CN113023900A - Method for degrading phenol in sewage by using pseudomonas paratyphi - Google Patents
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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
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- C12N1/20—Bacteria; Culture media therefor
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
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- C02F2101/00—Nature of the contaminant
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Abstract
The invention provides a method for degrading phenol in sewage by using a patent strain, namely pseudomonas paratyphi DTSP 2. The process comprises the following steps: (1) the separation and identification of Pseudomonas parabrevis DTSP2 (patent deposit number is GDMCC No. 61389); (2) phenol tolerance range evaluation; (3) measuring the degradation rate of phenol in the simulated sewage; the invention utilizes the biodegradation of the pseudomonas paratyphi to degrade phenol in the industrial wastewater, the optimal concentration of phenol degradation is 200mg/l, the degradation rate in 12 hours is 20-47%, the highest degradation rate is about 45%, and the pollution effect of phenol is effectively reduced.
Description
Technical Field
The invention belongs to the application of microbiology technology in the field of environmental protection, and the phenol in industrial wastewater is degraded and metabolized by utilizing the metabolism of microorganisms. The technology provides an efficient and universal mode for degrading phenol in sewage by using a biodegradation method. The degradation process adopts P.parapsilosis. Pseudomonas paraphaeformis (P.parafuulva) DTSP2 (patent deposit number GDMCC number 61389) is an indigenous bacterium isolated from river water, and the nutritional type belongs to chemoautotrophic heterotrophic bacteria.
Background
Phenol is a common pollutant emitted by human activities and can be widely detected in human living and living environments. The phenolic compounds are widely existed in the waste water of industries such as coking, pharmacy, papermaking, dye, petrochemical industry and the like, and can be discharged into the environment along with industrial waste water, waste gas and the like, so that the water body environment is polluted, and the biological safety is endangered. Phenol can directly enter human body through skin mucosa, respiratory tract and digestive tract, low concentration phenol can cause accumulation to cause chronic poisoning, and high concentration phenol can directly cause acute poisoning to cause coma and even death. If a water source polluted by phenol is drunk for a long time, anemia, dizziness and various nervous system diseases can be caused. In addition, toxic polychlorinated phenols are also formed when phenol-containing water is chlorinated. The use of phenol in a variety of fields results in the derivatization of other pollutants and the discharge of large amounts of phenol into the environment, mainly in rivers, industrial waste water, landfill leachate. The pollutants are stable in chemical property and difficult to remove, so that the pollutants are easy to accumulate in the environment, and the treatment effect of industrial production and sewage treatment plants is influenced. Phenol is toxic at low concentrations and is an important compound in the field of environmental research and is often classified as a typical contaminant. In China, phenol is listed in a water priority control pollutant blacklist, and the concentration of phenol in certain industrial wastewater is too high, so that the self-cleaning capability of the environment is difficult to load, and therefore, the removal of phenol in the environment and the reduction of the harm of phenol to organisms become problems which need to be solved urgently.
The existing method for removing phenol in the environment can be summarized as a physical method, a chemical method and a biological method. Compared with the physical and chemical treatment process, the biological treatment technology has the characteristics of low operation cost, simple operation, no secondary pollution, simple operation, large treatment capacity, economy, safety and the like, and gradually becomes a new trend for phenol degradation research. The microorganisms are rich in variety, various in metabolic pathway, various in metabolic type, relatively mild in reaction condition and reaction environment, and usually screened microorganisms are used for converting and metabolizing refractory pollutants by screening strains and domesticating. The main principle of microbial degradation of organic waste water is the co-metabolism of microbes, and the specific pollutants are treated intensively by enriching and domesticating organisms capable of specifically degrading certain pollutants by adding growth substrates (co-metabolism substrates) and pollutants (non-growth substrates) of the microbes. Many of the bacteria known to date have the capacity to degrade phenol, all with good degradation of phenol at a certain initial concentration, of which pseudomonas is the major dominating species.
The bacteria separated from the pseudomonas paratungensis DTSP2 river water are water samples from water treated by a sewage treatment plant, the collection sites are 500 meters upstream and downstream and are about 30 centimeters away from the water surface, and the bacteria belong to indigenous microorganisms. The degradation of phenol by the strain is realized by the catalysis of a series of enzymes. Firstly, phenol is hydroxylated into catechol under the action of hydroxylase, then unsaturated fatty acid is generated under the action of dioxygenase, and finally products after ring opening and cracking are continuously decomposed into micromolecular fatty acid and finally metabolized and degraded by microorganisms.
The biological method is a treatment technology which is generally applied to the treatment of low-concentration phenol wastewater at present. The organic matters in the wastewater are degraded into non-toxic micromolecular compounds such as carbon dioxide, water, nitrogen and the like through the metabolism of microorganisms. The biological method has wide application, large processing capacity, simple equipment and high economic benefit. Among the benzene-degrading bacteria reported so far, pseudomonas is the most important degrading bacteria in the environment, especially in the terrestrial environment. The Pseudomonas paraphuang DTSP2 can be used for realizing the rapid ring-opening cleavage of phenol and completing the decomposition and conversion of phenol.
The degradation effect of P.parapsilosis on phenol is related to the tolerance concentration and degradation efficiency. The phenol stress limit of the pseudomonas parapsilosis is detected by a gradient method. Under the phenol stress, the pseudomonas paratyphi (GDMCC number 61389) continuously increases under the condition that the maximum concentration of phenol is 900mg/l to 1000mg/l, the highest degradation efficiency occurs in the environment with the phenol concentration of 200mg/l, and the degradation efficiency is about 45 percent. Phenol is both a carbon source for the strain to grow and inhibits the growth of the strain. When the phenol concentration is higher than 1000mg/l, the bacteria grow slowly and phenol is hardly degraded.
The method for removing phenol is suitable for wastewater treatment in refineries or coking, oil refining, petrochemical plants and enterprises. Compared with physical and chemical treatment methods, the method can carry out in-situ remediation on the polluted site, is not limited by time and site, has simple metabolites and does not generate secondary pollution. The cost is low, the method is simple, the treatment difficulty is low, and most environmental protection requirements can be met. The method is also suitable for the soil polluted by phenol. The pollutants generated in industrial engineering can pose great threat to human body and environmental health, and population growth and industrial development provide development requirements of pollution-free environment for industrial process. By utilizing the specific degradation bacterial strain, a unique and effective solution is provided for the treatment of phenol industrial wastewater.
Disclosure of Invention
The method for degrading phenol in sewage by using bacteria is popularized and verified, and has obvious advantages in environmental protection. The degradation rate of phenol can reach about 45% by adding pseudomonas parapoxeroides into sewage, and the technical scheme of the invention is as follows:
a method for degrading phenol by using pseudomonas paratyphi is characterized by comprising the following process steps:
(1) screening of target Strain
Preparing a lactose bile salt culture medium (LBB): preparing a liquid culture medium according to the LBB standard proportion, and sterilizing for 20-40 min at 120-130 ℃. 15-20 g/l agar powder needs to be added into the solid culture medium, and other components are the same as those of the liquid culture medium.
Gram negative bacteria and benzene resistant bacteria are selectively enriched. And adding 5-10 mL of downstream water sample into 100mL of selective sterilization liquid culture medium. Adding benzene into LBB, wherein the density is 0.8-1.2 mg/ml. The inoculation bottle is shaken to be in a turbid state at the temperature of 25-35 ℃ at the speed of 100-120 revolutions per minute (rpm) for 10-12 h.
③ liquid cultures were streaked with benzene on solid LBB.
Preparing a Luria-Bertani (LB) culture medium: preparing a liquid culture medium according to LB standard proportion, sterilizing for 20-40 min at 120-130 ℃, adding 15-25 g/l agar powder into a solid culture medium, and keeping the other components the same as the liquid culture medium.
Fifthly, transferring the single colony containing the yellow pigment into a solid LB culture medium to increase the growth amount of the single colony.
(2) Isolation and identification of target bacteria
The 16S rRNA gene was subjected to targeted amplification using the universal primer 27F/1492R to obtain an amplicon of the isolate in (1). The purified amplicons were sequenced by Takara, Inc. The obtained sequence was queried against the 16S rRNA sequence database by BLASTn. The isolates were analyzed for phylogenetic development using Mega6.0 software based on the retrieved sequences, as shown in FIG. 2.
(3) Phenol degradation rate evaluation and application
Firstly, a gradient method is adopted to determine the phenol tolerance limit of the separating bacteria. Will be in exponential phase (density about 1X 10)7-1×108Colony forming units/mL) was inoculated into LB medium containing phenol. Setting phenol concentration gradient from 100-1200 mg/l. In order to avoid data fluctuation, three parallel samples are arranged for each concentration gradient, and the aim of repeating the concentration determination three times after degradation is fulfilled. All volumetric flasks were incubated at 25-35 ℃ for 10-12 h at 100-120 rpm. The phenol concentration was measured using High Performance Liquid Chromatography (HPLC). The Phenol Degradation Rate (PDR) is determined by the ratio of degradants to the initial amount.
② the practical application of phenol degrading bacteria. The simulated sample is used for detecting the phenol degradation capability of the strain, and the strain is tried to be used for environmental protection or restoration.
Drawings
FIG. 1 is a process flow diagram for degrading phenol by using Pseudomonas parachutista.
FIG. 2 is a graph showing the evolutionary relationship of the strain DTSP2(GDMCC number 61389) used in the present invention.
Pseudomonas paraphaeformis (P.parafuulva) DTSP2, which is classified and named as Pseudomonas parafuulva and deposited in the Guangdong province collection of microorganisms with the deposit number GDMCC No: 61389, preservation address: the preservation time of the No. 59 building 5 of the No. 100 college of the Xieli Zhonglu Guangzhou city is 12 months and 25 days in 2020.
Detailed Description
The water sample adopted by the embodiment of the invention is from 500 meters upstream and downstream of the Fine river in Fuxin City in Liaoning province.
(1) Screening, isolation and identification of target strains
Firstly, collecting a sample. And taking a water sample 20-30 cm away from the water surface, wherein two sampling points are respectively positioned at the upstream 500m and the downstream 500m of the water outlet of the Fuxin Mongolia glossy privet sewage treatment plant. 12 sterilized glass bottles of 1L capacity were used. Immediately after the sample collection, the sample was transferred to a refrigerator at a constant temperature of about 4 ℃. The microorganisms separated and extracted from the water sample belong to indigenous microorganisms of local river water, adapt to geological conditions of the local river water and have effectiveness on material decomposition and water body restoration of the environment.
② selective enrichment of gram-negative bacteria and benzene-resistant bacteria. LBB culture medium is prepared, and the LBB culture medium is sterilized for 20min to 40min at 120 ℃ to 130 ℃. 15-20 g/l agar powder needs to be added into the solid culture medium, and other components are the same as those of the liquid culture medium. Mixing 5-10 ml
Add water sample to 100ml of selectively sterilized LBB broth. To LBB, 0.8 to 1.2mg/ml of benzene is added.
The inoculation bottle is shaken to be turbid at the speed of 100 to 120 revolutions per minute at the temperature of between 25 and 35 ℃, and cultured for 10 to 12 hours.
③ liquid cultures were streaked on solid LBB with benzene.
Preparing a Luria-Bertani (LB) culture medium: preparing a standard LB culture medium, sterilizing for 20-40 min at 120-130 ℃, adding 15-25 g/l agar powder into a solid culture medium, and keeping the other components the same as those of a liquid culture medium.
Transferring the single colony with yellow pigment to solid LB culture medium to increase growth. The isolate was designated DTSP 2.
(2) Isolation and identification of P.parapsilosis
The PCR method amplifies the genus-specific gene of the isolate bacteria. The 16S rRNA gene is targeted and amplified by using a universal primer 27F/1492R. The purified amplicons were sequenced by Takara, Inc., of Dalian (Chinese Dalian). The obtained sequence was queried against the 16S rRNA sequence database by BLASTn to obtain the accession number MK 788360. On the basis of the search sequence, phylogenetic relationship analysis was performed on the isolate using Mega6.0 software. The sequence is matched with the 16S rRNA gene of P.parafuva72432, and the recognition rate is 99.65%. Therefore, isolate DTSP2 was initially identified as p. The evolution relationship is shown in the attached figure 2.
(3) Phenol degradation rate evaluation and application
Firstly, measuring phenol degradation efficiency
Preparation of a phenol stress system solution: strain DTSP2 was prepared in exponential order (approximately at a density of 1X 10)7-1×108Colony forming units/mL) were inoculated onto LB medium containing phenol. Setting phenol concentration gradient from 100-1200 mg/l. Each degradation assay was performed in triplicate. All the inoculation bottles are cultured for 10-12 h at the temperature of 25-35 ℃ and under the condition of 100-120 r/min.
And the phenol degradation efficiency was measured by High Performance Liquid Chromatography (HPLC). The P.parafuuvaGDMCC 61389 continuously grows under the stress of phenol when the maximum phenol concentration is 900-1000 mg/L, which is 2000 times of 1000-fold of the national sewage discharge standard CJ343-2010 (0.5mg/L or 1 mg/L). Based on HPLC data of 100, 200, 300, 400, 500 and 600mg/L, the mean values of PDRs were calculated to be 20.6%, 45.7%, 33.2%, 25.8%, 16.4% and 1.6%, respectively. When the concentration of the phenol is 200mg/L, the P.parafaulva GDMCC 61389 has the best effect of degrading the phenol within 10 to 12 hours.
Application of para-pseudomonas
The phenol degradation ability of the strain DTSP2 was tested using a mock sample, and environmental protection or remediation was attempted using this strain. According to the predicted phenol degradation rate, a water outlet sample of the large Tang (Sewage treatment plant, geographical location 121.594645.41.962196) is prepared into a simulated sample with the phenol concentration of 200mg/L, and the degradation experiment is carried out under the laboratory condition. And (3) culturing the P.parafuulva GDMCC No.61389 to an index period, respectively adding 1-5 ml of bacterial liquid into the simulated sample, and determining the phenol degradation rate by adopting the same culture, degradation detection and calculation method.
(4) Calculation of phenol degradation Rate
The phenol degradation rate is calculated by bringing the peak area of the sample obtained by HPLC analysis into a standard curve to obtain the residual phenol content in the sample, and the ratio of the reduced phenol amount to the original phenol amount is the degradation efficiency.
The method can effectively reduce the content of phenol in water, and can be applied to the environment with slight phenol pollution and the environment with serious phenol pollution. The pseudomonas paratyphi is the most common bacterium in water, widely exists in the environment, has the suitable survival temperature of 35-37 ℃, has obvious phenol degradation effect, and has the degradation range of 20-47 percent. Compared with physical and chemical methods, the method has the advantages of wide raw material source, low cost and no secondary pollution, thereby having wider application range.
The invention has the following characteristics:
(1) wide source, low cost, no toxicity and no harm. At present, the phenol degrading bacteria are selected from soil or water polluted by phenol organic matters, and mainly comprise pseudomonas, alcaligenes eutrophus, saccharomycetes, rhizobia, denitrifying bacteria and other phenol degrading bacteria. Compared with other phenol degrading bacteria, the pseudomonas paratuberans has the advantages of wide existence, easy extraction, simple culture environment requirement, strong environment tolerance and the like. Meanwhile, the strain has no pathogenicity, is safe to use, has simple metabolite and cannot cause secondary pollution.
(2) The isolated strain belongs to a local indigenous microorganism. Is suitable for local environment, has local advantages and strong stress resistance. Compared with foreign microorganisms, the pseudomonas paratyphi has strong adaptability to local physical and chemical factors and has larger potential for in-situ remediation application; no need of specific culture, stable biochemical property, and small variability and pathogenicity.
TABLE 1 degradation rates at different phenol concentrations
TABLE 2 statistic table of phenol degradation rate in Pseudomonas parapsilosis GDMCC No.61389 simulation experiment
Claims (2)
1. A method for degrading phenol in sewage by using Pseudomonas paradoxa (P.parafuulva) DTSP2 (the patent is preserved in Guangdong province microorganism culture collection center with the number of GDMCC NO.61389), which is characterized by comprising the following process steps:
(1) screening of target Strain
Preparing a lactose bile salt culture medium (LBB): preparing a liquid culture medium according to an LBB standard ratio, sterilizing for 20-40 min at 120-130 ℃, adding 15-25 g/L agar powder into a solid culture medium, and keeping the other components the same as those of the liquid culture medium;
gram-negative bacteria and benzene-resistant bacteria are selectively enriched: adding 5-10 mL of downstream water sample into 100mL of selective sterilization liquid culture medium, adding benzene into LBB (LBB) with the concentration of 0.8-1.2 mg/mL, and oscillating an inoculation bottle at the speed of 100-120 rpm at the temperature of 25-35 ℃ until the inoculation bottle is in a turbid state for 10-12 hours;
thirdly, the liquid culture is streaked on the solid LBB by benzene;
preparing a Luria-Bertani (LB) culture medium: preparing an LB liquid culture medium according to a standard ratio, sterilizing for 20-40 min at 120-130 ℃, adding 15-25 g/L agar powder into a solid culture medium, and keeping the other components the same as those of the liquid culture medium.
Transferring the single colony containing yellow pigment to solid LB culture medium to increase its growth;
(2) isolation and identification of target bacteria
The 16SrRNA gene was subjected to targeted amplification using the universal primer 27F/1492R to obtain species information of the isolate in (1). Sequencing the purified amplicon by Takara, and searching the 16SrRNA sequence database with BLASTn for the obtained sequence, and analyzing the phylogenetic relationship of the isolate according to the searched sequence by using Mega6.0 software;
(3) phenol degradation rate determination and application
The phenol tolerance limit of the separated bacteria is determined by adopting a gradient method: will be in exponential phase (density about 1X 10)7-1×108Flora forming unit/ml) is inoculated into an LB culture medium containing phenol, and the concentration gradient of phenol is set from 100 mg/l to 1200mg/l in order to avoid data wavesMeasuring degradation concentration three times each time; culturing all volumetric flasks for 10-12 h at 25-35 ℃ under the condition of 100-120 r/min, measuring the phenol concentration by using High Performance Liquid Chromatography (HPLC), and determining the Phenol Degradation Rate (PDR) by the ratio of degradation products to the initial amount;
② the practical application of phenol degrading bacteria: the simulated sample is used for detecting the phenol degradation capability of the strain, and the strain is tried to be used for environmental protection or restoration.
2. The method for degrading phenol in sewage by using Pseudomonas parapsilosis DTSP2 as claimed in claim 1, wherein the strain is screened and the concentration of the degrading bacteria with the highest phenol degradation rate is found by setting a phenol concentration gradient, so as to find the best application of the degrading bacteria.
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