at 28°C for 10 days. Gliese reagent was directly dripped onto the plates to confirm the nitrification activity, and a plate without bacteria was used as a blank control. Within 1 minute, the Gliese reagent developed a red color, indicating that nitrite was generated. Inoculate the bacteria on the plate again, repeat the verification, the color reaction is still positive, and it is initially confirmed as nitrifying active bacteria. (See Table 2). The PM medium was analyzed by high-pressure liquid chromatography, which showed that the contents of nitrite and nitrate were both trace amounts: no nitrite was detected, and the nitrate content was less than 0.2mg N L ^-1 , which would not cause positive interference to the results .

2.5 Re-screening and establishment of model strains

Select the representative strains with strong activity in the primary screening. Scrape the pure bacterial lawn grown on the PM plate into 30 ml of sterile water to make it fully dispersed and uniform to make a bacterial suspension. Inoculate 1 ml of bacterial suspension into 250 ml Erlenmeyer flasks containing 50 ml of NB medium containing different organic substances as carbon sources, and each group has three repetitions. And the medium without inoculation was used as blank control. After static culture at 28°C for 42 days, the culture solution was centrifuged at 5000g for 15min at 4°C, and the nitrite concentration in the supernatant was determined colorimetrically by the Gliese reagent method. When the difference between the colorimetric value of the culture supernatant of the strain sample minus the colorimetric value of the blank control supernatant was greater than 0.3 mg N L ^-1 , it was judged as heterotrophic nitrifying active bacteria (see Table 3).

Table 2 Classification of some active strains

Species (genus) name Representative strain

Arthrobacter WY-1, WR-2

Arthrobacter globiformis WR-2

Erwinia WT-1, XY-3

Corynebacterium WY-19

Micromonospora (Micromonospora) WH-1

Bacillus

Bacillus brevis WY-2, WY-21

Bacillus licheniformis WX-2

Bacillus circulans WR-8

Bacillus firmus WR-9

Table 3 Colorimetric values of each strain (expressed as nitrite, NO ₂ ^- -N mg L ^-1 )

Strain number Carbon source Sodium citrate Sodium acetate

XY-3 0.4 1.0

WY-2 0.0 1.7

WY-19 0.7 0.3

WY-20 0.0 0.4

WY-21 0.7 0.8

WR-2 0.6 8.2

WT-1 0.3 1.9

WH-1 0.0 0.4

CK 0.0 0.1

2.6 Physiological identification of active strains

Refer to Bergey's Manual of Bacterial Identification, Ninth Edition. See Table 4 and Table 5 for bacterial characteristics.

Example: Identification of 3 Arthrobacter globosa WR-2

1. Morphological characteristics of the colony: after 7 days of aerobic culture at a constant temperature of 28°C on the nutrient agar PM plate, a small colony is formed.

Table 4 Physiological and biochemical characteristics of bacteria of the genus Arthrobacter

Strain number Anaerobic growth Contact enzyme Starch hydrolysis Glucose fermentation Cellulose decomposition Indole experiment Nitrate reduction

B173 - + + + + + + - - - - +

B256 - + + + + + + - - - - +

B459 - + + + + + + - - - - +

B464 - + + + + + + - - - - +

B602 - + + + + + + - - - - +

B605 - + + + + + + - - - - +

WY-1 - + + + + + + - - - - +

WR-2 - + + + + + + - - - - +

Note: + indicates a positive reaction or can be used; - indicates a negative reaction or cannot be used.

Table 5 Individual morphological characteristics of bacteria of the genus Arthrobacter

Strain number

The colonies on the B173 PM plate are round, light yellow, slightly raised; Gram staining is positive, no endophytic spores,

   At 12 hours, there are bacilli of different lengths, and at 7 days, they are spherical cells with specific arrangements

B256 The colony on the PM plate is round, yellow, slightly raised; Gram staining is positive, no endophytic spores, 12 hours

    Straight or curved small bacilli, some with branches, club-shaped cells at 7 days

B459 The colony on the PM plate is round, light yellow, slightly raised; Gram staining is positive, no endophytic spores, 12 small

  It was a bacillus with two branches and irregular shape, and it was a coccus and a coccus at 7 days

B464 The colony on the PM plate is round, light yellow, slightly raised; Gram staining is positive, no endophytic spores, 12 small

  It was bacillus with branching at 7 days, it was cocci and coccus at 7 days

The colonies on the B602 PM plate are round, egg yellow, opaque, raised, and smooth; Gram staining is positive, without inner

   Spore-forming, bacilli at 12 hours, cocci at 7 days, spherical or ellipsoidal

The colonies on the B605 PM plate are round, egg yellow, raised, smooth; Gram stain positive, no endophytic spores, 12

    At 1 hour, it is a bacillus, slender and curved, and at 7 days it is a coccus, a coccus

The colonies on the WY-1 PM plate are round, yellow, raised, and smooth; Gram staining is positive, no endophytic spores,

   12 hours for bacilli, some with branches, 7 days for cocci, cocci

WR-2 can form very small colonies on PM plates, round, flat, and entire; the surface is not smooth and wrinkled,

    The surface of the lawn is khaki; Gram-positive; in the early stage of culture, the cells are irregular rod-shaped, and the culture time is prolonged

There is a spherical shape in the space, and the aging cells are almost all spherical; they are mostly arranged in chains; they do not move, do not form

    Endospores

The colony is round, flat, and entire; the surface is rough and wrinkled, the surface of the lawn is earthy yellow, and there is no obvious soluble pigment in the matrix.

2. Morphological characteristics of the bacteria: Gram-positive; in the early stage of culture, the cells are irregular rod-shaped, and after prolonged culture, they appear spherical, and the aged cells are almost all spherical; most of them are arranged in a chain form; they do not move, do not form Endospores.

3. The main physiological and biochemical characteristics are shown in Table 1. The optimum culture temperature is 28-32°C, pH 7.0-7.2, and aerobic.

4. Ammonia oxidation activity: With ammonium sulfate as the nitrogen source, the strain can grow well and have nitrification at an initial pH of 5.0-9.0 and a culture temperature of 15-35°C; cultured on a shaker at 90-100r/min The nitrite concentration accumulated in 14 days was higher than 60.0mg NO ₂ -N L ^-1 , and some individual plants could be as high as 95mg NO ₂ -NL ^-1 or more. When the initial pH is lower than 5, the bacteria have no growth and no nitrification activity.

5. Nitrogen removal activity: when sodium acetate is used as the carbon source, when the concentration is 0.075mol L ^-1 , C/N=5:1, cultivated for 28 days, the removal rate of ammonia nitrogen is 65%, and the removal rate of total nitrogen is 62 %; When pyruvate is the carbon source, when the concentration is 0.050mol L ^-1 , C/N=5:1, cultivated for 28 days, the removal rate of ammonia nitrogen is 90%, and the removal rate of total nitrogen is 68%. The remaining total nitrogen is almost bacterial cells.

According to the classification and identification of the ninth edition of Bergey's Bacteria Identification Manual, the strain is Arthrobacter globiformis. Therefore, it was named Arthrobacter globosa WR-2. Arthrobacter globosa WR-2 was deposited on November 5, 2002 at the China Center for Type Culture Collection (CCTCC), a depository designated by the China Patent Office, with a deposit number of CCTCC M202043. The cultural characteristics and individual morphological characteristics of the WR-2 strain are shown in A and B in Fig. 1.

Embodiment: 4 cultivation and biological activity performance of Arthrobacter globosa WR-2

4.1 Scrape the pure bacterial lawn grown on the PM plate into 20 ml of sterile water to make it fully dispersed and uniform to make a bacterial suspension. Inoculate 1 ml of bacterial suspension into 250 ml Erlenmeyer flasks containing ①sodium acetate, ②malic acid, ③pyruvate, ④sodium formate, ⑤sodium citrate, and 50 ml of NB medium as carbon sources, with three repetitions in each group . And the medium without inoculation was used as blank control. After static culture at 28°C for 42 days, the culture medium was centrifuged at 5000g for 15min at 4°C, and the nitrite concentration in the supernatant was colorimetrically determined by the Gliese reagent method. The results are shown in Table 6.

Table 6 The growth and nitrification activity of pure culture of WR-2 bacteria under different carbon sources

Carbon source Cell(Log N) Net NO ₂ ^- (mg N L ^-1 )

①Sodium acetate 8.8 8.2

②Malic acid 8.7 8.0

③Pyruvate 9.1 26.1

④Sodium formate 7.9 9.2

⑤Sodium citrate 6.0 0.3

CK 6.1 0.3

Note: CK means culture without adding any organic matter as carbon source

4.2 Transfer a ring of bacterial lawn from the PM slant into a 250ml Erlenmeyer flask containing 50ml of NB medium, and cultivate for 24 hours at 28-30°C with shaking at 120-130r/min. Inoculate according to 2% amount, connect 1 milliliter of bacterial liquid to the 250 milliliter Erlenmeyer flask that is equipped with 50 milliliter medium. The initial pH value of the medium was set as: 5.0, 6.0, 7.0, 8.0, and 9.0; the culture temperature was set as: 15°C, 25°C, 30°C, 30°C, 35°C, and 37°C; the concentration of iron ions was measured by FeSO ₄ 7H ₂ O meter is set to 0 g/L, 0.005 g/L, 0.01 g/L, 0.02 g/L, 0.03 g/L; carbon/nitrogen ratios are 1:10, 1:5, 2:1 , 5:1, 10:1, 20:1. A control group without inoculation was set up and repeated 3 times. After culturing for 7 days, 14 days, and 21 days, the culture solution was taken and centrifuged at 5000 g for 15 minutes at 4° C., and the nitrite concentration in the supernatant was determined colorimetrically by the Gliese reagent method. The results are shown in Tables 7.1, 7.2, 7.3, 7.4, 7.5, and 7.6.

Table 7.1 Growth and nitrite accumulation of strains under different initial pH conditions (14 days of shaker culture)

Initial pH 5.0 6.0 7.0 8.0 9.0

_OD600 1.12 1.18 1.17 1.14 1.12

_NO2 ^- 59.51 70.53 80.16 83.98 84.54

During the experiment, the temperature was 30°C, the iron ion concentration was 0.01g L ^- , and the carbon/nitrogen ratio was 2:1. In the table NO ₂ ^- expressed in mg N L ^-1

Table 7.2 Growth and nitrite accumulation of bacterial strains at different temperatures (static culture for 21 days)

Culture temperature 15℃ 25℃ 32℃ 35℃ 37℃

_OD600 0.52 1.10 1.00 0.92 0.83

NO ₂ ^- 4.45 17.43 29.91 10.0 0.031

During the experiment, the pH was 6.5, the iron ion concentration was 0.01g L ^- , and the carbon/nitrogen ratio was 2:1. In the table NO ₂ ^- expressed in mg N L ^-1

Table 7.3 Growth and nitrite accumulation of strains at different temperatures (14 days of static culture)

Culture temperature 28℃ 30℃ 35℃

_OD600 0.98 0.98 0.87

NO ₂ ^- 3.96 17.5 14.71

During the experiment, the pH was 6.5, the iron ion concentration was 0.01g L ^- , and the carbon/nitrogen ratio was 2:1. In the table NO ₂ ^- expressed in mg N L ^-1

Table 7.4 Growth and nitrite accumulation of strains under different iron ion concentrations (14 days of shaker culture)

FeSO ₄ ·7H ₂ O (gL ^-1 ) 0 0.005 0.01 0.02 0.03

_OD600 0.50 0.87 1.11 0.94 0.90

_NO2 ^- 1.18 17.3 52.6 36.2 19.2

During the experiment, the pH was 6.5, the temperature was 30°C, and the carbon/nitrogen ratio was 2:1. In the table NO ₂ ^- expressed in mg N L ^-1

      Table 7.5 Culture status of strains under different carbon-nitrogen ratios (30°C, static culture for 28 days)

Carbon nitrogen ratio 1:10 1:5 2:1 4:1 5:1 10:1 20:1

_OD600 0.18 0.30 0.80 1.09 1.12 1.08 0.90

_NO2 ^- 0.55 0.80 10.55 26.10 5.57 2.03 1.56

_NH4 ⁺ % 92.45% 89.22% 41.82% 18.36% 10.63% 19.63% 17.50%

T _N % 94.62% 93.81% 57.22% 39.19% 32.64% 37.61% 38.50%

Note: NO ₂ ^- expressed in mg N L ^-1

Ammonium nitrogen residual percentage (NH ₄ ⁺ %) = ammonium nitrogen concentration of inoculated culture / ammonium nitrogen concentration of non-inoculated control

Total nitrogen residual percentage (T _N %) = total nitrogen of inoculated culture/total nitrogen of non-inoculated control

The culture of different concentrations of carbon sources in each group had a decrease in total nitrogen. The denitrification effect is very good when the concentration is C/N=4:1, C/N=5:1, C/N=10:1, and the best is C/N=5:1: cultivated for 28 days, The removal rate of ammonia nitrogen is 90%, and the removal rate of total nitrogen is 68%. The remaining total nitrogen is almost bacterial cells, and the accumulated nitrite is below 5.57mg N L ^-1 . C/N=4:1 and C/N=10:1 have similar removal capacity of ammonia nitrogen and total nitrogen.

The optimum temperature for nitrification of Arthrobacter globosa WR-2 strain is 30℃, the optimum iron ion concentration is 0.01g L ^- , the optimum carbon/nitrogen ratio is 5∶1, and the optimum initial pH is pH9.0. .0, the ammonia volatilizes seriously, and the initial pH of the follow-up experiment is set at pH7.0.

Embodiment: the denitrification in water body of 5 Arthrobacter globosa WR-2

5.1 The denitrification effect of different concentrations of sodium acetate as carbon source

5.1.1 The medium is NB medium, with sodium acetate as the carbon source, the concentrations are 0.015mol L ^-1 , C/N=1:1; 0.045mol L ^-1 , C/N=3:1; 0.075mol L ^-1 , C/N=5:1; 0.15mol L ^-1 , C/N=10:1. The preparation method is the same as 1.3.

5.1.2 Culture process: Pre-cultivate a ring of bacterial lawn from the PM slant and put it into a 250ml Erlenmeyer flask containing 50ml NB medium with 0.015mol L ^-1 sodium acetate as carbon source, at 30°C, 120- 130r/min shaker shaking culture for 24 hours. Inoculate according to 2% amount, and connect 1 milliliter of the pre-cultivated bacterial solution to a 250 milliliter conical flask containing 50 milliliters of NB medium with different concentrations of sodium acetate as a carbon source. Set no inoculation control, repeated 3 times. 30°C, static culture. The results are shown in Table 8, Table 9, Figure 2, and Figure 3.

Table 8 Growth and nitrite accumulation of WR-2 strain with different concentrations of sodium acetate as carbon source

(30°C, static culture for 42 days)

Carbon nitrogen ratio 1:1 3:1 5:1 10:1

_OD600 0.50±0.010 0.78±0.020 0.90±0.021 0.83±0.018

NO ₂ ^- 7.26±0.050 0.41±0.098 0.12±0.020 0.09±0.016

NH ₄ ⁺ 16.38±5.37 5.62±0.22 4.59±1.34

Total Nitrogen 89.81±1.99

Note: C ₀ is expressed in mol L ^-1 ; total nitrogen, NH ₄ ⁺ , NO ₂ ^- are expressed in mg N L ^-1 ; data representation: mean ± standard error n=3.

             Table 9 Culture conditions when different concentrations of sodium acetate were used as carbon sources (30°C, static culture)

Carbon nitrogen ratio 1:1 3:1 5:1

28 days 42 days 28 days 48 days 28 days 38 days

NO ₂ ^- 1.19±0.29 4.15±1.33 0.074±0.021 0.41±0.19 0.094±0.010 0.090±0.016

_NH4 ⁺ % - 42.08±6.16% 37.4±1.90% - 34.5±1.84% 9.38±0.78%

T _N % 70.9±1.24% 54.7±3.03% 45.0±2.97% 23.6±1.85% 37.7±1.20% 22.3±1.82%

Note: NO ₂ ^- expressed in mg N L ^-1 .

Ammonium nitrogen residual percentage (NH ₄ ⁺ %) = ammonium nitrogen concentration of inoculated culture / ammonium nitrogen concentration of non-inoculated control

Total nitrogen residual percentage (T _N %) = total nitrogen of inoculated culture/total nitrogen of non-inoculated control

Data representation: mean ± standard error n = 3.

Arthrobacter globosa WR-2 has a good ability to remove ammonia nitrogen, and the measured nitrite concentration in the biological nitrogen removal process is only 0.1mg N L ^-1 , without nitrite accumulation. Among them, when the concentration of sodium acetate is 0.075mol L ^-1 , C/N is 5:1 and 0.15mol L ^-1 , C/N is 10:1, the ammonia nitrogen removal ability is strong, and the concentration of sodium acetate is 5:1. 0.045mol L ^-1 , followed by C/N ratio of 3:1.

Embodiment: 6 WR-2 and nitrifying bacteria bacillus cereus NBB-135, the mixed culture of CGMCC No.0560

6.1 The medium is NB medium with 0.015mol L ^-1 sodium acetate as the carbon source, and the preparation method is the same as 1.3.

6.2 Pre-cultivation:

6.2.1 Connect a ring of Bacillus cereus NBB-135, CGMCC No.0560 bacterial lawn from the PM slant into a 250 ml Erlenmeyer flask containing 50 ml of NB medium with 0.015 mol L ^-1 sodium acetate as carbon source In 30° C., 120-130 r/min shaker shake culture for 24 hours, the OD value of the bacterium was OD ₆₀₀ =0.40.

6.2.2 Pick up a lawn of Arthrobacter globularis WR-2 from the PM slant and put it into a 250ml Erlenmeyer flask containing 50ml NB medium with 0.015mol L ^-1 sodium acetate as carbon source, at 30°C, Shaking culture at 120-130 r/min for 24 hours, the OD value of the adjusted bacteria was OD ₆₀₀ =0.40.

6.3 Cultivation: NBB-135 (culture in 6.2.1) and WR-2 bacteria (culture in 6.2.2) were mixed at a ratio of 1:1 (volume ratio), and then inoculated with 1 ml of 2% inoculum mixture. Add 0.075 mol L ^-1 acetate as carbon source to the NB medium (the formula is the same as 1.3) at 30°C and culture it statically for 28 days. Ammonia nitrogen was measured by indophenol blue colorimetry, nitrite nitrogen by Gliese reagent colorimetry, and total nitrogen by Kelvin method.

6.4 Results: After 28 days of culture, the mixed culture removed 62.3% of the total nitrogen in the system, the ammonium nitrogen removal rate was 78.1%, and the nitrite concentration was only 0.52mg N L ^-1 .

Embodiment: the mixed culture of 7 WR-2 and denitrifying bacteria Brevibacterium plantarum WO-8, CCTCC M202044

7.1 The medium is NB medium with 0.015mol L ^-1 sodium acetate as the carbon source, and the preparation method is the same as 1.3.

7.2 Cultivation:

7.2.1 Connect a ring of Brevibacterium plantarum WO-8, CCTCC M202044 bacterial lawn from the PM slant into a 250 ml Erlenmeyer flask containing 50 ml of NB medium with 0.015 mol L ^-1 sodium acetate as carbon source. 30° C., 120-130 r/min shaker culture for 24 hours, the OD value of the adjusted bacteria was OD ₆₀₀ =0.40.

7.2.2 Pick up a lawn of Arthrobacter globularis WR-2 from the PM slant and put it into a 250ml Erlenmeyer flask containing 50ml NB medium with 0.015mol L ^-1 sodium acetate as carbon source, at 30°C, Shaking culture at 120-130 r/min for 24 hours, the OD value of the adjusted bacteria was OD ₆₀₀ =0.40.

7.2.3 WO-8 (culture in 7.2.1) and WR-2 bacteria (culture in 7.2.2) were mixed at a ratio of 1:2 (volume ratio), and 1 ml of the mixed bacteria liquid was inoculated with 2% inoculum. Add 0.075 mol L ^-1 acetate as carbon source to NB culture medium (the formula is the same as 1.3), and culture at 30°C for 28 days. Ammonia nitrogen was determined by indophenol blue colorimetric method, nitrite nitrogen was determined by Gliese reagent colorimetric method, and total nitrogen was determined by Kelvin method.

7.3 Results: After 28 days of cultivation, the mixed culture removed 68.2% of the total nitrogen in the system, the ammonium nitrogen removal rate was 79.7%, and the nitrite concentration was only 0.32mg N L ^-1 .

Example: 8 Denitrification of immobilized Arthrobacter globosa WR-2

8.1 Preparation of immobilized membrane of Arthrobacter globosa WR-2

8.1.1 Preparation of concentrated cells of Arthrobacter globosa WR-2

Put a ring of bacterial lawn from the PM slant into a 250ml Erlenmeyer flask containing 50ml of NB medium with 0.015mol L ^-1 sodium acetate as the carbon source, and culture at 30°C on a shaking table at 120-130r/min for 24 Hour. Inoculate according to 1% amount, connect the bacterial solution to a 500 ml Erlenmeyer flask containing 150 ml of NB medium with 0.015 mol L ^-1 sodium acetate as carbon source, and culture at 30°C, 120-130r/min shaker for 72 Hour. Centrifuge at 5000r/min at 4°C for 15 minutes, wash and centrifuge twice with saline, and suspend in 15ml of saline.

8.1.2 Immobilization of Arthrobacter globosa WR-2

Add the concentrated bacteria to the mixed solution of 20% PVA and 1.0mol L ^-1 CaCl ₂ , stir well and spread it on a plexiglass plate, put it in the refrigerator, freeze overnight at -20°C, and then thaw at room temperature . Repeat freezing and thawing for 3-4 times, and wash thoroughly with distilled water to obtain flat immobilized cell membranes.

8.1.3 Immobilized membrane reactor and nitrogen removal experiment

The resulting immobilized cell membrane was fixed with a flange and assembled into a biological denitrification reactor. The volume of the reactor is 1600 ml, and the NB medium with 0.015 mol L ^-1 acetate as the carbon source, in which the concentration of ammonium nitrogen is reduced to 100 mg N L ^-1 , is placed in a constant temperature incubator at 28°C for activation. After the cell activity was stabilized, the denitrification experiment was carried out. During the experiment, the dissolved oxygen concentration was controlled at 5-8 mg/L. Take a small amount of samples at regular intervals to analyze the concentration of ammonium nitrogen, nitrite nitrogen and nitrate nitrogen. The results showed that after 16 days of cultivation, the removal rate of ammonium nitrogen was 92.7%, and the concentration of nitrite was only 0.22mg N L ^-1 .

Example: Arthrobacter globosa WR-2 was used as a model strain to screen nitrification inhibitors.

9.1 Effects of different concentrations of ATC on the growth and nitrification activity of WR-2 bacteria

9.1.1 Connect a ring of bacterial lawn from the PM slope into a 250ml Erlenmeyer flask containing 50ml of NB medium with 0.020mol L ^-1 pyruvate as the carbon source, and place it on a shaker at 30°C and 120-130r/min Incubate with shaking for 24 hours. Inoculate the bacterial solution according to 2% amount into a 250 ml Erlenmeyer flask containing 50 ml of NB medium. Set no inoculation control, repeated 3 times. ATC set 4 concentration gradients as 0, 15mg L ^-1 , 75mg L ^-1 , 150mg L ^-1 respectively. ATC and carbon source are sterilized separately and mixed before use. At 30°C, culture statically.

9.1.2 The results are shown in Figure 4 and Figure 5. The results showed that ATC had no inhibitory effect on the growth and nitrification activity of the heterotrophic nitrification active strain WR-2 within the concentration range of 0-75 mg L ^-1 . At a high concentration of 150mg L ^-1 , ATC inhibited the growth and nitrification activity of the strain.

8.2 The effect of vitamin C on the growth and nitrification activity of WR-2 bacteria

8.2.1 Connect a ring of bacterial lawn from the PM slant into a 250ml Erlenmeyer flask containing 50ml NB medium with 0.015mol L ^-1 sodium acetate as the carbon source, at 30°C, 120-130r/min shaker Incubate with shaking for 24 hours. Inoculate according to 2% amount, and connect the bacterial solution to a 250 ml Erlenmeyer flask containing 50 ml of NB medium. Set no inoculation control, repeated 3 times. The concentration of vitamin C is 2.17 g/L; vitamin C and carbon source are sterilized separately and mixed before use. 30°C, static culture.

9.2.2 The results are shown in Table 11:

Table 11. Growth and nitrite accumulation of strains when Vc was added and cultured without Vc

  (30°C, static culture for 21 days)

Measurement items Add Vc Control (without Vc)

_OD600 0.020 0.51

_NO2 ^- 0.12 7.10

Note: NO ₂ ^- expressed in mg N L ^-1

The strain had no growth and no nitrification activity, indicating that the Vc concentration of 2.17 g/L could strongly inhibit the growth and nitrification activity of the strain.

                           

Strain number

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

WO-8 CCTCC M202044 Curtoacterium plantarum WO-8 Brevibacterium plantarum

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

Claims (8)

1. A bacterium with denitrification biological activity, characterized in that the bacterium is Arthrobacter globiformis WR-2, and its preservation registration number is CCTCC M202043. 2. A method for cultivating bacteria described in claim 1, the method is 5.0-9.0 at a) pH; b) temperature is 15-35 ° C; c) iron ion concentration is calculated as FeSO ₄ .7H ₂ O 0.005-0.02 g/L; d) Cultivate bacteria under the condition of carbon/nitrogen ratio of 1:10-20:1. 3. The method according to claim 2, wherein a) the pH is 6.0-8.0; b) the temperature is 20-35° C.; c) the iron ion concentration is 0.01-0.02 grams in terms of FeSO ₄ .7H ₂ O / liter; d) Cultivate bacteria under the condition of carbon/nitrogen ratio of 1:1-10:1. 4. The method according to claim 3, wherein a) pH is 7.0; b) temperature is 30° C.; c) iron ion concentration is 0.01 g/liter in terms of FeSO ₄ .7H ₂ O; d) carbon Bacteria were cultured at a nitrogen/nitrogen ratio of 5:1. 5. A bacterial composition for denitrification in water bodies, containing an effective amount of a bacterium as claimed in claim 1 and an acceptable carrier in water body biological denitrification. 6. the application of the bacterium described in a kind of claim 1 in water body biological denitrification. 7. The application of the bacterial composition in claim 5 in the biological denitrification of water bodies. 8. the application of bacterium described in a kind of claim 1 in screening nitrification inhibitor.

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