CN113430134A - Treatment process of petroleum hydrocarbon-containing wastewater - Google Patents

Treatment process of petroleum hydrocarbon-containing wastewater Download PDF

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Publication number
CN113430134A
CN113430134A CN202110735801.2A CN202110735801A CN113430134A CN 113430134 A CN113430134 A CN 113430134A CN 202110735801 A CN202110735801 A CN 202110735801A CN 113430134 A CN113430134 A CN 113430134A
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stenotrophomonas
ochrobactrum
petroleum hydrocarbon
petroleum
waste water
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CN113430134B (en
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梁国斌
吴晶晶
印霞棐
林伟
单旋
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Jiangsu University of Technology
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    • C—CHEMISTRY; METALLURGY
    • 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
    • C02F3/341—Consortia of bacteria
    • C—CHEMISTRY; METALLURGY
    • 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
    • C02F3/343—Biological treatment of water, waste water, or sewage characterised by the microorganisms used for digestion of grease, fat, oil
    • C—CHEMISTRY; METALLURGY
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2101/00—Nature of the contaminant
    • C02F2101/30—Organic compounds
    • C02F2101/32—Hydrocarbons, e.g. oil

Abstract

The invention discloses a process for treating 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%. 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 exploitation and transportation of offshore oil resources, the frequent occurrence of events such as oil leakage accidents and the like,seriously pollutes the marine environment, destroys the ecological balance of a marine system and threatens 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 km2The main overproof 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 (bioorganization), which utilizes microorganisms which have the capability of oxidizing and decomposing petroleum and naturally exist in oceans or soil, and can take petroleum hydrocarbon compounds as a carbon source and a very energy source to finally generate H2O and CO2The process of (1). 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 center at 5 days and 6 days in 2021, and the preservation number is CGMCC No. 22273.
The invention provides a Ochrobactrum, which has been preserved in the China general microbiological culture Collection 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 10 g/L.
In one embodiment, the concentration of petroleum hydrocarbons in the wastewater is greater than or equal to 20 g/L.
The invention also provides a method for degrading petroleum hydrocarbon, which is to add the stenotrophomonas acidiphila 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 bacterial concentration OD of stenotrophomonas acidophilus or ochrobactrum and/or bacterial agent6001.3 to 1.5.
In one embodiment, the time for degradation is 15-36 hours.
In one embodiment, the time for degradation is 24-30 hours.
In one embodiment of the present invention, the treatment conditions are all: the temperature is 25-40 ℃ and 0-200 r/min.
In one embodiment, the sample is a petroleum hydrocarbon-containing wastewater.
In one embodiment, the concentration of petroleum hydrocarbons in the wastewater is greater than or equal to 10 g/L.
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 the microbial inoculum in preparation of a sewage treatment agent.
The invention provides a culture method of stenotrophomonas acidiphila.
In one embodiment, the method comprises inoculating stenotrophomonas acidiphila into a culture medium at an inoculation amount of 1-5% by volume for 24-48 h.
In one embodiment, the culturing is carried out at a pH of 6.0 to 7.5, a rotation speed of 140 to 180r/min, and a temperature of 28 to 35 ℃.
The invention provides a culture method of ochrobactrum.
In one embodiment, the method comprises inoculating the ochrobactrum into a culture medium at an inoculation amount of 1-5% by volume for 24-48 h.
In one embodiment, the culturing is carried out at a pH of 6.0 to 7.0, a rotation speed of 140 to 180r/min, and a temperature of 28 to 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 have certain degradation effect on substances harmful to the environment in the waste water containing petroleum hydrocarbon, the degradation effect on engine oil substances is good, the degradation rate of the petroleum hydrocarbon of stenotrophomonas acideophila can reach 87.87% at most, and the degradation rate of the petroleum hydrocarbon of ochrobactrum can reach 42.41% at most. 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 the 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 map.
FIG. 2 is a diagram of the colony morphology and cell morphology of Ochrobactrum anthropi; a is colony morphology, B is cell morphology map.
Detailed Description
LB culture medium: tryptone (10g), yeast extract powder (5g), sodium chloride (10g), water (1L).
The method for measuring the content of the engine oil comprises the following steps: the method for measuring the oil content in the wastewater by a gravimetric method comprises the following steps of: +5mL 50% (v/v) sulfuric acid solution. ② pouring the acidified sample into a 250mL separating funnel, then adding 25mL petroleum ether (60-90 ℃), shaking for 2min, standing and layering. Collecting supernatant, and inoculating subnatant into the original conical flask. ③ repeatedly extracting the lower layer liquid with petroleum ether for 2 times, using 25mL of petroleum ether each time, and merging the upper layer liquid obtained by 3 times, namely the petroleum ether extracting solution. Fourthly, adding anhydrous sodium sulfate (dehydration) into the petroleum ether extract, slightly shaking until the mixture does not cake, and covering and placing for 1 hour. Fifthly, filtering the mixture by qualitative filter paper washed by petroleum ether in advance, and collecting filtrate in a 100mL beaker with constant drying weight. Sixthly, placing the beaker on a constant temperature water bath kettle at the temperature of 65 +/-1 ℃ to evaporate to be nearly dry, taking out the beaker to be dried, placing the beaker in an oven at the temperature of 65 +/-1 ℃ for 1-2h, cooling the beaker for 30min and weighing the beaker. Degradation rate ═ residual oil mass of blank-residual oil mass of sample x 100%/oil mass 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 the temperature of 30 ℃ at 140r/min, 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 acclimatizing 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
5mL of sludge was added to 100mL of acclimatized liquid medium, and the mixture was incubated at 37 ℃ and 140r/min, performing shake culture for 5 days, adding 5mL of culture solution into new domesticated liquid culture medium, shaking for 5 days, repeating for 5 times, and preparing 10 with cultured bacterial solution-1、10-2、10-3、10-4、10-5、10-6The 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 domestication, only two strains are finally obtained by separation and purification.
3. Strain preservation and identification
(1) And (3) 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) Inoculating stenotrophomonas acidophilus CGMCC No.22273 and Ochrobactrum CGMCC No.22274 obtained by screening and separating in the embodiment 1 into 100mL of LB liquid culture medium respectively according to the inoculum size of 2% in 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 contents600。
The OD of stenotrophomonas microacidophilus CGMCC No.22273 in culture media of different salinity is shown in Table 1600The 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 1 OD of stenotrophomonas acidiphila at different salinity600
Figure BDA0003141623280000041
As shown in Table 2, the strain of Ochrobactrum CGMCC No.22274 with different salinityOD in culture Medium600The 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 salinity600
Figure BDA0003141623280000042
Figure BDA0003141623280000051
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 48 h;
(3) OD measurements in media of different initial pH at 6h intervals600The value is obtained.
As shown in Table 3, stenotrophomonas acidiphila enters the logarithmic phase of growth earlier under the condition that the initial pH is 6.0-7.5, so that the more suitable initial pH range is 6.0-7.5.
TABLE 3 OD of stenotrophomonas acidithiophila at different initial pH600Value of
Figure BDA0003141623280000052
As shown in Table 4, the genus Ochrobactrum enters the logarithmic phase of growth earlier under the condition that the initial pH is 6.0-7.0, and thus the preferable initial pH range is 6.0-7.0.
TABLE 4 OD of Ochrobactrum at different initial pH600Value of
Figure BDA0003141623280000053
Figure BDA0003141623280000061
Example 4: culturing the strains at different rotation 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 6h600The value is obtained.
As shown in Table 5, stenotrophomonas acidophilus enters the logarithmic phase of microorganism growth at the earliest under the condition of 140r/min of rotation speed, so that the optimum rotation speed of the shaking table is 140 r/min.
TABLE 5 OD of stenotrophomonas acidiphila at different rotational speeds600Value of
Figure BDA0003141623280000062
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 140 r/min.
TABLE 6 OD of Ochrobactrum at different rotational speeds600Value of
Figure BDA0003141623280000071
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 48 h.
(3) OD measurement in media at different temperatures at 6h intervals600The 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 temperatures600Value of
Figure BDA0003141623280000072
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 temperatures600Value of
Figure BDA0003141623280000073
Figure BDA0003141623280000081
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 acidiphila prepared in the step (1) is 50% of the volume of the wastewater, the efficiency of degrading petroleum hydrocarbon substances reaches the maximum of 87.87%; the canebacterium also reached the maximum degradation rate of 42.41% when the addition amount was 50% 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
Figure BDA0003141623280000082
TABLE 10 degradation rate of stenotrophomonas acidiphila and Ochrobactrum according to the mixing amount of 1:1 of volume ratio to petroleum hydrocarbon
Figure BDA0003141623280000083
Figure BDA0003141623280000091
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 (10)

1. Stenotrophomonas acidaminiphila, has been deposited in the China general microbiological culture Collection center at 5 days and 6 days 2021 with the preservation number of CGMCC No. 22273.
2. Ochrobactrum daejeonense is preserved in the China general microbiological culture collection center at 5 days and 6 days in 2021, and the preservation number is CGMCC No. 22274.
3. A microbial preparation comprising the stenotrophomonas acidiphila according to claim 1 or the living microbial cell of the genus Ochrobactrum according to claim 2.
4. Use of stenotrophomonas microacidophilus according to claim 1 and/or of the bacterial agent according to claim 3 for degrading petroleum hydrocarbons.
5. Use of the ochrobactrum of claim 2 and/or the microbial inoculum of claim 3 for degrading petroleum hydrocarbons.
6. Use according to claim 4 or 5, wherein the petroleum hydrocarbon source is a waste water containing petroleum hydrocarbons; the concentration of the petroleum hydrocarbon in the wastewater is more than or equal to 10 g/L.
7. A method of degrading petroleum hydrocarbons, the method comprising: adding the stenotrophomonas microacidophilus of claim 1 or the ochrobactrum of claim 2 and/or the microbial inoculum of claim 3 to a petroleum hydrocarbon-containing sample in an amount of 10-50% by volume to degrade the petroleum hydrocarbon; a bacterial concentrate OD of stenotrophomonas acidophilus according to claim 1 or Ochrobactrum sp according to claim 2 and/or a bacterial agent according to claim 36001.3 to 1.5.
8. The method according to claim 7, wherein the degradation time is 15-36 h, the temperature is 25-40 ℃, and the temperature is 0-200 r/min.
9. The method of claim 7, wherein the sample is a waste water containing petroleum hydrocarbons.
10. Use of stenotrophomonas microacidophilus according to claim 1 or ochrobactrum according to claim 2 and/or bacterial agent according to claim 3 for the preparation of a wastewater treatment agent.
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CN108949634A (en) * 2018-08-08 2018-12-07 东南大学 The oil degradation bacteria and its separation method of a kind of degradable heavy crude and application
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