CN114276963A - Breeding method and application of deep-sea salt-tolerant phenol degrading bacteria - Google Patents
Breeding method and application of deep-sea salt-tolerant phenol degrading bacteria Download PDFInfo
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Abstract
The invention discloses a breeding method and application of deep sea halotolerant phenol degrading bacteria, wherein the method comprises the following steps of (1) enriching deep sea halotolerant bacteria; (1.1) preparing a culture medium; (1.2) enrichment of mixed bacterial liquid: adding a deep sea sediment sample into a culture medium for culturing for 5 days, then transferring the deep sea sediment sample into a fresh culture medium which is treated by the same method in an inoculation amount (v/v) of 10 percent, and continuously carrying out enrichment culture for four times; detecting the concentration of the strains in the enrichment solution of the fifth generation; when the fifth generation culture solution is inThe concentration of the bacteria is more than 1 × 10 after 5 days of culture9When the strain per mL is detected, finishing the enrichment culture to obtain an enrichment culture bacterial liquid; (2) and domesticating the salt tolerance of the mixed strain: and (3) performing acclimatization culture on the enrichment culture bacterial solution, and performing gradient acclimatization on the salt tolerance of the enrichment culture bacterial solution by adopting a method for gradually increasing the NaCl concentration in a culture medium of the acclimatization culture. The invention can solve the problem that the microorganism is difficult to degrade phenol in a high-salt environment.
Description
Technical Field
The invention relates to the technical field of microbial degradation, in particular to a breeding method and application of deep-sea salt-tolerant phenol degrading bacteria.
Background
Phenol belongs to a common phenolic substance, is widely applied to the production processes of bactericides, resins, petroleum, medicines and the like, and the discharge of phenol-containing wastewater not only causes serious pollution to the environment, but also harms the health of animals, plants and human beings. The long-term drinking of phenol-contaminated water or food by humans can damage liver and kidney functions, even leading to death. Therefore, the wastewater has the characteristics of wide source, large water quantity, large harm and high salinity.
As a pollutant to be preferentially controlled, methods for treating phenol-containing wastewater are mainly classified into physical methods, chemical methods and biological methods. Among them, biological methods are receiving more and more attention due to their advantages of economy, high efficiency, no secondary pollution, etc. Scientists have screened a variety of phenol-degrading bacteria, mainly including Bacillus (Bacillus sp.), Pseudomonas (Pseudomonas sp.), Acinetobacter (Acinetobacter sp.), Rhodococcus (Rhodococcus sp.) and the like, but the high salinity of the wastewater severely limits the ability of these microorganisms to degrade phenol. Therefore, the research on microorganisms which can efficiently degrade phenol and can tolerate high salinity is still the focus of the work of scholars at home and abroad.
The living environment of marine microorganisms is greatly different from that of terrestrial microorganisms, and the diversity and specificity of marine microorganisms are caused by the particularity of marine environment. Because the marine environment has the characteristics of high salt, high pressure, low temperature, oligotrophism, low illumination (even no illumination) and the like, marine microorganisms adapt to the complex marine environment for a long time to live, so that the marine microorganisms have the unique characteristics of halophilicity, pressure addiction, psychrophilia, low trophism, chemotaxis and attachment growth, polymorphism, luminescence and the like, and have great application potential in the field of environmental remediation. Therefore, the method provides a feasible idea for treating the high-salt organic industrial wastewater by screening the microbial strains which are salt-resistant and efficiently degrade phenol from the extreme environment of the deep sea.
Disclosure of Invention
The invention aims to solve the problems that: provides a breeding method of deep-sea salt-tolerant phenol degrading bacteria and application thereof, and can solve the problem that the phenol is difficult to degrade by microorganisms in a high-salt environment.
The technical scheme provided by the invention for solving the problems is as follows: a method for breeding deep-sea salt-tolerant phenol degrading bacteria comprises the following steps
(1) Enriching the deep sea halotolerant bacteria;
(1.1) preparing a culture medium;
(1.2) enrichment of mixed bacterial liquid: adding a deep sea sediment sample into a culture medium for culturing for 5 days, then transferring the deep sea sediment sample into a fresh culture medium which is treated by the same method in an inoculation amount (v/v) of 10 percent, and continuously carrying out enrichment culture for four times; detecting the concentration of the strains in the enrichment solution of the fifth generation; when the culture solution of the fifth generation is cultured for 5 days, the concentration of the bacteria is more than 1 multiplied by 109When the strain per mL is detected, finishing the enrichment culture to obtain an enrichment culture bacterial liquid;
(2) and domesticating the salt tolerance of the mixed strain: and (3) performing acclimatization culture on the enrichment culture bacterial solution, and performing gradient acclimatization on the salt tolerance of the enrichment culture bacterial solution by adopting a method for gradually increasing the NaCl concentration in a culture medium of the acclimatization culture.
Preferably, the formula of the culture medium in the step (1) is prepared from the following components in parts by weight: 5 parts of peptone, 1 part of yeast extract powder, 0.1 part of ferric citrate, 19.45 parts of NaCl, and MgCl25.98 parts of Na2SO43.24 parts of CaCl21.8 parts, KCl 0.55 parts, Na2CO30.16 part, 0.08 part of KBr and SrCl20.034 part of H3BO30.022 parts of Na2SiO30.04 part of NaF 0.0024 part of NH4NO30.0016 part of Na2HPO40.008 part of the extract is dissolved in distilled water under heating and then autoclaved.
Preferably, the autoclaving conditions are at a natural pH of 121 ℃ for 15 minutes.
Preferably, the culturing conditions in the step (1.2) are 30 ℃ and 150rpm shake culture for 5 days.
Preferably, the method for detecting the concentration of the strains in the fifth generation enrichment liquid in the step (1.2) specifically comprises the following steps
(1.2.1) preparation of a bacterial concentration Standard Curve
Counting the bacterial suspension cultured for 24 hours by using a blood cell counting plate, putting 1mL of the bacterial suspension into a centrifuge, centrifuging for 5min at 3000rpm, transferring the supernatant into another clean centrifuge tube with 1.5mL, centrifuging for 5min at 10000rpm, pouring off the supernatant, and respectively diluting with sterile water to adjust the concentration to 3.2 multiplied by 10 bacteria content per milliliter8、1.6×108、8×107、4×107、2×107And 1X 107Then determining the OD value of each tube at the wavelength of 660nm, taking the OD value as a longitudinal coordinate and the number of cells per milliliter as a horizontal coordinate to draw a bacteria concentration standard curve at the moment by taking sterile water as a blank control;
(1.2.2) measurement of actual cell concentration
Centrifuging 1mL of actual bacteria sample at 3000rpm for 5min, transferring the supernatant into another clean 1.5mL centrifuge tube, centrifuging at 10000rpm for 5min, pouring off the supernatant, washing the bacteria pellet at the bottom of the centrifuge tube with 1mL of sterile water, vortexing for 3min, and determining OD600And (4) calculating the concentration of the bacteria through a bacteria concentration standard curve.
Preferably, in the medium for acclimatization culture in the step (2), the concentration of NaCl is adjusted to 0% to 12%, and specifically may be 0%, 2%, 4%, 6%, 8%, 10%, 12%, or any value between any two of the above values.
Preferably, the conditions for the acclimatization culture in the step (2) comprise that the temperature is 28-30 ℃, the rotating speed is 120-150r/min, the culture time is 5-7 days, then the concentration of the strain in the culture medium is measured to determine whether the strain can tolerate the salt concentration under the setting, and each set of experiments is repeated for three times.
Preferably, the culture medium for acclimatization and culture in the step (2) further comprises the following components in parts by mass in addition to NaCl: heating and dissolving 5 parts of peptone and 1 part of yeast extract powder in distilled water, and carrying out autoclaving at 121 ℃ for 15 minutes at natural pH for later use.
The invention also aims to provide application of the deep-sea salt-tolerant phenol degrading bacteria obtained by breeding by any one of the breeding methods in high-salt organic industrial wastewater.
Compared with the prior art, the invention has the advantages that: the deep-sea salt-tolerant phenol degrading bacteria bred and obtained by the breeding method have the advantages of wide salinity adaptation range, high salt tolerance content, strong phenol degrading capability, high degrading speed and the like, and have great application potential in biological treatment of high-salt phenol wastewater.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 is a standard curve of the concentration of bacteria according to the present invention;
FIG. 2 is a graph showing the maximum NaCl tolerance of the strain of the present invention;
FIG. 3 is a graph of a phenol standard according to the present invention;
FIG. 4 is a graph showing the degradation of various concentrations of phenol by the strains of the present invention;
FIG. 5 is a genus-level species abundance ratio chart of the deep sea halotolerant bacteria acclimation culture solution of the present invention;
FIG. 6 is a diagram showing the effect of the deep sea halotolerant bacteria of the present invention on the actual wastewater treatment.
Detailed Description
The embodiments of the present invention will be described in detail with reference to the accompanying drawings and examples, so that how to implement the technical means for solving the technical problems and achieving the technical effects of the present invention can be fully understood and implemented.
It will be understood that the terms "comprises" and/or "comprising," when used in this specification and the appended claims, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.
It is also to be understood that the terminology used in the description of the embodiments of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the embodiments of the invention. As used in the description of embodiments of the present invention and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.
In the description of the present invention, it should be noted that, for the terms of orientation, such as "central", "lateral", "longitudinal", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "clockwise", "counterclockwise", etc., it indicates that the orientation and positional relationship shown in the drawings are based on the orientation or positional relationship shown in the drawings, and is only for the convenience of describing the present invention and simplifying the description, but does not indicate or imply that the device or element referred to must have a specific orientation, be constructed in a specific orientation, and be operated without limiting the specific scope of protection of the present invention.
Furthermore, if the terms "first" and "second" are used for descriptive purposes only, they are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features. Thus, a definition of "a first" or "a second" feature may explicitly or implicitly include one or more of the features, and in the description of the invention, "a number" means two or more unless explicitly defined otherwise.
In the present invention, unless otherwise expressly specified or limited, the terms "assembled", "connected", and "connected" are to be construed broadly, e.g., as meaning fixedly connected, detachably connected, or integrally connected; or may be a mechanical connection; the two elements can be directly connected or connected through an intermediate medium, and the two elements can be communicated with each other. The specific meanings of the above terms in the present invention can be understood by those of ordinary skill in the art according to specific situations.
The invention provides a method for degrading phenol by utilizing deep-sea halotolerant bacteria,
1. enrichment of deep sea halotolerant bacteria
(1) Preparation of culture medium
The formulation of 1L of 2216E liquid culture medium used in the step is as follows: 5g of peptone, 1g of yeast extract, 0.1g of ferric citrate, 19.45g of NaCl, and MgCl2 5.98g、Na2SO4 3.24g、CaCl2 1.8g、KCl 0.55g、Na2CO3 0.16g、KBr 0.08g、SrCl2 0.034g、H3BO3 0.022g、Na2SiO3 0.04g、NaF 0.0024g、NH4NO3 0.0016g、Na2HPO40.008g, heated and dissolved in 1000mL of distilled water, and autoclaved at 121 ℃ for 15 minutes at natural pH for later use.
(2) Enrichment of mixed bacteria liquid
A sample of deep sea bottom mud (1.0 g) was added to 100mL of a medium containing sea salt (hereinafter, 2216E) and cultured at 30 ℃ for 5 days with a shaker at 150rpm, and then transferred to a fresh medium treated in the same manner with an inoculum size of 10% (v/v) and continuously subjected to enrichment culture four times. And detecting the concentration of the strains in the enrichment solution of the fifth generation by adopting an ultraviolet spectrophotometer. When the fifth generation culture solution is cultured for 5 days, the bacterial concentration can be more than 1 × 109When the strain/mL is reached, the enrichment culture is finished, and the obtained enrichment culture bacterial liquid is named as NH-1.
(3) The bacterial concentration test method comprises the following steps: ultraviolet spectrophotometry
(I) preparation of a bacterial concentration Standard Curve
Counting the bacterial suspension cultured for 24 hours by using a blood cell counting plate, putting 1mL of the bacterial suspension into a centrifuge, centrifuging for 5min at 3000rpm, transferring the supernatant into another clean centrifuge tube with 1.5mL, centrifuging for 5min at 10000rpm, pouring off the supernatant, and respectively diluting with sterile water to adjust the concentration to 3.2 multiplied by 10 bacteria content per milliliter8、1.6×108、8×107、4×107、2×107And 1X 107Then determining the OD of each tube at 660nm, using sterile water as blank, and finally using the Optical Density (OD) value as ordinate and the number of cells per ml as abscissaAnd marking, and drawing a bacteria concentration standard curve.
(II) determination of actual bacterial concentration
Centrifuging 1mL of actual bacteria sample at 3000rpm for 5min, transferring the supernatant into another clean 1.5mL centrifuge tube, centrifuging at 10000rpm for 5min, pouring off the supernatant, washing the bacteria pellet at the bottom of the centrifuge tube with 1mL of sterile water, vortexing for 3min, and determining OD600And (4) calculating the concentration of the bacteria through a bacteria concentration standard curve.
2. Acclimatization of salt tolerance of mixed strain
And (3) performing acclimatization culture on the enrichment culture bacterial liquid, and performing gradient acclimation on the salt tolerance of the enrichment culture bacterial liquid (NH-1) by adopting a method for gradually increasing the NaCl concentration in a culture medium of the acclimatization culture.
In the culture medium of the acclimatization culture, the concentration of NaCl is adjusted to 0% to 12%, specifically 0%, 2%, 4%, 6%, 8%, 10%, 12%, or any value between any two of the above values.
Inoculating the enrichment culture bacterial liquid (NH-1) into a fresh domesticated culture medium with the inoculation amount of 10% (v/v), wherein the domesticated culture conditions comprise the temperature of 28-30 ℃, the rotating speed of 120-150r/min and the culture time of 5-7 days, then measuring the concentration of the strain in the culture medium to determine whether the strain can tolerate the salt concentration under the setting, and each group of experiment setting is repeated for three times.
The culture medium for domestication culture comprises the following components in addition to NaCl: 5g of peptone and 1g of yeast extract powder are heated and dissolved in 1000mL of distilled water, and sterilized for 15 minutes at 121 ℃ under high pressure at natural pH for later use.
As can be seen from fig. 2: with the increase of the salt concentration, the growth condition of the strains is inhibited to a certain degree, but the flora grows well under the salt concentration of 2% -10%, and the maximum bacteria concentration can reach 109cells/mL, which shows that the maximum NaCl tolerance of the deep sea enriched strain (NH-1) can reach 10% after the gradient acclimation.
3. Degradation capability of strain to phenol with different initial concentrations
Preparing phenol degradation culture medium with different concentrations, wherein the concentration of phenol is 200-1200mg/L, specifically 200mg/L, 400mg/L, 600mg/L, 800mg/L, 1000mg/L, 1200mg/L, or any value between any two values. Inoculating the enriched bacterial liquid (NH-1) into a freshly prepared phenol degradation culture medium in an inoculation amount of 10% (v/v), wherein the conditions of the phenol degradation culture comprise that the temperature is 28-30 ℃, the rotating speed is 120-150r/min, the culture time is 5-7 days, then measuring the degradation condition of the strain on the phenol, and setting each group of experiments for three times.
The phenol degradation culture medium comprises the following components in addition to phenol: 5g of peptone, 1g of yeast extract powder and 100g of NaCl, heating and dissolving in 1000mL of distilled water, and autoclaving at 121 ℃ for 15 minutes for later use.
(1) The method for measuring the phenol concentration comprises the following steps: ultraviolet spectrophotometry
The phenol has a stable maximum absorption peak at the wavelength of 270nm, and the concentration and OD270nmHas good linear relation (y is 15.34x-0.0011, wherein R is20.9998, y is OD270nm value, x is phenol concentration), so the phenol degradation rate was calculated as (non-inoculated control phenol concentration-inoculated culture phenol concentration)/non-inoculated control phenol concentration x 100% by measuring the phenol content in the inoculated culture solution and the non-inoculated control culture solution using ultraviolet spectrophotometry. Finally, repeating the experiment for three times to obtain the phenol degradation rate of the culture solution of the inoculated NH-1 strain.
(C0: phenol concentration was compared without inoculation; c: inoculation Medium phenol concentration)
As can be seen from fig. 4: the deep sea enrichment culture bacterial liquid (NH-1) has a good degradation effect on phenol, can resist phenol of 1000mg/L, has a good degradation effect within the range of 1000mg/L of phenol, and has a degradation rate of over 90 percent.
4. Target strain community composition analysis (Men and genus level)
When the deep sea salt-tolerant flora domestication culture solution is cultured, the change of microbial community structures in different culture periods is inspected. Inoculating the prepared deep-sea halotolerant bacteria domestication culture solution into a phenol degradation culture medium containing 10% NaCl and 1000mg/L phenol in an inoculation amount of 10%, and performing constant-temperature constant-speed culture at 29 ℃ at 120 r/min.
When the mixed bacteria microbial community is cultured to the 2 nd day, the 7 th day and the 10 th day, respectively taking 100mL of bacterial liquid, firstly centrifuging at the rotating speed of 3000rpm for 10min and at the rotating speed of 12000 rpm for 10min, then collecting thalli, placing the thalli in a refrigerator at the temperature of-80 ℃ for storage, and sending the thalli to Tianjinnuo and kinogenic organism detection company to detect the V4 area of 16S rRNA of the mixed bacteria microbial community, wherein the detection results are as follows:
the results of gene sequencing by 16S rRNA are shown in fig. 1: after the microbial community structure of the deep sea salt-tolerant flora is contacted with phenol, the microbial community structure is obviously changed along with the extension of treatment days, and the specific expression is as follows: in species with the genus level and the top 10 of species abundance, the abundance of the halomonas is always higher than 36%, and is positioned in the top two of species abundance ranks, the halomonas is reported in documents to be a type of salt-tolerant bacteria capable of growing under the condition that the NaCl concentration is 0.1% -32%, and the halomonas is distributed in extreme environments of salt lakes, salt fields, saline-alkali lands, sea ice and oceans, which explains why the mixed flora in the invention can tolerate salt stimulation with higher concentration. Compared with the halomonas, the abundance occupied by the microorganisms of the other 9 genera is improved to different degrees. The most obvious change is the bacteria of the marine genera and the marine genera, wherein the abundance of the marine genera rises from 23.1 percent to 43.6 percent in the middle period of treatment and 38.0 percent in the final period; the marine bacillus is increased from 0.54% to 0.61% (middle stage) and 6.8% (later stage). Both types of bacteria are offshore microorganisms, which, in addition to being able to withstand high salt environments, also have the ability to degrade organic contaminants. Therefore, the deep sea salt-tolerant flora can not only tolerate the salt stimulation with higher concentration, but also effectively degrade the phenol in the water.
5. Investigating the treatment effect of deep-sea salt-tolerant flora on different types of phenol-containing wastewater
(1) After the pH of three different phenol-containing waste waters (A, B, C) were measured, the pH of the phenol-containing waste water was adjusted to 6.5 to 7.5 with 0.1mol/L NaOH solution, and the waste water was diluted with sterile water to a suitable phenol concentration as shown in Table 1.
(2) Culturing the obtained deep-sea salt-tolerant flora domestication culture solution to logarithmic phase (with the bacteria concentration of 10)9cells/mL) is inoculated into three different phenol wastewater obtained in the step (1) by an inoculation amount of 20%, peptone and yeast extract are added into the phenol wastewater at a ratio of 0.1g/L to serve as exogenous nutrient substances, constant-temperature constant-speed culture is carried out for 10 days at 29 ℃ and 120r/min, the concentration of metal ions in the supernatant of each culture solution is detected every two days, and the test results are shown in Table 1.
(3) The removal rate of the deep sea halotolerant flora domestication culture solution to phenol along with time is shown in fig. 6, the deep sea halotolerant flora has an obvious treatment effect on three phenol-containing water bodies, the degradation rate of phenol is 92.4-100% after the deep sea halotolerant flora is cultured for 10 days, the domestication flora also has a good treatment effect on high-salt phenol wastewater, and 92.4% of phenol can be degraded under the conditions that the salt content is 8% and the phenol content is 750.0 mg/L.
The invention shows that the deep-sea halotolerant flora NH-1 has the advantages of wide salinity adaptation range, high salt tolerance content, strong phenol degradation capability, high degradation rate and the like, and has great application potential in the biological treatment of high-salt phenol wastewater.
TABLE 1 removal efficiency of salt-tolerant deep-sea flora after 10 days treatment of three phenol wastewaters
The foregoing is merely illustrative of the preferred embodiments of the present invention and is not to be construed as limiting the claims. The present invention is not limited to the above embodiments, and the specific structure thereof is allowed to vary. All changes which come within the scope of the invention as defined by the independent claims are intended to be embraced therein.
Claims (9)
1. A breeding method of deep sea salt-tolerant phenol degrading bacteria is characterized by comprising the following steps: the method comprises the following steps
(1) Enriching the deep sea halotolerant bacteria;
(1.1) preparing a culture medium;
(1.2) enrichment of mixed bacterial liquid: adding a deep sea sediment sample into a culture medium for culturing for 5 days, then transferring the deep sea sediment sample into a fresh culture medium which is treated by the same method in an inoculation amount (v/v) of 10 percent, and continuously carrying out enrichment culture for four times; detecting the concentration of the strains in the enrichment solution of the fifth generation; when the culture solution of the fifth generation is cultured for 5 days, the concentration of the bacteria is more than 1 multiplied by 109When the strain per mL is detected, finishing the enrichment culture to obtain an enrichment culture bacterial liquid;
(2) and domesticating the salt tolerance of the mixed strain: and (3) performing acclimatization culture on the enrichment culture bacterial solution, and performing gradient acclimatization on the salt tolerance of the enrichment culture bacterial solution by adopting a method for gradually increasing the NaCl concentration in a culture medium of the acclimatization culture.
2. The method for breeding the deep-sea salt-tolerant phenol degrading bacteria according to claim 1, characterized in that: the formula of the culture medium in the step (1) comprises the following components in parts by weight: 5 parts of peptone, 1 part of yeast extract powder, 0.1 part of ferric citrate, 19.45 parts of NaCl, and MgCl25.98 parts of Na2SO43.24 parts of CaCl21.8 parts, KCl 0.55 parts, Na2CO30.16 part, 0.08 part of KBr and SrCl20.034 part of H3BO30.022 parts of Na2SiO30.04 part of NaF 0.0024 part of NH4NO30.0016 part of Na2HPO40.008 part of the extract is dissolved in distilled water under heating and then autoclaved.
3. The breeding method of the deep-sea salt-tolerant phenol degrading bacteria according to claim 2, characterized in that: autoclaving was carried out at a natural pH of 121 ℃ for 15 minutes.
4. The breeding method of the deep-sea salt-tolerant phenol degrading bacteria according to claim 2, characterized in that: the culturing conditions in the step (1.2) are 30 ℃ and 150rpm shake culture for 5 days.
5. The method for breeding the deep-sea salt-tolerant phenol degrading bacteria according to claim 1, characterized in that: the method for detecting the concentration of the strains in the fifth generation enrichment liquid in the step (1.2) specifically comprises the following steps
(1.2.1) preparation of a bacterial concentration Standard Curve
Counting the bacterial suspension cultured for 24 hours by using a blood cell counting plate, putting 1mL of the bacterial suspension into a centrifuge, centrifuging for 5min at 3000rpm, transferring the supernatant into another clean centrifuge tube with 1.5mL, centrifuging for 5min at 10000rpm, pouring off the supernatant, and respectively diluting with sterile water to adjust the concentration to 3.2 multiplied by 10 bacteria content per milliliter8、1.6×108、8×107、4×107、2×107And 1X 107Then determining the OD value of each tube at the wavelength of 660nm, taking the OD value as a longitudinal coordinate and the number of cells per milliliter as a horizontal coordinate to draw a bacteria concentration standard curve at the moment by taking sterile water as a blank control;
(1.2.2) measurement of actual cell concentration
Centrifuging 1mL of actual bacteria sample at 3000rpm for 5min, transferring the supernatant into another clean 1.5mL centrifuge tube, centrifuging at 10000rpm for 5min, pouring off the supernatant, washing the bacteria pellet at the bottom of the centrifuge tube with 1mL of sterile water, vortexing for 3min, and determining OD600And (4) calculating the concentration of the bacteria through a bacteria concentration standard curve.
6. The method for breeding the deep-sea salt-tolerant phenol degrading bacteria according to claim 1, characterized in that: in the culture medium for acclimatization culture in the step (2), the concentration of NaCl is adjusted to 0% to 12%, specifically, 0%, 2%, 4%, 6%, 8%, 10%, 12%, or any value between any two of the above values.
7. The method for breeding the deep-sea salt-tolerant phenol degrading bacteria according to claim 1, characterized in that: the conditions of the domestication culture in the step (2) comprise that the temperature is 28-30 ℃, the rotating speed is 120-150r/min, the culture time is 5-7 days, then the concentration of the strain in the culture medium is measured to determine whether the strain can tolerate the salt concentration under the setting, and each group of experiment setting is repeated for three times.
8. The method for breeding the deep-sea salt-tolerant phenol degrading bacteria according to claim 1, characterized in that: the culture medium domesticated and cultured in the step (2) comprises the following components in parts by mass besides NaCl: heating and dissolving 5 parts of peptone and 1 part of yeast extract powder in distilled water, and carrying out autoclaving at 121 ℃ for 15 minutes at natural pH for later use.
9. The application of the deep sea salt tolerant phenol degrading bacteria bred by the breeding method according to any one of claims 1 to 8 in high salt organic industrial wastewater.
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| 黄中子等: "一株耐盐菌对苯酚的降解及其影响因素研究", 水处理技术 * |
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