CN111979140B - Bright photobacterium growth promoter for monitoring water pollutants and application thereof - Google Patents
Bright photobacterium growth promoter for monitoring water pollutants and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and discloses a luminous photobacterium growth promoter for monitoring water pollutants, which comprises the following components: sodium chloride, urea, glucose, magnesium chloride, oxaloacetic acid, adenosine, yeast extract and formic acid. The invention provides a bright photobacterium growth promoter for monitoring water pollutants, which can be used for rapidly recovering and enlarging culture, save monitoring time and reduce enterprise burden.
Description
Technical Field
The invention belongs to the technical field of biological environmental protection, and particularly relates to a bright photobacterium growth promoter for monitoring water pollutants and application thereof.
Background
The luminous bacteria have been applied to various fields in the last century, and foreign scientists firstly separate and screen luminous bacteria sensitive to the environment for detecting the water body biotoxicity. In recent years, the method has gradually become a simple and rapid biotoxicity detection means.
The luminous bacillus is a luminous bacterium which is widely applied, and the application steps are as follows: purchasing strain freeze-drying powder, recovering, diluting and monitoring pollutants. However, the application method has the problems that the survival rate and the luminous efficiency of the strains are reduced after recovery, and multiple times of sampling and monitoring at multiple time points are generally needed in pollutant monitoring, so that a large amount of bacteria products are needed, and the purchase cost of enterprises is high. Generally, photobacterium brightens is not very nutrient-demanding, and can grow under conditions of basic nutrients such as glucose, yeast extract, and sodium chloride, but the growth is slow, the luminous efficiency is reduced, and the activity is not stable enough. How to recover and expand the culture of the photobacterium brightens, and maintain better survival rate and luminous efficiency is a technical problem to be solved.
Prior art 1: the continuous culture condition of the photobacterium brightens is optimized, 2016 years is reported by university of east China science and technology, freeze-dried powder is revived, shake flask culture and fermentation tank culture are carried out, but the culture time of the method is long, the concentration of thalli and the luminous efficiency are both required to be improved, and the method is not suitable for being used by environment monitoring enterprises.
Prior art 2: "CN 201910137277" discloses a method for culturing luminous bacteria, which comprises the steps of placing luminous bacteria in a culture medium for inoculation, rapidly sealing a bottle opening with a polypropylene plastic film and latex ring, placing the inoculated culture medium in a constant temperature box at 20-28 ℃, and culturing for 2-3 days at constant temperature. The method also has the problem of too long culture time, and cannot be quickly applied to environmental monitoring.
Prior art 3: "CN 201010537039" discloses a rapid recovery method for bright photobacterium, which needs multiple centrifugal stirring, causes great damage to bacterial strains, obviously reduces the number of viable bacteria, and is complicated in process because recovery liquid is added for multiple times.
Prior art 4: CN201911074734 discloses a method for resuscitating photobacterium, which comprises freezing photobacterium and freeze-drying protective agent together, and then re-dissolving with equal amount of distilled water or salt solution, wherein the activity and recovery luminescence rate of bacteria are both about 80%; to be further lifted.
The price of the freeze-dried powder is high, and the monitoring efficiency and scale can be enlarged when the commercial freeze-dried powder is subjected to resuscitation and expansion culture. However, inappropriate recovery and culture are a significant cause of accelerated bacterial species decline and death. Poor recovery culture conditions such as nutrient components, temperature, pH value and the like can not only induce the death of strain cells, but also induce the appearance of degenerated cells, and once the degenerated cells rapidly reproduce, the number of the degenerated cells greatly exceeds that of normal cells, so that the strain degeneration can be caused. How to recover and rapidly expand the culture of the photobacterium lucidum freeze-dried powder is a technical problem to be solved in the field of biological environment monitoring.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides the bright photobacterium growth promoter for monitoring water pollutants and the application thereof, and the bright photobacterium growth promoter can be rapidly recovered and expanded for culture, save time and reduce the burden of enterprises.
The invention is realized by the following technical scheme.
The photobacterium brightening growth promoter for monitoring water pollutants is characterized by comprising the following components: sodium chloride, urea, glucose, magnesium chloride, oxaloacetic acid, adenosine, yeast extract and formic acid.
Further, the air conditioner is provided with a fan,
the growth promoter comprises the following components: 20-40g/L of sodium chloride, 1-10g/L of urea, 1-10g/L of glucose, 1-10g/L of magnesium chloride, 1-5g/L of oxaloacetic acid, 0.1-1g/L of adenosine, 0.1-1g/L of yeast extract and 1-50ml/L of formic acid.
Further, in the present invention,
the growth promoter comprises the following components: 25-35g/L of sodium chloride, 2-5g/L of urea, 5-10g/L of glucose, 3-4g/L of magnesium chloride, 1-2g/L of oxaloacetic acid, 0.5-1g/L of adenosine, 0.1-0.5g/L of yeast extract and 10-20ml/L of formic acid.
Preferably, the first and second electrodes are formed of a metal,
the growth promoter consists of the following components: 25-35g/L of sodium chloride, 2-5g/L of urea, 5-10g/L of glucose, 3-4g/L of magnesium chloride, 1-2g/L of oxaloacetic acid, 0.5-1g/L of adenosine, 0.1-0.5g/L of yeast extract and 10-20ml/L of formic acid.
More preferably still, the first and second liquid crystal compositions are,
the growth promoter consists of the following components: 30g/L of sodium chloride, 2g/L of urea, 5g/L of glucose, 4g/L of magnesium chloride, 1.5g/L of oxaloacetic acid, 0.7g/L of adenosine, 0.2g/L of yeast extract and 20ml/L of formic acid.
More preferably still, the first and second liquid crystal compositions are,
the growth promoter consists of the following components: 25g/L of sodium chloride, 3g/L of urea, 7g/L of glucose, 3g/L of magnesium chloride, 1.1g/L of oxaloacetic acid, 0.9g/L of adenosine, 0.4g/L of yeast extract and 15ml/L of formic acid.
More preferably still, the first and second liquid crystal compositions are,
the growth promoter consists of the following components: 22g/L of sodium chloride, 2g/L of urea, 6g/L of glucose, 4g/L of magnesium chloride, 1.3g/L of oxaloacetic acid, 0.6g/L of adenosine, 0.5g/L of yeast extract and 18ml/L of formic acid.
More preferably still, the first and second liquid crystal compositions are,
the growth promoter consists of the following components: 28g/L of sodium chloride, 4g/L of urea, 8g/L of glucose, 3g/L of magnesium chloride, 1.6g/L of oxaloacetic acid, 0.8g/L of adenosine, 0.3g/L of yeast extract and 19ml/L of formic acid.
More preferably still, the first and second liquid crystal compositions are,
the growth promoter is prepared according to the following steps: adding the above components into distilled water, stirring, and adjusting pH to 7.0-7.5.
The invention also claims the application of the growth promoter in monitoring water body pollutants by culturing the photogenic bacteria.
Compared with the prior art, the invention has at least the following beneficial effects:
glucose and urea as quick-acting carbon source and quick-acting nitrogen source can be quickly utilized by the photobacterium brightens, which is beneficial to quick recovery and proliferation. Magnesium ions are the most important metal ions influencing the activity of cells and the enzyme activity, and the proliferation speed of the bright photobacterium is increased along with the increase of the concentration of magnesium chloride in the range of 0-4 g/L; meanwhile, on the premise of sufficient adenosine, the luminous efficiency is also stably improved. The reason is probably that magnesium ions can activate enzymes related to ATP synthesis, the amount of ADP generated by conversion is increased under the culture condition of providing sufficient adenosine, and then the ADP is converted into ATP through substrate level phosphorylation, so that the strain activity is restored, and the luminous efficiency is improved. When the concentration of magnesium ions is too high, the luminous efficiency is rather lowered, and the specific reason is yet to be further clarified.
Formic acid in the concentration range of 0-20ml/L can improve the luminous efficiency of the photobacterium brightens, has no obvious influence on the proliferation of the bacterial cells, and continuously increases the concentration of formic acid, so that the proliferation of the bacterial cells is inhibited to a certain extent; it is possible that a relatively low concentration of formate can rapidly activate the activity of bacterial luciferase (monooxygenase). The oxaloacetate can increase the respiratory capacity of the chemoheterotrophic bacteria, improve the content of intracellular ATP and activate the rapid proliferation of somatic cells. The yeast extract can not only improve long-acting nitrogen source substances required by thallus proliferation, but also provide amino acid and vitamin for maintaining cell enzyme activity and metabolic efficiency.
Drawings
FIG. 1: examples 1-4 effects of growth promoters on the proliferation of strains;
FIG. 2: examples 1-4 effects of growth promoters on luminescence intensity;
FIG. 3: the influence of magnesium ions on biomass and specific luminous intensity;
FIG. 4: the effect of oxaloacetate on biomass and specific luminescence intensity;
FIG. 5: influence of formic acid on biomass and specific luminescence intensity.
Detailed Description
Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the products and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations and modifications, or appropriate alterations and combinations, of the products and methods described herein may be made and utilized without departing from the spirit, scope, and spirit of the invention. For a further understanding of the present invention, reference will now be made in detail to the following examples.
Example 1
The specific components and concentrations of the photobacterium brightening growth promoter for monitoring water pollutants are shown in table 1:
TABLE 1
| Components | Example 1 | Example 2 | Example 3 | Example 4 |
| Sodium chloride g/ |
30 | 25 | 22 | 28 |
| g/L of |
2 | 3 | 2 | 4 |
| Glucose g/ |
5 | 7 | 6 | 8 |
| Magnesium chloride g/ |
4 | 3 | 4 | 3 |
| Oxaloacetic acid g/L | 1.5 | 1.1 | 1.3 | 1.6 |
| Adenosine g/L | 0.7 | 0.9 | 0.6 | 0.8 |
| Yeast extract g/L | 0.2 | 0.4 | 0.5 | 0.3 |
| Formic acid ml/ |
20 | 15 | 18 | 19 |
The preparation method comprises the following steps: sequentially taking the components according to the concentration ratio, adding the components into distilled water, uniformly stirring, and adjusting the pH value to 7.0-7.5 to obtain the composition.
Comparative examples 1 to 5
The components of the photobacterium brightens growth promoter are shown in the table 2:
TABLE 2
Example 2
Application example of the photobacterium brilliant growth promoter.
The lyophilized powder is commercially available LUMINGHUAJUN lyophilized powder (ATCC11040) 1 g/tube; homemade freeze-dried powder can also be adopted.
The preparation method of the homemade freeze-dried powder comprises the following steps:
freeze-drying protective agent under aseptic condition, bright luminous rod bacterial liquid (concentration is 1 × 10)9cfu/ml) and lyoprotectant (parts by weight: 10% of skimmed milk powder, 8% of sucrose, 1% of sodium glutamate and sterile water as a solvent) according to the following ratio of 1: 1, mixing uniformly, and filling into a penicillin bottle; cooling to-70 deg.C within 60min, freezing for 12 hr, vacuum freeze-drying at-40 deg.C to obtain lyophilized powder, and storing at-20 deg.C.
The method for recovering and expanding culture of the photobacterium:
taking out the lyophilized powder, immediately placing in 38 deg.C water bath for thawing, shaking for 60s, after completely thawing, adding into shake flask containing 10 times volume of growth promoter, and shake culturing at 24 deg.C and 200rpm for 6h to obtain the final product.
Example 3
Examples 1-4 effects of growth promoters on the proliferation and luminescence intensity of strains.
The detection method comprises the following steps: the detection of the thallus concentration adopts a spectrophotometer method (OD 600); the luminous intensity of the photobacterium aurantiacae is detected by using a DXY-3 intelligent biotoxicity tester.
The test process comprises the following steps: referring to example 2, a commercially available lyophilized powder was used. As shown in figure 1, the growth promoter of examples 1-4 can realize rapid resuscitation and proliferation of Photobacterium brightens within 6 hours, which is beneficial to saving detection time, and obtains a large amount of bacteria liquid with stable luminous efficiency in a short time, thereby enlarging detection scale and reducing enterprise cost.
And (3) comparing the luminous intensity:
the conventional method comprises the following steps: taking out the lyophilized powder, immediately thawing in 38 deg.C water bath, shaking for 60s, adding appropriate amount of 3% sodium chloride solution, diluting, and controlling thallus concentration in the diluent to 1 × 104cfu/ml, detecting the luminous intensity, and marking as A;
the method comprises the following steps: taking out the lyophilized powder, immediately thawing in 38 deg.C water bath, shaking for 60s, completely thawing, adding into shake flask containing 10 times of growth promoter, shake-culturing at 24 deg.C and 200rpm for a period of time, stopping culturing, adjusting thallus concentration of 1 × 10 with 3% sodium chloride solution4cfu/ml, and the luminescence intensity was measured every 2 hours during the culture and recorded as B.
Calculating the formula: the specific luminous intensity is B/A.
As shown in figure 2, the activity of the recovered cells is not completely recovered, the luminous intensity is low, after the cells are cultured, the cell metabolism is vigorous, the activity of relevant enzymes such as luciferase and the like is greatly improved, the luminous intensity is also improved, the luminous efficiency is in a stable peak value at 6-8h, the specific luminous intensity is reduced after the cells are continuously cultured, and the reduction of the activity of the enzymes or the obstruction of the metabolism can be caused by the exhaustion of part of components in the promoter.
Example 4
Comparative examples 1-5 effects of promoters on the proliferation and luminous efficiency of the strains.
On the basis of comparative example 1 (conventional medium component) magnesium chloride was added at a concentration of: 0,1,2,3,4,5,6 in g/L, cultured for 6h in the same manner as in example 3, and the growth of the strain and the specific luminescence intensity were examined. As shown in FIG. 3, the concentration of magnesium chloride is positively correlated with the growth and specific luminous intensity of the strain, magnesium ions are the most important metal ions influencing the activity and the enzyme activity of the cell, and the proliferation speed and the luminous efficiency of the photobacterium brightens along with the increase of the magnesium chloride in the concentration range of 0-4 g/L; however, when the concentration of magnesium ions is too high, the luminous efficiency is rather lowered, and the specific reason is yet to be clarified.
The adding concentration of magnesium chloride is selected to be 4g/L, and the oxaloacetate adding concentration is verified to the growth condition of the strain and the specific luminous intensity. The oxaloacetate addition concentration is set to be 0,0.5,1,1.5,2,2.5 and 3, the unit is g/L, as shown in figure 4, the oxaloacetate has relatively consistent regulation effect on the growth quantity and the luminous intensity, has an obvious promotion effect on the growth quantity of the strain at 1g/L, gradually slows down in the later period, and has a certain promotion effect on the comparative luminous intensity. The reason for the analysis is that oxaloacetate can increase the respiratory capacity of chemoheterotrophic bacteria, increase the intracellular ATP content and activate the somatic cells to rapidly proliferate.
On the basis of the test 2, the oxaloacetate addition concentration is 1.5g/L for the next test, the influence of adenosine on the biomass and the specific luminous intensity of the thallus is verified, the test is carried out between the addition concentrations of 0 to 2g/L, the increase amplitude of the biomass of the thallus by the additional addition of adenosine is found to be small and is only 5 to 10 percent, the comparative luminous intensity has a certain increase effect between 0.5 and 1g/L, and the specific luminous intensity is 2.18 by taking the addition amount of 0.7g/L as an example; the yeast extract has a relatively obvious effect of improving the biomass of the thalli, when the addition amount is 0.2g/L, the improvement range is about 30%, and the contrast luminous intensity has no obvious influence.
On the basis of the above test 3, the addition amount of adenosine is 0.7g/L, the addition amount of yeast extract is 0.2g/L, the addition amount of formic acid is 0,5,10,15,20,25,30, and the unit is ml/L, as shown in FIG. 5, in the concentration range of 0-20ml/L, the activity of bacterial luciferase (monooxygenase) can be rapidly activated along with the addition amount of formic acid, so that the luminous efficiency of the photobacterium luminescens can be obviously improved, the proliferation of the bacterial cells is not obviously affected, the formic acid concentration is continuously increased, and the proliferation of the bacterial cells is inhibited to a certain extent.
The foregoing list is only illustrative of the preferred embodiments of the present invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
Claims (5)
1. The photobacterium brightening growth promoter for monitoring water pollutants is characterized by consisting of the following components: 25-35g/L of sodium chloride, 2-5g/L of urea, 5-10g/L of glucose, 3-4g/L of magnesium chloride, 1-2g/L of oxaloacetic acid, 0.5-1g/L of adenosine, 0.1-0.5g/L of yeast extract and 10-20ml/L of formic acid;
the growth promoter is prepared according to the following steps: adding the above components into distilled water, stirring, and adjusting pH to 7.0-7.5.
2. The growth promoter according to claim 1, characterized in that the growth promoter consists of: 30g/L of sodium chloride, 2g/L of urea, 5g/L of glucose, 4g/L of magnesium chloride, 1.5g/L of oxaloacetic acid, 0.7g/L of adenosine, 0.2g/L of yeast extract and 20ml/L of formic acid.
3. The growth promoter according to claim 1, characterized in that the growth promoter consists of: 25g/L of sodium chloride, 3g/L of urea, 7g/L of glucose, 3g/L of magnesium chloride, 1.1g/L of oxaloacetic acid, 0.9g/L of adenosine, 0.4g/L of yeast extract and 15ml/L of formic acid.
4. The growth promoter according to claim 1, characterized in that the growth promoter consists of: 22g/L of sodium chloride, 2g/L of urea, 6g/L of glucose, 4g/L of magnesium chloride, 1.3g/L of oxaloacetic acid, 0.6g/L of adenosine, 0.5g/L of yeast extract and 18ml/L of formic acid.
5. The growth promoter according to claim 1, characterized in that the growth promoter consists of: 28g/L of sodium chloride, 4g/L of urea, 8g/L of glucose, 3g/L of magnesium chloride, 1.6g/L of oxaloacetic acid, 0.8g/L of adenosine, 0.3g/L of yeast extract and 19ml/L of formic acid.
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