CN114350558A - Solid oil-removing microbial inoculum and preparation method and application thereof - Google Patents
Solid oil-removing microbial inoculum and preparation method and application thereof Download PDFInfo
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- Processing Of Solid Wastes (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a solid oil-removing microbial inoculum and a preparation method and application thereof. The solid oil-removing microbial inoculum is obtained by fermenting bacillus subtilis in a solid fermentation culture medium, wherein the solid fermentation culture medium comprises an enzymolysis product, wheat bran, corn flour, brown sugar, urea, inorganic salt, trace elements and water, and the enzymolysis product is one or more of corncobs, rice husks and sawdust bacteria residues which are subjected to enzymolysis by cellulase and xylanase. The solid oil-removing microbial inoculum prepared from the enzymatic hydrolysate can improve the effective viable bacteria amount, can be applied to the biological remediation of oil-contaminated soil, realizes the field planting and propagation of strains in the soil, is beneficial to the maintenance of water in the soil, reduces the compactness of the soil and increases the nutrition in the soil.
Description
Technical Field
The invention belongs to the field of biological fermentation, and particularly relates to a solid oil-removing microbial inoculum, and a preparation method and application thereof.
Background
With the rapid development of the petroleum industry, the development and use of petroleum and its products are increasing. The problem of soil petroleum pollution is gradually serious, so that serious threats and negative influences are generated on crop production and ecological safety, and the ecological environment bears huge pollution load. The petroleum has the characteristics of low density, strong adhesion and low emulsifying capacity, so that the petroleum is easy to adhere to soil particles in soil, blocks soil pores, influences the permeability of the soil, causes soil hardening, increases the compactness of the soil, reduces the water holding capacity of the soil, reduces the oxygen and nutrient level, hinders the respiration of plant roots and the absorption of nutrient substances, and seriously harms the growth of plants.
At present, the remediation methods for the petroleum-polluted soil are mainly divided into three main categories: physical repair methods, chemical repair methods, and biological repair methods. The bioremediation method has the advantages of low treatment cost, no secondary pollution and the like, and becomes a hot point of domestic and foreign research.
Disclosure of Invention
In view of the above, the present invention aims to provide a solid oil-removing microbial inoculum, and a preparation method and an application thereof, which are beneficial to increase of effective viable bacteria amount of the solid oil-removing microbial inoculum and remediation effect on petroleum-contaminated soil.
In order to achieve the aim, the technical scheme of the invention is to provide a solid oil-removing microbial inoculum, wherein the solid oil-removing microbial inoculum is obtained by fermenting bacillus subtilis in a solid fermentation culture medium; the solid fermentation medium comprises the following components in percentage by mass: 12-46% of an enzymolysis product, 10-20% of wheat bran, 3-10% of corn flour, 5-10% of brown sugar, 0-3% of urea, 0.5-2% of inorganic salt, 0.001-0.01% of trace elements and the balance of water; the enzymolysis product is one or more of corncob, rice hull and sawdust fungus residue which are subjected to enzymolysis by cellulase and xylanase.
According to the invention, the solid oil-removing microbial inoculum is obtained by inoculating bacillus subtilis into a solid fermentation culture medium for fermentation culture.
In one embodiment, the Bacillus subtilis is Bacillus subtilis with the preservation number of CGMCC NO.16167, is separated from oil stain soil of certain refining and chemical enterprises in Shandong, is identified as Bacillus subtilis through 16SrDNA, is preserved in the general microbiological center of China Committee for culture Collection of microorganisms in 7 months in 2018 and has the strain code YJY18-05, and the effective viable bacteria amount of the Bacillus subtilis in the solid oil-removing microbial inoculum is 3 to 108～8*109cfu/g, preferably 1 x 109～5*109cfu/g。
In the solid fermentation culture medium, the mass percentage of an enzymolysis product is 12-46%, preferably 25-46%, and the enzymolysis product is one or more of corncobs, rice husks and sawdust bacteria residues which are subjected to enzymolysis by cellulase and xylanase. The sawdust fungus residue is the sawdust fungus residue after edible fungi are cultured and fruiting, the edible fungi comprise one or more of agaric, mushroom and oyster mushroom, and preferably more than two of agaric, mushroom and oyster mushroom.
In one embodiment, the enzymolysis product is a product obtained by performing enzymolysis on corncobs, rice husks and sawdust bacteria residues through cellulase and xylanase, and the mass ratio of the corncobs, the rice husks and the sawdust bacteria residues is (1-21): 1-5): 10-20, preferably (10-21): 2-4): 15-20.
In one embodiment, the preparation method of the enzymatic hydrolysate specifically comprises the following steps: corncob, rice husk and sawdust bacteria residue are firstly subjected to enzymolysis by cellulase, and then are subjected to enzymolysis by xylanase, wherein the enzymolysis conditions of the cellulase are as follows: the enzymolysis temperature is 50-55 ℃, the preferable temperature is 52-55 ℃, the pH is 5.5-6.0, the preferable temperature is 5.8-6.0, and the time is 1-2 h, and the preferable time is 1.5-2 h; the enzymolysis conditions of the xylanase are as follows: the enzymolysis temperature is 50-60 ℃, preferably 50-55 ℃, the pH is 5.0-6.0, preferably 5.5-6.0, and the time is 1-2 h, preferably 1-1.2 h; the mass ratio of the cellulase to the xylanase is (0.1-5): 0.1-1, preferably (1-3): 0.3-0.5.
In the solid fermentation culture medium, the wheat bran accounts for 10-20% by mass, and preferably 18-20%; the mass percentage of the corn flour is 3-10%, preferably 5-10%; the mass percentage of the brown sugar is 5-10%, preferably 6-10%; the mass percentage of the urea is 0-3%, preferably 0.1-1.5%.
In the solid fermentation medium, the mass percentage of inorganic salt is 0.5-2%, preferably 0.9-1.5%, and the inorganic salt comprises one or more of monopotassium phosphate, dipotassium hydrogen phosphate, calcium chloride, calcium carbonate, calcium sulfate and magnesium chloride, preferably more than two of monopotassium phosphate, dipotassium hydrogen phosphate, calcium chloride, calcium carbonate, calcium sulfate and magnesium chloride.
In the solid fermentation medium, the mass percentage of the trace elements is 0.001-0.01%, preferably 0.004-0.008%, the trace elements comprise one or more of zinc sulfate, ferric trichloride, manganese chloride and copper sulfate, and preferably more than two of zinc sulfate, ferric trichloride, manganese chloride and copper sulfate.
The solid oil-removing microbial inoculum prepared by performing enzymolysis on one or more of corncobs, rice husks and sawdust bacteria residues with cellulase and xylanase has higher effective viable bacteria amount.
The invention also provides a preparation method of the solid oil-removing microbial inoculum, which comprises the following steps:
inoculating bacillus subtilis into a solid fermentation culture medium for fermentation culture;
the solid fermentation medium comprises the following components in percentage by mass: 12-46% of an enzymolysis product, 10-20% of wheat bran, 3-10% of corn flour, 5-10% of brown sugar, 0-3% of urea, 0.5-2% of inorganic salt, 0.001-0.01% of trace elements and the balance of water;
the enzymolysis product is one or more of corncob, rice hull and sawdust fungus residue which are subjected to enzymolysis by cellulase and xylanase.
The invention inoculates the bacillus subtilis in the liquid culture medium for culturing to obtain the liquid strain. In one embodiment, the effective viable bacterial count of the liquid bacterial species is 1 x 108～3*109cfu/mL, preferably 0.9 x 109～3*109cfu/mL. In one embodiment, the screened, separated and purified bacillus subtilis with the preservation number of CGMCC NO.16167 is inoculated into a liquid culture medium for culture to obtain a liquid strain, and the liquid culture medium is an LB liquid culture medium; the culture temperature is 28-37 ℃, and preferably 30-37 ℃; the rotating speed is 80-220 rpm, preferably 100-200 rpm; the time is 12-48 h, preferably 30-48 h.
The preparation method of the solid fermentation medium specifically comprises the following steps: preparing an enzymolysis product, and mixing the enzymolysis product with wheat bran, corn flour, brown sugar, urea, inorganic salt, trace elements and water to obtain the solid fermentation culture medium.
The enzymatic hydrolysis product is one or more products subjected to enzymatic hydrolysis by cellulase and xylanase in corncobs, rice husks and sawdust bacteria residues, and the pretreatment is preferably carried out before the enzymatic hydrolysis by the cellulase and the xylanase, wherein the pretreatment comprises the following steps: one or more of corncobs, rice husks and sawdust fungus residues are ground, sieved and mixed with water, and the material-liquid ratio is 1: 3-1: 8, preferably 1: 3-1: 4. In one embodiment, corncobs, rice hulls and sawdust bacteria residues are ground, sieved and mixed with water at a feed-liquid ratio of 1: 3-1: 4, and then subjected to enzymolysis by using cellulase and xylanase. In one embodiment, the cellulase employed in the present invention is a Novoxin acid cellulase and the xylanase employed is a Novoxin acid xylanase.
Pretreating one or more of corncobs, rice husks and sawdust bacteria residues, and then carrying out enzymolysis by adopting cellulase and xylanase. The cellulase and the xylanase can be subjected to enzymolysis simultaneously or sequentially, and the enzymolysis conditions of the cellulase and the xylanase are the enzymolysis conditions of the cellulase and the xylanase in the technical scheme, so that the details are not repeated.
The invention adopts cellulase and xylanase to carry out enzymolysis on one or more of corncobs, rice husks and sawdust bacteria residues to obtain a mixture of an enzymolysis product and water.
The mixture of the obtained enzymolysis product and water is preferably subjected to post-treatment, and the post-treatment is concentration, preferably vacuum reduced pressure concentration. After the mixture of the enzymolysis product and the water is concentrated, the mass ratio of the water to the enzymolysis product is lower than that of the water to the enzymolysis product in the solid oil-removing microbial inoculum.
And mixing the mixture of the post-treated enzymolysis product and water with wheat bran, corn flour, brown sugar, urea, inorganic salt, trace elements and water, and sterilizing to obtain the solid fermentation medium.
In one embodiment, the invention dissolves brown sugar, urea, inorganic salt and trace elements in water, mixes with wheat bran, corn flour, enzymolysis product and water mixture, sterilizes, gets the solid fermentation culture medium.
After obtaining liquid strains and a solid fermentation culture medium, inoculating the liquid strains into the solid fermentation culture medium under an aseptic condition, and carrying out fermentation culture. The volume-to-mass ratio of the liquid strain to the solid fermentation medium is 0.005-0.1: 1, and preferably 0.05-0.1: 1.
And inoculating the liquid strain to the solid fermentation medium, uniformly mixing, and culturing at the temperature of 28-37 ℃, preferably at the temperature of 30-37 ℃, more preferably at the temperature of 35-37 ℃ for 24-60 h, preferably at the temperature of 50-60 h, more preferably at the temperature of 55-60 h to obtain the solid oil-removing microbial inoculum.
The invention also provides a solid fermentation culture medium which comprises the following components in percentage by mass: 12-46% of an enzymolysis product, 10-20% of wheat bran, 3-10% of corn flour, 5-10% of brown sugar, 0-3% of urea, 0.5-2% of inorganic salt, 0.001-0.01% of trace elements and the balance of water; and the corncobs, the rice husks and the sawdust fungus residues are subjected to enzymolysis by adopting cellulase and xylanase. The enzymolysis product of the solid fermentation medium is the enzymolysis product in the technical scheme, and is not described herein again.
The invention also provides application of the solid oil-removing microbial inoculum in degrading petroleum hydrocarbon, wherein the solid oil-removing microbial inoculum can degrade the petroleum hydrocarbon existing in various forms, and the petroleum hydrocarbon can be petroleum hydrocarbon in polluted soil, petroleum hydrocarbon in sewage and petroleum hydrocarbon in gas. In one embodiment, a solid degreasing agent is used to degrade petroleum hydrocarbons in contaminated soil.
The invention also provides a method for restoring the petroleum-polluted soil by using the solid oil-removing microbial inoculum, which comprises the following steps:
mixing the solid oil-removing microbial inoculum with petroleum-polluted soil for remediation;
the solid oil-removing microbial inoculum is obtained by fermenting bacillus subtilis in a solid fermentation culture medium;
the solid fermentation medium comprises the following components in percentage by mass: 12-46% of an enzymolysis product, 10-20% of wheat bran, 3-10% of corn flour, 5-10% of brown sugar, 0-3% of urea, 0.5-2% of inorganic salt, 0.001-0.01% of trace elements and the balance of water;
the enzymolysis product is one or more of corncob, rice hull and sawdust fungus residue which are subjected to enzymolysis by cellulase and xylanase.
According to the invention, the soil polluted by petroleum is repaired by mixing the solid oil removing microbial inoculum and the soil polluted by petroleum according to any proportion, wherein the solid oil removing microbial inoculum, the solid fermentation culture medium and the enzymolysis product are the solid oil removing microbial inoculum, the solid fermentation culture medium and the enzymolysis product in the technical scheme, and are not described herein again. In one embodiment, the solid oil-removing microbial inoculum is mixed with the petroleum-polluted soil according to the mass ratio of 1:99, so that the petroleum-polluted soil is repaired.
The solid oil-removing microbial inoculum is applied to the remediation of the oil-contaminated soil, and can solve the problems of soil hardening, poor air permeability, compact soil, poor water holding capacity and reduced oxygen and nutrient levels in the oil-contaminated soil.
The invention provides a solid oil-removing microbial inoculum and a preparation method and application thereof, wherein the solid oil-removing microbial inoculum is obtained by fermenting bacillus subtilis in a solid fermentation culture medium. The experimental result shows that the effective viable bacteria amount of the solid oil-removing microbial inoculum prepared by adopting the enzymolysis product is improved by 20 times, and the solid oil-removing microbial inoculum has higher effective viable bacteria amount; the solid oil removing microbial inoculum is applied to the soil polluted by petroleum, after the soil is treated for 60 days, the oil content in the soil is reduced by 2.8 times, the soil polluted by petroleum is improved, meanwhile, the solid fermentation culture medium can provide nutrients for the bacterial strain and serve as a carrier for field planting of the bacterial strain in the soil polluted by petroleum, and can also effectively adsorb petroleum hydrocarbon in the soil polluted by petroleum, so that the maintenance of water in the soil is facilitated, the hardening condition of the soil is improved, the compactness of the soil is reduced, and the nutrition in the soil is increased. After the petroleum polluted soil is treated by the solid oil removing microbial inoculum, rich organic matters, inorganic salts, trace elements and other nutrient substances can be provided for later plant growth.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, but the present invention is not limited to the following embodiments, and all other embodiments obtained by a person of ordinary skill in the art without any creative work based on the embodiments of the present invention belong to the protection scope of the present invention.
Example 1:
preparing a solid oil-removing microbial inoculum:
inoculating the screened, separated and purified Bacillus subtilis with the preservation number of CGMCC NO.16167 into an LB liquid culture medium for culture at the culture temperature of 28 ℃, the rotation speed of 80rpm for 12 hours to obtain a liquid strain, wherein the effective viable count of the liquid strain is 0.9 x 108cfu/mL。
Weighing 15kg of sawdust fungus residues, 10kg of corncobs and 2kg of rice husks, crushing, sieving with a 20-mesh sieve, putting into an enzymolysis tank, adding 108kg of water, stirring, adjusting the pH to 5.5, adjusting the temperature of the enzymolysis tank to 55 ℃, adding 0.1% of cellulase by mass fraction, continuously stirring in the enzymolysis process, after 1 hour of enzymolysis, heating the enzymolysis tank to 80 ℃, keeping for 15 minutes, and performing enzyme deactivation treatment. And (3) cooling the enzymolysis tank to 50 ℃, adjusting the pH to 5.0, adding 0.05% of xylanase by mass, carrying out enzyme deactivation treatment after enzymolysis for 1.2h, and simultaneously carrying out vacuum reduced pressure concentration on the enzymolysis liquid to remove 32kg of water to obtain the corn cob, the rice hull and the sawdust bacteria residue after enzymolysis. Dissolving 5kg of brown sugar, 0.3kg of urea, 1kg of inorganic salt (0.8kg of monopotassium phosphate, 0.05kg of calcium chloride and 0.15kg of magnesium chloride) and 0.005kg of trace elements (0.001kg of zinc sulfate, 0.001kg of ferric trichloride and 0.003kg of manganese chloride) in a small amount of water, mixing with 20kg of wheat bran, 5kg of corn flour, corn cob after enzymolysis, rice hull and sawdust bacteria residue, uniformly mixing, loading into a sterilization device, and sterilizing at 121 ℃ for 1 h. And cooling the sterilized solid culture medium to 35-40 ℃, transferring the solid culture medium to a sterile stirring tank, and starting stirring.
Spraying liquid strains into a stirring tank, wherein the volume-to-mass ratio of the liquid strains to a solid fermentation culture medium is 0.05:1, stirring for 30min after inoculation, fully and uniformly mixing the solid fermentation culture medium and the strains, transferring the mixture to a sterile room for fermentation culture, introducing sterile air and stirring, wherein the fermentation temperature is 36 ℃, the fermentation time is 50h, obtaining a solid oil-removing microbial inoculum after fermentation, and determining the effective viable count of the obtained solid oil-removing microbial inoculum to be 1.3 x 109cfu/g。
Example 2:
preparing a solid oil-removing microbial inoculum:
inoculating the screened, separated and purified Bacillus subtilis with the preservation number of CGMCC NO.16167 into an LB liquid culture medium for culture at the culture temperature of 33 ℃, the rotation speed of 180rpm for 14 hours to obtain a liquid strain, wherein the effective viable count of the liquid strain is 1.3 x 109cfu/mL。
Weighing 18kg of sawdust fungus residues, 14kg of corncobs and 3kg of rice husks, crushing, sieving with a 20-mesh sieve, putting into an enzymolysis tank, adding 140kg of water, stirring, adjusting the pH to 5.8, adjusting the temperature of the enzymolysis tank to 55 ℃, adding 0.3% of cellulase by mass fraction, continuously stirring in the enzymolysis process, after 1.5 hours of enzymolysis, heating the enzymolysis tank to 80 ℃, keeping for 15min, and performing enzyme deactivation treatment. Cooling the enzymolysis tank to 50 ℃, adjusting the pH to 5.5, adding 0.03 mass percent of xylanase, carrying out enzyme deactivation treatment after 1h of enzymolysis, carrying out vacuum reduced pressure concentration on the enzymolysis liquid, and removing 48kg of water to obtain the corn cob, the rice hull and the sawdust bacteria residue after the enzymolysis. Dissolving 6kg of brown sugar, 1.2kg of urea, 0.9kg of inorganic salt (0.5kg of monopotassium phosphate, 0.3kg of dipotassium phosphate and 0.1kg of magnesium chloride) and 0.004kg of trace elements (0.002kg of zinc sulfate and 0.002kg of manganese chloride) in a small amount of water, mixing with 20kg of wheat bran, 6kg of corn flour, the corn cob after enzymolysis, the rice hull and sawdust bacteria residue, loading the mixture into a sterilization device after uniform mixing, and sterilizing for 1 hour at 121 ℃; and cooling the sterilized solid culture medium to 35-40 ℃, transferring the solid culture medium to a sterile stirring tank, and starting stirring.
Spraying liquid strains into a stirring tank, wherein the volume-to-mass ratio of the liquid strains to a solid fermentation culture medium is 0.08:1, stirring for 30min after inoculation, fully and uniformly mixing the solid fermentation culture medium and the strains, transferring the mixture to a sterile room for culture, introducing sterile air and stirring, wherein the fermentation temperature is 36 ℃, the fermentation time is 55h, obtaining a solid oil-removing microbial inoculum after fermentation, and determining the effective viable count of the obtained solid oil-removing microbial inoculum to be 2.8 x 109cfu/g。
Example 3:
preparing a solid oil-removing microbial inoculum:
inoculating the screened, separated and purified Bacillus subtilis with the preservation number of CGMCC NO.16167 into an LB liquid culture medium for culture at the culture temperature of 37 ℃ and the rotation speed of 220rpm for 16 hours to obtain a liquid strain, wherein the effective viable count of the liquid strain is 2.0 x 109cfu/mL。
Weighing 20kg of sawdust fungus residues, 20kg of corncobs and 4kg of rice husks, crushing, sieving with a 20-mesh sieve, putting the materials into an enzymolysis tank, adding 156kg of water, stirring, adjusting the pH to 6.0, adjusting the temperature of the enzymolysis tank to 55 ℃, adding 0.3% of cellulase by mass fraction, continuously stirring in the enzymolysis process, performing enzyme deactivation treatment after 2 hours of enzymolysis, and heating the enzymolysis tank to 80 ℃ for 15 minutes. And (3) cooling the enzymolysis tank to 55 ℃, adjusting the pH to 6.0, adding 0.05% of xylanase by mass, and carrying out enzyme deactivation treatment after enzymolysis for 1 h. And (3) carrying out vacuum reduced pressure concentration on the enzymolysis feed liquid, and removing 90kg of water to obtain the corn cob, the rice hull and the sawdust bacteria residue after enzymolysis. 10kg of brown sugar, 1kg of urea, 1.5kg of inorganic salt (0.6kg of monopotassium phosphate, 0.65kg of dipotassium phosphate, 0.05kg of calcium chloride, 0.1kg of calcium carbonate, 0.05kg of calcium sulfate and 0.05kg of magnesium chloride) and 0.008kg of trace elements (0.003kg of zinc sulfate, 0.002kg of ferric trichloride, 0.002kg of manganese chloride and 0.001kg of copper sulfate) are dissolved by a small amount of water, mixed with 18kg of wheat bran, 10kg of corn flour, the corn cob after enzymolysis, the rice hulls and sawdust bacteria residue, loaded into a sterilization device after being uniformly mixed, and sterilized for 1 hour at 121 ℃. And cooling the sterilized solid culture medium to 35-40 ℃, transferring the solid culture medium to a sterile stirring tank, and starting stirring.
Spraying liquid strains into a stirring tank, wherein the volume-to-mass ratio of the liquid strains to a solid fermentation culture medium is 0.1:1, stirring for 30min after inoculation to fully and uniformly mix solid materials and the strains, transferring the mixture to a sterile chamber for culture, introducing sterile air and stirring, wherein the fermentation temperature is 36 ℃, the fermentation time is 55h, obtaining a solid oil-removing microbial inoculum after fermentation, and determining the effective viable count of the obtained solid oil-removing microbial inoculum to be 4.5 x 109cfu/g。
Comparative example 1:
preparing a solid oil-removing microbial inoculum:
inoculating the screened, separated and purified Bacillus subtilis with the preservation number of CGMCC NO.16167 into an LB liquid culture medium for culture at the culture temperature of 37 ℃ and the rotation speed of 220rpm for 16 hours to obtain a liquid strain, wherein the effective viable count of the liquid strain is 2.0 x 109cfu/mL。
Weighing 20kg of sawdust fungus residue, 20kg of corncob and 4kg of rice hull, crushing, sieving with a 20-mesh sieve, mixing with 66kg of water, 10kg of brown sugar, 1kg of urea, 1.5kg of inorganic salt (comprising 0.6kg of monopotassium phosphate, 0.65kg of dipotassium hydrogen phosphate, 0.05kg of calcium chloride, 0.1kg of calcium carbonate, 0.05kg of calcium sulfate and 0.05kg of magnesium chloride), 0.008kg of trace elements (0.003kg of zinc sulfate, 0.002kg of ferric trichloride, 0.002kg of manganese chloride and 0.001kg of copper sulfate), 18kg of wheat bran and 10kg of corn flour, uniformly mixing, loading into a sterilization device, and sterilizing for 1h at 121 ℃; and cooling the sterilized solid culture medium to 35-40 ℃, transferring the solid culture medium to a sterile stirring tank, and starting stirring.
Spraying liquid strains into a stirring tank, wherein the volume-to-mass ratio of the liquid strains to a solid fermentation culture medium is 0.1:1, stirring for 30min after inoculation to fully and uniformly mix solid materials and the strains, transferring the mixture to a sterile chamber for culture, introducing sterile air and stirring, wherein the fermentation temperature is 36 ℃, the fermentation time is 55h, obtaining a solid oil-removing microbial inoculum after fermentation, and determining the effective viable count of the obtained solid oil-removing microbial inoculum to be 2.0 x 108cfu/g。
Example 4:
the solid oil-removing microbial inoculum prepared in the example 1, the example 2, the example 3 and the comparative example 1 is respectively mixed with petroleum-polluted soil and is used for repairing the petroleum-polluted soil, the mass fraction of the solid oil-removing microbial inoculum is 1%, the petroleum-polluted soil without the solid oil-removing microbial inoculum is used as a blank control, and the oil content, the total number of colonies, the water content and the compactness of the soil are measured by periodically sampling.
And (3) measuring the oil content of the soil: samples were taken periodically at 15d, 30d, 45d and 60d, respectively, and the oil content was determined using an infrared oil meter.
And (3) measuring the bacteria content in the soil: the assay was performed according to GB 4789.2-2010.
And (3) measuring the water content difference of the soil: taking 50g of each soil sample to be treated, respectively measuring the water content (X1), respectively spraying 10mL of water into each sample, uniformly stirring, flatly spreading in a container with the same volume, standing outdoors for 24h, uniformly stirring, measuring the water content, and calculating the water content difference. The larger the difference in water content, the better the water retention. The water content difference calculation formula is as follows:
the water content difference is X2-X1.
And (3) measuring the soil compactness: the soil compactness is a parameter for measuring the soil body compactness, and is measured at Xcm3The soil sample was charged into the container (2), and the net weight m (g) of the soil sample was measured to calculate the soil compaction degree. The soil compactness calculation formula is as follows:
soil compactness is m/X (g/cm)3)。
TABLE 1 determination of oil content of different sample soils
Table 1 shows the oil content of different sample soils, and the results show that the solid oil-removing microbial inoculum prepared from sawdust bacteria residue, corncobs and rice hulls which are subjected to enzymolysis by cellulase and xylanase can degrade petroleum hydrocarbon in the petroleum-polluted soil and reduce the oil content of the petroleum-polluted soil.
TABLE 2 determination of the bacteria content, the water content difference and the compactness of different sample soils
| Sample (I) | Bacterial content (cfu/g) | 24h Water content Difference (%) | Compactness (g/cm)3) |
| Blank space | 1.0*105 | 15.4 | 256 |
| Example 1 | 3.4*108 | 17.2 | 222 |
| Example 2 | 4.9*108 | 17.4 | 219 |
| Example 3 | 5.8*108 | 17.3 | 214 |
| Comparative example 1 | 4.3*106 | 16.2 | 238 |
Table 2 shows the determination of the content of bacteria, the water content difference and the compactness of different samples, and the results show that the solid oil removing microbial inoculum prepared from sawdust bacteria residue, corncobs and rice hulls which are subjected to enzymolysis by cellulase and xylanase is applied to soil remediation, so that the bacteria content of the soil is improved, the water in the soil is favorably maintained, the compactness of the soil is reduced, the looseness of the soil is increased, the hardening condition of the soil is improved, and the air permeability of the soil is increased, so that the oxygen content in the soil is increased, and the remediation of the petroleum-polluted soil is favorably realized.
The above description of the embodiments is only intended to facilitate the understanding of the method of the invention and its core idea. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.
Claims (10)
1. A solid oil-removing microbial inoculum is characterized in that the microbial inoculum is obtained by fermenting bacillus subtilis in a solid fermentation culture medium;
the solid fermentation medium comprises the following components in percentage by mass: 12-46% of an enzymolysis product, 10-20% of wheat bran, 3-10% of corn flour, 5-10% of brown sugar, 0-3% of urea, 0.5-2% of inorganic salt, 0.001-0.01% of trace elements and the balance of water;
the enzymolysis product is one or more of corncob, rice hull and sawdust fungus residue which are subjected to enzymolysis by cellulase and xylanase.
2. The solid oil-removing microbial inoculum according to claim 1, wherein the enzymolysis products are products obtained by enzymolysis of corncobs, rice husks and sawdust microbial dregs by cellulase and xylanase;
the mass ratio of the corncobs, the rice husks and the sawdust fungus residues is (1-21): (1-5): (10-20).
3. The solid oil-removing microbial inoculum according to claim 2, wherein the preparation method of the enzymolysis product specifically comprises the following steps: carrying out enzymolysis on corncobs, rice husks and sawdust fungus residues by using cellulase, and then carrying out enzymolysis by using xylanase;
the enzymolysis temperature of the cellulase is 50-55 ℃, the pH value is 5.5-6.0, and the enzymolysis time is 1-2 h;
the enzymolysis temperature of the xylanase is 50-60 ℃, the pH value is 5.0-6.0, and the time is 1-2 h;
the mass ratio of the cellulase to the xylanase is (0.1-5) to (0.1-1).
4. The solid oil-removing microbial inoculum according to claim 1, wherein the sawdust mushroom dregs are sawdust mushroom dregs obtained after edible mushroom cultivation and fruiting, and the edible mushroom comprises one or more of agaric, shiitake and oyster mushroom;
the inorganic salt comprises one or more of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, calcium chloride, calcium carbonate, calcium sulfate and magnesium chloride;
the microelements comprise one or more of zinc sulfate, ferric trichloride, manganese chloride and copper sulfate.
5. The solid oil-removing microbial inoculum according to claim 1, wherein the bacillus subtilis is bacillus subtilis with the preservation number of CGMCC NO. 16167.
6. The solid oil-removing microbial inoculum of claim 5, wherein the effective viable count of the bacillus subtilis is 3 x 108～8*109cfu/g。
7. A preparation method of a solid oil-removing microbial inoculum is characterized by comprising the following steps:
inoculating bacillus subtilis into a solid fermentation culture medium for fermentation culture;
the solid fermentation medium comprises the following components in percentage by mass: 12-46% of an enzymolysis product, 10-20% of wheat bran, 3-10% of corn flour, 5-10% of brown sugar, 0-3% of urea, 0.5-2% of inorganic salt, 0.001-0.01% of trace elements and the balance of water;
the enzymolysis product is one or more of corncob, rice hull and sawdust fungus residue which are subjected to enzymolysis by cellulase and xylanase.
8. The solid fermentation culture medium is characterized by comprising the following components in percentage by mass: 12-46% of an enzymolysis product, 10-20% of wheat bran, 3-10% of corn flour, 5-10% of brown sugar, 0-3% of urea, 0.5-2% of inorganic salt, 0.001-0.01% of trace elements and the balance of water;
the enzymolysis product is one or more of corncob, rice hull and sawdust fungus residue which are subjected to enzymolysis by cellulase and xylanase.
9. An application of a solid oil-removing microbial inoculum in degrading petroleum hydrocarbon.
10. A method for restoring petroleum-polluted soil by using a solid oil-removing microbial inoculum is characterized by comprising the following steps:
mixing the solid oil-removing microbial inoculum with petroleum-polluted soil for remediation;
the solid oil-removing microbial inoculum is obtained by fermenting bacillus subtilis in a solid fermentation culture medium;
the solid fermentation medium comprises the following components in percentage by mass: 12-46% of an enzymolysis product, 10-20% of wheat bran, 3-10% of corn flour, 5-10% of brown sugar, 0-3% of urea, 0.5-2% of inorganic salt, 0.001-0.01% of trace elements and the balance of water;
the enzymolysis product is one or more of corncob, rice hull and sawdust fungus residue which are subjected to enzymolysis by cellulase and xylanase.
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| CN109486725A (en) * | 2018-12-27 | 2019-03-19 | 黄河三角洲京博化工研究院有限公司 | Bacterial strain and its application of one plant of degradable benzene homologues and petroleum hydrocarbon |
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