CN114426934A - Lactobacillus plantarum for source wastewater biotoxicity detection and application thereof - Google Patents
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Abstract
The invention relates to the technical field of sewage treatment, and discloses lactobacillus plantarum for detecting biological toxicity of source wastewater and application thereof. The lactobacillus plantarum is named as CR3, is preserved in China general microbiological culture Collection center of China Committee for Culture Collection of Microorganisms (CCM) at 3 months and 15 days in 2021, has the address of No. 3 Siro No. 1 Hospital of Chaoyang area in Beijing, has the preservation number of CGMCC NO.22011, and is named as lactobacillus plantarum in a microorganism classification wayLactobacillus plantarum. The lactobacillus plantarum CR3 can hydrolyze esculin to generate black substances, has proper tolerance to wastewater toxicity, can intuitively reflect the intensity of wastewater toxicity through color development depth, has obvious and stable color development, and can be used for biological toxicity detection of source wastewater.
Description
Technical Field
The invention relates to the technical field of sewage treatment, in particular to lactobacillus plantarum for detecting biological toxicity of source wastewater and application thereof.
Background
With the rapid development of the industry in China, the types of pollutants in the water body are more and more complex. Many special pollutants can cause inhibition effect even destructive damage to biological treatment of wastewater after the concentration reaches a certain threshold value, such as heavy metal, chloroaniline, cyanide, nitrobenzene and the like, and the water quality condition of the wastewater cannot be objectively and accurately evaluated only by using conventional water quality indexes such as COD, BOD, TN, TP and the like, so that the current situation of industrial wastewater treatment cannot be met only by performing quality division and shunting according to the conventional water quality indexes, and source quality division and shunting based on wastewater toxicity are required.
The biotoxicity test (Bio-toxicity Tests) is a method for comprehensively evaluating the toxicity of a substance by evaluating the substance against a living body. Compared with a physical and chemical method, the biological method can evaluate the influence of unknown toxic and harmful pollutants in the wastewater on organisms and can reflect the complex interaction among a plurality of pollutants in the wastewater and the bioavailability of the pollutants, thereby playing an important role in wastewater toxicity control and management, being used for searching the safe concentration of a certain chemical substance or industrial wastewater on the organisms, providing scientific basis for formulating reasonable water quality standard and wastewater discharge standard, and also being used for evaluating the effective degree of wastewater treatment and screening a proper industrial wastewater toxicity reduction technology.
In industrial wastewater treatment, conventional activated sludge is an essential treatment unit, and activated sludge is microorganisms in nature, so the bacterial method is most suitable when developing a toxicity detection technology suitable for industrial wastewater to a rear-end conventional activated sludge treatment system. MIC (Minimum inhibition concentration) refers to the lowest concentration of a drug that can inhibit the visible growth of the granulosa of a bacterium to be tested, and is originally applied to the determination of drug sensitivity so as to accurately and effectively utilize the drug for treatment; and is also the traditional means for measuring the drug effect of antibiotics on various pathogenic bacteria. Biologically poisoning MIC The sex detection technology is applied to the biological toxicity detection of wastewater, and the toxicity of wastewater to microorganisms can be judged by mixing wastewater with a bacteria culture medium to culture for a period of time and finding the lowest wastewater concentration capable of inhibiting the growth of bacteria in the culture medium. For example, patent CN201910403966.2 discloses a differential management method for pharmaceutical wastewater based on MIC toxicity detection technology, which uses G with the first abundance ratio in activated sludge+The standard strains of the bacteria and the G-bacteria can judge the toxicity of the wastewater by observing the growth conditions of the two strains in wastewater with different dilutions, but the growth conditions of the strains can only be reflected by the abundance of the strains in the wastewater, and are difficult to observe intuitively.
Disclosure of Invention
In order to solve the technical problems, the invention provides a lactobacillus plantarum for source wastewater biotoxicity detection and an application thereof. The lactobacillus plantarum CR3 can hydrolyze esculin to generate black substances, has proper tolerance to wastewater toxicity, can intuitively reflect the intensity of wastewater toxicity through color development depth, has obvious and stable color development, and can be used for biological toxicity detection of source wastewater.
The specific technical scheme of the invention is as follows:
In a first aspect, the invention provides a Lactobacillus plantarum for source wastewater biotoxicity detection, which is named as CR3, and is preserved in China general microbiological culture Collection center at 3 months and 15 days 2021, wherein the address is No. 3 of Beijing Shangyang district North West Lu No. 1 institute, the preservation number is CGMCC NO.22011, and the microorganism classification is named as Lactobacillus plantarum.
The lactobacillus plantarum CR3 is easy to culture, can secrete esculin hydrolase, decomposes esculin into glucose and escin, generates a black compound after the escin reacts with iron ions, and the black is darker as the lactobacillus plantarum CR3 is more in number, so that the inhibition effect of the wastewater on the lactobacillus plantarum CR3 can be intuitively reflected by observing the color development depth. In addition, the lactobacillus plantarum CR3 has proper tolerance to the toxicity of the wastewater, can reflect the intensity of the toxicity of the wastewater, and has obvious and stable color development. Therefore, the lactobacillus plantarum CR3 can be used for biological toxicity detection of source wastewater, and the toxicity of the wastewater is judged according to the color development condition of a culture system, so as to judge whether the wastewater can directly enter a rear-end biochemical treatment system (usually, the wastewater is introduced into a rear-end comprehensive regulating tank, the wastewater treated by the comprehensive regulating tank is further subjected to toxicity detection, and the wastewater is introduced into a microorganism treatment unit such as an activated sludge treatment unit after the detection is qualified).
In a second aspect, the invention provides a lactobacillus plantarum mutant for source wastewater biotoxicity detection, wherein the mutant is obtained by subjecting lactobacillus plantarum to mutagenesis, domestication, genetic recombination or natural mutation.
In a third aspect, the invention provides a bacterial culture comprising said Lactobacillus plantarum or comprising said mutant.
Preferably, the bacterial cell culture is a bacterial solution or a bacterial agent.
In a fourth aspect, the invention provides an application of the lactobacillus plantarum or the mutant in biological toxicity detection of source wastewater.
Preferably, the application comprises the steps of:
(1) adding tryptone, yeast extract, ferric citrate and esculin into water, mixing well, and preparing into culture medium;
(2) adding the wastewater into a culture medium to prepare a mixed system; inoculating the lactobacillus plantarum or the mutant into a mixed system to prepare a culture system;
(3) and (3) culturing the culture system, observing the color development condition, wherein if the color development condition does not develop black, the wastewater cannot directly enter the rear-end biochemical treatment system, and if the color development condition develops black, the wastewater can directly enter the rear-end biochemical treatment system.
Preferably, in the step (2), the volume fraction of the wastewater in the mixed system is 25-35%; the inoculation amount of the lactobacillus plantarum or the mutant in a mixed system is 0.1-0.34 g/L.
According to experience, the wastewater concentration of 25-35% (i.e. the volume of the wastewater after dilution accounts for 25-35% of the total volume) is used as the critical point for judging that the wastewater has higher toxicity, and the wastewater concentration is preferably 30%. That is, when the MIC is less than 25-35%, the wastewater is highly toxic, has a large influence on the back-end biochemical treatment system, and cannot directly enter the back-end biochemical treatment system; when the MIC is more than 25-35%, the wastewater is non-highly toxic, has little influence on the back-end biochemical treatment system, and can directly enter the back-end biochemical treatment system.
Under the condition that the inoculation amount of the lactobacillus plantarum CR3 is 0.1-0.34g/L, when the concentration of the wastewater is 25-35%, highly toxic wastewater and non-highly toxic wastewater can be distinguished, namely, when the lactobacillus plantarum CR3 is cultured under the condition that the concentration of the wastewater is 25-35%, the highly toxic wastewater does not show black, but the highly toxic wastewater shows black. Therefore, the lactobacillus plantarum CR3 can be applied to biological toxicity detection of source wastewater, and can judge whether the wastewater can directly enter a rear-end biochemical treatment system or not according to the color development condition when the lactobacillus plantarum CR3 is cultured under the condition that the wastewater concentration is 25-35%.
In addition, when esculin and ferric citrate are added into a conventional lactic acid bacteria culture medium to culture lactobacillus plantarum CR3, a culture system cannot develop color, which indicates that the conventional lactic acid bacteria culture medium cannot be used for source wastewater biotoxicity detection. Therefore, the invention innovatively discloses a culture medium formula, and when the culture medium is adopted to culture lactobacillus plantarum CR3, a culture system can develop color, so that the culture medium can be used for source wastewater biotoxicity detection.
Preferably, in step (1), the concentrations of tryptone, yeast extract, ferric citrate and esculin in the medium are 3.3-10g/L, 1.67-5g/L, 0.5-0.6g/L and 1-1.2g/L, respectively, and more preferably 3.3g/L, 1.67g/L, 0.5g/L and 1 g/L.
The biological toxicity detection of source wastewater is influenced by too high or too low concentration of each component in the culture medium, and specifically: when the concentration is too low, the culture system cannot develop color, so that the intensity of the toxicity of the wastewater cannot be reflected; when the concentration is too high, the nutrient content in the culture medium is too high, and the influence of the nutrient content in the wastewater on the growth of the strain can be masked. Therefore, the concentrations of the tryptone, the yeast extract, the ferric citrate and the esculin are respectively set to be 3.3-10g/L, 1.67-5g/L, 0.5-0.6g/L and 1-1.2g/L, and the culture system can develop color in the ranges; further preferably 3.3g/L, 1.67g/L, 0.5g/L and 1g/L, at which the nutrient content in the medium is low and the effect of the nutrient content in the wastewater on the growth of the strain is not masked.
Compared with the prior art, the invention has the following advantages:
(1) the lactobacillus plantarum CR3 can hydrolyze esculin to generate black substances, has proper tolerance to wastewater toxicity, can intuitively reflect the intensity of the wastewater toxicity through color development, and has obvious and stable color development, so that the biological toxicity of source wastewater can be conveniently detected;
(2) the front-end wastewater biotoxicity detection method can take the wastewater concentration of 25-35% as the critical point of the wastewater with higher toxicity degree, distinguish the high-toxicity wastewater from the non-high-toxicity wastewater, and judge whether the wastewater can directly enter the back-end biochemical treatment system or not according to the critical point.
Detailed Description
The present invention will be further described with reference to the following examples.
Example 1: color development degree of different strains
By data investigation, a typical species with esculin hydrolase activity is enterococcus. Enterococcus is a pathogenic bacterium, and is not suitable for use. Enterococci belongs to the order of Lactobacillales (Lactobacillus) in the microbiological classification, and it is speculated that Lactobacillales strains may have similar biochemical characteristics, so that the model strains are focused on the lactobacilli.
Selecting 8 strains of lactic acid bacteria, enterococcus faecalis and escherichia coli from different sources as test bacteria, respectively inoculating bacterial powder of each strain to 2 nutrient culture media containing color development agent esculin according to the inoculation amount of 1% (namely 0.1g/L), placing the culture media in a constant-temperature shaking incubator at 30 ℃ for 24h, and observing the color development condition, wherein the results are shown in table 1.
The formula of the culture medium 1 is as follows: 10g/L of tryptone, 5g/L of Yeast Extract (YE), 0.5g/L of ferric citrate, 1g/L of esculin and water as a solvent; pH 6.2 ± 0.2.
The formula of the culture medium 2 is as follows: 10g/L of peptone, 5g/L of beef extract powder, 4g/L of YE, 20g/L of glucose and K2HPO42g/L, 2g/L of triammonium citrate, 5g/L of sodium acetate and MgSO4 0.2g/L，MnSO40.05g/L, Tween 801 g/L, ferric citrate 0.5g/L, esculin 1g/L and water as solvent; pH 6.2 ± 0.2.
TABLE 1 color development degree of different strains
From table 1, it can be seen that:
(1) the culture medium 1 is a culture medium designed by the inventor, and the culture medium 2 is a conventional lactic acid bacteria culture medium. No strains No. 1-8 developed color in the culture medium 2, which indicates that the conventional culture medium for lactic acid bacteria cannot be used as a culture medium for lactic acid bacteria in the source wastewater biotoxicity detection (probably because some color development inhibitors exist in the culture medium 2).
(2) Different strains have different degrees of color development in the culture medium 1, 1# -8# and enterococcus faecalis are gram-positive bacteria and can develop color, 1#, 6#, 7# and 8# develop color obviously, 11# is gram-negative bacteria and does not develop color, so 1#, 6#, 7# and 8# can be used as pseudo-model strains.
Example 2: influence of salinity on growth of strains
NaCl with different amounts is respectively added into the culture medium 1, the bacterial powder of each strain is inoculated according to the inoculation amount of 1g/L, then the mixture is placed in a constant-temperature shaking incubator at 30 ℃ for 24 hours, the growth condition of the strain is observed, and the turbidity of the bacterial liquid under the wavelength of 600nm is measured by a spectrophotometer, and the result is shown in Table 2.
TABLE 2 influence of salinity on growth of strains
The model strain needs to satisfy the following requirements: can grow normally and develop color within the normal salinity range (less than or equal to 3 weight percent); i.e., the series of techniques cannot characterize toxicity in the normal salinity range (< 3 wt%).
From table 2, it can be seen that: the addition of salinity has a relatively obvious influence on the growth of strains, and the growth amounts of the 1# strain and the 5# strain are always ranked in the front in the salinity range of 0-3 wt%, so the 1# strain and the 5# strain can be used as pseudo-model strains.
Example 3: influence of organic matter and inorganic ion on color development
Various substances which possibly have influence on color development in the culture medium 2 are respectively added into the culture medium 1, after various bacterial strains are inoculated into the bacterial powder according to the inoculation amount of 1g/L, the bacterial powder is placed in a constant-temperature shaking incubator at 30 ℃ for 24 hours, and the color development condition is observed, and the results are shown in table 3.
TABLE 3 Effect of the substances on the coloration of the strains
From table 3, it can be seen that: the addition of glucose can obviously inhibit the color development, and the addition of sodium acetate is beneficial to the color development, so the detection technology of the invention needs to be vigilant about whether the water sample contains glucose and sodium acetate. Magnesium sulfate and manganese sulfate have almost no influence on the 1# strain and the 8# strain, so the 1# strain and the 8# strain can be used as a pseudotyped strain.
Example 4: effect of wastewater concentration on color development
Combining the results of example 2 and example 3, 4 strains were preferred: 1#, 2#, 6#, 8 #. Wastewater with different contents is added into the culture medium 1, so that the final content of the wastewater in the system is 10%, 30% and 50% (v/v), 1#, 2#, 6# and 8# strain powder are respectively inoculated into the wastewater according to the inoculation amount of 1g/L, and after the wastewater is placed in a constant-temperature shaking incubator at 30 ℃ for 24 hours, the coloration condition is observed, and the results are shown in Table 4.
TABLE 4 MIC results for wastewater from four strains
| Serial number | 10％ | 30％ | 50％ |
| 1# | ++ | + | - |
| 2# | ++ | + | - |
| 6# | ++ | - | - |
| 8# | ++ | - | - |
Note: "+" represents color development and "-" represents no color development.
From table 4, it can be seen that: the strains No. 1, No. 2, No. 6 and No. 8 can show the toxicity of the wastewater.
Example 5: identification of strains
The results of the comprehensive examples 1-4 show that the strain No. 1 has obvious color development, the maximum growth amount and stable color development and can reflect the intensity of the toxicity of the wastewater. Subsequent experiments on a large amount of wastewater show that the toxicity of the 1# bacterium detected various types of wastewater is basically consistent with the early detection result in a laboratory. Therefore, the strain # 1 was selected as a model strain.
16S rRNA sequencing was performed on strain # 1 and the highest homology alignment was performed with known 16S rRNA genes in GenBank. The 1# strain was identified to belong to Lactobacillus plantarum and was named CR 3.
Example 6: effect of sodium acetate on MIC results of 1# Strain wastewater
Adding 5g/L sodium acetate into medium 1 containing esculin and medium not containing esculin to obtain 2 kinds of medium; selecting 3 representative waste water (LP3#, LP24#, and LP27#), adding waste water with different contents into two culture media, enabling the final content of the waste water in a system to be 10%, 30% and 50% (v/v), inoculating bacterial powder of the strain No. 1 to the waste water according to the inoculation amount of 1g/L, placing the waste water in a constant-temperature shaking incubator at 30 ℃ for 24 hours, observing the growth condition of bacteria in a culture system without esculin and the color development condition in the culture system with esculin, and comparing the results with the results of a conventional MIC method (namely the method in the patent CN 201910403966.2), wherein the results are shown in Table 5.
TABLE 5 Effect of sodium acetate addition on MIC test results of 1# bacteria wastewater
Note: "+" indicates that the strain grew, and "-" indicates that the strain did not grow.
From table 5, it can be seen that: the addition of sodium acetate can change the toxicity result of MIC detection of wastewater, so that the 1# strain cannot accurately reflect the toxicity of the wastewater, and the addition of sodium acetate in a culture medium is not considered in the follow-up process although the sodium acetate is beneficial to color development.
Example 7: optimization of addition amount of bacterial powder
Selecting 2 representative waste water (LP1# and LP2#), adding the waste water with different contents into the culture medium 1 to ensure that the final content of the waste water in the system is 10%, 30% and 50% (v/v); adding different amounts of 1# strain liquid into the mixed system respectively to obtain culture systems with the bacteria concentrations of 1.0g/L, 1.7g/L and 3.4g/L respectively; the culture system was placed in a 30 ℃ constant temperature shaking incubator for 24 hours, and then the color development was observed, and the results are shown in Table 6.
TABLE 6 Experimental results for different addition amounts of fungal powder
| LP1# waste water | LP2# waste water | |
| Results of conventional MIC method | 10％+，30％-，50％- | 10％+，30％-，50％- |
| The concentration of the bacteria is 1.0g/L | 30% black and greenish and 50% slight green | 30% black and greenish and 50% slight green |
| The concentration of the bacteria is 1.7g/L | 30% black and greenish and 50% slight green | 30% black and greenish and 50% slight green |
| The concentration of the bacteria is 3.4g/L | 30% black, 50% green | 30% black, 50% green |
Note: "+" indicates that the strain grew, and "-" indicates that the strain did not grow.
From table 6, it can be seen that: under three concentrations, the wastewater toxicity is inconsistent with the conventional results, and the MIC value is higher, which indicates that the concentration of the bacterial powder is too high. Therefore, the bacterial powder mixture was further diluted, and a research experiment was performed after the concentration of the bacterial powder was reduced, and the experimental results are shown in table 7.
TABLE 7 Experimental results for different addition amounts of fungal powder
| LP1# waste water | LP2# waste water | |
| Conventional MIC results | 10％+，30％- | 10％+，30％- |
| The concentration of the bacteria is 0.1g/L | 10% black to green and 30% slight green | 110% black and greenish and 30% slight green |
| The concentration of the bacteria is 0.17g/L | 10% black to green and 30% slight green | 10% black to green and 30% slight green |
| The concentration of the bacteria is 0.34g/L | 10% black, 30% green | 10% black, 30% green |
Note: "+" indicates that the strain grew, and "-" indicates that the strain did not grow.
From table 7, it can be seen that: under three concentrations, the toxicity of the waste water is consistent with the conventional result, so the inoculation amount of the bacterial powder can be 0.1-0.34 g/L.
Example 8: optimization of the concentration of the Components of the Medium
The concentrations of the components in the culture medium 1 are adjusted to different degrees to obtain 9 different culture media, strain powder of the strain No. 1 is inoculated according to the inoculation amount of 0.1g/L, the strain powder is placed in a constant-temperature shaking incubator at 30 ℃ for 24 hours, the color development condition is observed, and the experimental results are shown in Table 8.
TABLE 8 color development results after concentration change of each component in the medium
From table 8 it can be seen that: the strain # 1 was cultured using two media from experimental groups 1 and 2, which were able to develop a black color, and thus, in the source wastewater biotoxicity assay using # 1, these two media were selected. The culture medium of experimental group 2 is preferred because: the culture medium of the experimental group 1 has high content of nutrient components, and is easy to cover the influence of the nutrient components in the wastewater on the growth of the No. 1 strain.
Example 9: detection of source wastewater biotoxicity
LP1#, LP2#, LP3#, LP24# and LP27# waste water are diluted by using the culture medium of the experimental group 2 in the example 8 to obtain mixed systems with the waste water concentrations of 10%, 30% and 50%, the mixed systems are inoculated with 1# strain powder according to the inoculation amounts of 0.1g/L, 0.17g/L and 0.34g/L, and after the mixed systems are placed in a constant-temperature shaking incubator at 30 ℃ for 24 hours, the coloration condition is observed and compared with the results of the conventional MIC method, and the results are shown in Table 9.
TABLE 9 color development results of wastewater toxicity test after bacteria concentration change
Note: in the conventional MIC method, "+" represents that the strain grows, and "-" represents that the strain does not grow; in the method of the present invention, "+" represents color development and "-" represents no color development.
From table 9, it can be seen that: when the culture medium formula of the experimental group 2 in the example 8 is adopted and the inoculation amount of the strain No. 1 is 0.1-0.34g/L, the detection result is the same as that of the conventional MIC method, which shows that the culture medium formula and the inoculation amount can be used for detecting the biotoxicity of source wastewater.
The raw materials and equipment used in the invention are common raw materials and equipment in the field if not specified; the methods used in the present invention are conventional in the art unless otherwise specified.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and all simple modifications, alterations and equivalents of the above embodiments according to the technical spirit of the present invention are still within the protection scope of the technical solution of the present invention.
Claims (7)
1. The lactobacillus plantarum for source wastewater biotoxicity detection is characterized in that the lactobacillus plantarum is named as CR3 and is preserved in China general microbiological culture Collection center (CGMCC NO. 22011) 3, 15 and 2021 in 15 days, the preservation number is CGMCC NO.22011, and the classification name of the microorganism is lactobacillus plantarum Lactobacillus plantarum。
2. A mutant of Lactobacillus plantarum used for biotoxicity detection of source wastewater, characterized in that the mutant is obtained by subjecting the Lactobacillus plantarum defined in claim 1 to mutagenesis, domestication, genetic recombination or natural mutation.
3. A cell culture comprising the Lactobacillus plantarum strain of claim 1 or comprising the mutant of claim 2.
4. The bacterial culture according to claim 3, which is a bacterial solution or a bacterial agent.
5. Use of a lactobacillus plantarum as defined in claim 1 or a mutant as defined in claim 2 for the biotoxicity assay of source wastewater.
6. The use according to claim 5, comprising the steps of:
(1) adding tryptone, yeast extract, ferric citrate and esculin into water, mixing well, and preparing into culture medium;
(2) adding the wastewater into a culture medium to prepare a mixed system; inoculating the lactobacillus plantarum or the mutant into a mixed system to prepare a culture system;
(3) and (3) culturing the culture system, observing the color development condition, wherein if the color development condition does not develop black, the wastewater cannot directly enter the rear-end biochemical treatment system, and if the color development condition develops black, the wastewater can directly enter the rear-end biochemical treatment system.
7. The use of claim 6, wherein in step (2), the volume fraction of wastewater in the mixed system is 25-35%; the inoculation amount of the lactobacillus plantarum or the mutant in a mixed system is 0.1-0.34 g/L.
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| CN114456968A (en) * | 2021-10-21 | 2022-05-10 | 杭州秀川科技有限公司 | Liangbacterium and composite flora for treating wastewater generated in probenazole production and application of Liangbacterium and composite flora |
| CN114457140A (en) * | 2021-08-30 | 2022-05-10 | 杭州秀川科技有限公司 | Source wastewater biotoxicity detection kit and using method thereof |
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| CN105132322A (en) * | 2015-09-02 | 2015-12-09 | 广州格拉姆生物科技有限公司 | Lactobacillus plantarum and application thereof |
| CN107058166A (en) * | 2017-01-13 | 2017-08-18 | 东南大学 | The Lactobacillus plantarum of one plant of extracellular polysaccharide |
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