CN107129954B

CN107129954B - Staphylococcus for degrading phenol

Info

Publication number: CN107129954B
Application number: CN201710488862.7A
Authority: CN
Inventors: 许继飞; 赵吉; 宋晓雪; 武琳慧; 刘崎峰; 王川; 包智华
Original assignee: Inner Mongolia University
Current assignee: Inner Mongolia University
Priority date: 2016-06-27
Filing date: 2017-06-23
Publication date: 2020-08-25
Anticipated expiration: 2037-06-23
Also published as: CN107325981A; CN107164277A; CN107129954A; CN107164277B; CN107325981B

Abstract

The invention provides a Staphylococcus (Staphylococcus caprae) and application thereof. The strain is named as CL, and the preservation number is CCTCC NO: M2016305. The strain can be used for culturing liquid culture medium (10g MgSO) containing 200mg/L phenol in 36h₄·7H₂O、0.2g CaCl₂·2H₂O, 2-5g KCl, 2.5g tryptone, 10g yeast extract, NaCl 5%, distilled water to 1000 ml. Adjusting pH to about 7.2, sterilizing at 121 deg.C for 15min), and degrading phenol by over 90%. The staphylococcus CL disclosed by the invention has strong phenol degradation capability, can be used for bioremediation of phenol polluted environment, and has good economic value and application prospect.

Description

Staphylococcus for degrading phenol

Technical Field

The invention relates to the technical field of microorganisms and biodegradation, and particularly relates to staphylococcus and application thereof in phenol degradation.

Background

Phenol and its derivatives are highly toxic to humans and animals and plants and have been included in the environmental priority pollutants list. The phenol-containing wastewater mainly comes from industries such as papermaking, oil refining, synthetic fiber, synthetic rubber, pesticide and the like, and is a main pollutant in industrial wastewater discharge. In addition to phenol-containing pollutants, these phenol-containing wastewaters often contain large amounts of salts, which result in high-salt phenol-containing wastewaters, which can cause serious pollution to soil, surface waters and ground waters when discharged into the environment. The conventional biodegradation technology has a bottleneck problem in treating the wastewater because the high-salt environment has the inhibiting and poisoning effects on the growth of microorganisms. Halophilic bacteria can be directly used for removing organic pollutants in high-salinity industrial wastewater without reducing the salinity of the wastewater, so that the halophilic bacteria gradually receives wide attention of people.

Staphylococcus (Staphylococcus caprae) is spherical or slightly elliptical, has a diameter of about 1.0um, and is arranged in grape shape. Staphylococci are nonflagellated and unable to move. No spores, and generally no capsules except a few strains. Is easy to be colored by common basic dyes, and gram stain is positive. After it has aged, died or phagocytosed by leukocytes, and certain strains that are resistant to drugs can be gram-negative. Most staphylococci decompose glucose, maltose and sucrose, and produce acid without producing gas. Pathogenic strains are capable of decomposing mannitol.

The staphylococcus is widely distributed in nature, the skin, the feather, the eyelids, the mucous membrane, the intestinal tract and the like of healthy poultry all have the staphylococcus, and the staphylococcus is also a common microorganism in the environment of hatching, feeding and processing of poultry. The research on the degradation of the staphylococcus to pollutants is rarely reported, the research on the degradation of the staphylococcus to phenol can reach more than 80% in 36 hours in a non-salt environment under the same condition, but no relevant report on the research on the phenol in a high-salt environment is found.

Disclosure of Invention

The invention aims to provide a staphylococcus capable of degrading phenol.

The invention separates a strain capable of degrading phenol from the bottom mud of a salt lake. The strain is identified as Staphylococcus by strain morphological characteristics, physiological and biochemical characteristics and 16S rDNA sequence analysis, is named as CL, and is preserved in a China center for type culture Collection (short for, the address: Wuhan university, Wuhan dynasty, China, zip code 430072) in 2016, 6, 2, with the preservation number of CCTCC NO. M2016305 and the classification name of Staphylococcus Staphylococcus caprae.

The invention provides a microbial inoculum containing staphylococcus CL.

The present invention provides a biological cleanser containing staphylococcal CL.

The invention provides application of staphylococcus CL or microbial inoculum containing the staphylococcus CL or biological cleaning agent containing the staphylococcus CL in cleaning environment.

Further, the invention provides application of staphylococcus CL or microbial inoculum containing the staphylococcus CL or biological cleaning agent containing the staphylococcus CL in phenol degradation.

The invention provides application of staphylococcus CL or microbial inoculum containing the staphylococcus CL or biological cleaning agent containing the staphylococcus CL in treatment of industrial wastewater.

The invention provides application of staphylococcus CL or microbial inoculum containing the staphylococcus CL or biological cleaning agent containing the staphylococcus CL in treating medical wastewater.

The invention provides application of staphylococcus CL in preparation of a biological cleaning agent.

The invention provides application of staphylococcus CL in preparation of a biodegradation agent.

The biodegradation agent is a phenol degradation agent.

The staphylococcus CL can degrade phenol in a culture medium at a concentration of 200mg/L to over 90% within 36h when the salt concentration is 0-5%, and the degradation rate of phenol is over 80% when the salt concentration is 10%. The staphylococcus CL and the microbial inoculum thereof provided by the invention are pollution-free and nuisanceless in the using process, can be applied to bioremediation of phenol polluted environment, can be widely applied to the field of environmental cleaning, and the field of phenol-containing industrial wastewater or medical wastewater treatment, and have better economic value and application prospect.

Drawings

FIG. 1 shows a tree of evolutionary trees of strain CL and closely related species 16S rDNA sequences.

FIG. 2 is a graph showing the growth of strain CL and the degradation characteristics of phenol.

FIG. 3 is a graph showing the degradation of phenol by strain CL at different NaCl concentrations.

FIG. 4 is a standard curve (OD) of phenol concentration₂₇₀) Figure (a).

FIG. 5 is a graph of the degradation of strain CL to different initial phenol concentrations.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.

Unless otherwise specified, the chemical reagents used in the examples are all conventional commercially available reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.

Example 1 isolation and identification of Staphylococcus CL

1. Isolation of Staphylococcus CL

a. Dissolving 1-5g or 500ml of filtrate of solid sample (salt lake sludge, seabed sludge, high-salinity wastewater treatment sludge, etc.) in 10-50ml of liquid culture medium (10g MgSO 10)₄·7H₂O、0.2g CaCl₂·2H₂O, 2-5g KCl, 2.5g tryptone, 10g yeast extract, NaCl 15%, distilled water to 1000 ml. Adjusting pH to about 7.2, sterilizing at 121 deg.C for 15min), shaking at 80-160rpm for 30-120 min at room temperature, taking out, standing for 20-40 min, sucking 100 μ L with pipette, uniformly coating on culture plate of solid culture medium (obtained by adding 2% agar into the above liquid culture medium), and standing in 28 deg.C constant temperature incubator.

b. Picking a single colony by using an inoculating loop until a visible colony grows, and continuously scribing on the surface of a flat solid culture medium;

c. and performing streak culture repeatedly until a pure culture halophilic bacteria strain is obtained.

d. Selecting a pure cultured single colony, transferring the pure cultured single colony into a liquid culture medium containing phenol, and culturing in a constant-temperature shaking table at 28 ℃ and 150rpm until the bacterial liquid is turbid;

the formula of the liquid culture medium 1L in the step is NaCl 100g, Tris 2g, KCl 2g and KNO₃1g、NH₄Cl 5g、MgSO₄·7H₂O 24g、MnSO₄·H₂O 0.688g、CaCl₂0.12g, glucose 1g, distilled water added to 1000 ml. Adding sterilized 0.05mol/L Na into 100ml of the liquid culture medium₂HPO₄2ml、0.01mol/L FeSO₄2mL, 2mL of 10mg/mL yeast extract, 2mL of 10mg/mL tryptone, and 2mL of 10mg/mL phenol (200 mg/L phenol solution after conversion).

e. And (d) sucking 200 mu L of bacterial liquid by using a pipette, transferring the bacterial liquid into the liquid culture medium containing the phenol in the step d, repeating the steps for 5-8 times, and obtaining a halophilic bacterium which is stable and efficient and can degrade the phenol, wherein the halophilic bacterium is named as CL.

2. Morphological characteristics of the Strain

The bacterial strain CL is diluted and coated in a solid culture medium, bacterial colonies are observed to be golden yellow, round and bulged after 60 hours, the surface is wet and smooth, the edges of the bacterial colonies are neat, the bacterial colonies are purple after gram staining and are gram-positive cocci, and the cocci are connected into a string in a mode of more than two cocci.

3. 16S rDNA identification

The length of the 16S rDNA partial sequence of the strain is 1058bp, the sequence is subjected to Blast comparison analysis on NCBI website (http:// www.ncbi.nlm.nih.gov /), representative strains with the highest similarity to the CL homologous sequence of the strain are selected, a phylogenetic tree is constructed by adopting a Neighbor Joining method (Neighbor-Joining) and MEGA 6.0 software, and the phylogenetic tree is shown in figure 1 and is subjected to self-development test (bootstrap) for 1000 times. The strain CL is known to be Staphylococcus, and the sequence similarity with the currently published Staphylococcus (Staphylococcus caprae ATCC 35538) reaches 99 percent.

The strain CL is preserved in the China center for type culture Collection (the short term, the address: Wuhan university in Wuhan, China, zip code 430072) in 2016, 6, 2 days, the preservation number is CCTCC NO. M2016305, and the strain is classified as Staphylococcus aureus caprae.

Example 2 degradation of Staphylococcus CL phenol Performance test

And (3) detecting the degradation performance of the staphylococcus CL on phenol in a phenol-containing culture medium by adopting a high performance liquid chromatography. (culture medium 10g MgSO₄·7H₂O、0.2g CaCl₂·2H₂O, 2-5g KCl, 2.5g tryptone, 10g yeast extract, NaCl 5%, distilled water to 1000 ml. Adjusting pH to about 7.2, sterilizing at 121 deg.C for 15min, adding phenol according to the following experimental requirements)

1. Growth of Strain CL and degradation Properties of phenol

The growth and phenol concentration profiles of strain CL under optimal growth degradation conditions are shown in FIG. 2. The strain CL enters a logarithmic growth phase after about 12h of lag phase, during which the strain grows almost linearly with time, nearly 40h and later, the strain is in a stable growth phase, and the biomass OD of the strain is at the moment₆₀₀A maximum value of 1.6 was reached. As can be seen from the phenol concentration curve, OD is dependent on biomass₆₀₀The phenol concentration is continuously reduced with continuous increase, and the final phenol degradation rate is 83 percent.

2. Degradation of phenol by strain CL under different NaCl concentrations

It can be seen by analysis that changes in NaCl concentration have a significant effect on the growth of strain CL and the ability to degrade phenol (P)<0.01). As can be seen from FIG. 3, the growth of the strain CL and the degradation of phenol both tend to increase and decrease with increasing NaCl concentration. When the NaCl concentration is 5%, the growth of the bacterial strain CL and the degradation of phenol are maximum, and the degradation rate of phenol is 90%. As can be seen from multiple comparisons, the biomass (OD) of the strain CL was found when the NaCl concentration was varied from 5% to 10%₆₀₀) And no significant change in phenol degradation (P)<0.01)。

3. Inoculating the strain into liquid culture medium with phenol concentration of 0mg/L, 100mg/L, 200mg/L, 400mg/L, 600mg/L, 800mg/L(the formula of the liquid culture medium is the same as above), the pH is adjusted to be 7.0, and the culture is carried out in a constant temperature shaking table at the temperature of 30 ℃ at the rotating speed of 165r/min, and other conditions are not changed. The initial biomass and phenol concentration and the biomass (OD) after 72h incubation on a constant temperature shaker were determined₆₀₀) And phenol concentration.

The determination method comprises the following steps: sucking 1mL of culture solution after 0h and 60h of culture, adding the culture solution into a 5mL centrifuge tube, adding 3mL of sterile culture solution into the centrifuge tube, uniformly mixing, and detecting the biomass OD600 by using an ultraviolet-visible spectrophotometer. Meanwhile, sucking 1mL of culture solution after 0h and 60h of culture, adding the culture solution into a 1.5mL centrifuge tube, centrifuging the culture solution for 10min under the condition of 6000r/min, taking 0.8mL of supernatant, adding the supernatant into a 5mL centrifuge tube, adding 2.4mL of sterile culture solution into the centrifuge tube, uniformly mixing, setting the wavelength of an ultraviolet visible spectrophotometer to be 270nm, measuring the absorbance of phenol, and calculating the initial concentration of phenol and the concentration of 60h according to a phenol standard curve (shown in figure 4) so as to calculate the degradation rate of phenol.

And (4) analyzing results: it can be seen from data analysis that changes in phenol concentration have a significant effect on the growth of strain CL and the ability to degrade phenol (P < 0.01). As can be seen from FIG. 5, the growth of the strain and the degradation of phenol tended to increase and then decrease with increasing phenol concentration. When the concentration of the phenol is 200mg/L, the growth of the bacterial strain CL and the degradation of the phenol are both maximized, wherein the degradation rate of the phenol is about 85 percent. As can be seen from multiple comparative analysis, when the phenol concentration is more than or less than 200mg/L, the phenol concentration has a significant influence on the growth of the strain CL and phenol degrading bacteria (P < 0.01).

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. A Staphylococcus (Staphylococcus caprae) CL with a preservation number of CCTCC NO: M2016305.

2. A microbial preparation comprising the Staphylococcus CL as claimed in claim 1.

3. A biological detergent comprising the staphylococcal CL of claim 1.

4. Use of a staphylococcal CL of claim 1 or a bacterial agent of claim 2 or a biological cleaner of claim 3 in a clean environment with phenol.

5. Use of the staphylococcal CL of claim 1 or the bacterial agent of claim 2 or the biological detergent of claim 3 for degrading phenol.

6. Use of the staphylococcal CL of claim 1 or the bacterial agent of claim 2 or the biological detergent of claim 3 in the treatment of phenol in industrial wastewater.

7. Use of the staphylococcal CL of claim 1 or the bacterial agent of claim 2 or the biological detergent of claim 3 in the treatment of phenol in medical wastewater.

8. Use of the staphylococcal CL of claim 1 in the preparation of a biological cleaner for the degradation of phenol.

9. Use of the staphylococcal CL of claim 1 in the preparation of a biodegrading agent which is a phenol degradation agent.