CN112608857A

CN112608857A - Salt-tolerant bacillus pumilus SZG-NY-001 capable of repairing polluted water and application

Info

Publication number: CN112608857A
Application number: CN202011449747.7A
Authority: CN
Inventors: 荆峰洋; 吴定心; 朱海; 孟希
Original assignee: Wuhan Shuizhiguo Environmental Protection Technology Co ltd
Current assignee: Wuhan Shuizhiguo Environmental Protection Technology Co ltd
Priority date: 2020-12-09
Filing date: 2020-12-09
Publication date: 2021-04-06
Anticipated expiration: 2040-12-09
Also published as: CN112608857B

Abstract

The invention belongs to the technical field of pollution treatment, and particularly discloses a salt-tolerant bacillus pumilus SZG-NY-001 capable of repairing polluted water and an application thereof, wherein the preservation number of the strain is CCTCC NO: m2020550. The strain can be applied to effectively removing COD in salt-containing wastewater with the salt content of 1.0-16.0%. The method is not influenced by other factors in the wastewater, for example, the TN, ammonia nitrogen and TP in the wastewater have almost no influence on the COD degradation efficiency; and is particularly suitable for treating COD in mixed wastewater containing p-hydroxybenzaldehyde and tosyl chloride.

Description

Salt-tolerant bacillus pumilus SZG-NY-001 capable of repairing polluted water and application

Technical Field

The invention relates to the technical field of pollution treatment, in particular to salt-tolerant bacillus pumilus SZG-NY-001 capable of repairing polluted water and application thereof.

Background

The high-salinity wastewater refers to wastewater with the total salt content of more than or equal to 1 percent by mass. This type of wastewater contains, in addition to a large amount of inorganic salts, organic matter and at least 3.5% of total dissolved solids. The high-salt domestic wastewater and the high-salt industrial wastewater comprise two main sources: firstly, seawater is directly used in daily life and industrial production, such as the seawater is used for flushing toilets, roads, industrial cooling water and the like, so that a large amount of inorganic salt is also contained in discharged wastewater; secondly, a large amount of high-concentration inorganic salt wastewater is discharged in industries such as marine product, dairy product processing, meat processing, printing and dyeing, papermaking, chemical industry, pharmacy, petroleum, fermentation and the like. The existence of the salts has obvious inhibiting effect on the conventional biological treatment, and special microorganisms which can still degrade organic pollutants in a high-salt environment, namely, the salt-tolerant microorganisms have high competitive advantage of biological wastewater treatment and can achieve the aim of restoring the polluted water body.

Bacillus species are capable of degrading a variety of organic substances, exhibiting a wide variety of physiological capabilities, ranging from psychrophilic to thermophilic, acidophilic to alkalophilic, some strains are salt tolerant, some are halophilic, and bacillus has great potential in the biological treatment of wastewater because of its wide distribution and diverse stress tolerance. For example, patent CN111662848A discloses a method for culturing salt-tolerant bacillus licheniformis and its application, which mainly aims at the synthesis and utilization of residual glutamic acid in wastewater under high-salt environment, and solves the problem of high sugar content in glutamic acid wastewater. However, the action system of the strain is single, only aiming at glutamic acid waste liquid, and the effect on the remediation of other types of high COD waste water is not mentioned.

Most of the disclosed salt tolerant microorganisms are generally tolerant to salinity of 10% and less. For example, CN106635858A discloses a paracoccus and the culture application thereof, the salt tolerance range is 1.0 wt% -8.0 wt%; CN106190875A discloses a halotolerant strain in a high-salt tolerance environment, a screening method and application thereof, wherein the salt tolerance of the halotolerant strain is that the NaCl mass concentration is 8%; CN106635860A discloses a salt-tolerant microbial agent and a preparation method and application thereof, wherein the salt tolerance of paracoccus, northern microrod and Pseudomonas stutzeri is 1 wt% -10 wt%.

Disclosure of Invention

The invention aims to solve the problems of great difficulty in the existing high-salt polluted water body restoration technology and low efficiency of a common bioremediation method in the environment, and provides a salt-tolerant Bacillus pumilus (Bacillus pumilus) SZG-NY-001 capable of restoring polluted water bodies, wherein the preservation number of the strain is CCTCC NO: M2020550.

The invention also aims to provide the application of the bacillus pumilus SZG-NY-001 in treating high-salinity wastewater.

In order to achieve the purpose, the invention adopts the following technical scheme:

obtaining the Bacillus pumilus SZG-NY-001:

diluting fresh sludge in certain salt lake in North Hu to obtain 10^-4，10^-5，10^-6Three gradient spreads, one day at 37 ℃ and single colonies picked and inoculated onto the slant.

The method comprises the following steps of streak-culturing by using LB solid culture medium with salt concentration of 5.0% (namely, 5g of sodium chloride is added into 100g of water), 10.0%, 15.0%, 20.0%, 25.0% and 30.0%, culturing for 3 days at 30 ℃, observing growth conditions, selecting 3 strains with better salt resistance, performing attribution identification on the strains, and finally identifying two strains of salt-resistant Bacillus pumilus.

One of the strains is delivered to China center for type culture Collection at 28/09/2020, and is classified and named as follows: the Bacillus pumilus SZG-NY-001 has a preservation number of CCTCC NO of M2020550, and is also called Bacillus pumilus 001 or Bacillus pumilus SZG-NY-001.

The bacillus pumilus is gram-positive bacteria, grows aerobically, hydrolyzes starch, has positive catalase reaction and negative oxidase and lecithinase reaction, and has the growth temperature of 10-45 ℃; colony characteristics: the strain is in a short rod shape under an optical microscope, and is white, wet, neat, round, small and convex in edge on an agar plate culture medium.

The application of the bacillus pumilus SZG-NY-001 comprises the step of reducing the COD of industrial wastewater by using the bacillus pumilus or the step of preparing an industrial wastewater COD degrading agent by using the bacillus pumilus as an active ingredient or one of the active ingredients.

In the above applications, the industrial wastewater includes, but is not limited to, inorganic salt industrial wastewater discharged from industrial production such as meat processing, printing and dyeing, paper making, chemical industry, pharmacy, petroleum or fermentation.

In the above application, preferably, the industrial wastewater is high-salt industrial wastewater, and the salinity of the wastewater is 13-165 g/L; in the above application, preferably, the salinity is 120 g/L-165 g/L;

in the application, the bacillus pumilus SZG-NY-001 and the bacillus pumilus SZG-NY-002 are compounded according to the effective bacteria concentration of 1:1, and then COD of the high-salt industrial wastewater is degraded, wherein the preservation number of the bacillus pumilus SZG-NY-002 is CCTCC NO: M2020549.

Compared with the prior art, the invention has the following advantages:

1) compared with the known halotolerant bacteria, the Bacillus pumilus SZG-NY-001 provided by the invention can exert the COD degradation capability at a higher salinity, and has potential advantages for environmental application.

2) The bacillus pumilus SZG-NY-001 provided by the invention has extremely high COD degradation capability under the condition of high salinity, and is not influenced by other factors in the wastewater, such as TN, ammonia nitrogen and TP in the wastewater, and almost has no influence on the COD degradation efficiency; and is particularly suitable for treating COD in mixed wastewater containing p-hydroxybenzaldehyde and tosyl chloride.

3) After the bacillus pumilus SZG-NY-001 and the bacillus pumilus SZG-NY-002 are compounded, the degradation rate of organic pollutants is accelerated by utilizing the synergistic effect among bacterial strains, the tolerance to various toxic substances is strong, the flora composed of a plurality of bacterial strains has more stable structure and stronger adaptability, and the defects of the prior art are overcome.

Detailed Description

The technical solutions of the embodiments of the present invention will be described clearly and completely below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments; all other embodiments, which can be derived by a person skilled in the art from the embodiments of the present invention without any inventive step, are within the scope of the present invention.

The technical schemes of the invention are conventional schemes in the field if not particularly stated; the reagents or materials, if not specifically mentioned, are commercially available. In the embodiment of the invention, the detection method of the soluble CODcr, TN, ammonia nitrogen, TP and salinity (g/L) of the industrial saline wastewater refers to a rapid digestion method for determining the chemical oxygen demand of HJ399-2007, an HJ 636-2012 alkaline potassium persulfate digestion ultraviolet spectrophotometry, an HJ 535-2009 nNer reagent spectrophotometry, an ammonium molybdate spectrophotometry for determining the total phosphorus of GB11893-89 and a determination method for the mineralization degree of the national standard water body SL 79-1994.

Example 1:

obtaining Bacillus pumilus (Bacillus pumilus) SZG-NY-001 and Bacillus pumilus SZG-NY-002:

taking fresh sludge of a salt lake in the North of a lake, diluting and taking 10^-4，10^-5，10^-6Three gradient coats were applied, incubated at 37 ℃ for one day, and single colonies were picked and inoculated onto the slant.

The method comprises the steps of streak-culturing on an LB solid culture medium containing 5.0% of sodium chloride salt (namely, 5g of sodium chloride is added into 100g of water), 10.0%, 15.0%, 20.0%, 25.0% and 30.0%, culturing for 3 days at 30 ℃, observing the growth condition, selecting 3 strains with better salt resistance, performing attribution identification on the strains, and finally identifying two strains of salt-resistant Bacillus pumilus.

The two strains are sent to China center for type culture Collection at 28/09/2020, and are classified and named: bacillus pumilus SZG-NY-001 with the preservation number of CCTCC NO: M2020550, address: in Wuhan university in Wuhan, China, the invention is also called Bacillus pumilus 001; classification designation of another strain of bacillus pumilus: bacillus pumilus SZG-NY-002 with the preservation number of CCTCC NO: M2020549, address: the invention is also called Bacillus pumilus 002 at Wuhan university in China.

Example 2:

fermentation culture of Bacillus pumilus (Bacillus pumilus) SZG-NY-001 and Bacillus pumilus SZG-NY-002:

shake flask culture medium: 10g/L of peptone, 5g/L of yeast extract, 5g/L of casein, 2g/L of KCl, 3g/L of sodium citrate and MgSO₄`7H₂20g/L of O, 80g/L of NaCl, 50mg/L of nystatin, pH8.0 and the balance of water.

Selecting single bacterial colony of Bacillus pumilus SZG-NY-001 or SZG-NY-002 on the plate by using an inoculating loop, inoculating the single bacterial colony into the culture medium, and performing shake culture at 30 ℃ and 200rpm/min for 24 hours to obtain liquid microbial inoculum seed liquid, wherein the effective bacterial concentration of the seed liquid of the two bacteria is 1 multiplied by 10⁸cfu/mL。

Example 3:

and (3) detecting the salt tolerance of the Bacillus pumilus SZG-NY-001 and SZG-NY-002:

inoculating 5% Bacillus pumilus SZG-NY-001 or SZG-NY-002 seed solution into LB culture medium containing NaCl with concentration of 0%, 12%, 14%, 16%, 18% or 20%, culturing for 24h, and counting plates, the results are as follows:

growth of Bacillus brevis 001 in culture media with different NaCl concentrations

Concentration of NaCl	Bacterial concentration (cfu/mL)
		0％	1×10⁸
12％	9×10⁷
		14％	8×10⁷
16％	5×10⁶
		18％	3×10⁵
20％	4×10⁴

The results show that the Bacillus pumilus 001 has no inhibiting effect on the growth of the invention when the NaCl concentration is not higher than 14%; when the NaCl concentration is more than 16%, the growth inhibition effect is obvious, and the bacteria concentration is greatly reduced; when the NaCl concentration is 18%, the bacteria concentration is only 3X 10⁵cfu/m. Thus, it is tolerant up to 16% of the salinity of sodium chloride.

Growth of Bacillus brevis 002 in culture medium with different NaCl concentration

Concentration of NaCl	Bacterial concentration (cfu/mL)
		0％	1×10⁸
12％	7×10⁷
		14％	8×10⁶
16％	5×10⁵
		18％	2×10⁵
20％	1×10⁴

The results show that the Bacillus pumilus 002 has no substantial inhibition effect on the growth of the present invention when the NaCl concentration is not higher than 14%; when the NaCl concentration is more than 14%, the growth inhibition effect is obvious, and the bacteria concentration is greatly reduced; when the NaCl concentration is 16%, the bacteria concentration is only 5X 10⁵cfu/mL. Therefore, it is tolerant up to 14% of the salinity of sodium chloride.

Example 4:

the application of the single bacillus pumilus SZG-NY-001 preparation in the treatment of high-salt wastewater comprises the following steps:

100ml of industrial saline wastewater is put into a 250ml triangular flask, and the activated Bacillus pumilus (SZG-NY-001 seed liquid (effective bacteria concentration is 10%) is inoculated according to the volume ratio of 1 percent⁸cfu/ml), incubated at 30 ℃ at 200rpm, and the soluble COD was measured daily.

Water quality condition (mg/L) of industrial salt-containing wastewater to be treated

In the above table, the "mixed wastewater of p-hydroxybenzaldehyde and tosyl chloride" is the wastewater of dye and dye intermediate.

The p-toluenesulfonamide waste water is the waste water of the dye intermediate.

The chemical wastewater is mixed wastewater of a plasticizer, a rubber foaming agent and a dye intermediate.

COD degradation condition (mg/L) of wastewater to be treated after being put into Bacillus pumius SZG-NY-001

As can be seen from the table above, the Bacillus pumilus SZG-NY-001 can tolerate the wastewater with the industrial salt content of about 16.5%, and the COD reduction effect after the strain feeding is obvious.

Example 5:

the application of the single bacillus pumilus SZG-NY-002 preparation in the treatment of high-salt wastewater:

100ml of industrial saline wastewater is put into a 250ml triangular flask and inoculated with activated Bacillus pumilus (SZG-NY-002) (effective bacteria concentration is 10%) according to the volume ratio of 1 percent⁸cfu/ml), incubated at 30 ℃ at 200rpm, and the soluble COD was measured daily.

Water quality condition (mg/L) of salt-containing wastewater to be treated

COD degradation condition (mg/L) after treatment of wastewater and feeding of Bacillus pumilus SZG-NY-002

Although the tolerance degree of the bacillus pumilus SZG-NY-002 to sodium chloride is 14 percent in a laboratory test, the bacillus pumilus SZG-NY-002 still has a good COD treatment effect on the wastewater with the industrial salinity of 16.5 percent due to the complexity of the industrial wastewater salinity.

Example 6:

the application of the composite microbial inoculum of the bacillus pumilus SZG-NY-001 and SZG-NY-002 in the treatment of high-salinity wastewater:

the bacillus pumilus SZG-NY-001 and the bacillus pumilus SZG-NY-002 are mixed according to the effective bacteria concentration of 1:1, and the effective average concentration of the obtained mixed bacteria liquid is 10⁸cfu/ml；

100ml of wastewater is taken and placed in a 250ml triangular flask, the compound bacterial liquid is inoculated according to the volume ratio of 1 percent, the culture is carried out at the temperature of 30 ℃ and the rpm is 200, and the soluble COD is detected every day.

Water quality condition (mg/L) of salt-containing wastewater to be treated

COD degradation condition (mg/L) after treatment of wastewater added with complex microbial inoculum

From the results, it can be seen that after the Bacillus pumilus SZG-NY-001 and SZG-NY-002 are mixed, the same wastewater is treated by the same bacteria concentration and the same inoculation amount, and the degradation rate of COD is obviously improved.

Example 7:

the preparation and application of the bacillus pumilus composite bacterial agent are as follows:

mixing Bacillus pumilus SZG-NY-001 and Bacillus pumilus SZG-NY-002 at effective bacteria concentration of 1:1, dissolving 5g in 100mL of brown sugar water containing 1%, and aerating for 30min to obtain 1 hundred million cfu/mL of composite microbial agent activation solution; before mixing, the effective bacteria concentration of two strains of Bacillus pumilus is 100 hundred million cfu/g.

Adding 1mL of compound microbial inoculum activation solution into a saline wastewater water body with the total volume of 200mL, and taking urea as a nitrogen source and dipotassium phosphate as a phosphorus source to supplement nitrogen and phosphorus nutrition to carbon: nitrogen: phosphorus 100:5:1 and the shake flask was placed in a shaker at 30 ℃ and a simulated degradation experiment was performed at 200 rpm/min.

Numbering	Sample (I)	Soluble CODcr	TN	Ammonia nitrogen	TP	Salinity (g/L)
							1	Hubei petrochemical salt-containing wastewater	371.1	10.58	7.08	0.5	78.53
2	High-salt mother liquor wastewater from printing and dyeing	60360	73515	60609	8.14	132.65
							3	Hebei rubber wastewater	3130	189.05	168.25	51.3	32.69
4	Ten-weir landfill leachate	4420	1393.5	1362.17	8.85	45.68
							5	Pharmaceutical wastewater	3540	865.75	156.89	0.12	67.59

Actual wastewater treatment effect (mg/L)

Numbering	Sample (I)	Duration of treatment	Initial COD	COD after treatment	Rate of degradation
						1	Salt-containing wastewater of certain petrochemical in Hubei province	48h	371.1	109.3	70.54％
2	High-salt mother liquor wastewater from printing and dyeing	96h	60360	48964	18.89％
						3	Hebei rubber wastewater	48h	3130	492.5	84.27％
4	Ten-weir landfill leachate	96h	4420	1715.6	61.19％
						5	Pharmaceutical wastewater	72h	3540	1520	57.06％

The adaptability and treatment effect of the bacillus pumilus SZG-NY-001 and the composite microbial inoculum thereof on the actual salt-containing wastewater are obviously better than those of the common conditions, the bacillus pumilus SZG-NY-001 still has a strong COD degradation function under the high-salt condition, can be used for treating the salt-containing wastewater by adopting a pure strain alone, can also be used as an exogenous functional microorganism to be added into the existing process for use, and can be used for enhancing the COD degradation capability of the whole system in cooperation with the original strain.

Claims

1. An isolated strain of Bacillus pumilus (B.) (Bacillus pumilus) The preservation number of the Bacillus pumilus is CCTCC NO: M2020550.

2. The use of the Bacillus pumilus of claim 1 for treating industrial wastewater.

3. The use of the bacillus pumilus of claim 1 in the preparation of an industrial wastewater treatment agent.

4. The use of claim 2 or 3, wherein the industrial wastewater is inorganic salt industrial wastewater discharged from meat processing, printing and dyeing, paper making, chemical industry, pharmaceutical industry, petroleum industry or fermentation industry.

5. The application of the industrial wastewater according to claim 4, wherein the salinity of the industrial wastewater is 13-165 g/L.

6. The use according to claim 5, wherein the salinity is 120-165 g/L.