CN110452836B

CN110452836B - Nutrient psychrophilic bacillus for degrading ammonia nitrogen and application thereof

Info

Publication number: CN110452836B
Application number: CN201910639946.5A
Authority: CN
Inventors: 刘金波; 孙欣; 于志刚
Original assignee: Weihai Coni Lihe Environmental Protection Technology Co ltd
Current assignee: Weihai Coni Lihe Environmental Protection Technology Co ltd
Priority date: 2019-07-16
Filing date: 2019-07-16
Publication date: 2022-03-18
Anticipated expiration: 2039-07-16
Also published as: CN110452836A

Abstract

The application provides a microbial strain for degrading ammonia nitrogen, wherein the strain is a culture material bacillus psychrophilus (Psychrobacter cibarius) Z-XWW G, and the culture material bacillus psychrophilus (Psychrobacter cibarius) Z-XWW G is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No. 17515. The application also provides the application of the strain and the compound microorganism containing the strain in degrading ammonia nitrogen.

Description

Nutrient psychrophilic bacillus for degrading ammonia nitrogen and application thereof

Technical Field

The application relates to the technical field of microorganisms, in particular to a microorganism capable of degrading ammonia nitrogen and application thereof.

Background

With the continuous growth of the population and the progress of the economic level in China, the demand of people on meat and milk products is continuously increased, and the livestock breeding scale is continuously enlarged. A large amount of livestock manure is generated while the output of meat products is met, the manure can be accumulated on land under the condition of not being reasonably treated or enters the sea along with household garbage, and the released malodorous gas causes great pollution to the land and the sea environment.

Data from national environmental management platforms show that the national complaints of malodor/odor increase year by year, and the problem of malodor pollution becomes increasingly prominent. The number of malodorous compounds is large, more than 168, and H₂S and NH₃As the most important component in malodorous gases, it has become a main object of control.

The microbial deodorization technology is an environment treatment technology which is developed rapidly at present by virtue of the advantages of low price, high efficiency, environmental friendliness and the like. The microbial deodorization technology is utilized to treat the environment, so that pollutants can be converted into pollution-free stable substances, and reasonable analysis of nutrient substances can be met. In addition, the deodorizing microorganism can be used for manufacturing deodorizing materials, and can maintain a better deodorizing effect for a longer time and improve the sustainable management of the environment on the premise of reasonable use.

The marine ammonia nitrogen nitrifying bacteria not only can degrade and utilize the odorous gas on the land by virtue of high salt tolerance, but also is the first choice for treating the marine odorous pollution. The method is to screen out high-efficiency and stable ammonia nitrogen nitrifying strains from the ocean, and meets the urgent requirements of land and ocean environment treatment and sustainable development.

Disclosure of Invention

The invention provides a microbial strain capable of degrading ammonia nitrogen, which takes the ammonia nitrogen content as a detection index and evaluates the ammonia nitrogen degradation capability of 196 strains from the ocean.

In one aspect, the invention provides a microbial strain for degrading ammonia nitrogen, wherein the strain is a nutrient Bacillus Psychrobacter (Psychrobacter cibarius) Z-XWW G or Bacillus Zhangzhou (Bacillus zhangzhouensis) Z-XWW 77.

Wherein the culture material Bacillus Psychrobacter (Psychrobacter cibarius) Z-XWW G is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No. 17515.

The Zhangzhou Bacillus (Bacillus zhangzhuensis) Z-XWW 77 is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 17516.

If not specifically stated, the culture material Bacillus psychrophilus (Psychrobacter cibarius) Z-XWW G with the preservation number of CGMCC No.17515 can be replaced by a strain G; bacillus Zhangzhzhou (Bacillus zhangzhuensis) Z-XWW 77 with the preservation number of CGMCC No.17516 can be replaced by a strain 77.

On the other hand, the invention provides a compound microorganism strain for degrading ammonia nitrogen.

In one embodiment, the complex microbial strain comprises Acidovorax fastidiosa (Psychrobacter cibarius) Z-XWW G and other ammonia nitrogen degrading microorganisms; preferably, the other ammonia nitrogen degrading microorganisms are selected from Bacillus Zhangzhuensis Z-XWW 77.

In other embodiments, the complex microbial strains include Bacillus Zhangzhuensis (Bacillus zhangzhou) Z-XWW 77 and other ammonia nitrogen degrading microorganisms; preferably, the other ammonia nitrogen degrading microorganisms are selected from the group consisting of Haemophilus fastidiosa (Psychrobacter cibarius) Z-XWW G.

On the other hand, the invention provides a material for degrading ammonia nitrogen, which comprises the microbial strain. In one embodiment, the material may be a powder or a liquid formulation.

On the other hand, the invention also provides the application of the microorganism or the material in ammonia nitrogen degradation.

In another aspect, the invention also provides the use of the above-mentioned microorganism strain or the above-mentioned material for the treatment of a sample to be treated containing ammonia.

The sample to be treated containing ammonia is preferably a pollutant containing ammonia, such as sewage, wastewater, household garbage, animal waste, which may be human waste.

In another aspect, the invention also provides a method for degrading ammonia nitrogen in a sample to be treated, wherein the method comprises the step of treating the sample by using the microorganism or the material.

Further, the sample to be treated contains ammonia, preferably ammonia-containing pollutants, such as sewage, wastewater, household garbage and animal wastes, and the animal wastes can be human wastes.

In one embodiment, the sample is treated with the Bacillus psychrophilus (Psychrobacter cibarius) Z-XWW G under the temperature of 25-40 deg.C, preferably 28-37 deg.C, more preferably 32 deg.C; the pH is 6.5-8, preferably, 7-7.5, more preferably, 7.

In another embodiment, when the sample is treated by using Bacillus Zhangzhuhuanensis Z-XWW 77, the treatment condition is that the temperature is 25-45 ℃, preferably, 32-42 ℃, and more preferably, 37 ℃; the pH is 6 to 7.5, preferably 6.5 to 7, more preferably 7.

Biological material preservation instructions

Biological material (strain): Z-XWW G;

and (3) classification and naming: psychrobacter auxotropha (Psychrobacter cibarius);

the name of the depository: china general microbiological culture Collection center;

the preservation unit is abbreviated as: CGMCC;

the address of the depository: western road No.1, north chen west road, north kyo, chaoyang, institute of microbiology, china academy of sciences, zip code 100101;

the preservation date is as follows: 4 months and 2 days 2019;

the preservation number is: CGMCC No. 17515;

biological material (strain): Z-XWW 77;

and (3) classification and naming: bacillus Zhangzhuensis (Bacillus zhangzhou);

the preservation unit is abbreviated as: CGMCC;

the preservation date is as follows: 4 months and 2 days 2019;

the preservation number is: CGMCC No. 17516.

Drawings

The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the application and together with the description serve to explain the application and not to limit the application. In the drawings:

FIG. 1. Effect of temperature on Ammonia Nitrogen conversion of strains.

Figure 2 effect of pH on ammonia nitrogen conversion of strains.

FIG. 3 scanning electron microscope for strain 77 morphology.

FIG. 4 scanning electron microscope for strain G morphology.

Detailed Description

In order to more clearly explain the overall concept of the present application, the following detailed description is given by way of example in conjunction with the accompanying drawings.

So that the manner in which the above recited objects, features and advantages of the present application can be understood in detail, a more particular description of the invention, briefly summarized above, may be had by reference to the embodiments thereof which are illustrated in the appended drawings. It should be noted that the embodiments and features of the embodiments of the present application may be combined with each other without conflict.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application, however, the present application may be practiced in other ways than those described herein, and therefore the scope of the present application is not limited by the specific embodiments disclosed below.

Example one, materials and methods

1. The microbial source is as follows: 196 strains of marine microorganisms which are preserved by a laboratory of the marine microorganism resource center of Shandong province.

2. Culture medium

2216E solid Medium: 1L of old seawater, 5g of peptone, 1g of yeast extract powder, 0.01g of ferric phosphate and 20g of agar powder.

Seed liquid culture medium: 1L of old seawater, 5g of peptone, 1g of yeast extract powder and 0.01g of high-iron phosphate.

Urea culture medium: peptone 1g sodium chloride 5g, potassium dihydrogen phosphate 2g, glucose 1g, distilled water 1000ml, 0.4% phenol red 3ml, 20% urea 100ml, 20 g; the preparation method comprises sterilizing urea and other culture medium components separately, adjusting pH of other culture medium components, sterilizing at 121 deg.C for 20min in autoclave, cooling to 50 deg.C, adding urea solution filtered by filter membrane to make concentration of urea in culture medium be 2%, and pH be 7.2.

Ammonia nitrogen fermentation liquor: 10.0g of sucrose, 2.0g of ammonium sulfate, 1.0g of sodium chloride, 0.5g of monopotassium phosphate, 0.5g of dipotassium phosphate, 0.12g of magnesium sulfate and 1000ml of distilled water.

3. Ammonia nitrogen degrading bacterial strain preliminary screening test

Inoculating the activated strain on a urea culture medium in a streaking mode, culturing at the constant temperature of 28 ℃ for 48h in a biochemical incubator, setting a blank control, and observing the color change of a plate after 48 h.

The urea is decomposed into ammonia and calcium carbonate under the action of urease, so that the pH value of the culture medium is increased, and the experimental result can be judged by the color change caused by the change of the pH value of the culture medium.

4. Detection method

5 colorimetric tubes of 25ml are taken, ammonia standard use solution is absorbed according to the table 1 and the table 2 to prepare a series of standard solutions, and a sodium hypochlorite-salicylic acid spectrophotometry method and a nano-scale reagent method are respectively drawn according to corresponding steps to respectively draw standard curves.

TABLE 1 sodium hypochlorite-salicylic acid spectrophotometry ammonia standard use solution preparation table

1.0ml of salicylic acid-potassium sodium tartrate solution, 0.4ml of sodium nitrosoferricyanide and 0.4ml of sodium hypochlorite solution are respectively added into a colorimetric tube, diluted to 25ml and shaken up, and then kept stand for 1 h. Adjusting the wavelength of a spectrophotometer to be 697nm, measuring the absorbance, and drawing a standard curve.

TABLE 2 Ner reagent method ammonia standard use liquid preparation table

Adding 1.0ml of sodium potassium tartrate solution and 1.0ml of Nashi reagent into a colorimetric tube respectively, shaking up, and adding water to a constant volume of 25 ml. Adjusting the wavelength of spectrophotometer to 420nm, measuring absorbance, and drawing standard curve

5. Re-screening test of nitrobacter ammoniacal strain

Inoculating the strain obtained by primary screening with inoculating loop according to 1% inoculum size into 100ml liquid 2216E culture medium, shake culturing at 28 deg.C for 48h, and rotating speed of 150r/min. Measuring absorbance of the seed liquid at 600nm with spectrophotometer, and controlling OD₆₀₀0.5. Inoculating into 200ml ammonia nitrogen fermentation liquid with an inoculum size of 5%, and performing shake culture at 28 deg.C and 150r/min for 24h, and repeating for multiple times. And after 24h, detecting the ammonia nitrogen content in the ammonia nitrogen fermentation liquor by using a Nashiner reagent colorimetric method, analyzing the variance of the data, and establishing a histogram to show the ammonia nitrogen conversion rate of each strain.

Calculating the formula: ammonia nitrogen conversion rate (blank control ammonia nitrogen content-test group ammonia nitrogen content)/blank control group ammonia nitrogen content.

6. Optimum fermentation conditions of strain

Preparing the strain to be detected into seed liquid for later use. The strain is inoculated in ammonia nitrogen fermentation liquor to carry out temperature, pH and inoculum size tests respectively. Optimum fermentation temperature test was carried out at 28 ℃, 32 ℃, 37 ℃ and 42 ℃ under the conditions of pH 7.5 and inoculum size of 5%. The optimum fermentation pH test is that the pH of the fermentation medium is set to 6, 6.5, 7, 7.5 and 8 under the conditions that the temperature is 28 ℃ and the inoculation amount is 5 percent. In the optimum fermentation inoculation amount test, the inoculation amount is set to be 1%, 3% and 5% under the conditions that the pH value is 7.5 and the temperature is 28 ℃. The effect of the inoculum size on the ammonia nitrogen conversion was examined.

7. Identification, physiology, biochemistry and morphology of strains

16SrDNA sequence analysis, bacterial colony PCR is carried out on the strain to be detected, the DNA amplification product is sequenced and sent to Beijing Liuhe Huada Gene science and technology limited company to obtain a gene sequence, and homologous sequence comparison is carried out to construct a phylogenetic evolutionary tree. And the strains with stronger ammonia nitrogen conversion capability are preserved. Performing physiological and biochemical analysis on the marine ammonia nitrogen degrading bacteria, and performing B-galactosidase, arginine double-hydrolytic enzyme, lysine decarboxylase, ornithine decarboxylation, zinc citrate utilization, H2S generation, urease, tryptophan deaminase, indole generation, 3-hydroxy butanone generation, and carbon source oxidation physiological and biochemical performance detection on the to-be-detected strain by using an API20E kit.

And (3) observing colony morphological characteristics, namely fixing the strain by glutaraldehyde, and observing the strain by a scanning electron microscope of Qingdao university.

Example II Primary screening test results for Ammonia Nitrogen degrading strains

The 196 experimental strains to be screened are 90 strains of fish epiphytic bacteria, 54 strains of swan intestinal tract and 52 strains of sea anemone epiphytic bacteria respectively.

The preliminary screening test is divided into three grades, the plate does not change color to be grade 2, the plate is light red to be grade 1, the plate is dark red to be grade 0, and the preliminary screening test results are shown in table 3.

TABLE 3 urea plate discoloration rating

As is clear from Table 3, 23 strains having the expected effects were selected in the preliminary screening. Wherein 13 strains are screened from the epiphytic bacteria of the fishes; screening swan intestinal flora to obtain 9 swans; screening the epiphytic bacteria of sea anemone to obtain 1 strain.

The standard curve is measured by sodium hypochlorite-salicylic acid spectrophotometry: r²0.9402, the regression equation: y is 0.0812 x-0.0952; the standard curve is measured by a Nassler reagent method: r²0.9997, regression equation: y is 0.0151x + 0.006. By comparison, R of the Nr reagent method²The value is very close to 1, the fitting degree of a regression line to an observed value is better, and the sodium hypochlorite-salicylic acid spectrophotometry R is²Relatively small, the regression line generally fits to the observed values.

The upper limit of the measurement of the Nashin reagent method is 2mg/l, the upper limit of the measurement of the sodium hypochlorite-salicylic acid spectrophotometry is 10mg/l, the upper limit of the measurement of the experiment is expected to be in the range of 1.5-1.8mg/l, and the Nashin reagent method is selected to obtain higher precision; the developing time of the NanSei reagent method is 10min, and the developing time of the sodium hypochlorite-salicylic acid spectrophotometry method is 1h, so that the NanSei reagent has higher efficiency.

Example III results of re-screening test of Ammonia nitrogen degrading strains

Carrying out constant-temperature shaking culture at 28 ℃ and 150r/min for 24h, preparing 23 strain seed solutions obtained by primary screening, inoculating ammonia nitrogen fermentation liquor, measuring the conversion rate of ammonia nitrogen after 24h, and calculating the conversion rate of ammonia nitrogen, wherein the result is based on the average value +/-standard deviation.

The re-screening result shows that 20 strains with ammonia nitrogen conversion capability obtained by re-screening 23 strains obtained by preliminary screening, and 3 strains do not show ammonia nitrogen degradation capability. Among them, the strains numbered G and 77 have good effects in primary screening and secondary screening, and the conversion rate of ammonia nitrogen is more than 45%.

The optimum fermentation conditions were tested for strain 77 and strain G.

As shown in figure 1, the ammonia nitrogen conversion rate of the strain 77 is better within the temperature range of 37-42 ℃, and the ammonia nitrogen conversion rate of the strain is the highest at 37 ℃ and is 57.63%; the ammonia-nitrogen conversion rate of the strain G is good within the temperature range of 28-42 ℃, and is 51.27% when the ammonia-nitrogen conversion rate of the strain G is highest at 32 ℃; the optimum temperatures of strain 77 and strain G were 37 ℃ and 32 ℃, respectively.

As shown in fig. 2, the ammonia nitrogen conversion rate of the strain 77 was good in the Ph range of 6.5 to 7, and the highest ammonia nitrogen conversion rate of the strain was 55.09% when Ph was 7; the ammonia-nitrogen conversion rate of the strain G is good in the range of pH7-8, and reaches the highest at pH7, namely 56.78%. The optimum pH of strain 77 and strain G was 7.

The result of the inoculation amount test shows that the ammonia nitrogen degradation rate is more than 50% when the strains 77 and G are inoculated at the inoculation amount of 1% -5%. Wherein, the highest ammonia nitrogen conversion rate of the strain 77 is up to 55.93% when the inoculation amount is 3%; when the inoculation amount of the strain G is 3%, the ammonia nitrogen conversion rate is highest and can reach 55.09%. The optimum inoculum size for strain 77 and strain G was 3%.

EXAMPLE four Ammonia Nitrogen degrading Strain identification

4.1 colony feature Observation

The color, edges, and other morphological features of the colonies of strain 77 and strain G were observed and gram stained and reported in Table 4.

TABLE 4 morphological characterization of the strains

The results of electron microscope photograph observation of the strain 77 and the strain G are shown in FIGS. 3 to 4.

As can be seen from fig. 3-4: the 77 strain is rod-shaped, the cell wall is complete, the edges of the strain are clear, the whole body has flagella, and the cytoplasm density is uniform; the strain G has rod-shaped thallus, complete cell wall, fuzzy thallus edge and uniform cytoplasm density.

4.2 physiological and Biochemical assays

The physiological and biochemical performance of the strain 77 and the strain G were measured, and the results are shown in Table 5.

TABLE 5 measurement results of physiological and biochemical Properties of Strain 77 and Strain G

4.316 SrDNA sequence analysis

Extracting genome DNA of the strain 77 and the strain G, amplifying a 16SrDNA sequence by using the extracted genome DNA as a template, performing similarity analysis on the obtained 16SrDNA sequence with a GenBank database by using BLAST software, and performing multi-sequence alignment analysis on the 16SrDNA sequence and similar sequences in the GenBank by using ClustalW of Mega 6.0. By constructing a primary phylogenetic evolution tree, the strain 77 and the strain G are respectively determined to be Zhangzhou Bacillus (Bacillus zhangzhouensis) and culture material Bacillus (Psychrobacter cibarius).

The strain 77 and the strain G are respectively preserved in China general microbiological culture Collection center (CGMCC), and the preservation numbers are respectively CGMCC No.17516 and CGMCC No. 17515.

The bacterial strain 77 and the bacterial strain G have good degradation effect on ammonia nitrogen in primary screening and secondary screening, and the conversion rate of ammonia nitrogen can reach more than 45%. Through strain identification, the strain 77 is determined to be Zhangzhou Bacillus (Bacillus zhangzhuensis), and the ammonia nitrogen conversion capability of the strain is higher than that of other Bacillus in the prior art. The strain G is a food Psychrobacter (Psychrobacter cibarius), and the current reports of the Psychrobacter indicate that the strain G can secrete low-temperature lipase and is widely applied to the fields of medical treatment, pharmacy and the like, but the ammonia nitrogen conversion capability is not mentioned, and the discovery can improve the application range of the Psychrobacter.

The embodiments in the present specification are described in a progressive manner, and the same and similar parts among the embodiments are referred to each other, and each embodiment focuses on the differences from the other embodiments. In particular, for the system embodiment, since it is substantially similar to the method embodiment, the description is simple, and for the relevant points, reference may be made to the partial description of the method embodiment.

The above description is only an example of the present application and is not intended to limit the present application. Various modifications and changes may occur to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

Claims

1. A microbial strain for degrading ammonia nitrogen is a culture material bacillus cibarius Z-XWW G, and is characterized in that the culture material bacillus cibarius Z-XWW G is preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 17515.

2. A composite microorganism for degrading ammonia nitrogen, which comprises the microorganism strain of claim 1 and other ammonia nitrogen degrading microorganisms.

3. The compound microorganism as claimed in claim 2, wherein the other ammonia nitrogen degrading microorganism is Bacillus Zhangzhou (Bacillus zhangzhuensis) Z-XWW 77, and the Bacillus Zhangzhou (Bacillus zhangzhuensis) Z-XWW 77 is deposited in China general microbiological culture Collection center with the deposit number of CGMCC No. 17516.

4. A material for degrading ammonia nitrogen, the material comprising the microbial strain of claim 1 or the complex microorganism of any one of claims 2 to 3.

5. A material as claimed in claim 4, wherein the material is in a powder or liquid formulation.

6. Use of a microorganism strain according to claim 1, a complex microorganism according to any one of claims 2 to 3 or a material according to any one of claims 4 to 5 for ammonia nitrogen degradation or for the treatment of a sample to be treated containing ammonia.

7. The use according to claim 6, wherein the sample to be treated containing ammonia is a contaminant containing ammonia.

8. Use according to claim 7, wherein the ammonia-containing contaminant is sewage, wastewater, household waste or animal waste.

9. Use according to claim 8, wherein the animal faeces are human faeces.

10. A method for degrading ammonia nitrogen in a sample to be treated, comprising the step of treating the sample to be treated with the microorganism strain of claim 1, the complex microorganism of any one of claims 2 to 3, or the material of any one of claims 4 to 5.

11. The method of claim 10, wherein the sample to be treated is a sample to be treated that contains ammonia.

12. The method of claim 11, wherein the sample to be treated containing ammonia is a contaminant containing ammonia.

13. The method of claim 12, wherein the ammonia-containing contaminant is sewage, wastewater, household waste, or animal waste.

14. The method of claim 13, wherein the animal waste is human waste.

15. The method according to any one of claims 10 to 14, wherein the process conditions are as follows: the temperature is 25-40 ℃; the pH value is 6.5-8.

16. The method of claim 15, wherein the processing conditions are as follows: the temperature is 28-37 ℃; the pH is 7-7.5.

17. The method of claim 16, wherein the processing conditions are as follows: the temperature is 32 ℃; the pH was 7.