CN113430134B

CN113430134B - Treatment process of petroleum hydrocarbon-containing wastewater

Info

Publication number: CN113430134B
Application number: CN202110735801.2A
Authority: CN
Inventors: 梁国斌; 吴晶晶; 印霞棐; 林伟; 单旋
Original assignee: Jiangsu University of Technology
Current assignee: Jiangsu University of Technology
Priority date: 2021-06-30
Filing date: 2021-06-30
Publication date: 2023-01-31
Anticipated expiration: 2041-06-30
Also published as: CN113430134A

Abstract

The invention discloses a treatment process of waste water containing petroleum hydrocarbon, which belongs to the field of waste water treatment by a microbiological method, and the two strains can adapt to the environment containing petroleum hydrocarbon, have good growth and reproduction, have good degradation and removal effects on engine oil components in the waste water, and have the highest degradation rate of 87.87 percent. Therefore, the two strains of the invention can adapt to the severe environment of the waste water containing petroleum hydrocarbon, can be applied to the treatment of the waste water containing petroleum hydrocarbon, and is beneficial to the sustainable development of the ecological environment.

Description

Treatment process of petroleum hydrocarbon-containing wastewater

Technical Field

The invention relates to a treatment process of petroleum hydrocarbon-containing wastewater, belonging to the field of wastewater treatment by a microbiological method.

Background

With the rapid development of the world industry, petroleum resources are widely utilized. The frequent events of offshore oil resource exploitation and transportation, oil leakage accidents and the like seriously pollute the marine environment, destroy the ecological balance of a marine system and threaten the life health of human beings. In the publication of' 2018 China Marine eco-Environment Condition, the ministry of ecological environmental protection states that the sea area of four poor water qualities in 2018 summer is about 3.3 km ² The main standard exceeding substances are active phosphate, inorganic nitrogen and petroleum substances; in the ocean section of the publication of 2018 on the ecological environmental conditions of China, petroleum substances are mentioned as one of the important pollutants in seawater, and seriously threaten the ecological balance of the ocean.

At present, the commonly used degradation treatment methods for marine petroleum hydrocarbon pollutants can be divided into three major types, namely physical methods, chemical methods and biological methods. The biological method mainly refers to biOremediation (bioroediation) which utilizes the oxidation and decomposition of petroleumThe microorganisms naturally existing in the ocean or the soil and can take petroleum hydrocarbon compounds as carbon sources and very energy sources to finally generate H ₂ O and CO ₂ The process of (2). Biological methods are important ways to remove oil contamination completely, compared with physical and chemical methods [10 ]]The influence on human and environment is small, and the repairing treatment cost is only 30% or 50% of that of the traditional physical and chemical treatment. Biological methods are considered as the most promising environmental management means with the advantages of small investment, no secondary pollution and capability of site-directed remediation.

Disclosure of Invention

The invention provides stenotrophomonas acidiphila, which has been preserved in China general microbiological culture collection management center at 5 days and 6 days in 2021 with the preservation number of CGMCC No.22273.

The invention provides an ochrobactrum, which has been preserved in China general microbiological culture collection management center at 5 days and 6 days in 2021, and the preservation number is CGMCC No.22274.

The invention also provides a microbial inoculum containing the stenotrophomonas acidiphila or the live bacteria of the genus Ochrobactrum.

The invention also provides application of the stenotrophomonas acidiphila and/or the microbial inoculum in degrading petroleum hydrocarbon.

The invention also provides application of the ochrobactrum and/or the microbial inoculum in degrading petroleum hydrocarbon.

In one embodiment, the petroleum hydrocarbon source is a wastewater containing petroleum hydrocarbons.

In one embodiment, the concentration of petroleum hydrocarbons in the wastewater is greater than or equal to 10g/L.

In one embodiment, the concentration of petroleum hydrocarbons in the wastewater is greater than or equal to 20g/L.

The invention also provides a method for degrading petroleum hydrocarbon, which is to add the stenotrophomonas acidophilus or the ochrobactrum and/or the microbial inoculum into a sample containing the petroleum hydrocarbon according to the addition amount of 10-50% of the volume ratio so as to degrade the petroleum hydrocarbon.

In one embodiment, the stenotrophomonas acidophilus is providedConcentrated OD of bacteria or ochrobactrum and/or microbial inoculum ₆₀₀ Is 1.3 to 1.5.

In one embodiment, the time for degradation is 15 to 36 hours.

In one embodiment, the time for degradation is 24 to 30 hours.

In one embodiment of the present invention, the treatment conditions are all: the temperature is 25-40 ℃, and the speed is 0-200 r/min.

In one embodiment, the sample is a petroleum hydrocarbon-containing wastewater.

The invention also provides application of the stenotrophomonas acidiphila and/or the microbial inoculum in preparation of a sewage treatment agent.

The invention also provides application of the ochrobactrum and/or microbial inoculum in preparation of a sewage treatment agent.

The invention provides a culture method of stenotrophomonas acidiphila.

In one embodiment, the method is to inoculate stenotrophomonas acidiphila into the culture medium for 24-48 h at an inoculation amount of 1-5% by volume.

In one embodiment, the culturing conditions are pH 6.0-7.5, rotation speed 140-180 r/min, and temperature 28-35 ℃.

The invention provides a culture method of ochrobactrum.

In one embodiment, the method is to inoculate the ochrobactrum into the culture medium for 24-48 h at an inoculation amount of 1-5% by volume.

In one embodiment, the culturing conditions are pH 6.0-7.0, rotation speed 140-180 r/min, and temperature 28-32 ℃.

The invention has the beneficial effects that:

the invention separates two strains from the waste water containing petroleum hydrocarbon, the two strains can grow and reproduce in the environment containing petroleum hydrocarbon, and the two strains can degrade substances harmful to the environment in the waste water containing petroleum hydrocarbon to a certain extent, and have good degradation effect on engine oil substances, the degradation rate of the petroleum hydrocarbon of stenotrophomonas acidiphilic can reach 87.87% as high as possible, and the degradation rate of the petroleum hydrocarbon of ochrobactrum can reach 42.41% as high as possible. Therefore, the two strains of the invention can adapt to the severe environment of the petroleum hydrocarbon-containing wastewater, and are beneficial to the sustainable development of ecological environment.

Biological material preservation

1. One strain of Stenotrophomonas acidophilus, which is classified and named Stenotrophormonas acidophilus, has been preserved in China general microbiological culture collection center at 5 days and 6 days in 2021, the preservation number is CGMCC No.22273, and the preservation address is the institute of microbiology, china academy of sciences.

2. The Ochrobactrum daejeonense is classified and named as Ochrobactrum daejeonense, is preserved in the China general microbiological culture Collection center at 5 days and 6 days in 2021, has the preservation number of CGMCC No.22274, and has the preservation address of the institute of microbiology of China academy of sciences.

Drawings

FIG. 1 is a diagram of colony morphology and cell morphology of stenotrophomonas microacida; a is colony morphology, B is cell morphology.

FIG. 2 is a diagram of the colony morphology and cell morphology of Ochrobactrum anthropi; a is colony morphology, B is cell morphology.

Detailed Description

LB culture medium: tryptone (10 g), yeast extract powder (5 g), sodium chloride (10 g), water (1L).

The method for measuring the content of the engine oil comprises the following steps: the method for determining the oil content in the wastewater by a gravimetric method comprises the following steps of (1) acidifying a sample by sulfuric acid: +5mL of a 50% (v/v) sulfuric acid solution. (2) The acidified sample was poured into a 250mL separatory funnel, then +25mL petroleum ether (60 deg.C-90 deg.C), shaken for 2min and allowed to stand with it to separate. Collecting supernatant, and inoculating subnatant into the original conical flask. (3) Extracting the lower layer liquid with petroleum ether for 2 times, each time with 25mL petroleum ether, and mixing the upper layer liquid obtained 3 times, i.e. petroleum ether extractive solution. (4) Adding anhydrous sodium sulfate (dehydrated) into petroleum ether extractive solution, shaking gently until no agglomeration, and standing for 1 hr with cover. (5) The filtrate was collected in a dry constant weight 100mL beaker by filtration through qualitative filter paper previously washed with petroleum ether. (6) Putting the beaker on a constant temperature water bath kettle at 65 +/-1 ℃ to evaporate to be nearly dry, taking out and wiping the beaker, putting the beaker in an oven at 65 +/-1 ℃ for 1-2h, cooling for 30min and weighing. (7) Degradation rate = (blank residual oil amount-sample residual oil amount) × 100%/oil amount initially charged.

Example 1: screening of strains

1. Sample collection

Sludge obtained from sewage treatment plants in Changzhou city is put into seawater simulating petroleum hydrocarbon pollutants for screening, separating, domesticating and culturing.

The method comprises the following specific steps: weighing 1g of petroleum-contaminated soil and 5mL of activated sludge, adding the petroleum-contaminated soil and 5mL of activated sludge into 100mL of sterilized liquid culture medium with the oil content of 2%, shaking and culturing for 5d at 140r/min and 30 ℃, transferring 4mL of suspension from the sterilized liquid culture medium, adding the suspension into 100mL of new sterilized liquid culture medium with the oil content of 2%, continuing to culture, and continuously and repeatedly acclimating for 6 times. Taking bacterial liquid after repeated acclimatization and culture for 6 times, diluting, coating on a solid LB culture medium plate, culturing in a constant temperature incubator at 37 ℃ for 24h, observing, selecting single bacterial colonies with different forms and colors, continuously performing streak culture on a new solid LB culture medium plate for 3 times, selecting single bacterial colonies obtained by separation, and continuously culturing in a new sterilized liquid culture medium.

2. Separation and purification of bacterial strains

Adding 5mL of sludge into 100mL of acclimatized liquid culture medium, performing shake culture at 37 deg.C and 140r/min for 5d, shaking 5mL of culture solution into new acclimatized liquid culture medium for 5d, repeating for 5 times, and preparing 10 from cultured bacteria solution ^-1 、10 ^-2 、10 ^-3 、10 ^-4 、10 ^-5 、10 ^-6 The diluted solution of (4 d) was applied onto LB solid medium and single colonies were obtained by inverted culture. Selecting the strain with superior growth vigor and storing the strain in glycerin tube at the temperature of minus 80 ℃. After multiple domestications, only two strains are obtained by separation and purification.

3. Strain preservation and identification

(1) Strain preservation: the inclined plane is preserved in the institute of microbiology of Chinese academy of sciences.

(2) And (3) strain identification: the strain is identified as stenotrophomonas acidophilus and Ochrobactrum through 16s RNA by the company Limited in the biological engineering (Shanghai), and the colony morphology and the cell morphology are shown in the figure 1 and the figure 2.

Example 2: optimization of salt tolerance of strain

(1) Respectively inoculating the stenotrophomonas acidophilus CGMCC No.22273 and the ochrobactrum CGMCC No.22274 obtained by screening and separating in the embodiment 1 into 100mL of LB liquid culture medium by the inoculum size of 2% of the volume ratio, and culturing under the natural pH condition of 35 ℃ and 180 r/min;

(2) Shaking and continuously culturing for 48h in culture medium with salt content of 1%, 2%, 3%, 4% and 5%, respectively;

(3) OD was measured every 6h in media with different salt contents ₆₀₀ 。

The OD of stenotrophomonas microacidophilus CGMCC No.22273 in culture media of different salinity is shown in Table 1 ₆₀₀ The value, in comparison, enters the log phase earlier under the condition that the salinity is 1% -3%, so the optimum salinity is 1% -3%.

TABLE 1 OD of stenotrophomonas acidithiophila at different salinities ₆₀₀

OD of Ochrobactrum CGMCC No.22274 in culture medium with different salinity as shown in Table 2 ₆₀₀ The value, in comparison, enters the logarithmic phase earlier under the condition that the salinity is 1% -3%, so the optimum salinity is between 1% -3%.

TABLE 2 OD of Ochrobactrum strain at different salinity ₆₀₀

Example 3: the strains were cultured at different pH

(1) Inoculating the two strains obtained in example 1 into 100mL LB liquid culture medium at a volume ratio of 2%, respectively, and culturing at 35 deg.C, 180r/min and salinity of 3%;

(2) Continuously shaking and culturing in LB liquid culture medium with initial pH of 6.0, 6.5, 7.0, 7.5 and 8.0 for 48h;

(3) OD measurements in media of different initial pH at 6h intervals ₆₀₀ The value is obtained.

Stenotrophomonas microacidophila enters the log phase of growth earlier under conditions of initial pH of 6.0 to 7.5 as shown in table 3, and therefore it is preferable that the initial pH range is between 6.0 and 7.5.

TABLE 3 OD of stenotrophomonas acidiphila at different initial pH ₆₀₀ Value of

As shown in Table 4, the genus Ochrobactrum enters the logarithmic phase of growth earlier at an initial pH of 6.0 to 7.0, and therefore, it is preferable that the initial pH is in the range of 6.0 to 7.0.

TABLE 4 OD of Ochrobactrum at different initial pH ₆₀₀ Value of

Example 4: culturing strains at different rotating speeds

(1) The two strains obtained in example 1 were inoculated into 100mL of LB liquid medium at a volume ratio of 2%, respectively, and cultured at 35 ℃ and a salinity of 3%;

(2) Continuously shaking and culturing for 48h in LB liquid culture medium with shaking table rotation speed of 0r/min, 100r/min, 140r/min and 180 r/min.

(3) Measuring OD in culture medium with different table rotation speeds at intervals of 6h ₆₀₀ The value is obtained.

As shown in Table 5, stenotrophomonas acidophilus enters the log phase of microbial growth at the earliest under the condition of 140r/min of rotation speed, so that the rotation speed of the shaking table is the most suitable for 140r/min.

TABLE 5 OD of stenotrophomonas acidiphila at different rotational speeds ₆₀₀ Value of

As shown in Table 6, the genus Ochrobactrum enters the logarithmic phase of growth of the microorganism at the earliest under the condition of 140r/min, and thus the optimum shaking table rotation speed is 140r/min.

TABLE 6 OD of Ochrobactrum at different rotational speeds ₆₀₀ Value of

Example 5: the strains were cultured at different temperatures

(1) Inoculating the strains obtained in example 1 into 100mL LB liquid culture medium with the inoculation amount of 2% by volume, and culturing at the salinity of 3% and the rotation speed of 140 r/min;

(2) Continuously shaking and culturing in LB liquid culture medium at 25 deg.C, 28 deg.C, 30 deg.C, 32 deg.C and 35 deg.C for 48h.

(3) OD measurement in media at different temperatures at 6h intervals ₆₀₀ The value is obtained.

As shown in Table 7, stenotrophomonas microacidophilus enters the log phase of growth earlier at a temperature of 28 ℃ to 35 ℃ and therefore a suitable temperature range is 28 ℃ to 35 ℃ in comparison.

TABLE 7 OD of stenotrophomonas acidiphila at different temperatures ₆₀₀ Value of

As shown in Table 8, the genus Ochrobactrum enters the logarithmic phase of growth earlier at a temperature of 28 ℃ to 32 ℃ and thus a suitable temperature range is 28 ℃ to 32 ℃ in comparison.

TABLE 8 OD of Ochrobactrum at different temperatures ₆₀₀ Value of

Example 6: application of bacterial strain in degradation of petroleum hydrocarbon wastewater

(1) Strain culture: respectively inoculating the stenotrophomonas microacidophilus and the ochrobactrum obtained in the embodiment 1 into 100mL of LB liquid culture medium by the inoculation amount of 2% of the volume ratio, carrying out shake culture at the conditions of 32 ℃, 3% of salinity and 140r/min of rotation speed, and culturing for 48 hours to enable the concentration of bacterial liquid in the culture medium to reach about OD600 of 1.5;

(2) Wastewater treatment: preparing water with the engine oil content of 6927mg/L to simulate the waste water containing petroleum hydrocarbon, and adding the separate bacterial liquid of stenotrophomonas acidophilus and ochrobactrum cultured for 48 hours in the step (1) and the bacterial liquid mixed according to the equal volume into the waste water containing petroleum hydrocarbon according to the amount of 10%, 30% and 50% of the volume of the waste water containing petroleum hydrocarbon respectively for treatment.

(3) And (4) determining the result: and (3) measuring the change of the oil content before and after the sewage, calculating to obtain the degradation rate of the petroleum hydrocarbon, and selecting the optimal strain combination.

As can be seen from tables 9 and 10, when the addition amount of the stenotrophomonas microaerophila prepared in step (1) is 50% of the volume of the wastewater, the efficiency of degrading petroleum hydrocarbon substances reaches the maximum, and is 87.87%; the degrading rate of the ochrobactrum is the maximum and is 42.41 percent when the adding amount is 50 percent of the volume of the wastewater. The degradation efficiency of the mixed bacteria is not ideal as that of a single strain, so that the optimal strain combination for treating the petroleum hydrocarbon-containing wastewater is that two strains are respectively subjected to degradation treatment.

TABLE 9 degradation Rate (%) of stenotrophomonas acidiphila and Ochrobactrum xanthii after 48h

Table 10 degradation rate of stenotrophomonas acidophilus and ochrobactrum on petroleum hydrocarbon by mixing amount of 1 by volume

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. Ochrobactrum daejeonense is preserved in China general microbiological culture Collection center on 5 days and 6 days in 2021, and the preservation number is CGMCC No.22274.

2. A microbial preparation comprising the live ochrobactrum of claim 1.

3. Use of the ochrobactrum of claim 1 and/or the microbial inoculum of claim 2 for degrading petroleum hydrocarbons.

4. The use of claim 3, wherein the source of petroleum hydrocarbon is waste water containing petroleum hydrocarbon; the concentration of the petroleum hydrocarbon in the wastewater is more than or equal to 10g/L.

5. A method of degrading a petroleum hydrocarbon, the method comprising: adding the ochrobactrum of claim 1 and/or the microbial inoculum of claim 2 to a petroleum hydrocarbon-containing sample in an amount of 10-50% by volume to degrade the petroleum hydrocarbon; ochrobactrum species of claim 1 and/or microbial agent of claim 2 ₆₀₀ Is 1.3 to 1.5.

6. The method according to claim 5, wherein the degradation time is 15-36 h, the temperature is 25-40 ℃, and the speed is 0-200 r/min.

7. The method of claim 5, wherein the sample is a petroleum hydrocarbon-containing wastewater.

8. Use of the ochrobactrum of claim 1 and/or the microbial agent of claim 2 in the preparation of a sewage treatment agent.