CN112795525A - Acinetobacter calcoaceticus culture medium and application thereof in degradation of bisphenol A - Google Patents

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a calcium acetate acinetobacter culture medium and application thereof in degradation of bisphenol A. The Acinetobacter calcoaceticus culture medium comprises a carbon source, a nitrogen source and inorganic phosphate, the culture method of the Acinetobacter calcoaceticus in the culture medium comprises the steps of culturing the Acinetobacter calcoaceticus 5d-6d under the conditions that the pH value of a solution is 6.0-8.0, the inoculation amount is 10% -15%, the culture temperature is 25-35 ℃, and the rotating speed of a shaking table is 150r/min-250r/min, and verifying that the optimal carbon source is 0.01% of glucose by mass fraction, the optimal nitrogen source is 0.01% of peptone and the optimal phosphorus source is 0.02% of KH₂PO₄+Na₂HPO₄Mass ratio ofIs 1: 1, the method effectively improves the yield of the fermentation product, and can ensure that the acinetobacter calcoaceticus is more sufficiently applied to actual life, such as removing phosphorus in water, degrading dibutyl phthalate, repairing eutrophic landscape water and the like.

Description

Acinetobacter calcoaceticus culture medium and application thereof in degradation of bisphenol A

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to a calcium acetate acinetobacter culture medium and application thereof in degradation of bisphenol A.

Background

There are groups of microorganisms in nature that degrade various contaminants. Microorganisms capable of degrading self-toxic substances have been isolated from various environments. Some bacteria of the genus acinetobacter can use autotoxic substances as energy source substances and degrade them. Some organizations and individuals at home and abroad are developing the use of acinetobacter calcoaceticus to degrade pollutants, acinetobacter calcoaceticus TS2H is separated from the environment in spring, and the like, and the strain can efficiently degrade dibutyl phthalate (DBP); acinetobacter calcoaceticus PHEA-2 separated from oil refinery sewage by aging and the like has stronger degradation capability on phenol; acinetobacter calcoaceticus T3 isolated from industrial wastewater in Laershance et al can maintain high degradation efficiency for benzoic acid and naphthalene.

Microbial fermentation is a complex biological process and is influenced by various factors, wherein the composition and the proportion of a culture medium are one of key factors. The requirements of different microorganisms for the nutrient composition of the medium are different, as are the raw materials required for different fermentative productions. Whether the composition and the proportion of the culture medium are proper or not can greatly influence the growth and the propagation of the production strains, the fermentation titer of target products, the subsequent refining process of the fermentation products and the quality and the purity of final products. The proper culture medium can fully exert the biosynthesis capacity of the engineering strains, and the maximum production efficiency can be achieved by matching with effective culture conditions. The carbon source in the fermentation medium can provide energy for the growth and metabolism of the thalli, and is a key factor for forming a cytoskeleton; the nitrogen source provides nitrogen elements for cells to synthesize nitrogen-containing substances such as proteins and nucleic acids; the inorganic salt can regulate osmotic pressure and enzyme activity of thallus. The common carbon source mainly comprises soluble starch, maltose, sucrose, glucose, corn flour and the like; the commonly used nitrogen sources mainly comprise ammonium nitrate, soybean meal, ammonium sulfate, ammonium chloride, urea and the like; the inorganic salt is selected from calcium carbonate, magnesium sulfate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, copper sulfate, etc.

Currently, single factor and orthogonal design are often combined to optimize the medium. Firstly, screening out the optimal carbon source, nitrogen source and inorganic salt by single-factor screening. Through orthogonal design, the adding proportion of the carbon source, the nitrogen source and the inorganic salt under the mutual influence condition is determined, and the optimal adding amount of the three is found out. The optimal combination screened by the orthogonal design can only screen out a rough range. The invention aims to research an optimal formula so as to improve the fermentation titer, reduce the cost, reduce the energy consumption and reduce the interference to the ecological environment through the optimization of a culture medium, so that the selection of a proper culture medium and the design of a reasonable proportion have very important significance to fermentation production.

Disclosure of Invention

The invention aims to provide a calcium acetate acinetobacter culture medium and application thereof in degrading bisphenol A, so that the calcium acetate acinetobacter is expected to improve fermentation titer, reduce cost, reduce energy consumption and reduce interference on ecological environment through culture medium optimization.

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

a calcium acetate immortal bacillus culture medium comprises the following components of a carbon source, a nitrogen source and inorganic phosphate, wherein the carbon source is 0.01% of glucose by mass fraction, the nitrogen source is 0.01% of peptone, and the phosphorus source is 0.02% of KH₂PO₄And Na₂HPO₄。

The culture medium for the calcium acetate acinetobacter is 0.02 percent KH by mass₂PO₄：Na₂HPO₄＝1:1。

The application of a calcium acetate acinetobacter culture medium in degrading bisphenol A is as follows: inoculating the acinetobacter calcoaceticus into the calcium acetate immotile culture medium containing bisphenol A for culturing for 5d-6d under the conditions that the pH of the culture medium solution is 6.0-8.0, the inoculation amount is 10% -15%, the culture temperature is 25-35 ℃ and the rotating speed of a shaking table is 150-250 r/min.

The calcium acetate acinetobacter culture medium is applied to degrading bisphenol A, the pH value of a culture medium solution is 7.0, the inoculation amount of the calcium acetate acinetobacter is 10%, the culture medium solution is inoculated into the calcium acetate acinetobacter culture medium containing the bisphenol A, the degradation temperature is 30 ℃, and the shaking table rotating speed is 150r/min, and the culture medium solution is vibrated and degraded for 6 d.

The calcium acetate acinetobacter culture medium is applied to degrading bisphenol A, and the initial mass concentration of bisphenol A is 60 mg/L.

The degradation rate of bisphenol A after the optimization of the culture medium at the 5d is improved to 61.3% from 52.6% compared with that before the optimization, and the culture medium after the optimization is proved to be capable of effectively improving the yield of the acinetobacter calcoaceticus.

The acinetobacter calcoaceticus colony is in a round shape with regular edge, faint yellow and opaque, is convex, moist, smooth and glossy in surface, has no flagella, no spores and a rod shape, is round in stationary period, and has the size of (0.7-1.3) Mumx (0.3-2.1) mum; gram staining is negative;

the invention has the beneficial effects that: the invention provides a method for optimizing a calcium acetate acinetobacter culture medium, which improves the yield of the calcium acetate acinetobacter on the original basis so as to achieve the aim of producing the maximum fermentation product, and cultures the calcium acetate acinetobacter into a microorganism group which can degrade certain pollutants in a targeted manner.

Drawings

FIG. 1 is a graph showing the growth of A.calcoaceticus in a conventional medium;

FIG. 2 is a graph showing the effect of different carbon sources on the growth of A.calcoaceticus;

FIG. 3 is a graph showing the effect of different nitrogen sources on the growth of A.calcoaceticus;

FIG. 4 is a graph of the effect of different inorganic salts on the growth of A.calcoaceticus;

FIG. 5 is a graph comparing the degradation of bisphenol A before and after optimization of the calcium acetate immortal medium.

Detailed Description

In order that the objects, advantages and effects of the invention will become more apparent, the invention will be further described in detail with reference to the accompanying drawings in conjunction with the embodiments.

Example 1

A growth curve of Acinetobacter calcoaceticus was prepared by culturing Acinetobacter calcoaceticus on a sterilized inorganic salt medium consisting of 1000mL of distilled water and 0.1g of NH₄Cl、1.0g NaCl、1.0g KH₂PO₄And 10.0g peptone. Culturing a week of Acinetobacter calcoaceticus (three parallel samples are set) under shaking at pH 7.0, inoculum size 10%, culture temperature 30 deg.C and shaking table rotation speed 150r/min, and determining OD every 1d₆₀₀The measured data were used to generate the growth curve shown in FIG. 1.

The figure shows that the growth lag phase of the acinetobacter calcoaceticus is 0-2 d, and the increase of the thallus concentration is slow; 2-4 d is the logarithmic growth period of the strain, and the strain cells are propagated in large quantity; the growth period is 4-5 days, the growth curve trend is stable, and the variation of the thallus concentration is small; after 7d, the growth state of the strain gradually worsens, and the strain enters a decay period, so that the inoculation culture medium at 5d is selected.

Example 2 optimization of carbon sources in culture Medium for Acinetobacter calcoaceticus

Respectively adding 3 carbon sources commonly used in laboratories, namely glucose, lactose and sucrose with the mass fraction of 0.1 percent respectively into a sterilized inorganic salt culture medium for carbon source optimization, wherein the other components of the culture medium comprise 1000mL of distilled water and 0.1g of NH₄Cl、1.0g NaCl、1.0g KH₂PO₄5.0g of beef extract and 10.0g of peptone, and determining OD by shake culture for 5d under the conditions of solution pH of 7.0, inoculum size of 10%, culture temperature of 30 ℃ and shaking table rotation speed of 150r/min₆₀₀The results are shown in FIG. 2.

As can be seen from FIG. 2, of these 3 additional carbon sources, A.calcoaceticus grew best in a medium containing 0.1% glucose by mass, OD₆₀₀The maximum is 0.66, so glucose is selected as the optimal carbon source for subsequent fermentation medium optimization.

Example 3 optimization of Nitrogen Source in culture Medium for Acinetobacter calcoaceticus

Respectively adding 3 common laboratory nitrogen sources, namely peptone, urea and ammonium chloride with the mass fraction of 0.1% into a sterilized inorganic salt culture medium for nitrogen source optimization, wherein the other components of the culture medium comprise 1000mL of distilled water, 1.0g of NaCl and 1.0g of KH₂PO₄5.0g of beef extract, 10.0g of peptone and 0.1 percent of glucose added by mass percent, and performing shake culture for 5d under the conditions that the pH of the solution is 7.0, the inoculum size is 10 percent, the culture temperature is 30 ℃ and the rotating speed of a shaking table is 150r/min to determine the OD₆₀₀The results are shown in FIG. 3.

As can be seen from FIG. 3, of these 3 additional nitrogen sources, A.calcoaceticus grew best in a medium containing 0.1% by mass of peptone, OD₆₀₀The highest content is 0.71, and the subsequent fermentation medium optimization is further carried out by taking peptone with the mass fraction of 0.1% as an optimal nitrogen source.

Example 4 optimization of the culture Medium for Acinetobacter calcoaceticus by phosphorus Source

Adding common inorganic salts of potassium dihydrogen phosphate and sodium monohydrogen phosphate into sterilized culture medium respectively, i.e. adding KH with mass fraction of 0.02%₂PO₄、Na₂HPO₄、KH₂PO₄+Na₂HPO₄(mixing at a mass ratio of 1: 1) to optimize the carbon source, wherein the other components of the culture medium comprise 1000mL of distilled water and 0.1g of NH₄Cl, 1.0g NaCl, 5.0g beef extract, 10.0g peptone, 0.1% glucose by mass fraction and 0.1% peptone by mass fraction, and determining OD by shake culture for 5d under the conditions of solution pH of 7.0, inoculum size of 10%, culture temperature of 30 ℃ and shaking table rotation speed of 150r/min₆₀₀The results are shown in FIG. 4.

As can be seen from FIG. 4, 0.02% KH was added to each of the mixtures₂PO₄、Na₂HPO₄、KH₂PO₄+Na₂HPO₄(mixed in a mass ratio of 1: 1) in inorganic salt, Acinetobacter calcoaceticus in KH₂PO₄+Na₂HPO₄Best growth (mixed at a mass ratio of 1: 1), OD₆₀₀Up to 0.68.

Example 5 degradation of bisphenol A by Acinetobacter calcoaceticus

The composition of the optimized pre-culture medium is 1000mL of distilled water and 0.1g of NH₄Cl、1.0g NaCl、1.0g KH₂PO₄And 10.0g of peptone, in order to further prove that the yield of the acinetobacter calcoaceticus after the optimization of the culture medium is improved, the experiment of degrading A is carried out on the culture medium before and after the optimization, the initial mass concentration of bisphenol A is selected to be 60mg/L, the vibration degradation is carried out for 6d under the conditions that the degradation pH is 7.0, the inoculation amount of the acinetobacter calcoaceticus is 10%, the degradation temperature is 30 ℃ and the rotating speed of a shaking table is 150r/min, and the degradation rate of the bisphenol A is shown in figure 5 under the optimal condition.

As shown in FIG. 5, when the growth stationary phase of Acinetobacter calcoaceticus was reached at the 5d, the degradation rate of bisphenol A after the optimization of the culture medium was increased from 52.6% to 61.3% as compared with that before the optimization, and thus, the yield of Acinetobacter calcoaceticus was effectively increased by the optimized culture medium.

What has been described above is merely a preferred embodiment of the invention. It should be noted that variations and modifications can be made by those skilled in the art without departing from the principle of the present invention, and they should also be considered as falling within the scope of the present invention.

Claims (5)

1. The calcium acetate immortal culture medium is characterized by comprising the following components of a carbon source, a nitrogen source and inorganic phosphate, wherein the carbon source is 0.01% of glucose in mass fraction, the nitrogen source is 0.01% of peptone, and the phosphorus source is 0.02% of KH₂PO₄And Na₂HPO₄。

2. The culture medium of claim 1, wherein the culture medium comprises 0.02% KH by mass₂PO₄：Na₂HPO₄＝1:1。

3. The application of the calcium acetate acinetobacter culture medium in degrading bisphenol A is characterized in that the method comprises the following steps: inoculating acinetobacter calcoaceticus into the acinetobacter calcoaceticus culture medium containing bisphenol A according to claim 1 or 2 for culturing for 5d-6d under the conditions that the pH of the culture medium solution is 6.0-8.0, the inoculation amount is 10% -15%, the culture temperature is 25-35 ℃ and the rotating speed of a shaking table is 150r/min-250 r/min.

4. The use of the Acinetobacter calcoaceticus culture medium according to claim 3 for degrading bisphenol A, characterized in that the pH of the culture medium solution is 7.0, the inoculation amount of Acinetobacter calcoaceticus is 10%, the culture medium of Acinetobacter calcoaceticus according to claim 1 or 2 containing bisphenol A is inoculated, and the culture medium is degraded by shaking at 30 ℃ and the rotation speed of the shaking table of 150r/min for 6 d.

5. The use of the culture medium of Acinetobacter calcoaceticus according to claim 4 for degrading bisphenol A, wherein the initial concentration of bisphenol A is 60 mg/L.

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