CN104195048A - Heterotrophic-nitrification fungus capable of producing mycelium pellets, and culture method and application thereof - Google Patents

Abstract

A heterotrophic nitrification and mycelium-producing fungus is Penicillium sp.L1, which is preserved in the General Microbiology Center of China Microbiological Culture Collection Management Committee; the preservation number is: CGMCC No.8615; the cultivation method of the fungus is: Penicillium sp. .L1 is inoculated in the culture medium, shaken at 20-38°C and 80-150rpm to obtain Penicillium sp.L1; this fungus is used to remove ammonia nitrogen in water by a single plant in microbial sewage containing ammonia nitrogen, and can be used in organic or Nitrogen removal is carried out under the condition of inorganic carbon source. The strain L1 can better use ammonia nitrogen as the sole nitrogen source under aerobic conditions, carry out nitrification activities, and finally convert the nitrogen form into nitrogen gas.

Description

A heterotrophic nitrifying, mycelium-producing fungus and its cultivation method and application

technical field

The present invention relates to a heterotrophic nitrification, mycelium-producing fungus and its cultivation method and application, furthermore, it is a strain of heterotrophic nitrification fungi for treating ammonia-nitrogen-containing microorganisms in waste water, its cultivation method and application . the

Background technique

Nitrogen-containing wastewater is a major problem in environmental pollution control at present, and biological denitrification is currently recognized as one of the most economical and effective methods for wastewater denitrification. The characteristic of biological denitrification technology commonly used at present is that the denitrification process is converted from ammonia nitrogen into nitrogen gas by two different types of microorganisms, autotrophic nitrifying bacteria and heterotrophic denitrifying bacteria, through the coupled metabolism of nitrification and denitrification. the

In recent decades, a variety of heterotrophic microorganisms with nitrification activity have been isolated from soil, deep-sea craters, sludge, lake water, etc., including bacteria, actinomycetes, and fungi, which are called heterotrophic nitrifiers. This is a class of microbial resources with important application value. They can use many substrates, including inorganic nitrogen and organic nitrogen, such as ammonium, amine, amide, N-alkyl hydroxylamine, oxime, hydroxamic acid and aromatic nitro compounds. the

Heterotrophic nitrifying bacteria are easy to cultivate, proliferate quickly, and have a wide range of substrate utilization. They can exist stably in wastewater biological nitrogen removal systems. Compared with the autotrophic nitrification process, the heterotrophic nitrification process can occur at a lower temperature and dissolved oxygen concentration (0.5~2.8mg/L), and has wider requirements on the water quality of the wastewater, and the C/N ratio is above 2.0. Therefore, heterotrophic nitrification denitrification has certain advantages compared with autotrophic nitrification denitrification in sewage treatment with high organic matter concentration and ammonia nitrogen concentration. Therefore, the use of heterotrophic nitrifying bacteria to develop and design a simple and simple wastewater denitrification new process can realize the rapid start-up and stable operation of the biological denitrification process, improve the denitrification efficiency, and reduce the operating cost. Efficient and economical denitrification contributes to solving the increasingly serious problem of environmental pollution caused by nitrogen-containing compounds. the

And some fungi grow in aqueous solution accompanied by stirring or shaking, and their mycelia will be entangled together to form tight balls, commonly known as mycelium balls. Mycelium balls have some superior properties that are different from other strains. Such as adsorption performance, when mixed with bacteria, the bacteria can be adsorbed on the mycelium balls, so that the bacteria are not easy to lose, thereby enhancing certain degradation properties of the bacteria; bio-sedimentation, when the environment is left still, the mycelium balls will settle At the bottom, it is easy to separate from the water body; in addition, it also has the functions of decolorization and metal ion removal. the

Contents of the invention

Aiming at the deficiencies of the above-mentioned prior art, the present invention provides a heterotrophic nitrification, mycophagium-producing fungus and its cultivation method and application. the

A heterotrophic nitrification provided by the present invention, mycelium-producing fungus, said fungus is Penicillium sp . L1, which is preserved in the General Microorganism Center of China Microbiological Culture Collection Management Committee; the preservation number is: CGMCC No. 8615.

The present invention provides a method for cultivating the heterotrophic nitrification and hyphae-producing fungi described above. The method is as follows: inoculate Penicillium sp . Cultivate; Wherein said culture medium is:

Beef extract 3 g/L, NaCl 5 g/L, peptone 10 g/L, distilled water 1000 mL, adjust the pH to 7.2-7.4, and sterilize at 121°C for 20 minutes; or

Carbon source sucrose 0.5-5 g/L, nitrogen source ammonium sulfate 0.1-2 g/L, other components K ₂ HPO ₄ 1 g/L, KCl 0.5 g/L, MgSO ₄ 7H ₂ O 0.5 g/L, FeSO ₄ 7H ₂ O 0.01g/L, H ₂ O 1000mL, adjust the pH to 7.0-7.4, and sterilize at 121°C for 20 minutes; or

Carbon source sucrose 0.5-10 g/L, aniline 0.1-2 g/L, nitrogen source ammonium sulfate 0.1-2 g/L, other components K ₂ HPO ₄ 1g/L, KCl 0.5g/L, MgSO ₄ 7H ₂ O 0.5g/L, FeSO ₄ 7H ₂ O 0.01g/L, H ₂ O 1000mL, adjust the pH to 7.0-7.4, and sterilize at 121°C for 20 minutes; or

Carbon source sucrose 0.5-10g/L, phenol 0.1-1 g/L, nitrogen source ammonium sulfate 0.1-2 g/L, other components K ₂ HPO ₄ 1g/L, KCl 0.5g/L, MgSO ₄ 7H ₂ O 0.5g/L, FeSO ₄ ·7H ₂ O 0.01g/L, H ₂ O 1000mL, adjust the pH to 7.0-7.4, and sterilize at 121°C for 20 minutes.

As above-mentioned a kind of heterotrophic nitrification, the cultivation method of producing mycelium fungus, the bacterium colony that Penicillium sp . After 3-5 days, it becomes dark green and powdery on the surface; the diameter of the colony is about 3.5-3.8cm, and the thickness is 1-2mm.

The bacterium colony that Penicillium sp . L1 presents on the solid plate culture medium in the described culture method is hyphae, and mycelium is well developed, and tool separates, and branching is arranged; The conidiophores are shorter; , varying in number, with broom-like branches.

The Penicillium sp. L1 in the culture method produces a certain number of mycelial balls in the liquid culture medium, ranging in size, with a maximum diameter of about 2.5mm, which will sink into the bottom of the bottle when left to stand, and the culture solution is transparent and clear. the

The application of the above-mentioned heterotrophic nitrification and hyphae-producing fungus provided by the present invention is to remove ammonia nitrogen in sewage; and it can use organic nitrogen source or inorganic nitrogen source for growth. the

Such as the application of a heterotrophic nitrification and mycelium-producing fungus described above, the optimal denitrification conditions for the application are sucrose as the carbon source and ammonium sulfate as the nitrogen source, the suitable C/N is 8-80, and the suitable pH is 3.0~10.0, temperature 20~40°C, shaker speed 120 rpm. the

For the application of a heterotrophic nitrification and mycelium-producing fungi mentioned above, the optimal temperature for the application is further 30° C., and the optimal initial pH value is further 7.0. the

For the above-mentioned application of a heterotrophic nitrification and mycelium-producing fungus, the optimal C/N of the application is 41. the

Description of drawings

Figure 1 is a picture of the L1 plate of the present invention. the

Fig. 2 is a picture of the L1 mycelium ball of the present invention. the

Fig. 3 is the spore picture under the microscope of the present invention. the

Detailed ways

The specific implementation manners of the present invention will be further described below. the

A strain of heterotrophic nitrification provided by the present invention, mycelium-producing fungus is isolated from the activated sludge of the wastewater pond of Taiyuan Coking Wastewater Treatment Plant, and is named as L1. Specifically, the bacterial strain is Penicillium Penicillium sp . L1, has been preserved in the General Microbiology Center of China Microbiological Culture Collection Management Committee, preservation number: CGMCC No.8615; preservation place: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; Post Code: 100101; preservation time is December 20, 2013.

The bacterial strain in the present invention is a fungus with denitrification biological activity, and a single strain can remove ammonia nitrogen in water and can remove nitrogen under the condition of organic or inorganic carbon source. During denitrification, the accumulation of nitrite and nitrate was not detected. the

The present invention is a method for cultivating the heterotrophic nitrification and mycelium-producing fungus, which is obtained by inoculating Penicillium sp . L1 in the culture medium and vibrating at 20-38°C at 80-150rpm.

Wherein said culture medium is:

A. Beef extract peptone medium: 3g beef extract, 5g NaCl, 10g peptone, 1000ml distilled water, sterilized at 121°C for 20 minutes.

B. Caspian medium (3g sucrose, 0.3g NaNO ₃ , 0.1g K ₂ HPO _{4 ,} 0.05g KCl, 0.05g MgSO ₄ 7H ₂ O, 0.001g FeSO ₄ 7H ₂ O, 100mL H ₂ O. When in use, add streptomycin solution to the melted medium to inhibit the growth of bacteria and actinomycetes, add 0.3ml of 1% streptomycin solution per 100ml medium)

C. Heterotrophic ammoniation medium: improved Chapei medium (sucrose 0.9782g, (NH ₄ ) ₂ SO ₄ 0.472g, K ₂ HPO ₄ 0.1g, KCl 0.05g, MgSO ₄ 7H ₂ O 0.05g, FeSO ₄ 7H ₂ O 0.001g, H ₂ O 100ml, adjust the pH to 7.0-7.4.)

If the above medium is made into a solid medium, add 2.0% agar.

Take the activated sludge from the wastewater tank of the coking wastewater treatment plant as a sample, take 10mL of sludge, put it into the sterilized beef extract peptone A above, and shake and enrich in a shaker at 30°C and 120r/min for three days. Then, they were inserted into the above-mentioned Chapei medium B, and cultured for three days. Take 1ml of the enriched cultured bacterial suspension in a 10ml colorimetric tube, add sterile water to the marked line, mix well, and dilute to a gradient of 10 ^-1 . Then take 1ml from gradient 10 ^-1 into a 10ml colorimetric tube, add sterile water to the marked line, mix well, dilute to gradient 10 ^-2 , and so on. Dilute to ten gradients of 10 ^-1 ~10 ^-10 , each take 0.2 ml of the diluted bacterial solution and smear it on the heterotrophic ammoniation agar plate medium. Put the coated plate into a biochemical incubator and incubate at 30° C. for three days. Select a plate with a suitable concentration gradient, and under aseptic conditions, pick each type of colony on the plate into a small test tube containing a heterotrophic ammonification liquid medium, and continue culturing in a shaker at 30°C and 120r/min.

Every 24 hours, take 0.5ml culture solution from the above-mentioned small test tube into a clean white porcelain plate, drop a drop of Nessler's reagent, Griess-Ilosvay reagent and diphenylamine reagent respectively, and use the blank culture medium without bacteria as a control. Confirm denitrification and nitrification activities. If the Nessler's reagent is added, the lighter the yellow color is, the less the remaining ammonia nitrogen content in the water sample is, that is, the better the degradation or conversion effect of the bacterial ammonia nitrogen is. If the Griess-Ilosvay reagent is dropped into red or even brown, it means that there is nitrite in the water sample, that is, nitrite is produced by the bacteria through nitrification, and the darker the color, the higher the nitrite content. Drop in the diphenylamine reagent, if it turns blue, it means that there is nitrate, that is, nitrate is produced during the nitrification process of the bacteria, and the darker the blue, the higher the nitrate content. Experimental data were recorded and preliminary qualitative analysis was carried out to confirm the heterotrophic nitrifying bacteria with better denitrification effect. After enrichment, separation and purification experiments, a batch of bacterial strains with good ammonia nitrogen removal effect and a small amount of NO _x -N can be obtained during the nitrification process. One of the strains with the best denitrification effect was selected, and the gene sequence analysis of the 26S rDNA-ITS region performed by Dalian Bao Biological Co., Ltd. identified the strain as Penicillium sp. and named it L1.

Morphological characteristics of Penicillium sp . L1 colonies (attached Figure 1): The colony characteristics of the strain L1 on solid plate medium: the colonies are round, white at the beginning, loose in texture, and dark green after 3 to 5 days. In powder form. The diameter of the colony is about 3.5-3.8cm, and the thickness is 1-2mm.

Morphological characteristics of Penicillium sp . L1 (attached figure 2): developed hyphae, septated and branched; conidiophores are short; body branch.

Morphological characteristics of Penicillium sp . L1 (attached figure 3): In the liquid culture medium, a certain number of mycelial balls are produced, ranging in size, with a maximum diameter of about 2.5mm. It is transparent and clear.

Denitrification activity of strain L1 : Strain L1 has the ability of heterotrophic denitrification, and hardly accumulates nitrate and nitrite during the denitrification process.

Optimum denitrification conditions: sucrose as carbon source, ammonium sulfate as nitrogen source, optimum C/N is 41, optimum pH is 7.0, temperature is 30°C, and shaker speed is 120 rpm. the

Strain L1 can use ammonia nitrogen as the sole nitrogen source under aerobic conditions, carry out nitrification activities, and finally convert nitrogen forms into nitrogen gas. the

The bacterial strain L1 described in the present invention is applied in the field of sewage treatment. In an environment with an ammonia nitrogen concentration of 130 mg/L, after 36 hours of growth, it can degrade ammonia nitrogen to 5.61 mg/L, and the removal rate of ammonia nitrogen is as high as 95.68%. The fastest removal rate is 13.80mg/L/h, there is almost no accumulation of nitrite nitrogen and nitrate nitrogen in the whole process.

When the ammonia nitrogen in the solution is 100mg/L, and 1000mg/L of phenol exists at the same time, after 5 days of degradation, the concentration of ammonia nitrogen in the solution remains 10.61mg/L, and the concentration of phenol remains 843.84mg/L. Courseware L1 has good tolerance to high concentration of phenol, and has a certain ability to degrade phenol. the

When the ammonia nitrogen in the solution is 100mg/L and aniline 1000mg/L is present at the same time, after 5 days of degradation, the concentration of ammonia nitrogen in the solution remains 17.94mg/L, and the concentration of aniline remains 352.56mg/L. It shows that L1 has good tolerance to high concentration of aniline, and has a good ability to degrade it. the

Sewage usually contains some toxic organic substances, and L1 has good tolerance to benzene, aniline, etc., and has a good application prospect in practical applications. the

Detection method

NH ₄ ⁺ -N: Nessler's reagent photometric method

NO ₂ ^- -N: N-(1-naphthyl)-ethylenediamine spectrophotometry

NO ₃ ^- -N: by UV spectrophotometry

TN: TOC-TN Meter

The above methods all refer to "Water and Wastewater Detection and Analysis Methods" (fourth edition) published by China Environmental Science Press.

the

Application examples are as follows:

(1) The nitrification performance of Penicillium sp . L1 was tested as follows:

Prepare simulated wastewater I, with ammonium sulfate as the only nitrogen source, with an initial concentration of 130 mg/L, a carbon-to-nitrogen mass ratio of 16, and a pH of 7.0 to 7.4, inoculate 100 mL of fresh liquid medium with an inoculum size of 1%, at 30°C , 120r/min shaking culture in the shaker. After 24 hours of cultivation, the ammonia nitrogen was degraded to 101.57mg/L; after 36 hours, the final ammonia nitrogen concentration was reduced to 5.61mg/L, the degradation rate was 95.68%, and the fastest degradation rate was as high as 13.8mg/L/h.

Simulated wastewater I: sucrose 4.891 g/L, (NH ₄ ) ₂ SO ₄ 0.472 g/L, K ₂ HPO ₄ 0.5 g/L, NaCl 0.5 g/L, MgSO ₄ 7H ₂ O 0.5 g/L, FeSO ₄ · 7H ₂ O 0. 01 g/L, adjust the pH to 7.0-7.4.

(2) Effect of carbon-nitrogen ratio on the nitrification performance of Penicillium sp . L1

 The nitrogen source concentration in the culture medium was kept constant at 130 mg/L, and the carbon source quality in the culture medium was adjusted so that the carbon-nitrogen ratios were 24, 32, 41, 48, and 56, respectively, and the rest of the conditions remained unchanged. Inoculate 100 mL of fresh liquid medium with a 2% inoculum size, and culture in a shaker at 30° C. and 120 r/min. After 32 hours, measure the remaining concentration of ammonia nitrogen in the solution. The corresponding concentrations are 63.42 mg/L, 30.57 mg/L, 14.78 mg/L, 13.77 mg/L and 12.89 mg/L, respectively. It shows that L1 has a better degradation effect on ammonia nitrogen at a higher carbon to nitrogen ratio.

(3) Simultaneous degradation of ammonia nitrogen and aniline by Penicillium sp . L1

 Add aniline to the simulated wastewater I to make the aniline concentrations 200mg/L, 400mg/L, 600mg/L, 800mg/L and 1000mg/L respectively. Inoculate with 2% inoculation amount, shake culture in a shaker at 30°C and 120r/min. After 5 days, measure the concentration of ammonia nitrogen and aniline in the solution. Ammonia nitrogen concentrations are 3.18mg/L, 12.43mg/L, 13.74mg/L, 16.55mg/L, 17.94mg/L; aniline concentrations are 149.67mg/L, 328.78mg/L, 545.18mg/L, 490.49mg /L, 352.56mg/L. It shows that L1 has a good ability to degrade ammonia nitrogen and aniline at the same time.

(4) Simultaneous degradation of ammonia nitrogen and phenol by Penicillium sp . L1

Phenol was additionally added to the simulated wastewater I to make the phenol concentration respectively 200mg/L, 600mg/L and 1000mg/L. Inoculate with 2% inoculation amount, shake culture in a shaker at 30°C and 120r/min. After 2 days, the concentrations of ammonia nitrogen in the solution remained 9.85 mg/L, 11.37 mg/L, and 10.61 mg/L respectively; after 5 days, the concentrations of phenol in the solution remained 95.27 mg/L, 391.59 mg/L, and 843.84 mg/L, respectively. It shows that L1 still maintains a good ability to degrade ammonia nitrogen even in the presence of high concentrations of phenol, and has a certain ability to degrade phenol.

sequence listing

<110> Taiyuan University of Technology

<120> A heterotrophic nitrifying fungus and its application method and use

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the

the

Claims (9)

1. A strain of heterotrophic nitrifying, mycelium-producing fungus, said fungus is Penicillium sp . L1, preserved in the General Microorganism Center of China Committee for the Collection of Microbial Cultures; the preservation number is: CGMCC No.8615. 2. A method for cultivating heterotrophic nitrification and producing mycelium fungi according to claim 1, wherein said method is: inoculating Penicillium sp . L1 in the culture medium, 20-38°C, 80-150rpm Shaking culture; wherein said culture medium is: Beef extract 3 g/L, NaCl 5 g/L, peptone 10 g/L, distilled water 1000 mL, adjust the pH to 7.2-7.4, and sterilize at 121°C for 20 minutes; or Carbon source sucrose 0.5-5 g/L, nitrogen source ammonium sulfate 0.1-2 g/L, other components K ₂ HPO ₄ 1 g/L, KCl 0.5 g/L, MgSO ₄ 7H ₂ O 0.5 g/L, FeSO ₄ 7H ₂ O 0.01g/L, H ₂ O 1000mL, adjust the pH to 7.0-7.4, and sterilize at 121°C for 20 minutes; or Carbon source sucrose 0.5-10 g/L, aniline 0.1-2 g/L, nitrogen source ammonium sulfate 0.1-2 g/L, other components K ₂ HPO ₄ 1g/L, KCl 0.5g/L, MgSO ₄ 7H ₂ O 0.5g/L, FeSO ₄ 7H ₂ O 0.01g/L, H ₂ O 1000mL, adjust the pH to 7.0-7.4, and sterilize at 121°C for 20 minutes; or Carbon source sucrose 0.5-10g/L, phenol 0.1-1 g/L, nitrogen source ammonium sulfate 0.1-2 g/L, other components K ₂ HPO ₄ 1g/L, KCl 0.5g/L, MgSO ₄ 7H ₂ O 0.5g/L, FeSO ₄ ·7H ₂ O 0.01g/L, H ₂ O 1000mL, adjust the pH to 7.0-7.4, and sterilize at 121°C for 20 minutes. 3. a kind of heterotrophic nitrification as claimed in claim 2, the cultivation method of producing mycelium fungus, the bacterium colony that its described Penicillium sp . L1 presents on the solid plate medium is circular, and the initial stage is white, The texture is loose, and after 3 to 5 days, it is dark green and the surface is powdery; the diameter of the colony is about 3.5-3.8cm, and the thickness is 1-2mm. 4. a kind of heterotrophic nitrification as claimed in claim 2, the cultivation method of producing mycelium fungus, the bacterium colony that its described penicillium Penicillium sp . Septate and branched; conidiophores are short; conidia overflow from the top of the small stalks, ranging in number, with bristle-like branches. 5. a kind of heterotrophic nitrification as claimed in claim 2, the cultivation method of producing mycelium fungi, its described Penicillium sp . L1 produces a certain number of mycelium balls in liquid medium, ranging in size, maximum The diameter is about 2.5mm, and it will sink to the bottom of the bottle when standing still, and the culture solution is transparent and clear. 6. The application of a strain of heterotrophic nitrification as claimed in claim 1, producing mycocymosis fungus, its described application is to remove ammonia nitrogen in sewage; and can utilize organic nitrogen source or inorganic nitrogen source to grow. 7. the application of a heterotrophic nitrification as claimed in claim 6, the mycelium fungus that produces, the optimal denitrification condition of its described application is that sucrose is made carbon source, and ammonium sulfate is made nitrogen source, and suitable C/N is 8~80, the suitable pH is 3.0~10.0, the temperature is 20~40°C, and the shaking table rotates at 120 rpm. 8. The application of a strain of heterotrophic nitrification and mycelium-producing fungus as claimed in claim 7, the optimum temperature for said application is further 30° C., and the optimum initial pH value is further 7.0. 9. The application of a strain of heterotrophic nitrification and mycelium-producing fungi as claimed in claim 7, the best C/N of its described application is 41.