CN113979548B

CN113979548B - Use of Arthrobacter TY2 for water treatment and microbial inoculum for water treatment

Info

Publication number: CN113979548B
Application number: CN202111320646.4A
Authority: CN
Inventors: 迟冉; 孙晨; 阮龙燕
Original assignee: SHANGHAI SHUIYUANDI CONSTRUCTION DEVELOPMENT CO LTD
Current assignee: SHANGHAI SHUIYUANDI CONSTRUCTION DEVELOPMENT CO LTD
Priority date: 2021-11-09
Filing date: 2021-11-09
Publication date: 2023-09-08
Anticipated expiration: 2041-11-09
Also published as: CN113979548A

Abstract

The application provides an application of Arthrobacter TY2 in water treatment, and the preservation number of Arthrobacter TY2 is CGMCC No1.18866. The Arthrobacter TY2 is used for water treatment, can denitrify water, remove ammonia nitrogen, nitrate nitrogen, nitrite nitrogen, total nitrogen and the like in the water, and can reduce chemical oxygen demand in the water.

Description

Use of Arthrobacter TY2 for water treatment and microbial inoculum for water treatment

Technical Field

The application relates to the technical field of microorganisms, in particular to application of Arthrobacter TY2 in water treatment and a microbial inoculum for water treatment.

Background

The river basin water resource is an important resource for human to survive, and because of population increase, rapid economic development and lag in water pollution treatment, a large amount of sewage is directly discharged into river and lake water bodies without treatment, the ecological systems of lakes and the river basin are seriously damaged, water quality pollution is caused, eutrophication is gradually serious, the water resource crisis of the river basin is increasingly aggravated, and the ecological environment of the river basin and the sustainable development of social economy are directly influenced.

At present, the river pollution treatment mode mainly adopts a physical method, a chemical method, a biological method and the like, wherein the biological method mainly recovers the diversity of aquatic organisms through a near-natural repair process, thereby realizing river treatment, and the river pollution treatment method has the advantages of low investment, low energy consumption, simple process, convenient operation and maintenance, high water outlet safety, remarkable ecological environment benefit and the like and is receiving more and more attention. For example, the artificial wetland treatment technology combines the functions of chemical, biological and physical aspects in an ecological system to achieve the function of purifying water, and microorganisms in the artificial wetland are main factors for realizing the purification of water.

Disclosure of Invention

The embodiment of the application aims to provide the application of the Arthrobacter TY2 for water treatment and the microbial inoculum for water treatment, so as to realize denitrification treatment of water and reduce the chemical oxygen demand in the water. The specific technical scheme is as follows:

the first aspect of the application provides an application of Arthrobacter TY2 in water treatment, wherein the preservation number of Arthrobacter TY2 is CGMCC No1.18866.

The second aspect of the application provides a microbial inoculum for water treatment, which comprises Arthrobacter TY2, wherein the preservation number of the Arthrobacter TY2 is CGMCC No1.18866.

Preferably, the microbial inoculum further comprises at least one of bacillus, saccharomyces, actinomycete and pseudomonas.

In a third aspect, the application provides the use of the microbial inoculum described above for water treatment.

Preferably, the water treatment comprises water treatment by adopting an artificial wetland, through a bearing device or directly adding.

Preferably, the water treatment comprises denitrification for a body of water.

More preferably, the denitrification of the water body includes removal of at least one of ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen in the water body.

Preferably, the water treatment includes reducing chemical oxygen demand in the body of water.

In a fourth aspect, the present application provides a method for water treatment using Arthrobacter TY2, said Arthrobacter TY2 being used at a concentration of 10 ⁵ cfu/L to 10 ⁸ cfu/L, wherein the preservation number of the Arthrobacter TY2 is CGMCC No1.18866.

In a fifth aspect, the present application provides a method for water treatment using the microbial inoculum described above, wherein the amount of the microbial inoculum is 1g/m, based on the Arthrobacter TY2 ³ To 20g/m ³ 。

The Arthrobacter TY2 is used for water treatment, can be used for denitrification of water, can remove ammonia nitrogen, nitrate nitrogen, nitrite nitrogen, total nitrogen and the like in the water, and can reduce Chemical Oxygen Demand (COD) in the water to realize purification of the water.

Preservation description

Strain name: TY2

Latin name: arthrobacter sp.

Classification naming: arthrobacter bacteria

Preservation number: CGMCC No1.18866

Preservation mechanism: china general microbiological culture Collection center (China Committee for culture Collection of microorganisms)

The preservation organization is abbreviated as: CGMCC

Address: beijing city, chaoyang area, north Chenli No. 1, no. 3

Preservation date: 2021, 10, 15

Drawings

In order to more clearly illustrate the embodiments of the application or the technical solutions in the prior art, the drawings used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the application, and other embodiments may be obtained according to these drawings to those skilled in the art.

FIG. 1 is a photomicrograph of Arthrobacter TY2 after gram staining;

FIG. 2 is a graph of the optical density change of water during the treatment of Arthrobacter TY2 water;

FIG. 3 is a graph showing the concentration variation of Arthrobacter TY2 for degrading ammonia nitrogen in water;

FIG. 4 is a graph showing the concentration variation of total nitrogen in water of the degradation of Arthrobacter TY 2;

FIG. 5 is a graph showing the concentration change of nitrate nitrogen in water of the Arthrobacter TY2 degradation;

FIG. 6 is a graph showing the concentration change of nitrous nitrogen in Arthrobacter TY 2-degraded water;

FIG. 7 is a graph showing the variation of chemical oxygen demand in water for the degradation of Arthrobacter TY 2;

FIG. 8 is a schematic diagram of the mechanism of denitrification of a body of water by Arthrobacter TY2.

Detailed Description

The following description of the embodiments of the present application will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present application, but not all embodiments. Based on the embodiments of the present application, all other embodiments obtained by the person skilled in the art based on the present application are included in the scope of protection of the present application.

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The Arthrobacter TY2 is preserved in China general microbiological culture Collection center (CGMCC) No. 3 of the national institute of microbiological culture Collection center (China general microbiological culture Collection center) in the North Chen Lu No. 1, korea of Beijing, 10 months and 15 days 2021, and the preservation number is CGMCC No. 1.18866. The Arthrobacter TY2 is used for water treatment, can be used for denitrification of water, can remove ammonia nitrogen, nitrate nitrogen, nitrite nitrogen, total nitrogen and the like in the water, can reduce COD in the water, and can realize purification of the water.

The method of enrichment culture and separation purification is not particularly limited as long as the purpose of the application can be achieved. For example, the enrichment culture and isolation purification steps of Arthrobacter TY2 may include, but are not limited to: adding at least one of river sludge, sewage plant biochemical pool bottom sludge or garbage treatment plant aerobic pool sludge into heterotrophic nitrification culture medium, measuring ammonia nitrogen content in the culture medium at regular intervals, and adding new culture medium into the culture medium for enrichment culture for 7-10 days if the ammonia nitrogen content is found to be reduced to 0 mg/L. Then separating and purifying by a gradient dilution method and a plate lineation method to obtain the Arthrobacter TY2. Among them, the heterotrophic nitrification medium known in the art may be used, and the present application is not particularly limited as long as the object of the present application can be achieved, for example, the heterotrophic nitrification medium may include, but is not limited to, the following: ammonium acetate, naOH, KH ₂ PO ₄ 、MgSO ₄ 、KCl、CuSO ₄ 、ZnSO ₄ And FeCl ₃ . In the present application, the plate means a culture containing a culture mediumThe dish.

The gradient dilution method may employ a gradient dilution method known in the art, for example, the gradient dilution method may include, but is not limited to, the following steps: subjecting the enriched sludge to gradient dilution, e.g. 10-10 times ⁵ Multiple times. Wherein the gradient dilution is 10 times to 10 ⁵ Double means respectively diluting 10 times, 10 ² Multiple of 10 ³ Multiple of 10 ⁴ Multiple of 10 ⁵ Multiple times.

The flat scribing method may employ a flat scribing method known in the art, for example, the flat scribing method may include, but is not limited to, the following steps: and (3) streaking and culturing the solution subjected to gradient dilution on the surface of the flat plate by using an inoculating loop, and repeating for a plurality of times until colonies growing on the surface of the flat plate are uniform.

The separated and purified Arthrobacter TY2 can be cultured in a culture medium, and then fermented and cultured under the following conditions: the culture temperature is 30-35 ℃, the culture pH is 8-9, and the aeration quantity is 1m ³ /h to 3m ³ And/h, the stirring speed is 120r/min to 250r/min. The medium for growth of Arthrobacter TY2 is not particularly limited in the present application, as long as the object of the present application can be achieved. For example, culture agents include, but are not limited to, the following: ammonium sulfate, sodium succinate, microelement solution, water and the like, wherein the mass ratio of the ammonium sulfate to the sodium succinate to the microelement solution is 1:10:100 to 1:20:150. The foregoing trace elements may include, but are not limited to, at least one of iron, iodine, zinc, selenium.

The second aspect of the application provides a microbial inoculum for water treatment, which comprises the Arthrobacter TY2 with a preservation number of CGMCC No1.18866. The microbial inoculum provided by the application can be used for denitrification of water, removal of ammonia nitrogen, nitrate nitrogen, nitrite nitrogen, total nitrogen and the like in the water, and simultaneously can reduce COD in the water, thereby realizing purification of the water.

In one embodiment of the present application, the microbial inoculum provided by the present application may further include at least one of bacillus, saccharomyces, actinomycete, and pseudomonas. Specifically, the genus bacillus may include, but is not limited to, at least one of bacillus mucilaginosus, bacillus licheniformis, bacillus subtilis, bacillus amyloliquefaciens. The genus Saccharomyces may include, but is not limited to, at least one of Saccharomyces cerevisiae, and Rhodotorula. Actinomycetes may include, but are not limited to, at least one of Streptomyces fradiae, streptomyces sulphenesulfonate, and Streptomyces kateli. The genus Pseudomonas may include, but is not limited to, at least one of rhodopseudomonas haemolytica, rhodopseudomonas globosa, rhodopseudomonas capsulata. For example, the microbial inoculum includes Arthrobacter TY2 and other bacteria, and the mass ratio of Arthrobacter TY2 to other bacteria is 1:1 to 5:1. Wherein the other genus is at least one of the genus Bacillus, genus Saccharomyces, genus Actinomyces and genus Pseudomonas.

In one embodiment of the present application, the microbial inoculum provided by the present application may further comprise a carrier, and the present application is not particularly limited in kind as long as the object of the present application can be achieved. For example, the carrier may be, but is not limited to, a solid carrier or a liquid carrier. Preferably, the solid carrier may be, but is not limited to, a mineral material, a plant material, or a polymer compound, etc., specifically, a mineral material including at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, diatomaceous earth, volcanic rock, magic frame, gravel, and ceramsite; the plant material comprises at least one of corn flour, bean flour and starch; the high molecular compound includes polyvinyl alcohol and/or polyglycol. Preferably, the liquid carrier may be, but is not limited to, vegetable oil, mineral oil, or water, among others.

In one embodiment of the present application, the microbial agents provided herein may also include surfactants, binders, stabilizers, pH adjusters, and the like. The surfactant, binder, stabilizer and pH adjuster of the present application are not particularly limited, and surfactants, binders, stabilizers and pH adjusters known in the art may be selected as long as the object of the present application can be achieved.

The method for producing the microbial inoculum is not particularly limited, and any method known in the art can be used as long as the object of the present application can be achieved. For example, the preparation method of the microbial inoculum may be, but is not limited to, liquid fermentation culture, solid fermentation culture or semisolid fermentation culture.

In particular, the arthrobacter TY2 or the microbial inoculum provided by the application can be used for denitrification of water and/or reduction of chemical oxygen demand in water during water treatment. Wherein the denitrification of the water body comprises removing at least one of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen in the water body. The form of the microbial inoculum when used in water treatment may be, but is not limited to, liquid, solid powder, granules, etc.

The form of the Arthrobacter TY2 and the microbial inoculum provided by the application for water treatment is not limited, and for example, the water treatment is carried out by adopting an artificial wetland, through a bearing device or by directly adding. The supporting device of the present application is not particularly limited as long as it can support the Arthrobacter TY2 or the microbial inoculum containing Arthrobacter TY2, and for example, it may be one of an integrated in-situ restoration device in the patent No. ZL202021371784.6, a black and odorous water body treatment device in the patent No. ZL201920100813.6, a solar power type microorganism device in the patent No. ZL201830642433.6, a microorganism activation device in the patent No. ZL201830642431.7, and the like.

In a fourth aspect, the present application provides a method for water treatment using Arthrobacter TY2, wherein Arthrobacter TY2 is used at a concentration of 10 ⁵ cfu/L to 10 ⁸ cfu/L, and the preservation number of the Arthrobacter TY2 is CGMCC No. 1.18866. When the use concentration of Arthrobacter TY2 is too small (e.g., less than 10 ⁵ cfu/L), the water treatment effect is poor; when the concentration of Arthrobacter TY2 used is too large (e.g., greater than 10 ⁸ cfu/L), the utilization rate of the Arthrobacter TY2 is low. cfu/L refers to the total number of bacterial colonies contained in each liter of sample.

In a fifth aspect, the present application provides a method for water treatment using the microbial inoculum described above, wherein the amount of the microbial inoculum is 1g/m, based on the Arthrobacter TY2 ³ To 20g/m ³ Preferably 4g/m ³ To 20g/m ³ . When the amount of Arthrobacter TY2 used is too small (e.g., less than 1g/m ³ ) The water treatment effect is poor; when the amount of Arthrobacter TY2 used is too large (e.g., more than 20g/m ³ ) The utilization rate of the microbial inoculum is low. For example, the water treatment is in the form of artificial wetland, and the use amount is expressed as Arthrobacter TY25g/m ³ . The water treatment mode is direct addition, the consumption is 5g/L by counting the Arthrobacter TY2.

Example 1

Culturing and fermenting Arthrobacter TY 2:

inoculating arthrobacter TY2 into a culture medium for fermentation culture, wherein the fermentation culture conditions are as follows: the fermentation temperature is 30 ℃ and the aeration rate is 2m ³ And/h, pH is 8-9, stirring speed is 160r/min, culturing is carried out for 48h, and the concentration of the arthrobacter TY2 in the culture solution is 10 ⁸ cfu/mL. Wherein, the culture medium of the arthrobacter TY2 is: ammonium sulfate: 0.47g; sodium succinate: 5.62g; 50mL of microelement solution; after dissolution, water was added to a constant volume of 1L. Wherein each liter of microelement solution contains 5.0g of K ₂ HPO ₄ 2.5g of MgSO ₄ ·7H ₂ O, 2.5g NaCl, 0.05g FeSO ₄ ·7H ₂ O, 0.05g MnSO ₄ ·4H ₂ O。

Identification of Arthrobacter TY 2:

1. identification of 16s rDNA sequence homology

DNA of Arthrobacter TY2 was extracted by alkaline denaturation, and PCR amplification of the 16S-strain conserved sequence was performed using 16S-universal primers (shown in Table 1) under the amplification conditions shown in Table 2. The PCR amplified product is detected for yield and specificity by 1% agarose gel electrophoresis, and is purified by a DNA purification kit, and the purified PCR product is sent to a biological engineering Co-Ltd for upstream and downstream sequencing, and the two-way result is spliced to obtain a DNA sequence. By comparison with the series on GenBank, it was determined that Arthrobacter TY2 was Arthrobacter.

TABLE 1PCR amplification primers

TABLE 2PCR cycle conditions

2. Morphological identification

The morphology of the cells of Arthrobacter TY2 in the logarithmic growth phase was observed by a light microscope after smear staining. And observe colony states in the logarithmic growth phase and stable in colony size, mainly including colony size, color, transparency, wettability, colony surface state, colony edge state, etc.

The morphology of Arthrobacter TY2 is as follows: is in the shape of irregular rod or sphere, (0.8-1.2) μm x (1.3-6.0) μm. End circles are arranged in a V shape, but no filar and no spore exists. Single, paired, irregular piled without flagella.

The morphology of the colony of Arthrobacter TY2 is as follows: the colony is round, light yellow and opaque, has smooth surface, no halo and bulge, and has a diameter of 0.8mm to 1mm.

3. Gram staining

The gram staining result of the Arthrobacter TY2 is shown in FIG. 1, and the gram staining result shows that TY2 is in purple by gram microscopic examination, and Arthrobacter TY2 is judged to be gram positive bacteria.

Example 2

Water treatment test of Arthrobacter TY 2:

the culture solution containing Arthrobacter TY2 of example 1 and a water sample were mixed at a volume ratio of 1:100, and denitrification performance test was conducted according to national standard GB11894-1989 method for determination of total Nitrogen of Water quality by alkaline Potassium persulfate digestion ultraviolet spectrophotometry, including Total Nitrogen (TN), ammonia Nitrogen (NH) ₄ -N), nitro nitrogen (NO ₃ -N) and nitrous (NO ₂ -N); COD reduction test is carried out according to national standard HJ 828-2017 'bichromate method for measuring water quality chemical oxygen demand'; the optical density (OD 600) of the water sample was measured using a spectrophotometer. Wherein, the initial total nitrogen of the water sample is 77.61mg/L, the ammonia nitrogen is 57.46mg/L, the nitrate nitrogen is 1.45mg/L, the nitrite nitrogen is 0.49mg/L, the initial COD is 201mg/L, and the initial optical density is 0.093.OD600 refers to the absorbance of a solution at a wavelength of 600 nm. The test results are shown in Table 3, and the results according to Table 3 are shown for OD600 and NH ₄ -N concentration, TN concentration, NO ₃ -N concentration, NO ₂ The N concentration and COD are plotted as a function of time as shown in figures 2 to 7.

TABLE 3 Water treatment test results for Arthrobacter TY2

As shown in FIG. 2, the delayed period of 8 hours after the passage of Arthrobacter TY2 was an exponential phase, indicating that the delayed period of Arthrobacter TY2 was short. The gradual increase of OD600 of the water body indicates that the bacterial cells are propagated and reach the maximum value of 0.801 at 32h, and the OD600 value is reduced to a certain extent along with the reaction, and the method is not limited by any theory, and can be that the Arthrobacter TY2 autolyzes and releases NH ₄ -N.

As shown in FIG. 3, the Arthrobacter TY2 pair NH in water ₄ Degradation of N is the fastest during 8-24h, with a degradation rate of 3.2mg/L. Without being limited to any theory, this may be due to the fact that part of the ammonia nitrogen is consumed by the growth of the first Arthrobacter TY2. At the same time, at 24h, NH ₄ -N degradation rate of 91.6%; at 40h, NH ₄ The degradation rate of the-N reaches 95.8 percent. With the progress of the reaction, NH for 40 to 48 hours ₄ The increase in N may be due to the formation of small amounts of ammonia nitrogen.

As shown in FIG. 4, the degradation rate of TN was 79.7% already at 24 hours. Meanwhile, as shown in fig. 5 and 6, during degradation, intermediate NO ₃ -N and NO ₂ Although a small amount of N accumulates, it is rapidly degraded, so that TN removal efficiency is substantially the same as NH ₄ -N is uniform.

As shown in FIG. 7, the COD value in the water body gradually decreases with the increase of the treatment time, and the degradation rate of the Arthrobacter TY2 on the COD does not change much with the lapse of time. At 48h, the removal rate of COD is 82.1%, which indicates that the Arthrobacter TY2 can effectively reduce the COD value in the water body.

The inventor finds that the microbial inoculum provided by the application can effectively denitrify a water body, and is not limited by any theory, and the possible reasons are as follows: nitrate in the culture solution is reduced into NO by nitrate reductase (Nar) ₂ ^- ，NO ₂ ^- Reduced to NO by nitrite reductase (Nir) and then finally by other enzymesBecomes N ₂ Escaping. Ammonia nitrogen is converted into hydroxylamine by the action of related enzymes, and hydroxylamine is further oxidized into NO by hydroxylamine oxidase (HAO) ₂ ^- ，NO ₂ ^- Then Nir is reduced to NO, and finally N is formed ₂ Escaping and denitriding is shown in figure 8. It can be seen that the nitrogen element in the water body is finally represented by N ₂ And the water body is effectively denitrified.

In combination, the Arthrobacter TY2 can be used for denitrification of water, removing ammonia nitrogen, nitrate nitrogen, nitrite nitrogen and total nitrogen in the water, and simultaneously can reduce COD in the water, thereby realizing purification of the water.

It should be noted that, in this document, the term "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a method or article that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such method or article.

In this specification, each embodiment is described in a related manner, and identical and similar parts of each embodiment are all referred to each other, and each embodiment mainly describes differences from other embodiments.

The foregoing description is only of the preferred embodiments of the present application and is not intended to limit the scope of the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application are included in the protection scope of the present application.

Claims

1. The application of the Arthrobacter TY2 in denitrification or reduction of COD in water is characterized in that the preservation number of the Arthrobacter TY2 is CGMCC No1.18866, the preservation organization of the Arthrobacter TY2 is China general microbiological culture Collection center (China Committee for culture Collection of microorganisms), and the preservation date of the Arthrobacter TY2 is 2021, 10 months and 15 days.

2. The use according to claim 1, characterized in that the Arthrobacter TY2 is used for denitrification or reduction of COD in water body by means of a carrying device or direct feeding.

3. The microbial agent for denitrification or reduction of COD in water is characterized by comprising Arthrobacter TY2, wherein the preservation number of the Arthrobacter TY2 is CGMCC No1.18866, the preservation organization of the Arthrobacter TY2 is China general microbiological culture Collection center (China Committee for culture Collection of microorganisms), and the preservation date of the Arthrobacter TY2 is 2021, 10 months and 15 days.

4. The microbial agent of claim 3, further comprising at least one of bacillus, saccharomyces, actinomycete, pseudomonas.

5. Use of the microbial agent of claim 3 or 4 for denitrification or reduction of COD in a water body.

6. The use according to claim 5, wherein the microbial inoculum is denitrified or reduced in COD in the water by means of a carrier device or direct dosing.

7. The use according to claim 1 or 5, wherein the denitrification of the body of water comprises removal of at least one of ammonia nitrogen, nitrate nitrogen, nitrite nitrogen in the body of water.

8. A method for denitrifying water body or reducing COD of water body by adopting Arthrobacter TY2 is characterized in that the use concentration of Arthrobacter TY2 is 10 ⁵ cfu/L to 10 ⁸ cfu/L, wherein the preservation number of the Arthrobacter TY2 is CGMCC No1.18866, the preservation organization of the Arthrobacter TY2 is China general microbiological culture Collection center (China Committee for culture Collection of microorganisms), and the preservation date of the Arthrobacter TY2 is 2021, 10 months and 15 days.

9. A method for denitrifying or reducing COD in water by using the microbial inoculum of claim 3 or 4, characterized in that the use amount is 1g/m based on the Arthrobacter TY2 ³ To 20 tog/m ³ 。