CN117866802A

CN117866802A - Bacillus sand and application thereof

Info

Publication number: CN117866802A
Application number: CN202311630368.1A
Authority: CN
Inventors: 郑素娟; 赵琬玫; 闫明英; 王锦梅; 温胜国; 王文红; 仲伟华; 刘圣鹏
Original assignee: Qingdao Weilan Saide Biotechnology Co ltd
Current assignee: Qingdao Weilan Saide Biotechnology Co ltd
Priority date: 2023-11-30
Filing date: 2023-11-30
Publication date: 2024-04-12

Abstract

The invention relates to a bacillus sand (Bacillus safensis) CY1, which is preserved in the China general microbiological culture Collection center (China Committee) for culture Collection of microorganisms, and has the following addresses: the collection number of the national institute of microbiology, national academy of sciences, no. 3, north Chen West Lu 1, chao yang, beijing, is: CGMCC No.27876, the strain has high degradation efficiency on diesel oil, and the degradation rate of the diesel oil in 72 hours reaches more than 99 percent aiming at the diesel oil content below 1 percent in an evaluation culture medium at 30 ℃; and moderately salt-tolerant, the number of viable bacteria is hardly affected when growing under the salinity condition of less than 5%.

Description

Bacillus sand and application thereof

Technical Field

The invention relates to bacillus sand and application thereof in the field of wastewater and soil treatment and restoration, and belongs to the technical field of environmental microorganisms.

Background

Petroleum hydrocarbons are one of the most important components in petroleum, mainly composed of alkanes and aromatic hydrocarbons, and also contain small amounts of sulfur-and nitrogen-containing compounds. The problems of petroleum hydrocarbon pollution of surrounding environment are inevitably caused by improper management or sudden leakage accidents in the processes of exploration, exploitation, smelting, transportation, use, storage and the like of petroleum, the alkane in the petroleum hydrocarbon is gradually deposited into soil, underground water and sea, the persistent environmental pollution is extremely easy to cause, the polycyclic aromatic hydrocarbon in the aromatic hydrocarbon is difficult to degrade and has carcinogenicity, and the polycyclic aromatic hydrocarbon can enter the human body through a food chain, thereby threatening the life and health of human beings.

The source of the oily wastewater is very wide, and besides the oil exploitation and processing industry which discharges a large amount of oily wastewater, the oily wastewater can be discharged by solid fuel thermal processing, wool washing wastewater in textile industry, leather making wastewater in light industry, railway and transportation industry, slaughtering and food processing industry, and emulsion generated by turning process in mechanical industry, and the like.

Currently, treatment methods for petroleum hydrocarbon pollution are largely classified into physical methods, chemical methods, and biological methods. The biological treatment technology has the advantages of small secondary pollution, high removal efficiency, thorough pollutant removal, high economic performance and the like, so that the biological treatment technology becomes a research hot spot of the modern wastewater treatment technology and is receiving more and more attention.

Currently, scientists have screened various strains with petroleum hydrocarbon degradation function, such as common petroleum hydrocarbon degradation bacteria including Acinetobacter (Acinetobacter sp.), pseudomonas sp., corynebacterium sp., micrococcus sp., arthrobacter sp., alcaligenes sp., etc. However, these strains are still not sufficiently effective in degrading petroleum hydrocarbon, and thus it is necessary to screen or construct strains having more remarkable effect in degrading petroleum hydrocarbon.

Disclosure of Invention

Aiming at the current situation that the effect of the existing petroleum hydrocarbon degradation strain is not enough, the invention provides a bacillus sand and the application thereof in the field of wastewater and soil treatment and restoration.

Bacillus sand (Bacillus safensis) CY1 is preserved in China general microbiological culture Collection center (China Committee) with the address: the collection number of the national institute of microbiology, national academy of sciences, no. 3, north Chen West Lu 1, chao yang, beijing, is: CGMCC No.27876, the preservation date is: 2023, 7 and 13 days, 16s rDNA of the strain is shown as SEQ ID NO. 1, and the bacillus subtilis disclosed by the invention refers to bacillus subtilis CY1 strain under the condition that NO special description exists.

The bacillus safoci provided by the invention has the beneficial effects that:

1) The strain has high degradation efficiency on diesel oil, and the degradation rate of the diesel oil in 72 hours reaches more than 99 percent aiming at the diesel oil content below 1 percent in an evaluation culture medium at the temperature of 30 ℃;

2) The strain has high-efficiency petroleum hydrocarbon degradation capability, the petroleum hydrocarbon degradation rate is 96% in five days for wax printing wastewater with the initial oil content of 110mg/L, and 98% in five weeks for petrochemical sludge contaminated soil with the initial oil content of 2%;

3) The strain is moderately salt-tolerant, and when the strain grows under the salinity condition of less than 5%, the number of viable bacteria is hardly affected, but the strain cannot tolerate the salinity of more than 6%;

4) The strain has the advantages of simple culture method, high growth speed, strong environmental adaptability, high safety, no damage to the original environment and no secondary pollution.

The petroleum hydrocarbons described in the present invention include, but are not limited to, normal paraffins, branched paraffins, naphthenes, and aromatics.

The invention also discloses a microbial agent containing the bacillus sarefolius.

Preferably, the fermentation method of the bacillus subtilis comprises the following steps:

(1) Primary seed culture: inoculating bacillus safoci into enrichment medium under aseptic condition, culturing at 25-35deg.C and 150-300rpm for 12-48 hr to obtain first seed culture solution of bacillus safoci;

(2) Secondary seed culture: inoculating the first-stage seed culture solution of bacillus subtilis into an enrichment culture medium according to an inoculum size of 1-10vol% under a sterile condition, and culturing for 12-48h at 25-35 ℃ and 150-300rpm to obtain a second-stage seed culture solution of bacillus subtilis;

(3) Fermentation: and (3) after the fermentation medium in the fermentation tank is disinfected, inoculating the second-level seed culture solution of the bacillus halofop obtained in the step (2) into the fermentation medium according to the inoculum size of 5-10vol%, controlling the temperature to be 25-35 ℃ and the rotating speed to be 150-300rpm, fermenting under the condition that the aeration ratio is 1 (1-2), and stopping fermenting when dissolved oxygen starts to rise to obtain the fermentation solution of the bacillus halofop.

The aeration ratio in the present invention means the ratio of the volume of air introduced into the fermenter per minute to the total volume of the fermentation liquid.

Wherein the composition of the enrichment medium is: 5-15g/L peptone, 3-8g/L yeast extract or yeast powder, 5-15g/L sodium chloride, water as solvent, and pH=6-8.

Preferably, the composition of the enrichment medium is: 10g/L of tryptone, 5g/L of yeast extract, 10g/L of sodium chloride and water as a solvent, wherein the pH=7-7.5.

Further, the composition of the fermentation medium is: 10-30g/L of carbon source and 5-20g/L, PO of nitrogen source ₄ ^3- 0.8-1.5g/L、K ⁺ 0.5-1.0g/L、Mg ²⁺ 0.05-0.5g/L、Na ⁺ 0.1-0.5g/L、Mn ²⁺ 0.03-0.2g/L、Ca ²⁺ 0.01-0.05g/L, water as solvent, pH=6-8.

Preferably, the PO ₄ ^3- The source of the catalyst is one or more of dipotassium hydrogen phosphate, potassium dihydrogen phosphate and diammonium hydrogen phosphate,the K is ⁺ The source of the catalyst is one or more of dipotassium hydrogen phosphate, potassium dihydrogen phosphate, potassium sulfate, potassium chloride and potassium nitrate, and the Mg ²⁺ Is one or more of magnesium sulfate and magnesium chloride, and the Na is ⁺ Is one or more of sodium chloride, sodium nitrate or sodium sulfate, the Mn ²⁺ The source of the Ca is one or more of manganese sulfate monohydrate, manganese sulfate tetrahydrate, manganese nitrate or manganese chloride ²⁺ The source of the catalyst is one or more of calcium chloride and calcium nitrate.

Further, the carbon source is selected from one or more of glucose, sucrose, starch, sodium acetate or sodium citrate.

Further, the nitrogen source is selected from one or more of yeast extract powder, peptone, corn steep liquor dry powder, ammonium sulfate, diammonium phosphate or potassium nitrate.

In the practical application process, the form of the final bacillus saxifrage product can be determined according to the practical use and storage requirements, when a liquid product is required to be used, the fermentation liquor can be diluted to the required concentration for direct use, when a solid product is required to be used, bacterial mud can be obtained after the fermentation liquor is centrifuged, and then the solid bacterial powder is prepared by adopting a spray drying or freeze drying process.

The invention also claims a method for purifying sewage or soil by using the activated liquid or microbial agent of the bacillus safteus, which comprises the step of applying the activated liquid of the bacillus safteus or the microbial agent containing the bacillus safteus to the sewage or the soil.

Further, the concentration of petroleum hydrocarbon in the sewage and wastewater is 1% or less, preferably 0.5% or less.

Further, the salinity of the sewage and wastewater is 5% or less, preferably 3% or less, and most preferably 2% or less.

Further, the inoculation amount of the activated liquid of bacillus subtilis or the microbial agent is more than 1000ppm, preferably 1000-20000ppm, and most preferably 1000-10000ppm.

Further, the concentration of petroleum hydrocarbon in the soil is 10% or less, preferably 5% or less, and most preferably 2% or less.

The invention also claims the application of the bacillus safoci and the microbial agent in the field of treatment and restoration of sewage and wastewater or soil.

Preferably, the bacillus subtilis and the microbial agent are used for degrading petroleum hydrocarbons in sewage or soil; more preferably, the bacillus subtilis and microbial agent are used for degrading the diesel oil in the sewage or soil.

Drawings

FIG. 1 is a photograph of a colony of Bacillus safoci;

FIG. 2 is a photograph of a colony of Bacillus saxifrage magnified by a 100-fold microscope.

Detailed Description

The principles and features of the present invention are described below in connection with examples, which are set forth only to illustrate the present invention and not to limit the scope of the invention.

The respective media used in the examples were composed as follows:

enrichment medium: 1g of dipotassium hydrogen phosphate, 1g of monopotassium phosphate, 1g of ammonium sulfate, 0.2g of magnesium sulfate, 1g of potassium nitrate, 0.01g of calcium chloride and 1mL of microelement solution, and fixing the volume to 1L, wherein pH=7;

basal medium: 10g of peptone, 5g of yeast powder, 10g of sodium chloride, 1000ml of water and pH=7;

evaluation of the medium: 1g of dipotassium hydrogen phosphate, 1g of monopotassium phosphate, 1g of ammonium sulfate, 0.2g of magnesium sulfate, 1g of potassium nitrate, 0.01g of calcium chloride and 1mL of microelement solution, and adjusting the concentration of different diesel oil according to the requirement, wherein the volume is fixed to 1L, and the pH=7;

trace element solution: 1.5g of ferric chloride, 0.1g of manganese sulfate, 70mg of zinc chloride, 2mg of copper chloride, 30mg of nickel chloride, 200mg of cobalt chloride, 5mg of sodium molybdate and the volume to 1L, wherein pH=7.

EXAMPLE 1 screening separation and purification of Bacillus Adefolius

1. Water sample enrichment

Taking 100mL of a water sample from an aerobic tank of a certain chemical plant in Shandong province, inoculating 10mL of the water sample into a triangular flask filled with 100mL of enrichment medium, adding 0.05vol% diesel oil into the enrichment medium, performing shake culture at 30 ℃ for 5 days under the condition of 200r/min, measuring the content of the diesel oil every other day, and adjusting pH; when the diesel oil content is reduced to below 50% of the original concentration, transferring the next stage of enrichment, namely taking 10mL of the cultured culture solution and inoculating the culture solution into 100mL of enrichment medium again, increasing the concentration of the transferred diesel oil by 0.05vol% each time, and repeatedly culturing for 4 times until the concentration of the diesel oil in the enrichment medium is 0.2vol%.

2. Screening and isolation of strains

Taking 0.5mL of the culture solution of the last stage, and carrying out gradient dilution by using sterile water to respectively dilute to 10 ^-4 ，10 ^-5 ，10 ^-6 ，10 ^-7 And 10 ^-8 The diluted culture solution is coated on a nutrient solid culture medium flat plate, and then the culture solution is placed in a 30 ℃ incubator for culture, after the colony grows to a proper size, the morphological characteristics of the colony are observed, single colony is selected for streak purification, after three generations of purification, inclined-plane preservation at 4 ℃ is carried out, 6 strains are separated out, and the strains are named CY 1-CY 5 respectively.

3. Double screen

The 5 strains obtained by primary screening are respectively inoculated into a basic culture medium, shake culture is carried out for 24 hours at 30 ℃, 10mL of culture solution is respectively taken and centrifuged for 10 minutes at 5000rpm, supernatant is discarded, each strain is respectively inoculated into 100mL of evaluation culture medium (diesel oil content is 0.2 vol%) and shake culture is carried out at 30 ℃, three groups of strains are arranged in parallel, sterile water is used as a blank control, and the change of the diesel oil content in the culture medium is periodically detected, and the results are shown in table 1.

Table 1 evaluation results of the strain obtained by preliminary screening on diesel oil

From the data in table 1, strain CY1 has the strongest effect of removing diesel oil, and its degradation ability to diesel oil is significantly better than that of other 4 strains.

Example 2 identification of species

1. Morphological observation

Strain CY1 was inoculated on a nutrient solid medium plate and the morphological characteristics of the colonies were observed. The colony of CY1 strain is shown in figure 1, the 100 times microscopic photograph is shown in figure 2, and the colony is punctiform, opaque, milky white, neat in edge, clear and smooth and dry in surface.

2. Molecular biological identification

Genomic DNA of strain CY1 was extracted and 16S rDNA was amplified using this as a template.

16S rDNA primer:

16S rDNA-F:5’-TATGTTGCCCTTGTCGTGG-3’，

16S rDNA-R:5’-CCGCGTTTGTAGGCCCTCGT-3’。

the PCR products were then extracted and DNA sequenced using a sequencer ABI 3730-XL. The spliced sequence file is compared with data in NCBI 16S database by NCBI Blast program to obtain species information with the maximum sequence similarity with the species to be detected, the species information is found to belong to the genus Bacillus, identified as bacillus safoci (Bacillus safensis), and named as bacillus safoci CY1.

EXAMPLE 3 salinity tolerance test of Bacillus Adefolius

Under the aseptic condition, bacillus safoci CY1 is inoculated into a 250mL conical flask containing 100mL of basic culture medium, and is subjected to shaking culture for 24 hours at 30 ℃ in a shaking table at 200rpm to perform strain activation, so that an activated liquid of bacillus safoci is obtained, and the number of viable bacteria of the activated liquid is 83 hundred million CFU/mL through test.

Based on the basal medium (salinity: 1%), 0g, 1g, 2g, 3g, 4g, 5g, 6g, 7g, 8g, 9g of NaCl was additionally added to each of the flasks containing 100mL of basal medium, and then salinity gradients of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% and 10% were set. Respectively taking 5mL of each activating solution of bacillus safoci, respectively inoculating into culture mediums with different salinity gradients, placing into a shaking table at 30deg.C for shake culture, and periodically measuring OD ₆₀₀ Values and viable count to determine the extent of strain growth, the results are shown in Table 2.

TABLE 2 OD of Bacillus Adefolius cultured under different salinity conditions ₆₀₀ Value and viable count

As can be seen from the data in Table 2, strain CY1 can grow normally at a salinity of 1-5%, 1% is the most suitable salinity for growth, and the strain growth condition is significantly better than that at a salinity of more than 5% when the salinity is less than 5%.

EXAMPLE 4 determination of degradation efficiency of Bacillus Adefolius at different Diesel oil contents

Under the aseptic condition, bacillus safoci CY1 is inoculated into a 250mL conical flask containing 100mL of basic culture medium, and is subjected to shaking culture for 24 hours at 30 ℃ in a shaking table at 200rpm to perform strain activation, so that an activated liquid of bacillus safoci is obtained, and the number of viable bacteria of the activated liquid is 87 hundred million CFU/mL through test.

Evaluation media with diesel oil volume contents of 0.5%, 1%, 2%, 3%, 4% and 5% were prepared, respectively, the activated liquid of bacillus safoci was inoculated into 100mL of each sterilized evaluation medium in an inoculum size of 1vol%, and then placed in shaking tables at 30 ℃ and 200rpm for shaking culture, 3 parallel groups were set, sterile water was added to the evaluation medium as a blank control, the experimental group arrangement was as shown in table 3, and the diesel oil content in each medium was measured every 24 hours, and the results are shown in table 4.

Table 3 each experimental group evaluates diesel content in the medium

Table 4 each experimental and control group evaluated the change in diesel content of the medium

As can be seen from the data in Table 4, when the diesel oil content was 1% or less, bacillus saxifragus CY1 exhibited excellent degradation effects, and the diesel oil degradation rate at 72 hours was 99% or more. However, when the diesel content is above 1%, the degradation rate is only 53.1% at maximum for 72 hours, and the degradation efficiency of CY1 gradually decreases as the diesel content increases. Thus, the result shows that the bacillus subtilis can tolerate 1% of diesel oil, but can tolerate less than 1% of diesel oil. EXAMPLE 5 fermentation Process of Bacillus Adefolius

The fermentation method of the bacillus safoci comprises the following steps:

1) Picking slant seeds of bacillus subtilis by an inoculating loop, inoculating the slant seeds into 100mL of basic culture medium, and culturing for 18 hours at 25-35 ℃ and 150-300rpm to obtain primary seed liquid;

2) Inoculating the cultured primary seed liquid into 1L of basic culture medium according to the inoculum size of 1-10vol%, and culturing at 25-35 ℃ and 150-300rpm for 18h to obtain secondary seed liquid;

3) Under the aseptic condition, inoculating the secondary seed liquid into a fermentation medium according to an inoculum size of 5-10vol%, wherein the formula of the fermentation medium is as follows: 10g/L of glucose, 10g/L of sucrose, 5g/L of yeast extract powder, 1.5g/L of ammonium sulfate, 0.5g/L of diamine hydrogen phosphate, 1g/L of monopotassium phosphate, 0.5g/L of magnesium sulfate, 0.5g/L of sodium chloride, 0.2g/L of manganese sulfate and 0.05g/L of calcium chloride, fermenting at the temperature of 25-35 ℃ and the rotating speed of 150-300rpm under the condition that the ventilation ratio is 1 (1-2), stopping fermenting when dissolved oxygen starts to rise, and obtaining fermentation liquor of bacillus sarefolius, wherein the viable count of the fermentation liquor is 194 hundred million CFU/mL through test.

Example 6 effects of Bacillus Adefolius on treatment of oily petrochemical wastewater

Bacillus safoci CY1 is inoculated into a 250mL conical flask containing 100mL of basic culture medium under the aseptic condition, and is subjected to shaking culture for 24 hours at 30 ℃ in a shaking table at 200rpm to perform strain activation, so that an activated liquid of the bacillus safoci is obtained, and the number of viable bacteria of the activated liquid is 86 hundred million CFU/mL through test.

The initial oil content of petrochemical oily wastewater in eastern mountain camping is 2890mg/L before treatment, and pH=7.8. 100mL of the oily wastewater is respectively placed in two identical 250mL conical flasks, an experimental group is inoculated with strain activation solution according to 1% of the volume of the oily wastewater, a control group is inoculated with sterile water with the same volume, the control condition is that the temperature is 30 ℃, the shaking treatment is carried out in a shaking table at 200rpm, and the pH value is natural. During the reaction, samples were taken every 48 hours after 24 hours, and the results are shown in Table 5.

Wherein, the oil detection method is implemented according to GB/T16488-1996 infrared photometry for determining oil and animal and vegetable oil.

TABLE 5 time-dependent oil content in petrochemical oily wastewater

As can be seen from the data in Table 5, the oil degradation rate was 54% within six days after the addition of the strain activation solution, which indicates that the Bacillus saxifrage of the present invention has a certain high concentration petroleum hydrocarbon degradation ability.

EXAMPLE 7 Effect of Bacillus Adefolius on treatment of oily wax printing wastewater

Bacillus safoci CY1 is inoculated into a 250mL conical flask containing 100mL of basic culture medium under the aseptic condition, and is subjected to shaking culture for 24 hours at 30 ℃ in a shaking table at 200rpm to obtain an activated liquid, and the activated liquid has 86 hundred million CFU/mL of viable bacteria.

The initial oil content of the oil-containing wax printing wastewater of Shandong province is 110mg/L before treatment, and the pH=7.8. 100mL of the oily wastewater is respectively placed in two identical 250mL conical flasks, an experimental group is inoculated with strain activation liquid according to 1% of the volume of the wastewater, a control group is inoculated with sterile water with the same volume, the control condition is that the temperature is 30 ℃, the shaking treatment is carried out in a shaking table at 200rpm, and the pH value is natural. Samples were taken every 24 hours during the reaction and the results are shown in table 6.

TABLE 6 variation of oil content in oil-containing wax printing wastewater with time

As can be seen from the data in Table 6, the degradation rate of petroleum hydrocarbon was 96% in five days after the addition of the strain-activating solution, which indicates that Bacillus saxifrage of the present invention has a high-efficiency petroleum hydrocarbon degradation ability.

EXAMPLE 8 Effect of Bacillus Adefolius on treatment of oil-containing soil

Bacillus safoci CY1 is inoculated into a 250mL conical flask containing 100mL of basic culture medium under the aseptic condition, and is subjected to shaking culture for 24 hours at 30 ℃ in a shaking table at 200rpm to obtain an activated liquid of the bacillus safoci, and the activated liquid has the viable count of 88 hundred million CFU/mL through test.

The soil polluted by some petrochemical sludge on the smoke table has the initial oil content of 2 percent before treatment, the detection value of 20200mg/L and the pH=8.2.

100g of the oily soil is respectively placed in two identical 250mL beakers, an experimental group is inoculated with strain activation liquid according to 10% of the mass of the oily soil, sterile water with the same mass is inoculated into a control group, the control condition is that the treatment is carried out in a constant temperature incubator at 30 ℃, the pH value is adjusted to be 7-8 before the bacteria are added, and the later-period pH is natural. And supplementing water at any time in the reaction process, and supplementing nitrogen and phosphorus to ensure that the proportion of CNP is 100:10:1. Samples were taken every 7 days and the results are shown in table 7.

TABLE 7 variation of oil content in oil-containing soil over time

As shown in the data in Table 7, five weeks after the addition of the strain activation solution, the petroleum hydrocarbon degradation rate was 97.9%, which indicates that the Bacillus saxifrage provided by the invention has high-efficiency petroleum hydrocarbon degradation capability.

The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.

Claims

1. A strain of bacillus sand (Bacillus safensis) deposited with the China general microbiological culture Collection center, having a deposit number: cgmccno.27876.

2. A microbial agent comprising the Bacillus saxifrage of claim 1 as an active ingredient.

3. The fermentation method of bacillus sarsashimi according to claim 1, which comprises the following steps:

4. A fermentation process according to claim 3, wherein the enrichment medium has a composition of: 5-15g/L of peptone, 3-8g/L of yeast extract or yeast powder, 5-15g/L of sodium chloride, water as solvent, and pH=6-8;

the composition of the fermentation medium is as follows: 10-30g/L of carbon source,Nitrogen source 5-20g/L, PO ₄ ^3- 0.8-1.5g/L、K ⁺ 0.5-1.0g/L、Mg ²⁺ 0.05-0.5g/L、Na ⁺ 0.1-0.5g/L、Mn ²⁺ 0.03-0.2g/L、Ca ²⁺ 0.01-0.05g/L, water as solvent, pH=6-8.

5. The fermentation process of claim 4, wherein the carbon source is selected from one or more of glucose, sucrose, starch, sodium acetate or sodium citrate;

the nitrogen source is selected from one or more of yeast extract powder, peptone, corn steep liquor dry powder, ammonium sulfate, diamine hydrogen phosphate or potassium nitrate.

6. A method for purifying waste water or soil, comprising the step of applying the activated liquid of bacillus safoci according to claim 1 or the microbial agent according to claim 2 to the waste water or soil.

7. The method according to claim 6, wherein the concentration of petroleum hydrocarbons in the waste water is 1% or less, preferably 0.5% or less.

8. The method according to claim 6, wherein the concentration of petroleum hydrocarbons in the soil is 10% or less, preferably 5% or less, most preferably 2% or less.

9. A method according to claim 6 or 7, characterized in that the salinity of the sewage water is below 5%, preferably below 3%, most preferably below 2%.

10. The bacillus subtilis of claim 1 and the application of the microbial agent of claim 2 in the field of treatment and restoration of sewage or soil; preferably, the bacillus subtilis and the microbial agent are used for degrading petroleum hydrocarbons in sewage or soil; more preferably, the bacillus subtilis and microbial agent are used for degrading the diesel oil in the sewage or soil.