CN114958816A - Biological repair material and raw material composition, preparation method and application thereof - Google Patents
Biological repair material and raw material composition, preparation method and application thereof Download PDFInfo
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
- C02F3/344—Biological treatment of water, waste water, or sewage characterised by the microorganisms used for digestion of mineral oil
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- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
- C02F3/347—Use of yeasts or fungi
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Abstract
The invention discloses a bioremediation material, a raw material composition thereof, a preparation method and application. The raw material composition comprises a Phanerochaete chrysosporium (Phanerochaete chrysosporium Burdsall) bacterial liquid and a biomass carrier; the biomass carrier is a biomass carrier containing lignin; the OD value of the phanerochaete chrysosporium bacterial liquid is 1.5-2.5; the ratio of the volume mL of the Phanerochaete chrysosporium bacterial liquid to the mass g of the biomass carrier is (0.5-1.5): 10. The bioremediation material is constructed by using Phanerochaete chrysosporium and a biomass carrier, overcomes the secondary pollution caused by physical and chemical remediation in the process of remedying the rice dumplings in the petroleum industry, and improves the bioremediation efficiency; the device can play a role in stopping the diffusion of spilled oil on the ocean surface, and can degrade pollutants on the basis of cutting the oil film of the spilled oil in a short time.
Description
Technical Field
The invention relates to a bioremediation material, a raw material composition thereof, a preparation method and application.
Background
Petroleum is one of the world important energy sources, and the demand of the petroleum is high along with the economic development. With the rapid increase of the oil usage amount, the inevitable oil overflow pollution in the offshore oil exploitation, extraction, transportation, loading and unloading and application processes poses great threat to the environment. In recent years, the offshore oil spill events are frequent, and under the action of ocean currents, tides and wind waves, the spilled oil not only pollutes the ocean, but also drifts to a coastal zone, so that the pollution is caused to the mudflat. The treatment and remediation of petroleum pollution become a problem to be solved urgently.
The process of oil spill pollution mainly has the actions of diffusion and drifting, evaporation (volatilization), dissolution, emulsification, adsorption and sedimentation, photooxidation, microbial degradation and the like, the migration and conversion process is influenced by the factors of the surrounding marine environment, the physical property and the chemical property can be changed, serious negative effects are generated on the ecological environment of the marine environment, particularly, the rapidly expanded oil spill oil film covers the sea surface in a large area, the gas exchange between the atmosphere and the sea water is blocked, and the fatal influence on marine organisms and birds can be caused in the past.
Petroleum and petroleum products are mainly mixtures of hydrocarbons such as alkanes, cycloalkanes and aromatics, wherein the petroleum aromatics mainly comprise Polycyclic Aromatic Hydrocarbons (PAHs), homologues of benzene and various high molecular weight, difficult-to-degrade and toxic substances. Most of the pollutants are very stable in the environment, have long retention time, have strong carcinogenic, teratogenic and mutagenic effects, belong to persistent toxic pollutants, and form great harm to human health and the safety of the whole ecological system.
Degradation or elimination pathways of polycyclic aromatic hydrocarbon in the environment mainly comprise volatilization, photooxidation, chemical oxidation, biological adsorption, microbial degradation and the like. The traditional disposal method mainly adopts a physical and chemical method, which not only has little effect, but also causes secondary pollution. The microbial remediation technology mainly degrades petroleum pollutants into nontoxic and stable products such as water, carbon dioxide, inorganic substances and the like through the action of microbes, and has the advantages of high economic benefit, environmental friendliness, obvious action effect and the like. The bioremediation technology of polycyclic aromatic hydrocarbons is one of the key points of international research.
Generally, a large amount of microorganisms capable of degrading polycyclic aromatic hydrocarbons exist in water bodies polluted by polycyclic aromatic hydrocarbons, and researchers at home and abroad perform screening work of a large amount of degradation organisms. However, most of the oleaginous microorganisms are suitable for living in a specific stable environment under severe living conditions, and most of the patents and documents in the microbial degradation method are utilized to research and screen high-efficiency degradation strains, but most of the degradation rates are low, the degradation period is long, and the operation cost is high.
Therefore, it is an urgent technical problem in the art to provide a petroleum degradation method and material with high petroleum degradation efficiency, short degradation period and low degradation cost.
Disclosure of Invention
The invention aims to overcome the defects of low efficiency and non-economical economy of oil degradation by microorganisms in the prior art, and provides a bioremediation material, and a raw material composition, a preparation method and application thereof. The bioremediation material is constructed by using the phanerochaete chrysosporium and the biomass carrier, overcomes the secondary pollution caused by physical and chemical remediation in the process of remedying the rice dumplings in the petroleum industry, improves the bioremediation efficiency, has plasticity and can be repeatedly reused; the device can play a role in stopping the diffusion of spilled oil on the ocean surface, and can degrade pollutants on the basis of cutting the oil film of the spilled oil in a short time.
The inventor finds that most of the prior methods for solving the marine oil spill pollution by means of microbial degradation utilize thalli to directly degrade, however, the thalli do not have nutrient substances capable of continuously supporting activity, are difficult to survive in severe environment, have low degradation rate and are difficult to continuously and effectively realize petroleum degradation. Based on the above, the inventor utilizes the phanerochaete chrysosporium and the biomass carrier to construct and obtain the bioremediation material, the bioremediation material can play a role in blocking the diffusion of oil spill on the ocean surface oil spill pollution, the removal rate of the petroleum pollutant is more than or equal to 70%, and pollutants can be effectively degraded.
The invention provides a raw material composition of a bioremediation material, which comprises a Phanerochaete chrysosporium (Phanerochaete chrysosporium Burdsall) bacterial liquid and a biomass carrier;
the biomass carrier is a biomass carrier containing lignin;
the OD value of the phanerochaete chrysosporium bacterial liquid is 1.5-2.5;
the ratio of the volume mL of the Phanerochaete chrysosporium bacterial liquid to the mass g of the biomass carrier is (0.5-1.5): 10.
In the present invention, the Phanerochaete chrysosporium (Phanerochaete chrysosporium Burdsall) may be a Phanerochaete chrysosporium conventional in the art, for example, a Phanerochaete chrysosporium available from northern Nay.
In the present invention, the Phanerochaete chrysosporium (Phanerochaete chrysosporium Burdsall) bacterial solution can be prepared by a method conventional in the art, for example: inoculating spore of Phanerochaete chrysosporium (Phanerochaete chrysosporium Burdsall) in liquid culture medium, and culturing.
The spores of Phanerochaete chrysosporium can be prepared by conventional cultivation methods in the art, for example, by inoculating Phanerochaete chrysosporium (Phanerochaete chrysosporium) into a solid medium and cultivating.
The solid medium may be a solid medium for culturing Phanerochaete chrysosporium (Phanerochaete chrysosporium Burdsall) which is conventional in the art, such as a potato solid medium.
The potato solid medium can be a potato solid medium conventional in the art, such as model P8931 potato solid medium available from Solarbio.
In the method for culturing spores of Phanerochaete chrysosporium, the culturing conditions are preferably as follows: culturing under illumination for 3-4 days.
In the preparation method of the phanerochaete chrysosporium bacterial liquid, the liquid medium can be a medium which is used for culturing the phanerochaete chrysosporium to obtain the bacterial liquid, such as a liquid potato medium, and is conventional in the field.
The liquid potato medium can be a liquid potato medium conventional in the art, such as model P9240 liquid potato medium available from Solarbio.
In the preparation method of the phanerochaete chrysosporium bacterial liquid, the cultivation temperature is preferably 36-38 ℃, for example 37 ℃.
In the preparation method of the phanerochaete chrysosporium bacterial liquid, the cultivation is preferably carried out in a shaking table. The shaker speed may be 70-140rpm, for example 80rpm or 120 rpm.
In the preparation method of the phanerochaete chrysosporium bacterial liquid, the cultivation time can be 36-60h, such as 48 h.
In the present invention, the phanerochaete chrysosporium liquid generally contains mycelium pellets, and for the purposes of the present invention, the OD value of the phanerochaete chrysosporium liquid is ensured to be in the range of 1.5-2.5, and the diameter of the mycelium pellets can be 0.5-1.5cm, such as 0.8-1.2cm, and further such as 1 cm.
In the present invention, the OD value of the Phanerochaete chrysosporium bacterial liquid is preferably 1.5 to 2, for example 1.5 or 2.
In the present invention, the biomass carrier may be a lignin-containing biomass carrier, such as lignin-containing crop waste, which is conventional in the art.
Wherein the crop waste material may be lignin-containing crop waste material as is conventional in the art, such as crop straw and/or rice hulls.
The crop straw may be conventional crop straw in the art, and generally refers to a general term for the stem and leaf (ear) part of a mature crop, such as the remaining part of wheat, rice, corn, potatoes, rape, cotton, sugar cane and other crops (usually coarse grain) after harvesting seeds.
In the present invention, the biomass carrier may be subjected to pretreatment such as drying, pulverization, and sterilization according to a conventional operation in the art. Generally, drying, pulverization, and sterilization processes may be sequentially performed.
Among them, preferably, the sterilization treatment may be a sterilization treatment conventional in the art, for example, a treatment at 120 ℃ for 20 minutes.
In the present invention, the humidity of the biomass carrier is preferably 30 to 40%, for example 30% or 40%, and the percentage refers to the mass percentage of water in the biomass carrier.
In the present invention, the ratio of the volume mL of the Phanerochaete chrysosporium bacterial liquid to the mass g of the biomass carrier is preferably (1-1.2):10, for example, 1:10 or 1.2: 10.
In a preferred embodiment of the present invention, in the raw material composition of the bioremediation material:
the biomass carrier is crop straws and/or rice husks;
the OD value of the phanerochaete chrysosporium bacterial liquid is 1.5-2;
the ratio of the volume mL of the phanerochaete chrysosporium bacterial liquid to the mass g of the biomass carrier is (1-1.2): 10.
The invention also provides a preparation method of the biological repair material, which comprises the following steps: mixing the phanerochaete chrysosporium bacterial liquid with the biomass carrier, and culturing in a liquid culture medium A until the coverage rate of mycelia on the surface of the biomass carrier is more than 90%; the liquid medium A contains KH 2 PO 4 And MgSO 4 ·7H 2 O。
In the present invention, preferably, the incubation is performed under light conditions.
In the present invention, preferably, the liquid medium a is sprayed in a mixture of the phanerochaete chrysosporium bacterial liquid and the biomass carrier.
In the present invention, the KH is 2 PO 4 The concentration in the liquid medium A is preferably 2-4g/L, for example 3 g/L.
In the present invention, the MgSO 4 ·7H 2 The concentration of O in the liquid medium A is preferably 1 to 2g/L, for example 1.5 g/L.
In the present invention, the liquid medium A may further comprise other components for culturing the Pieris chrysosporium mycelial pellets, which are conventional in the art, such as a liquid potato medium.
Wherein the concentration of the liquid potato medium in the liquid medium A is preferably 8-12mg/L, such as 10 mg/L.
In the present invention, the temperature of incubation in the liquid medium A is preferably 24 ℃ to 31 ℃.
In the present invention, the incubation time in the liquid medium a may be 10 days.
In the present invention, the humidity of cultivation is preferably 30 to 40%, for example 30% or 40%, in the cultivation process in the liquid medium a, and the percentage refers to the mass percentage of water in the bioremediation material.
Wherein, in the cultivation process, the cultivation humidity can be maintained by spraying the liquid medium A.
The invention also provides a biological repair material prepared by the method.
The invention also provides application of the bioremediation material as a petroleum degradation agent in the remediation of the rice dumpling land in the petroleum industry.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.
The reagents and starting materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows:
the bioremediation material is constructed by using Phanerochaete chrysosporium and a biomass carrier (such as crop waste), a good carrier is provided for the growth of microorganisms through the organic combination of the microorganisms and the biomass carrier, and the lignin provides nutrients required by the growth of the Phanerochaete chrysosporium. The bioremediation material can play a role in blocking the diffusion of spilled oil on ocean surface, is easy to salvage for subsequent treatment after degradation, and can not cause secondary pollution. The removal rate of the petroleum pollutants is more than or equal to 70 percent, the pollutants can be effectively degraded, and the petroleum degradation capability of the phanerochaete chrysosporium is improved.
Drawings
FIG. 1 shows a suspension of a Phanerochaete chrysosporium pellet prepared in step (1) of example 2 (OD value: 2).
FIG. 2 is a block of bioremediation material formed after 10 days of culture in step (3) of example 2.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the invention thereto. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
In the following examples and comparative examples:
phanerochaete chrysosporium (Phanerochaete chrysosporium Burdsall) from North Nara;
the crop waste straw is corn straw;
potato solid medium was purchased from Solarbio (P8931);
liquid potato medium was purchased from Solarbio (P9240); the liquid potato culture medium is sold in a solid powder state, and the concentration of the liquid potato culture medium of 10mg/L is 10mg/L based on the solid powder;
the detection method of the OD value comprises the following steps: after a small amount of bacterial liquid is taken to rinse a quartz cuvette, absorbing 3/4 which is the approximate volume of the cuvette and is occupied by a proper amount of bacterial liquid, and measuring the absorbance A of the cuvette at 600nm by using UV-Vis, wherein the absorbance A is the OD value;
the preparation method of the liquid culture medium A comprises the following steps: mixing KH with water 2 PO 4 、MgSO 4 ·7H 2 O and liquid potato culture medium according to 3g/L KH 2 PO 4 、1.5g/L MgSO 4 ·7H 2 Mixing O with 10mg/L liquid potato culture medium powder, and water in balance; the specific operation is as follows: the powder is prepared, water is added to the powder to a constant volume, and 3g KH is weighed 2 PO 4 、1.5g MgSO 4 ·7H 2 O and 10mg PDB powder (liquid potato medium powder) dissolved in 1L water).
The percentage in humidity refers to the mass percentage of water in each material.
Example 1 mycelium pellet solution having OD value of 1.5
(1) Inoculating Phanerochaete chrysosporium Pc (strain from Beina biological company) into a potato solid culture medium, forming white powdery conidia in 3-4 days under the illumination condition, transferring the conidia into a liquid potato culture medium under the aseptic environment, fully dispersing in a sterilized triangular flask, oscillating at 37 ℃ and 120rpm for 48 hours to form milky hypha ball suspension (OD value is 1.5) with uniform size and diameter of about 1cm, and storing at 4 ℃ for later use.
(2) Drying crop waste straw (corn straw), pulverizing, mixing with testa oryzae, sterilizing at 120 deg.C for 20 min, adding liquid potato culture medium into crop mixture, and maintaining its humidity at 30%.
(3) Uniformly adding Pc mycelium pellet liquid into the crop fertilizer mixture in the step (2), adding 50mL of the mycelium pellet liquid with the OD value of 1.5 in the step (1) into every 500g of the crop mixture, wherein the mycelium pellets with the diameter of about 1cm are contained according to 3g/L KH 2 PO 4 、1.5g/L MgSO 4 ·7H 2 Preparing a liquid culture medium A from O and 10mg/L of liquid potato culture medium, spraying 20mL of the liquid culture medium A into the mixture of the mycelium pellet liquid and the crop waste, and culturing at 24-31 ℃ for 10 days under the illumination condition, wherein the humidity of the material is maintained at 30% by adding the liquid culture medium A.
Example 2 mycelial pellet solution having OD value of 2
(1) Inoculating Phanerochaete chrysosporium Pc (strain from Beina biological company) into a potato solid culture medium, forming white powdery conidia in 3-4 days under the illumination condition, transferring the conidia into a liquid potato culture medium under the aseptic environment, fully dispersing in a sterilized triangular flask, oscillating at 37 ℃ and 120rpm for 48 hours to form milky hypha ball suspension (OD value is 2) with uniform size and diameter of about 1cm, and storing at 4 ℃ for later use.
(2) Drying crop waste straw (corn straw), pulverizing, mixing with rice hull, sterilizing at 120 deg.C for 20 min, adding liquid potato culture medium into crop mixture, and maintaining the humidity at 30-40%.
(3) Uniformly adding Pc mycelium pellet liquid into the crop fertilizer mixture in the step (2), and adding 50mL of the mycelium pellet liquid with the OD value of 2 in the step (1) containing mycelium pellets with the diameter of about 1cm into every 500g of the crop fertilizer mixture according to the ratio of 3g/L KH 2 PO 4 、1.5g/L MgSO 4 ·7H 2 Preparing a liquid culture medium A from O and 10mg/L of liquid potato culture medium, spraying 20mL of the liquid culture medium A into the mixture of the mycelium pellet liquid and the crop waste, and culturing at 24-31 ℃ for 10 days under the illumination condition, wherein the humidity of the material is maintained at 30% by adding the liquid culture medium A.
Example 3 dark conditions
(1) Inoculating Phanerochaete chrysosporium Pc (strain from Beina biological company) into a potato solid culture medium, forming white powdery conidia in 3-4 days under the illumination condition, transferring the conidia into a liquid potato culture medium under the aseptic environment, fully dispersing in a sterilized triangular flask, oscillating at 37 ℃ and 120rpm for 48 hours to form milky hypha ball suspension (OD value is 2) with uniform size and diameter of about 1cm, and storing at 4 ℃ for later use.
(2) Drying crop waste straw (corn straw), pulverizing, mixing with testa oryzae, sterilizing at 120 deg.C for 20 min, adding liquid potato culture medium into crop mixture, and maintaining its humidity at 30%.
(3) Uniformly adding Pc mycelium pellet liquid into the crop fertilizer mixture in the step (2), and adding 50mL of the mycelium pellet liquid with the OD value of 2 in the step (1) containing mycelium pellets with the diameter of about 1cm into every 500g of the crop fertilizer mixture according to the ratio of 3g/L KH 2 PO 4 、1.5g/L MgSO 4 ·7H 2 Preparing a liquid culture medium A from O and 10mg/L of liquid potato culture medium, spraying 20mL of the liquid culture medium A into the mixture of mycelium pellet liquid and crop waste, and culturing at 24-31 ℃ for 10 days in the absence of illumination, wherein the humidity of the material is maintained at 30-40% by adding the liquid culture medium A.
Example 4
(1) Inoculating Phanerochaete chrysosporium Pc (strain from Beina biological company) into a solid potato culture medium, forming white powdery conidia in 3-4 days under illumination, transferring the conidia into the liquid potato culture medium under sterile environment, fully dispersing in a sterilized triangular flask, oscillating at 37 ℃ and 80rpm for 48 hours to form milky hypha ball suspension (OD value is 2) with uniform size and diameter of about 1cm, and storing at 4 ℃ for later use.
(2) Drying crop waste straw (corn straw), pulverizing, mixing with rice hull, sterilizing at 120 deg.C for 20 min, adding liquid potato culture medium into crop mixture, and maintaining the humidity at 30-40%.
(3) Uniformly adding Pc mycelium pellet liquid into the crop fertilizer mixture in the step (2), and adding 50mL of the mycelium pellet liquid with the OD value of 2 in the step (1) containing mycelium pellets with the diameter of about 1cm into every 500g of the crop fertilizer mixture according to the ratio of 3g/L KH 2 PO 4 、1.5g/L MgSO 4 ·7H 2 Preparing a liquid culture medium A from O and 10mg/L of liquid potato culture medium, spraying 20mL of the liquid culture medium A into the mixture of the mycelium pellet liquid and the crop waste, and culturing at 24-31 ℃ for 10 days under the illumination condition, wherein the humidity of the material is maintained at 30% by adding the liquid culture medium A.
Example 5
(1) Inoculating Phanerochaete chrysosporium Pc (strain from Beina biological company) into a potato solid culture medium, forming white powdery conidia in 3-4 days under the illumination condition, transferring the conidia into a liquid potato culture medium under the aseptic environment, fully dispersing in a sterilized triangular flask, oscillating at 37 ℃ and 120rpm for 48 hours to form milky hypha ball suspension (OD value is 2) with uniform size and diameter of about 1cm, and storing at 4 ℃ for later use.
(2) Drying crop waste straw (corn straw), pulverizing, mixing with rice hull, sterilizing at 120 deg.C for 20 min, adding liquid potato culture medium into crop mixture, and maintaining the humidity at 30% -40%.
(3) Uniformly adding Pc mycelium pellet liquid into the crop fertilizer mixture in the step (2), and adding 60mL of the mycelium pellet liquid with the OD value of 2 in the step (1) into every 500g of the crop mixture, wherein the mycelium pellet liquid contains the mycelium pellets with the diameter of about 1cmMycelium pellets in a ratio of 3g/L KH 2 PO 4 、1.5g/L MgSO 4 ·7H 2 Preparing a liquid culture medium A from O and 10mg/L of liquid potato culture medium, spraying 20mL of the liquid culture medium A into the mixture of the mycelium pellet liquid and the crop waste, and culturing at 24-31 ℃ for 10 days under the illumination condition, wherein the humidity of the material is maintained at 30% by adding the liquid culture medium A.
Comparative example 1
(1) Inoculating Phanerochaete chrysosporium Pc (strain from Beina biological company) into a potato solid culture medium, forming white powdery conidia in 3-4 days under the illumination condition, transferring the conidia into a liquid potato culture medium under the aseptic environment, fully dispersing in a sterilized triangular flask, oscillating at 37 ℃ and 120rpm for 48 hours to form milky hypha ball suspension (OD value is 1) with uniform size and diameter of about 1cm, and storing at 4 ℃ for later use.
(2) Drying crop waste straw (corn straw), pulverizing, mixing with rice hull, sterilizing at 120 deg.C for 20 min, adding liquid potato culture medium into crop mixture, and maintaining the humidity at 30-40%.
(3) Uniformly adding Pc mycelium pellet solution into the crop fertilizer mixture in step (2), adding 50mL of the mycelium pellet solution with OD value of 1 in step (1) containing mycelium pellets with diameter of about 1cm according to 3g/L KH per 500g of crop mixture 2 PO 4 、1.5g/L MgSO 4 ·7H 2 Preparing a liquid culture medium A from O and 10mg/L of liquid potato culture medium, spraying 20mL of the liquid culture medium A into the mixture of the mycelium pellet liquid and the crop waste, and culturing at 24-31 ℃ for 10 days under the illumination condition, wherein the humidity of the material is maintained at 30% by adding the liquid culture medium A.
Comparative example 2
(1) Inoculating Phanerochaete chrysosporium Pc (strain from Beina biological company) into a potato solid culture medium, forming white powdery conidia in 3-4 days under the illumination condition, transferring the conidia into a liquid potato culture medium under the aseptic environment, fully dispersing in a sterilized triangular flask, oscillating at 37 ℃ and 120rpm for 48 hours to form milky hypha ball suspension (OD value is 3) with uniform size and diameter of about 1cm, and storing at 4 ℃ for later use.
(2) Drying crop waste straw (corn straw), pulverizing, mixing with rice hull, sterilizing at 120 deg.C for 20 min, adding liquid potato culture medium into crop mixture, and maintaining the humidity at 30-40%.
(3) Uniformly adding Pc mycelium pellet solution into the crop fertilizer mixture in step (2), adding 50mL of mycelium pellet solution with OD value of 3 in step (1) containing mycelium pellets with diameter of about 1cm according to 3g/L KH per 500g of crop mixture 2 PO 4 、1.5g/L MgSO 4 ·7H 2 Preparing a liquid culture medium A from O and 10mg/L of liquid potato culture medium, spraying 20mL of the liquid culture medium A into the mixture of the mycelium pellet liquid and the crop waste, and culturing at 24-31 ℃ for 10 days under the illumination condition, wherein the humidity of the material is maintained at 30% by adding the liquid culture medium A.
Comparative example 3
(1) Inoculating Phanerochaete chrysosporium Pc (strain from Beina biological company) into a potato solid culture medium, forming white powdery conidia in 3-4 days under the illumination condition, transferring the conidia into a liquid potato culture medium under the aseptic environment, fully dispersing in a sterilized triangular flask, oscillating at 37 ℃ and 120rpm for 48 hours to form milky hypha ball suspension (OD value is 2) with uniform size and diameter of about 1cm, and storing at 4 ℃ for later use.
(2) Drying crop waste straw (corn straw), pulverizing, mixing with rice hull, sterilizing at 120 deg.C for 20 min, adding liquid potato culture medium into crop mixture, and maintaining the humidity at 30% -40%.
(3) Uniformly adding Pc mycelium pellet liquid into the crop fertilizer mixture in the step (2), adding 100mL of the mycelium pellet liquid with the OD value of 2 in the step (1) into every 500g of the crop mixture, wherein the mycelium pellet liquid contains mycelium pellets with the diameter of about 1cm according to 3g/L KH 2 PO 4 、1.5g/L MgSO 4 ·7H 2 Preparing liquid culture medium A from O and 10mg/L liquid potato culture medium, spraying 20mL liquid culture medium A into mixture of mycelium pellet liquid and crop waste under illumination conditionNext, the culture was carried out at 24 ℃ to 31 ℃ for 10 days, during which the humidity of the material was maintained at 30% by adding liquid medium A.
Comparative example 4
(1) Inoculating Phanerochaete chrysosporium Pc (strain from Beina biological company) into a potato solid culture medium, forming white powdery conidia in 3-4 days under the illumination condition, transferring the conidia into a liquid potato culture medium under the aseptic environment, fully dispersing in a sterilized triangular flask, oscillating at 37 ℃ and 120rpm for 48 hours to form milky hypha ball suspension (OD value is 2) with uniform size and diameter of about 1cm, and storing at 4 ℃ for later use.
(2) Drying crop waste straw (corn straw), pulverizing, mixing with rice hull, sterilizing at 120 deg.C for 20 min, adding liquid potato culture medium into crop mixture, and maintaining the humidity at 30-40%.
(3) Uniformly adding Pc mycelium pellet liquid into the crop fertilizer mixture in the step (2), and adding 20mL of the mycelium pellet liquid with the OD value of 2 in the step (1) into every 500g of the crop mixture, wherein the mycelium pellet liquid contains mycelium pellets with the diameter of about 1cm according to 3g/L KH 2 PO 4 、1.5g/L MgSO 4 ·7H 2 Preparing a liquid culture medium A from O and 10mg/L of liquid potato culture medium, spraying 20mL of the liquid culture medium A into the mixture of the mycelium pellet liquid and the crop waste, and culturing at 24-31 ℃ for 10 days under the illumination condition, wherein the humidity of the material is maintained at 30% by adding the liquid culture medium A.
Comparative example 5 without KH 2 PO 4 And MgSO 4
(1) Inoculating Phanerochaete chrysosporium Pc (strain from Beina biological company) into a potato solid culture medium, forming white powdery conidia in 3-4 days under the illumination condition, transferring the conidia into a liquid potato culture medium under the aseptic environment, fully dispersing in a sterilized triangular flask, oscillating at 37 ℃ and 120rpm for 48 hours to form milky hypha ball suspension (OD value is 2) with uniform size and diameter of about 1cm, and storing at 4 ℃ for later use.
(2) Drying crop waste straw (corn straw), pulverizing, mixing with rice hull, sterilizing at 120 deg.C for 20 min, adding liquid potato culture medium into crop mixture, and maintaining the humidity at 30-40%.
(3) Adding Pc mycelium pellet liquid into the crop fertilizer mixture in the step (2) uniformly, adding 50mL of the mycelium pellet liquid with the OD value of 2 in the step (1) into every 500g of the crop mixture, wherein the mycelium pellet liquid contains mycelium pellets with the diameter of about 1cm, preparing a liquid culture medium A according to the concentration of the liquid potato culture medium of 10mg/L, spraying 20mL of the liquid culture medium A into the mixture of the mycelium pellet liquid and the crop waste, and culturing at 24-31 ℃ for 10 days under the illumination condition, wherein the humidity of the material is maintained at 30% by adding the liquid culture medium A.
Effect example 1
The petroleum degradation rate of the biomaterials prepared in examples 1-5 and comparative examples 1-5 was measured.
The determination method is a gravimetric method for determining the oil content, and comprises the following specific steps:
(1) extracting 50mL seawater containing petroleum (the oil content in the seawater is 50%, the percentage is weight percentage) with 10mL n-hexane for 2 times, collecting and combining upper organic phase, performing rotary evaporation and drying at 50 deg.C, cooling in a drier to constant weight, and weighing M 0 ；
(2) Respectively co-culturing 50mL of seawater containing petroleum (the oil content in the seawater is 50%, and the percentage refers to the mass percentage) and the prepared bioremediation bricks (the biological materials prepared in the steps (3) of the examples 1-5 and the comparative examples 1-5) for 5 days, and weighing the biological materials as M after the same treatment in the step (1);
(3) the biomaterial composites not inoculated with Pc mycelial liquid in examples 1-5 and comparative examples 1-5 were treated as controls through the above steps; specifically, the method comprises the following steps:
extracting 50mL seawater containing petroleum (the oil content in the seawater is 50%, the percentage is weight percentage) with 10mL n-hexane for 2 times, collecting and combining upper organic phase, performing rotary evaporation and drying at 50 deg.C, cooling in a drier to constant weight, and weighing M c0 (quality (g) of petroleum before control treatment);
50mL of seawater containing petroleum (the oil content in the seawater is 50%, and the percentage refers to the mass percentage) is subjected toThe biomaterial composites not inoculated with Pc mycelial pellet solutions of examples 1 to 5 and comparative examples 1 to 5 were co-cultured for 5 days, respectively, and then weighed as M after the same treatment as in step (1) c (residual oil mass (g) after control treatment).
The oil removal rate was calculated according to equation (1):
η＝((M 0 -M)-(M c0 -M c ))/M 0 ×100％ (1)
in the formula (1), M 0 The quality (g) of petroleum before the sample is treated by the biological brick; m is the mass (g) of residual oil after the sample is treated by the biological bricks; m c0 The mass (g) of the petroleum before the control sample treatment; m c The residual oil mass (g) after the control treatment was obtained.
Specific data are shown in table 1 below.
TABLE 1
| Numbering | Oil pollutant removal rate (oil removal rate) |
| Example 1 | 80％ |
| Example 2 | 95％ |
| Example 3 | 70％ |
| Example 4 | 75％ |
| Example 5 | 90％ |
| Comparative example 1 | 29％ |
| Comparative example 2 | 27％ |
| Comparative example 3 | 21％ |
| Comparative example 4 | 8％ |
| Comparative example 5 | 12％ |
Claims (10)
1. A raw material composition of a bioremediation material, which comprises a Phanerochaete chrysosporium (Phanerochaete chrysosporium Burdsall) bacterial liquid and a biomass carrier;
the biomass carrier is a biomass carrier containing lignin;
the OD value of the phanerochaete chrysosporium bacterial liquid is 1.5-2.5;
the ratio of the volume mL of the Phanerochaete chrysosporium bacterial liquid to the mass g of the biomass carrier is (0.5-1.5): 10.
2. The raw material composition for bioremediation material of claim 1, wherein the Phanerochaete chrysosporium (Phanerochaete chrysosporium Burdsall) is a Phanerochaete chrysosporium available from beina bio-corporation;
and/or, the Phanerochaete chrysosporium (Phanerochaete chrysosporium Burdsall) bacterial liquid is prepared by the following method: inoculating spore of Phanerochaete chrysosporium (Phanerochaete chrysosporium Burdsall) in liquid culture medium, and culturing to obtain the final product;
and/or the biomass carrier is sequentially subjected to drying, crushing and sterilization pretreatment;
and/or the humidity of the biomass carrier is 30-40%, and the percentage refers to the mass percentage of water in the biomass carrier.
3. The raw material composition for bioremediation materials according to claim 2, wherein the spores of Phanerochaete chrysosporium are obtained by inoculating Phanerochaete chrysosporium (Phanerochaete chrysosporium Burdsall) in a solid medium and culturing; the solid medium can be a potato solid medium, such as model P8931 potato solid medium available from Solarbio; the incubation conditions are preferably: culturing for 3-4 days under illumination;
and/or, in the preparation method of the phanerochaete chrysosporium liquid, the liquid culture medium is a liquid potato culture medium, such as a liquid potato culture medium which is purchased from Solarbio company and has the model number of P9240;
and/or in the preparation method of the phanerochaete chrysosporium bacterial liquid, the cultivation temperature is 36-38 ℃, such as 37 ℃;
and/or in the preparation method of the phanerochaete chrysosporium bacterial liquid, the cultivation is carried out in a shaking table; the shaker may have a speed of 70-140rpm, for example 80rpm or 120 rpm;
and/or in the preparation method of the phanerochaete chrysosporium bacterial liquid, the cultivation time is 36-60h, such as 48 h;
and/or the sterilization pretreatment is treatment at 120 ℃ for 20 minutes.
4. The raw material composition for bioremediation material of claim 1 wherein said phanerochaete chrysosporium broth contains mycelium pellets;
and/or the OD value of the phanerochaete chrysosporium bacterial liquid is 1.5-2;
and/or the biomass carrier is crop waste containing lignin;
and/or the ratio of the volume mL of the phanerochaete chrysosporium bacterial liquid to the mass g of the biomass carrier is (1-1.2): 10.
5. The raw material composition for bioremediation material according to claim 4, wherein the mycelium pellet has a diameter of 0.5 to 1.5 cm;
and/or the OD value of the phanerochaete chrysosporium bacterial liquid is 1.5 or 2;
and/or the crop waste is crop straw and/or rice husk.
6. A feedstock composition for bioremediation material according to claim 5, wherein said mycelium pellets have a diameter of 0.8-1.2cm, such as 1 cm.
7. A preparation method of a biological repair material is characterized by comprising the following steps: mixing the raw material composition of the bioremediation material of any one of claims 1-6, and incubating in a liquid medium A until the coverage of mycelia on the surface of the biomass carriers is > 90%; the liquid medium A contains KH 2 PO 4 And MgSO 4 ·7H 2 O。
8. The method for producing the bioremediation material of claim 7, wherein the incubation in the liquid medium A is performed under light conditions;
and/or, said KH 2 PO 4 The concentration in the liquid medium A is 2-4g/L, such as 3 g/L;
and/or, the MgSO 4 ·7H 2 The concentration of O in the liquid medium A is 1-2g/L, for example 1.5 g/L;
and/or the liquid culture medium A further comprises a liquid potato culture medium, and the concentration of the liquid potato culture medium in the liquid culture medium A can be 8-12mg/L, such as 10 mg/L;
and/or the temperature of incubation in the liquid medium a is between 24 ℃ and 31 ℃;
and/or the incubation in the liquid medium A is for a period of 10 days;
and/or in the process of culturing in the liquid culture medium A, the humidity of the culture is 30-40%, and the percentage refers to the mass percentage of water in the bioremediation material.
9. A bioremediation material made by the method for making a bioremediation material as recited in claim 7 or 8.
10. Use of the bioremediation material of claim 9 as a petroleum degradant in the remediation of petroleum industry rice-pudding land.
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| CN103409382A (en) * | 2013-07-25 | 2013-11-27 | 江苏大学 | Method used for accelerating lignin degradation in phanerochaete chrysosporium solid state fermentation |
| CN111826288A (en) * | 2020-03-18 | 2020-10-27 | 广东省生态环境技术研究所 | Method for degrading tricresyl phosphate by using Phanerochaete chrysosporium and application of method |
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| CN103409382A (en) * | 2013-07-25 | 2013-11-27 | 江苏大学 | Method used for accelerating lignin degradation in phanerochaete chrysosporium solid state fermentation |
| CN111826288A (en) * | 2020-03-18 | 2020-10-27 | 广东省生态环境技术研究所 | Method for degrading tricresyl phosphate by using Phanerochaete chrysosporium and application of method |
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