CN111088187A - Charcoal-based immobilized polycyclic aromatic hydrocarbon efficient degradation microbial inoculum and preparation method and application thereof - Google Patents
Charcoal-based immobilized polycyclic aromatic hydrocarbon efficient degradation microbial inoculum and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a high-efficiency degrading microbial inoculum for fixed polycyclic aromatic hydrocarbon based on biochar, which comprises a biochar-based material and a bacterial strain adsorbed on the biochar-based material. The biochar curing microbial inoculum is used for degrading polycyclic aromatic hydrocarbon. According to the invention, the biochar curing microbial inoculum with the maximum attached microbial biomass and the best removal effect is prepared by optimizing the curing culture medium and the curing time, and the removal rates of the 2 microbial inocula for 16h to phenanthrene can respectively reach 93.08% and 66.36%; compared with free bacteria which basically lose activity after 7 days at room temperature, the biochar solidifying microbial inoculum still has a good removal effect, the removal rate of phenanthrene can still reach 71.78% and 40.72% respectively, and obviously, the biochar can keep the activity of the bacteria and prolong the survival time of microorganisms.
Description
Technical Field
The invention belongs to the technical field of microbial agents, and particularly relates to a biochar-based immobilized polycyclic aromatic hydrocarbon efficient degrading microbial agent as well as a preparation method and application thereof.
Background
Because oil exploitation causes severe Polycyclic Aromatic Hydrocarbons (PAHs) pollution to soil around oil fields, the PAHs are typical persistent organic pollutants in the soil, and toxicity and carcinogenicity of the PAHs have great harm to the environment, so the PAHs are prioritized to control pollutants by the United states environmental protection agency and the European Union. Therefore, a method for exploring how to treat PAHs efficiently, effectively, economically and economically under high-salt conditions has become one of the hotspots of the current environmental protection.
Currently, the soil PAHs are degraded by microorganisms, which is considered to be the best way for biologically repairing the pollution of the soil PAHs at present, for example, Chinese patent CN 107881126A discloses a moderately halophilic bacterium for efficiently degrading multiple polycyclic aromatic hydrocarbons and application thereof, the moderately halophilic bacterium is enriched and screened from polycyclic aromatic hydrocarbon polluted saline-alkali soil in a Shengli oil field, identified and named as Martelella sp.AD-3(CCTCC M2011218), and the strain is preserved in China Center for Type Culture Collection (CCTCC) for 6-30 days in 2011. The strain can efficiently degrade various pollutants such as biphenyl, acenaphthylene, fluorene, phenanthrene, anthracene, fluoranthene, benzo [ a ] anthracene and the like in the salinity of 0.5-15% and the pH value of 6-10. Within 36h, the degradation rate of the fluorene with the concentration of 100mg/L can reach 100%. The optimal degradation condition of the AD-3 bacteria in the soil is pH9.0, the salt content is 15g/kg, the water content is 25 percent, and the temperature is 30 ℃. Under the optimal conditions, the AD-3 bacteria have obvious removal effect on acenaphthylene, biphenyl, fluorene, phenanthrene, anthracene and the like in soil, wherein the removal rate of acenaphthylene and biphenyl reaches 100%.
However, in addition to the structural specificity of PAHs and their low water solubility which limits their microbial degradation, PAHs are also involved in their difficult transfer from soil to bacterial cells, high molecular weight PAHs are difficult to transfer from soil to bacterial cells and have low bioavailability.
The biochar has larger specific surface area and rich porosity, can provide better habitat for the growth and the propagation of microorganisms, and provides nutrient substances for the microorganisms attached to the biochar, so the biochar can be used as a carrier for immobilizing bacteria, and can more effectively promote the removal of polycyclic aromatic hydrocarbons in soil. The use of biochar to fix microorganisms for remediation of organic contaminated soil has been widely focused, but there are few studies related to screening and optimization of curing conditions of biochar as a curing carrier.
Xiong (Xiong B, Zhang Y, Hou Y et al. enhanced biological degradation of PAHs in biological contaminated soil by M. gilvum in encapsulated biological J. Chemosphere,2017,182: 316-.
Garcia-Delgado C (Garcia-Delgado C, Alfaro-Barta I, Eymar E. combination of biochemical and biochemical analysis for multicyclic aromatic hydrosynthesis and biodegradation in biological contaminated soil [ J ]. Journal of hazardous Materials,2015,285(285): 259) 266.) uses the biochar in combination with P.ostreatus method to remediate PAHs contaminated soil, Phe, Ant degradation rates in soil after remediation 42 were 62%, 79%, respectively, in combination with fungi.
The maClear (maClear. biochar-based immobilized microorganism and repair research on petroleum-polluted soil [ D ].2017.) degrades petroleum hydrocarbon pollutants by solidifying microorganisms with three biochar including corncobs, straws and sawdust, wherein the oil removal rates of the three biochar microbial inoculums are 59.3% -70.7%, 48.7% -58.3% and 45.9% -54.7% respectively.
Chinese patent CN109628353A discloses a biochar-based immobilized microbial agent and a preparation method and application thereof. The biochar-based immobilized microbial agent comprises a biochar-based material, and thalli and a nutrient which are adsorbed on the biochar-based material, wherein the thalli is a Mycobacterium CSC-6(Mycobacterium sp.CSC-6), is preserved in the China Center for Type Culture Collection (CCTCC) in 2017, 11 and 27 months, and has the preservation number: CCTCC No. M2017726. The biochar-based immobilized microbial agent is used for degrading petroleum.
In summary, the current research mainly focuses on the application of the biochar immobilized microorganisms, and the screening indexes of the biochar carriers, the preparation process and the optimization process of the biochar immobilized microorganisms pay less attention.
Disclosure of Invention
The invention aims to provide a high-efficiency degradation microbial inoculum for biochar-based fixed polycyclic aromatic hydrocarbons, and a preparation method and application thereof, so as to obtain the high-efficiency degradation microbial inoculum for biochar-based fixed polycyclic aromatic hydrocarbons with good activity and degradation effect, and further solve the problem of low degradation rate of polycyclic aromatic hydrocarbons in the prior art.
The purpose of the invention can be realized by the following technical scheme:
a high-efficiency degrading microbial inoculum for fixed polycyclic aromatic hydrocarbons on a charcoal base comprises a charcoal base material and a bacterial strain adsorbed on the charcoal base material, wherein the bacterial strain is a moderate halophilic bacterium Martelella sp.AD-3 with the preservation number of CCTCC M2011218, and the bacterial strain is preserved in a Chinese typical culture preservation center in 2011 for 6 and 30 days, and the addresses are as follows: wuhan university school of eight-channel 299 # in Wuchang area of Wuhan city, Hubei province. The strain is screened from petroleum-contaminated soil. The strain is disclosed in Chinese patent CN 107881126A.
The biochar-based material comprises cotton stalk biochar and rice hull biochar.
The preparation method of the charcoal-based fixed polycyclic aromatic hydrocarbon high-efficiency degradation microbial inoculum comprises the following steps:
selecting the strain to activate, transferring the activated bacterial liquid into 3% LB containing phenanthrene, and performing enlarged culture to obtain a seed liquid;
adding the seed solution subjected to the expanded culture into a liquid culture medium containing a biochar-based material, mixing and incubating, and removing excess culture medium to obtain the biochar-based fixed polycyclic aromatic hydrocarbon efficient degrading microbial inoculum.
Further, 15-30mL, preferably 20mL, of AD-3 bacterial liquid needed for curing per 1g of the biochar-based material, namely OD (oxygen-free radical) bacterial liquid600In the range of 0.6-1.5, the amount of the contained bacteria is 20-40 × 107CFU/mL。
Further, the preparation method of the charcoal-based fixed polycyclic aromatic hydrocarbon high-efficiency degradation microbial inoculum comprises the following specific steps:
firstly, selecting a strain from an LB flat plate for storing Martelella sp.AD-3 by using an inoculating loop, inoculating the strain into 5-10mL of nutrient solution, and activating for 24-48h in a shaking culture box at the temperature of 20-35 ℃ and the rotating speed of 120-150 r/min;
secondly, inoculating the activated bacterial liquid into a 3% LB culture medium containing 50-100mg/L phenanthrene for amplification culture according to an inoculation amount of 1-5% of the volume ratio, and activating for 36-48h in a shaking culture box at 20-35 ℃ and a rotation speed of 120-150r/min to obtain a seed liquid; the phenanthrene is included to provide a certain PAHs selection pressure.
And (III) taking a quantitative seed solution, suspending thallus according to the solidification culture medium, adding a charcoal base material, and solidifying for 24-72 hours in an oscillation culture box at the temperature of 20-35 ℃ and the rotating speed of 100-150r/min to obtain the charcoal base fixed polycyclic aromatic hydrocarbon high-efficiency degrading microbial inoculum.
The nutrient solution in the step (I) comprises the following components (g/L): peptone 8.0-12.0; yeast powder 4.0-6.0; and (5) NaCl 8-12.
In the step (III), the solidifying medium is selected from 1# solidifying medium, 2# solidifying medium or 3# solidifying medium, preferably 2# solidifying medium,
the composition of No. 1 solidified medium (g/L) is as follows: peptone 8.0-12.0; yeast powder 4.0-6.0; 24-36 parts of NaCl; the bacterial load of AD-3 is 20-40 × 107CFU/mL；
The composition of No. 2 solidified medium (g/L) is: peptone 8.0-12.0; yeast powder 4.0-6.0; 24-36 parts of NaCl; the bacterial load of AD-3 is 20-40 × 106CFU/mL；
The composition of No. 3 solidified medium (g/L) is as follows: 15-30 parts of NaCl; NH (NH)4Cl 0.5-2.0；MgCl2·6H2O 2-5；MgSO4·7H2O 3-10；CaCl20.1-1；KCl 0.5-2；NaHCO30.01-0.1; 0.1-0.2 NaBr, 3-5% total salinity, adding microelement solution at a ratio of 0.5-1.0mL/L, pH 8.0-9.0, and AD-3 bacteria amount of 20-40 × 107CFU/mL，
Microelement solution (g/L): ca (NO)3)2·4H2O 2-5；FeSO4·7H2O 1-3；CuSO4·5H2O，0.1-0.5；ZnSO4·7H2O 0.1-0.5；MnSO4·4H2O 0.1-0.5；NaMO4·2H2O 0.01-0.1；HBO30.01-0.1。
In the invention, the 2# culture medium has a good effect, which is probably because the 3# culture medium mainly adopts inorganic salt which cannot provide enough nutrition for the production of the microorganism, so the microorganism can only be adsorbed onto the biochar from the bacterial liquid in the 3# culture medium, and the microorganism cannot be rapidly propagated on the biochar in a large quantity. Although the 1# culture medium can provide enough nutrition for the growth of microorganisms, the SEM picture shows that the amount of microorganisms attached to the surface of the biochar microbial inoculum prepared by the 1# culture medium is significantly smaller than that of the 2# culture medium, which is probably because the amount of microorganisms in the 1# culture medium is much larger than that of the 2# culture medium, so that the microorganisms adsorbed on the biochar cannot obtain enough nutrition for growth and propagation, and the 2# culture medium has the best effect.
In the step (three), the curing time is preferably 48 hours.
The microbial inoculum is the most in surface attached microbial biomass when solidified for 2d, and a great amount of AD-3 microbial cells attached to the surface of the biochar can be seen from an SEM image of the biochar microbial inoculum solidified for 2 d; AD-3 bacteria are sporadically attached to the surfaces of the biochar bacteria of 1d and 3d in a curing mode, and the attached microbial biomass is obviously smaller than that of the solidified bacteria of 2d in a curing mode. Duyong [ Duyong, research on removal of phenol in water by using biochar immobilized microorganisms [ D ]. Chongqing university, 2012] research shows that the biochar immobilized microorganisms are mainly in the initial adsorption stage, the adsorption of the microorganisms is saturated and the adsorption is completed, and the microorganisms start to metabolize and secrete extracellular polymers on the surfaces of the biochar to form biofilms. The lower amount of microorganisms in the electron micrograph of the cured 1d sample according to the present invention is probably because the slow growth phase of microorganisms is being established at the initial stage of adsorption, and the lower amount of microorganisms in the system results in a smaller amount of microorganisms that can attach to the system. The number of microorganisms visible in the electron micrograph of the cured 3d sample is also small, and the curing 2d sample has the best effect probably because the microorganism is in the death phase of growth at 72 h.
In the step (III), the adding amount of the biochar-based material is 1-10% by weight of the biochar-based material and volume of the culture medium.
In the step (III), the biochar-based material is sterilized before being added, and one optional mode of the sterilization treatment is as follows: and (3) placing the conical flask made of the biochar-based material in a high-temperature and high-pressure sterilization mode at the temperature of 121 ℃ for 20min, and cooling to the normal temperature.
In the step (III), after solidification culture, filtering out a biochar microbial inoculum by using a 200-mesh filter screen, washing the microbial inoculum by using 0.2mol/L phosphate buffer (pH is 7.4), adding a stationary liquid for fixation for 4 hours, centrifuging to remove a supernatant, washing for 2 times, placing a sample in 30%, 50%, 70%, 90% and 100% ethanol solutions for gradient dehydration, and freeze-drying to obtain the biochar-based fixed polycyclic aromatic hydrocarbon efficient degrading microbial inoculum.
The fixative solution may be a glutaraldehyde solution. The fixing liquid (glutaraldehyde) is used for fixing the structural appearance of the cells and preventing the cells from deforming and breaking in the processes of dehydration and freeze drying. .
The reason for gradient dehydration is: because ethanol is an organic solvent with low surface tension, free water in tissue cells can be replaced, so that the surface tension of the sample is reduced during drying, and the damage to the surface of the sample is reduced to the maximum extent. If dehydrated sharply, shrinkage is caused. Therefore, the water is removed by adopting an ethanol gradient dehydration method from low concentration to high concentration
The invention preliminarily screens 2 biochar-based materials of rice hulls and cotton stalks as curing carriers from a plurality of biochar in a laboratory through the specific surface area, the porosity, the C/H and the removal rate of biochar curing AD-3 microbial inoculum to phenanthrene and anthracene, researches microbial masses attached to the microbial inoculum prepared by different curing media and different curing time, and preferably selects optimal preparation conditions. Meanwhile, the activity change of the microbial inoculum stored in a refrigerator at 4 ℃ and at room temperature is investigated, and a scientific reference basis is provided for further application of the immobilized microbial inoculum to the polluted soil.
The biochar-cured polycyclic aromatic hydrocarbon efficient degrading microbial inoculum prepared by optimizing the curing culture medium and the curing time has higher degrading capability on polycyclic aromatic hydrocarbon, and the degrading rates of the cotton stalk curing microbial inoculum and the rice hull curing microbial inoculum on high-concentration sewage containing 200mg/L phenanthrene in 16 hours can respectively reach: 93.08% and 66.36%. After the microbial inoculum is stored in a refrigerator at 4 ℃ for 14 days, the activity of 2 biochar curing microbial inocula is not reduced basically, and the degradation rate of the 1# microbial inoculum is reduced by 25.5%; the microbial inoculum is stored at room temperature, the 1# microbial inoculum has no activity basically after 7 days, and the cotton stalk solidified microbial inoculum and the rice hull solidified microbial inoculum still have activity, which indicates that the charcoal can keep the activity of the bacteria and prolong the survival time of microorganisms.
Compared with the prior art, the invention has the beneficial effects that:
(1) the biochar-based solidified microbial inoculant has high degradation rate on polycyclic aromatic hydrocarbon, and the removal rate of the cotton stalk solidified inoculant and the rice hull solidified inoculant to phenanthrene can reach 93.08% and 66.36% respectively within 16 h.
(2) The charcoal-based solidified microbial inoculum optimizes the solidified microbial inoculum from a solidified culture medium and solidified time, and obtains the optimal conditions for preparing the microbial inoculum: the biochar solidified AD-3 microbial inoculum prepared by the 2# culture medium and the solidified 2d has the best removal effect.
(3) The biochar-based solidified microbial inoculant has good activity, after the solidified microbial inoculant and free bacteria are placed at room temperature for 7 days, the free bacteria are degraded and have no degradation activity basically, the solidified microbial inoculant still has good activity, and the removal rates of the cotton stalk and the rice hull biochar inoculant to phenanthrene after 7 days are 71.78% and 40.72% respectively; therefore, compared with free bacteria, the charcoal can maintain the activity of the bacteria and prolong the survival time of the microorganisms.
The preparation method and the preparation condition of the biochar-based solidified microbial agent can be optimized, so that the preparation method has practical significance for preparing the biochar-based solidified microbial agent.
Drawings
FIG. 1 is a scanning electron microscope image of a cotton stalk biochar-based material, a cotton stalk biochar material and a # 1 microbial inoculum;
in fig. 1, (a) surface structure of cotton stalk biochar (x 3500) (B) surface structure of rice hull biochar (x 3500) (C)1# microbial inoculum free AD-3 bacteria (x 3500).
FIG. 2 is a scanning electron microscope image of 2# microbial inoculum and 3# microbial inoculum prepared at different curing times;
in fig. 2, (a) a 2# microbial inoculum surface structure cured for 1D (× 3500) · (B) a 2# microbial inoculum surface structure cured for 2D (× 3500) · (C) a 2# microbial inoculum surface structure cured for 3D (× 3500) · (D) a 3# microbial inoculum surface structure cured for 1D (× 3500) · (E) a 3# microbial inoculum surface structure cured for 2D (× 3500) · (F) a 3# microbial inoculum surface structure cured for 3D (× 3500).
FIG. 3 is a scanning electron microscope image of 2# microbial inoculum and 3# microbial inoculum prepared by different solidified culture mediums;
(A) preparing a 2# microbial inoculum surface structure (X3500) by using a 1# solidification medium, (B) preparing a 2# microbial inoculum surface structure (X3500) by using a 2# solidification medium, (C) preparing a 2# microbial inoculum surface structure (X3500) by using a 3# solidification medium, (D) preparing a 3# microbial inoculum surface structure (X3500) by using a 1# solidification medium, (E) preparing a 3# microbial inoculum surface structure (X3500) by using a 2# solidification medium, (F) preparing a 3# microbial inoculum surface structure (X3500) by using a 3# solidification medium.
FIG. 4 shows the activity of the charcoal-immobilized microbial inoculum after being stored in a refrigerator at 4 ℃ for 7 days and 14 days;
FIG. 5 shows the activity of the biological charcoal solidified microbial inoculum after being stored for 7 days and 14 days at room temperature.
Detailed Description
The invention is described in detail below with reference to the figures and specific embodiments.
Example 1
Preparation of biochar-based solidified microbial agent
A strain is selected from LB plate for storing AD-3(Martelella sp. AD-3) by using an inoculating loop and inoculated into a nutrient solution of 5m L, wherein the nutrient solution comprises the following components (g/L): peptone 8.0-12.0; yeast powder 4.0-6.0; NaCl8-12, and activating for 36 hours in a shaking incubator at the temperature of 30 ℃ and the rotating speed of 150 r/min;
inoculating 5m of activated bacterial liquid into 200mL of 3% LB for amplification culture, adding 50mg/L phenanthrene to provide a certain PAHs selection pressure, culturing at 30 ℃ and a rotation speed of 150r/min for 48h, taking out a conical flask, and repeatedly cleaning the thalli for 3 times.
Taking the strain liquid after the amplification culture according to the inoculation amount of 10 percent of the volume ratio, adding the strain liquid into a conical flask filled with 20mL of 3 percent LB, and culturing under the conditions that: the rotating speed is 100r/min, the temperature is 30 ℃, the culture time is 48h, after the culture is finished, the culture is centrifuged on a high-speed centrifuge of 8000r/min, and the supernatant is removed, thus obtaining the No. 1 microbial inoculum. The scanning electron microscope images of the cotton stalk biochar-based material, the cotton stalk biochar material and the No. 1 microbial inoculum are shown in figure 1.
Weighing 1.0g of cotton stalk biochar, placing the cotton stalk biochar in a 100mL conical flask, sterilizing the cotton stalk biochar at the high temperature of 121 ℃ for 20min under high pressure, cooling the cotton stalk biochar to the normal temperature, adding 20mL of different solidification culture media with resuspended thalli, placing the cotton stalk biochar in a constant-temperature oscillation incubator at the temperature of 30 ℃ and the rotating speed of 100r/min for fixing for different times, filtering the biochar microbial inoculum by using a 200-mesh filter screen, cleaning the suspended thalli on the surface by using sterilized saline water, and repeating the steps for 2 times to obtain.
Weighing 1.0g of rice hull biochar in a 100mL conical flask, placing the conical flask in the conical flask, sterilizing the conical flask at the temperature of 121 ℃ for 20min at high temperature and high pressure, cooling the conical flask to the normal temperature, adding 20mL of different solidification culture media with resuspended thalli, placing the conical flask in a constant-temperature oscillation incubator at the temperature of 30 ℃ and the rotating speed of 100r/min for fixing for different times, filtering the biochar microbial inoculum by using a 200-mesh filter screen, cleaning the suspended thalli on the surface by using sterilized saline water, and repeating the steps for 2 times to obtain the.
Example 2
Optimization of biochar-based solidified microbial agent solidified culture medium
The effect of different solidification media on the acquisition of the inoculum was examined according to the method of example 1.
Wherein, the 1# microbial inoculum is not fixed on the biochar-based material, the 2# microbial inoculum is fixed on the cotton stalk biochar-based material, and the 3# microbial inoculum is fixed on the rice husk biochar-based material.
Respectively selecting a 1# solidification culture medium, a 2# solidification culture medium and a 3# solidification culture medium to prepare the cotton stalk and rice hull biochar solidification microbial inoculum, namely weighing 1.0g of cotton stalk biochar or rice hull biochar in a 100mL conical flask, placing the conical flask in the conical flask, sterilizing at the temperature of 121 ℃ for 20min at high temperature and high pressure, cooling to normal temperature, adding 20mL of the 1# solidification culture medium, the 2# solidification culture medium or the 3# solidification culture medium with suspended bacteria, placing the conical flask in a constant-temperature oscillation culture box at the temperature of 30 ℃ and the rotating speed of 100r/min for fixing for 48h, and filtering the biochar microbial inoculum by using a 200-mesh filter screen.
Then, the microbial inoculum was washed with 0.2mol/L phosphate buffer (pH 7.4), then fixed with a fixing solution (glutaraldehyde) for 4 hours, centrifuged to remove the supernatant, washed 2 times, and the sample was subjected to gradient dehydration in 30%, 50%, 70%, 90%, and 100% ethanol solution, and freeze-dried.
After gold spraying, the number of bacteria on the surfaces of the cotton stalk biochar microbial inoculum and the rice hull biochar microbial inoculum is observed through a scanning electron microscope, and the results are shown in fig. 3, the research result shows that the number of microorganisms on the surfaces of the biochar-based curing microbial inoculum prepared by the 2# curing culture medium is the largest, and the curing culture medium of the biochar-based curing microorganisms is determined to be the 2# curing culture medium.
The composition of No. 1 solidified medium (g/L) is as follows: peptone 10; 5.0 of yeast powder; NaCl 30; the bacterial load of AD-3 is 20-40 × 107CFU/mL；
The composition of No. 2 solidified medium (g/L) is: peptone 10; 5.0 of yeast powder; NaCl 30; the bacterial load of AD-3 is 20-40 × 106CFU/mL；
The composition of No. 3 solidified medium (g/L) is as follows: 23.84 parts of NaCl; NH (NH)4Cl 1.0；MgCl2·6H2O 3.85；MgSO4·7H2O 5.23；CaCl20.38；KCl 1.1；NaHCO30.05; NaBr 0.13; 0.2 of yeast powder. The total salinity is 3%, and trace element solution with pH of 9.0 and AD-3 bacteria content of 20-40 × 10 is added at a ratio of 0.5mL/L7CFU/mL. Microelement solution (g/L): ca (NO)3)2·4H2O 3.2；FeSO4·7H2O 1-3；CuSO4·5H2O，0.32；ZnSO4·7H2O0.16；MnSO4·4H2O 0.16；NaMO4·2H2O 0.032；HBO30.024。
Example 3
Optimization of biochar-based solidified microbial agent solidification time
The effect of different curing times on the acquisition of the inoculum was examined as in example 1.
Wherein, the 1# microbial inoculum is not fixed on the biochar-based material, the 2# microbial inoculum is fixed on the cotton stalk biochar-based material, and the 3# microbial inoculum is fixed on the rice husk biochar-based material.
1d, 2d and 3d are respectively selected for curing time to prepare the solidified microbial agent of cotton stalk and rice hull biochar, namely 1.0g of cotton stalk biochar or rice hull biochar is weighed into a 100mL conical flask, the conical flask is placed at 121 ℃ for high-temperature high-pressure sterilization for 20min, the conical flask is cooled to normal temperature, 20mL of 2# curing culture medium with suspended bacteria is added, the conical flask is placed in a constant-temperature oscillation incubator at 30 ℃ and the rotating speed of 100r/min for respectively fixing for 1d, 2d and 3d, and a 200-mesh filter screen is used for filtering the microbial agent of biochar.
Then, the microbial inoculum was washed with 0.2mol/L phosphate buffer (pH 7.4), then fixed with a fixing solution (glutaraldehyde) for 4 hours, centrifuged to remove the supernatant, washed 2 times, and the sample was subjected to gradient dehydration in 30%, 50%, 70%, 90%, and 100% ethanol solution, and freeze-dried.
After gold spraying, the number of bacteria on the surfaces of the cotton stalk biochar microbial inoculum and the rice hull biochar microbial inoculum is observed through a scanning electron microscope, the result is shown in fig. 2, the research result shows that the number of microorganisms on the surfaces of the biochar-based curing microbial inocula prepared by curing for 2d is the largest, and the curing time of the biochar-based curing microorganisms is determined to be 2 d.
Example 4
The charcoal curing microbial inoculum is respectively stored for 7 days and 14 days at the room temperature in a refrigerator at the temperature of 4 ℃ and then the activity of the microbial inoculum is obtained
Preparing a 1# microbial inoculum, a 2# microbial inoculum and a 3# microbial inoculum under an optimal solidification culture medium (2# solidification culture medium) and an optimal solidification time (2d),
preparation of # 1 microbial inoculum was performed as in example 1.
Weighing 1.0g of cotton stalk biochar in a 100mL conical flask, placing the conical flask in the conical flask, sterilizing the conical flask at the temperature of 121 ℃ for 20min at high temperature and high pressure, cooling the conical flask to the normal temperature, adding 20mL of 2# solidified culture medium with resuspended thalli, placing the conical flask in a constant-temperature oscillation incubator at the temperature of 30 ℃ and the rotating speed of 100r/min for fixing for 2d, and filtering the biochar microbial inoculum by using a 200-mesh filter screen. Then, the microbial inoculum is washed by 0.2mol/L phosphate buffer (pH 7.4), then fixing solution (glutaraldehyde) is added for fixing for 4h, the supernatant is removed by centrifugation, the sample is washed for 2 times, and the sample is placed in 30%, 50%, 70%, 90% and 100% ethanol solution for gradient dehydration and freeze drying to obtain the 2# microbial inoculum.
Weighing 1.0g of rice hull biochar in a 100mL conical flask, placing the conical flask in the conical flask, sterilizing the conical flask at the temperature of 121 ℃ for 20min at high temperature and high pressure, cooling the conical flask to the normal temperature, adding 20mL of 2# solidified culture medium with resuspended thalli, placing the conical flask in a constant-temperature oscillation incubator at the temperature of 30 ℃ and the rotating speed of 100r/min for fixing for 2d, and filtering the biochar microbial inoculum by using a 200-mesh filter screen. Then, the microbial inoculum was washed with 0.2mol/L phosphate buffer (pH 7.4), then the fixative (glutaraldehyde) was added for fixation for 4 hours, the supernatant was centrifuged off, the sample was washed 2 times, and the sample was placed in 30%, 50%, 70%, 90%, 100% ethanol solution for gradient dehydration and freeze-dried to obtain # 3 microbial inoculum.
Respectively storing the 1# microbial inoculum, the 2# microbial inoculum and the 3# microbial inoculum in a refrigerator at 4 ℃ and at room temperature, respectively applying the 1# microbial inoculum, the 2# microbial inoculum and the 3# microbial inoculum to a 3% SSDM culture medium added with 200mg/L phenanthrene after 0d, 7d and 14d, sampling, extracting, blowing nitrogen, filtering for 16h, measuring the content of polycyclic aromatic hydrocarbon in a system by using a high performance liquid chromatography, and calculating the removal rate of the 1# microbial inoculum, the 2# microbial inoculum and the 3# microbial inoculum to the phenanthrene.
Referring to fig. 4 and 5, the removal rates of the cotton stalk and the rice hull biochar fungicide prepared by the 2# solidified culture medium and the solidified 2d in 16h can reach 93.08% and 66.36% respectively; under the same condition, the degradation rate of the 1# microbial inoculum to phenanthrene is 88.91%. The degradation rate of the microbial inoculum after 7d and 14d is 64.72 percent and 62.41 percent when the microbial inoculum is placed in a refrigerator at 4 ℃, and is slightly reduced compared with that of a fresh microbial inoculum; the degradation rate of the 2# microbial inoculum is basically not reduced after 7 days and 14 days, and the degradation rates are 92.40 percent and 92.43 percent respectively; the degradation rate of the 3# microbial inoculum is not reduced basically after 7d and 14d, and the degradation rate is 66.88 percent and 66.26 percent respectively.
After the microbial inoculum is placed at room temperature for 7 days, the activity of the biochar curing microbial inoculum is reduced compared with that of a fresh microbial inoculum, the removal rates of MG and DK biochar curing microbial inoculum to phenanthrene are 71.78% and 40.72%, and the removal rates are respectively reduced by 21.30% and 25.65% compared with that of the fresh microbial inoculum; the removal rate of the free bacteria to phenanthrene at room temperature for 7d is only 2.00 percent, and basically no degradation activity exists.
Thus, the biochar immobilizes the microorganisms to a certain extent, relatively speaking, prolonging the survival time of the microorganisms, maintaining the activity of the microorganisms.
The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.
Claims (10)
1. The high-efficiency degrading microbial inoculum for the biochar-based fixed polycyclic aromatic hydrocarbon is characterized by comprising a biochar-based material and a bacterial strain adsorbed on the biochar-based material, wherein the bacterial strain is a moderately halophilic bacterium Martelella sp.AD-3, the preservation number of the bacterial strain is CCTCC M2011218, the bacterial strain is preserved in the China center for typical culture preservation in 2011 for 30 days 6 and 30, and the addresses are as follows: wuhan university school of eight-channel 299 # in Wuchang area of Wuhan city, Hubei province.
2. The biological carbon-based fixed polycyclic aromatic hydrocarbon high-efficiency degrading microbial inoculum according to claim 1, wherein the biological carbon-based material comprises cotton stalk biochar and rice hull biochar.
3. The preparation method of the biochar-based fixed polycyclic aromatic hydrocarbon high-efficiency degrading microbial inoculum as claimed in claim 1 or 2, which is characterized by comprising the following steps:
selecting the strain to activate, transferring the activated bacterial liquid into 3% LB containing phenanthrene, and performing enlarged culture to obtain a seed liquid;
adding the seed solution subjected to the expanded culture into a liquid culture medium containing a biochar-based material, mixing and incubating, and removing excess culture medium to obtain the biochar-based fixed polycyclic aromatic hydrocarbon efficient degrading microbial inoculum.
4. The method for preparing the biochar-based immobilized polycyclic aromatic hydrocarbon high-efficiency degrading microbial inoculum according to claim 3, wherein 15-30mL of AD-3 bacterial liquid is required for curing every 1g of biochar-based material, and OD (oxygen demand) of the bacterial liquid600In the range of 0.6-1.5.
5. The preparation method of the biochar-based immobilized polycyclic aromatic hydrocarbon high-efficiency degrading microbial inoculum according to claim 3, which is characterized by comprising the following steps:
firstly, selecting a strain from an LB flat plate for storing Martelella sp.AD-3 by using an inoculating loop, inoculating the strain into 5-10mL of nutrient solution, and activating for 24-48h in a shaking culture box at the temperature of 20-35 ℃ and the rotating speed of 120-150 r/min;
secondly, inoculating the activated bacterial liquid into a 3% LB culture medium containing 50-100mg/L phenanthrene for amplification culture according to an inoculation amount of 1-5% of the volume ratio, and activating for 36-48h in a shaking culture box at 20-35 ℃ and a rotation speed of 120-150r/min to obtain a seed liquid;
and (III) taking a quantitative seed solution, suspending thallus according to the solidification culture medium, adding a charcoal base material, and solidifying for 24-72 hours in an oscillation culture box at the temperature of 20-35 ℃ and the rotating speed of 100-150r/min to obtain the charcoal base fixed polycyclic aromatic hydrocarbon high-efficiency degrading microbial inoculum.
6. The preparation method of the biochar-based immobilized polycyclic aromatic hydrocarbon high-efficiency degrading microbial inoculum according to claim 5, wherein in the step (three), the solidification culture medium is selected from a 1# solidification culture medium, a 2# solidification culture medium or a 3# solidification culture medium, preferably the 2# solidification culture medium,
the composition of No. 1 solidified medium (g/L) is as follows: peptone 8.0-12.0; yeast powder 4.0-6.0; 24-36 parts of NaCl; the bacterial load of AD-3 is 20-40 × 107CFU/mL；
The composition of No. 2 solidified medium (g/L) is: peptone 8.0-12.0; yeast powder 4.0-6.0; 24-36 parts of NaCl; the bacterial load of AD-3 is 20-40 × 106CFU/mL；
The composition of No. 3 solidified medium (g/L) is as follows: 15-30 parts of NaCl; NH (NH)4Cl 0.5-2.0；MgCl2·6H2O 2-5；MgSO4·7H2O 3-10；CaCl20.1-1；KCl 0.5-2；NaHCO30.01-0.1; 0.1-0.2 NaBr, 3-5% total salinity, adding microelement solution at a ratio of 0.5-1.0mL/L, pH 8.0-9.0, and AD-3 bacteria amount of 20-40 × 107CFU/mL，
Microelement solution (g/L): ca (NO)3)2·4H2O 2-5；FeSO4·7H2O 1-3；CuSO4·5H2O，0.1-0.5；ZnSO4·7H2O 0.1-0.5；MnSO4·4H2O 0.1-0.5；NaMO4·2H2O 0.01-0.1；HBO30.01-0.1。
7. The preparation method of the biochar-based immobilized polycyclic aromatic hydrocarbon high-efficiency degrading microbial inoculum according to claim 5, wherein in the step (III), the curing time is 48 hours.
8. The method for preparing the biochar-based immobilized polycyclic aromatic hydrocarbon high-efficiency degrading microbial inoculum according to claim 5, wherein in the step (three), the adding amount of the biochar-based material is 1% -10% by the weight of the biochar-based material and the volume ratio of the culture medium.
9. The method for preparing the biochar-based immobilized polycyclic aromatic hydrocarbon high-efficiency degrading microbial inoculum according to claim 5, wherein in the step (III), the biochar-based material is sterilized before being added.
10. The preparation method of the biochar-based immobilized polycyclic aromatic hydrocarbon high-efficiency degrading microbial inoculum according to claim 5, wherein in the step (three), after solidification culture, a 200-mesh filter screen is used for filtering the biochar microbial inoculum, then, 0.2mol/L phosphate buffer solution is used for washing the microbial inoculum, then, a stationary solution is added for fixation for 4 hours, supernatant is removed by centrifugation, washing is carried out for 2 times, and a sample is placed in 30%, 50%, 70%, 90% and 100% ethanol solution for gradient dehydration and freeze drying to obtain the biochar-based immobilized polycyclic aromatic hydrocarbon high-efficiency degrading microbial inoculum.
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