CN115247139B - Pseudomonas capable of utilizing methyl mercaptan as unique carbon source and application thereof - Google Patents
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Abstract
The invention relates to the technical field of methyl mercaptan degradation, and discloses pseudomonas capable of utilizing methyl mercaptan as a unique carbon source and application thereof. The Pseudomonas is named as SJ-1 and is preserved in China Center for Type Culture Collection (CCTCC) in 2021, 4 months and 19 days, and the preservation number is CCTCC No: m2021406, microorganism Classification designated PseudomonasPseudomonassp. The pseudomonas SJ-1 can grow and reproduce by taking methyl mercaptan as the only carbon source and energy source, can degrade methyl mercaptan efficiently, and can adapt to a wider temperature and pH range. In addition, the pseudomonas SJ-1 can also take the propanethiol as the only carbon source and energy source to efficiently degrade the propanethiol.
Description
Technical Field
The invention relates to the technical field of methyl mercaptan degradation, in particular to pseudomonas capable of utilizing methyl mercaptan as a unique carbon source and application thereof.
Background
Methyl mercaptan is a malodorous gas, has the characteristics of low boiling point, low odor threshold value and easy volatilization, is insoluble in water and easy to dissolve in organic solvents such as ethanol, diethyl ether and the like, and is widely used as raw materials, catalysts and the like of organic synthesis, jet additives and pesticides. Methyl mercaptan is toxic and highly irritating, and can cause anesthesia after being inhaled into higher concentration poisoning, with cyanosis, cramps, headache, nausea and the like, can stimulate the lung, and can cause lung injury even respiratory paralysis for serious people.
Therefore, efficient degradation of methyl mercaptan in research environments is essential for human health. The bacterial CZ05 utilizing methyl mercaptan is separated in China and identified as bacillus polymyxa, and further experiments show that the bacterial can effectively remove high-concentration methyl mercaptan; jiang Anxi et al, a strain of bacteria JLL isolated has good effect on the degradation of methyl mercaptan and was identified as Flavobacterium. The degradation of methyl mercaptan studied by Raquel et al abroad has achieved good results, indicating that the use of microorganisms to degrade methyl mercaptan is a viable research direction.
At present, although some strains of pseudomonas are reported to degrade methyl mercaptan, the strains cannot take methyl mercaptan as the only carbon source to realize high-efficiency degradation of methyl mercaptan, so that the application of pseudomonas in methyl mercaptan biological evolution engineering is limited. For example, the literature "dominant bacteria mix inoculation peat trickling tower to remove mixed sulfur systems" (Jiang Anxi, wang Xiaohui, ma Li, liu Bo. University of Harbin industry journal, 2004 (02): 140-142+146.) discloses a strain of heterotrophic Pseudomonas Hm-6 which is inoculated into a peat biofilm carrier tower, and the peat is used to provide a carbon source for the strain, enabling the degradation of methyl mercaptan.
Disclosure of Invention
In order to solve the technical problems, the invention provides pseudomonas which can utilize methyl mercaptan as a unique carbon source and application thereof. The pseudomonas SJ-1 can take methyl mercaptan as the only carbon source and energy source, realizes the high-efficiency degradation of the methyl mercaptan, and has important significance for engineering application of purifying the methyl mercaptan by a biological method.
The specific technical scheme of the invention is as follows:
in a first aspect, the invention provides a pseudomonas strain which can utilize methyl mercaptan as a unique carbon source, wherein the pseudomonas strain is named as SJ-1 and is preserved in China Center for Type Culture Collection (CCTCC) No: m2021406, the preservation unit address is China, the university of Wuhan; the microorganism classification is named Pseudomonas sp.
The pseudomonas is separated from activated sludge of a sewage treatment plant in Zhejiang Zhoushan island north, is aerobic gram-negative bacteria, and has small spots, white color, opacity, full shape, smoothness and wetness, easy picking up and growth of lawn along a streak. After 16S rDNA sequencing, it was judged to be a new species in Pseudomonas and designated Pseudomonas sp.SJ-1.
The pseudomonas SJ-1 can grow and reproduce by taking methyl mercaptan as the only carbon source and energy source, can degrade methyl mercaptan efficiently, and can adapt to a wider temperature and pH range. In addition, the pseudomonas SJ-1 can also take the propanethiol as the only carbon source and energy source to efficiently degrade the propanethiol.
In a second aspect, the present invention provides a mutant of Pseudomonas which can utilize methyl mercaptan as a sole carbon source, the mutant being obtained by mutagenesis, domestication, gene recombination or natural mutation of the Pseudomonas.
In a third aspect, the invention provides the use of said pseudomonas or said mutant for degrading an organic contaminant, said organic contaminant being methylthio alcohol and/or propylmercaptan.
Preferably, the application comprises the steps of: the bacterial liquid containing the pseudomonas or the mutant is used as an enzyme source, the organic pollutant is used as a substrate, inorganic nutrition is provided, and degradation reaction is carried out, so that the degradation of the organic pollutant is realized.
Preferably, the application specifically comprises the following steps: adding bacterial liquid containing the pseudomonas or the mutant and organic pollutants into an inorganic culture medium with pH of 4-8 to prepare a mixed liquid; and (3) placing the mixed solution in a temperature of 20-40 ℃ for degradation reaction to realize degradation of organic pollutants.
Further, the mixed solution contains methyl mercaptan with a concentration of 87 to 435mg/L and/or propyl mercaptan with a concentration of 84 to 420mg/L, preferably 87 to 261 mg/L.
When the methyl mercaptan content is 87-261 mg/L, the pseudomonas SJ-1 can grow and reproduce rapidly in a short time, so that the high-efficiency degradation of the methyl mercaptan is realized.
Preferably, the application specifically comprises the following steps: inoculating bacterial liquid containing the pseudomonas or the mutant and organic pollutants into a bioreactor, and introducing the organic pollutants and inorganic nutrient solution with pH of 4-8 into the bioreactor for degradation reaction to degrade the organic pollutants.
Further, the bioreactor is a biotrickling filter; the organic pollutants are introduced from the bottom of the tower, and the inorganic nutrient solution is sprayed downwards from the top of the tower. Preferably, the pH of the inorganic medium or inorganic nutrient solution is 7.0 to 8.0.
Preferably, the temperature of the degradation reaction is 20 to 35 ℃.
When the initial pH is 7.0-8.0 and the temperature is 20-35 ℃, the Pseudomonas SJ-1 has a faster propagation speed, and can realize the rapid degradation of methyl mercaptan.
Preferably, the preparation method of the bacterial liquid containing the pseudomonas or the mutant comprises the following steps:
1) Slant culture: inoculating the pseudomonas or the mutant into an inclined plane LB solid culture medium, and culturing for 24-36 h at the temperature of 30-35 ℃ to obtain inclined plane thalli;
2) And (3) performing expansion culture: inoculating the slant thallus obtained in the step 1) into LB liquid medium by using an inoculating loop, and culturing for 24-36 h at 30-35 ℃ to obtain OD 600 Bacterial liquid of =0.1 to 0.2, namely bacterial liquid containing the pseudomonas or the mutant.
Preferably, the inorganic salt culture medium or the inorganic nutrient solution comprises the following components: na (Na) 2 HPO 4 ·12H 2 O 4.0～ 5.0g/L、KH 2 PO 4 0.8～1.2g/L，(NH 4 ) 2 SO 4 2.3～2.8g/L，MgSO 4 ·7H 2 O0.18-0.23 g/L, anhydrous CaCl 2 0.022-0.24 g/L, 0.8-1.2 mL/L of trace element mother liquor and deionized water as solvent.
Preferably, the trace element mother liquor comprises the following components: feSO 4 ·7H 2 O 0.8～1.2g/L、CuSO 4 ·5H 2 O 0.015～0.025g/L、H 3 BO 3 0.013～0.015g/L、MnSO 4 ·4H 2 O 0.08～0.13g/L、ZnSO 4 ·7H 2 O 0.08～ 0.13g/L、Na 2 MoO 4 ·2H 2 O 0.015～0.025g/L、CoCl 2 ·6H 2 O0.015-0.025 g/L, and deionized water as solvent.
Compared with the prior art, the invention has the following advantages:
(1) The pseudomonas SJ-1 can take methyl mercaptan or propanethiol as the only carbon source and energy source, so that the high-efficiency degradation of the methyl mercaptan and the propanethiol is realized, the degradation rate of the methyl mercaptan in 20 hours can reach more than 99%, and the degradation rate of the propanethiol in 24 hours can reach more than 67%;
(2) The pseudomonas SJ-1 can adapt to a wide temperature and pH range, can grow and propagate at 20-40 ℃ and pH 4-8, and can degrade methyl mercaptan to a certain extent.
Drawings
FIG. 1 is a phylogenetic tree of Pseudomonas SJ-1;
FIG. 2 is a graph showing the growth of Pseudomonas SJ-1 cells and the degradation of methyl mercaptan;
FIG. 3 is a bar graph showing the effect of different pH culture solutions on methyl mercaptan degradation (panel A), growth (panel B) and mineralization (panel C) of Pseudomonas SJ-1;
FIG. 4 is a bar graph showing the effect of different temperatures on methyl mercaptan degradation (panel A), growth (panel B) and mineralization (panel C) of Pseudomonas SJ-1;
FIG. 5 is a graph of degradation of Pseudomonas SJ-1 against various initial concentrations of methyl mercaptan;
FIG. 6 shows the growth curves of Pseudomonas SJ-1 at different initial methyl mercaptan concentrations.
Detailed Description
The invention is further described below with reference to examples.
The ingredients of each medium used in the following examples were as follows:
inorganic salt medium or inorganic nutrient solution: na (Na) 2 HPO 4 ·12H 2 O 4.5g/L、KH 2 PO 4 1.0g/L、(NH 4 ) 2 SO 4 2.5g/L、 MgSO 4 ·7H 2 O0.2 g/L, anhydrous CaCl 2 0.023g/L, 1mL/L of trace element mother liquor and pH7.0, the solvent is deionized water; the trace element mother liquor comprises the following components: feSO 4 ·7H 2 O 1.0g/L、CuSO 4 ·5H 2 O 0.02g/L、H 3 BO 3 0.014g/L、 MnSO 4 ·4H 2 O 0.10g/L、ZnSO 4 ·7H 2 O 0.10g/L、Na 2 MoO 4 ·2H 2 O 0.02g/L、CoCl 2 ·6H 2 O0.02 g/L, and deionized water as solvent.
LB solid medium: 10g/L of NaCl, 10g/L of tryptone, 5g/L of yeast powder and 20g/L of agar, wherein the solvent is deionized water, and the pH value is natural.
LB liquid medium: 10g/L NaCl, 10g/L tryptone, 5g/L yeast powder, deionized water as solvent and natural pH value.
Example 1: isolation, purification and identification of Pseudomonas SJ-1
(1) Isolation and purification of Pseudomonas SJ-1
The method comprises the steps of collecting activated sludge of a north sewage treatment plant in Zhejiang Zhoushan island on site, mixing the lower sludge after standing with an inorganic salt culture medium according to a ratio of 1:2 (v/v), inoculating 3L of the mixture into a 5L sludge acclimation tank (see Jin Xiaojun. Separation and identification of new dioxane degradation strains, degradation characteristics and bacterial preparation research [ D ]. Zhejiang industrial university, 2012.), taking methyl mercaptan as a substrate and performing acclimation culture at room temperature, taking 5mL of sludge from the acclimation tank after approximately 20 days, adding the sludge into a shake flask filled with 50mL of inorganic salt culture medium, and finding that the acclimation sludge can stably degrade 87mg/L methyl mercaptan daily in the shake flask, wherein the degradation rate can reach 80-90% (shake flask experimental conditions: 30 ℃ and 160 r/min), so as to obtain acclimation samples.
1mL of sludge sample in the shake flask is respectively taken and 2mL of sludge sample is transferred into other shake flasks for further testing performance, the transferred sludge is found to be capable of degrading 87mg/L methyl mercaptan stably every day in the shake flask, the sludge sample is continuously transferred and enriched for 6 generations (1 mL is transferred each time), and then the sludge after the 6 generations is enriched is processed according to 10 percent -1 ～10 -6 And (3) performing plate coating multiple times, selecting single bacterial colony, taking methyl mercaptan as a substrate, performing degradation activity measurement, and separating and purifying to obtain a strain SJ-1 with methyl mercaptan degradation activity.
(2) Identification of Pseudomonas SJ-1
Bacterial colony of strain SJ-1 is in small dot shape, white, opaque, full in shape, smooth and moist, easy to pick up and fungus coating grows along a streak.
The physiological and biochemical characteristics of the strain SJ-1 are as follows: aerobic, gram staining was negative.
The strain SJ-1 was subjected to 16S rDNA sequencing, which was performed by Shanghai Yuan-Shen biological medicine technologies Co. The 16S rDNA sequence of the strain SJ-1 is uploaded to an Ezbiocloud website, the strain is compared with a standard strain on the website, a MEGA5.05 software is used for constructing a bacterial development evolutionary tree by adopting a Neighbor-training method, a Bootstrap method (repeated 1000 times) is used for evaluating the bacterial development evolutionary tree, the constructed phylogenetic tree is shown in figure 1, and therefore the strain SJ-1 is determined to be a new species in Pseudomonas and is named as Pseudomonas sp SJ-1, and the strain is preserved in China center for type culture Collection with the preservation date of 2021 for 4 months and 19 days and the preservation number of CCTCC No: m2021406, address: chinese, university of Wuhan, post code 430072.
Example 2: enlarged culture of Pseudomonas SJ-1
After the pseudomonas SJ-1 is subjected to expansion culture, bacterial liquid containing the pseudomonas is obtained, and the specific process is as follows:
1) Slant culture: pseudomonas SJ-1 is inoculated in a slant LB solid culture medium and cultured for 24 hours at 30 ℃ to obtain slant thalli.
2) And (3) performing expansion culture: inoculating the slant thallus obtained in the step 1) into LB liquid medium by using an inoculating loop, culturing at 30 ℃ for 24 hours to obtain OD 600 Bacterial liquid=0.1.
Example 3: performance of Pseudomonas SJ-1 to degrade methyl mercaptan
The bacterial liquid obtained in example 2 was inoculated into fresh 50mL of an inorganic salt medium (pH=7) containing 87mg/L of methyl mercaptan to give an initial bacterial cell concentration of OD 600 Calculated as 0.03. 2 replicates and a blank without strain were designed. Culturing in shaking table at 30deg.C and rotation speed of 160r/min, sampling every 2 hr to measure methyl mercaptan degradation rate, extracting part of bacterial liquid with 5mL syringe, and measuring bacterial OD value2。
As can be seen from fig. 2: with the lapse of time, the cell concentration gradually increased, and the cell concentration was cultured for 20 hours to the maximum (about OD 600 =1.5), the methyl mercaptan degradation rate reaches more than 99%. The pseudomonas SJ-1 can utilize methyl mercaptan as the only carbon source and energy source for growth and reproduction, and has the capability of stably and efficiently degrading the methyl mercaptan.
Example 4: effect of initial pH on Pseudomonas SJ-1 degradation of methyl mercaptan
With 1mol/L NaOH aqueous solution or 1mol/L H 2 SO 4 The inorganic salt culture medium is adjusted to different pH values (4.0, 5.0, 6.0, 7.0 and 8.0) by aqueous solution; the bacterial liquid obtained in example 2 was inoculated into a fresh 50mL pH-adjusted inorganic salt medium containing 87mg/L methyl mercaptan to give an initial bacterial cell concentration of OD 600 Calculated as 0.03. A blank group of 3 replicates and one strain-free was designed. Shaking culture of the sample in a shaking table at 30deg.C and 160r/min for 20 hr, sampling, and measuring methyl mercaptan degradation rate and OD in the reaction solution 600 Value and CO 2 Values, results are shown in FIG. 3.
As can be seen from fig. 3: within the pH=4.0-8.0, pseudomonas SJ-1 can grow and propagate by taking methyl mercaptan as the only carbon source and energy source, and degrade the methyl mercaptan to a certain extent; within the pH=7.0-8.0, pseudomonas SJ-1 has high degradation rate (more than 99%) on methyl mercaptan. At the same time, the growth amount of the pseudomonas SJ-1 and the generated CO 2 The values also tended to be consistent with methyl mercaptan degradation rates.
Example 5: effect of temperature on Pseudomonas SJ-1 degradation of methyl mercaptan
The bacterial liquid obtained in example 2 was inoculated into fresh 50mL of an inorganic salt medium (pH=7) containing 87mg/L of methyl mercaptan to give an initial bacterial cell concentration of OD 600 Calculated as 0.03. Each sample was cultured by shaking in a shaker at a constant temperature of 20℃at 25℃at 30℃at 35℃and 40℃respectively (shaking rotation speed of shaker was 160 r/min). At each temperature 2 replicates and a blank without strain were designed. After culturing for 20 hours, sampling is carried out, and the degradation rate and OD of methyl mercaptan in the reaction liquid are measured 600 Value and CO 2 Value, knotSee fig. 4.
As can be seen from fig. 4: within the temperature range of 20-40 ℃, pseudomonas SJ-1 can grow and reproduce by taking methyl mercaptan as the only carbon source and energy source, and degrade the methyl mercaptan to a certain extent; the pseudomonas SJ-1 has higher degradation rate (more than 99 percent) to methyl mercaptan in the temperature range of 20-35 ℃, and simultaneously the growth amount of the pseudomonas SJ-1 and the generated CO 2 The values also tended to be consistent with methyl mercaptan degradation rates.
Example 6: effect of substrate concentration on Pseudomonas SJ-1 degradation of methyl mercaptan
Adding different concentrations of substrate methyl mercaptan into fresh inorganic salt culture medium to make initial concentrations of substrate 87, 174, 261, 348 and 435mg/L, respectively inoculating bacterial solutions prepared in example 2, and making initial bacterial body concentration be OD 600 Calculated as 0.03. 2 replicates and a blank without strain were designed. Culturing at 30deg.C in a shaker at 160r/min, sampling at regular time, and determining OD of Pseudomonas SJ-1 600 And methyl mercaptan concentration, the results are shown in FIGS. 5 and 6, respectively.
As can be seen from fig. 6: when the concentration of methyl mercaptan is 87-261 mg/L, the pseudomonas SJ-1 can be rapidly propagated within 15h, and the methyl mercaptan can be almost completely converted into CO after 10h 2 And H 2 O; when the concentration of methyl mercaptan reaches 348mg/L, the degradation rate of pseudomonas SJ-1 to methyl mercaptan reaches about 80 percent, so that the methyl mercaptan is not degraded.
Example 7: degradation ability of Pseudomonas SJ-1 to propanethiol
Adding propanethiol into fresh inorganic salt culture medium to make initial concentration of propanethiol 84mg/L, inoculating bacterial liquid obtained in example 2, making initial bacterial body concentration be OD 600 Calculated as 0.03. Culturing at 30deg.C in shaking table with rotation speed of 160r/min under constant temperature and shaking, culturing for 24 hr, and detecting OD 600 And the degradation rate of propanethiol, and the results are shown in Table 1.
TABLE 1 degradation capability of Pseudomonas SJ-1 on propanethiol
As can be seen from table 1: pseudomonas SJ-1 can efficiently degrade propanethiol by taking propanethiol as the only carbon source and energy source.
Example 8: treatment of methyl mercaptan waste gas by simulated biological trickling filtration tower
Inoculating the bacterial liquid of Pseudomonas SJ-1 prepared in the method of example 2 to a biotrickling filter (see Zhang Dingfeng, fang Junyi, she Jiexu, etc.. Biotrickling filter for investigation of purification of multicomponent exhaust gas [ J)]Environmental science, 2013.) for purifying exhaust gas containing methyl mercaptan. Methyl mercaptan at 200mg/m 3 Introducing the gas inlet concentration from the bottom of the tower, spraying the inorganic nutrient solution with the pH value of 7.0 from the top of the tower downwards, starting the film forming after 20 days, and enabling the methyl mercaptan removal rate to reach more than 90% under the condition that the residence time is 36s, wherein the system can stably operate all the time. The pseudomonas SJ-1 has stable performance of continuously treating high-concentration methyl mercaptan waste gas in a biological trickling filtration tower and high purification efficiency.
The raw materials and equipment used in the invention are common raw materials and equipment in the field unless specified otherwise; the methods used in the present invention are conventional in the art unless otherwise specified.
The foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and any simple modification, variation and equivalent transformation of the above embodiment according to the technical substance of the present invention still fall within the scope of the technical solution of the present invention.
Claims (9)
1. The Pseudomonas capable of utilizing methyl mercaptan as the sole carbon source is characterized in that the Pseudomonas is named as SJ-1 and is preserved in China center for type culture Collection (CCTCC No) in the year 2021, month 4 and day 19: m2021406, microorganism Classification designated PseudomonasPseudomonas sp.。
2. Use of pseudomonas in degrading organic contaminants according to claim 1, wherein the organic contaminants are methylthio alcohol and/or propanethiol.
3. The use according to claim 2, comprising the steps of: the bacterial liquid containing the pseudomonas is used as an enzyme source, organic pollutants are used as substrates, inorganic nutrition is provided, and degradation reaction is carried out, so that the degradation of the organic pollutants is realized.
4. The use according to claim 3, characterized in that it comprises in particular the following steps: adding the bacterial liquid containing the pseudomonas and the organic pollutants into an inorganic culture medium with the pH value of 4-8 to prepare a mixed liquid; and (3) placing the mixed solution in a temperature of 20-40 ℃ for degradation reaction to realize degradation of organic pollutants.
5. The use according to claim 3, characterized in that it comprises in particular the following steps: inoculating bacterial liquid containing the pseudomonas and organic pollutants into a bioreactor, and introducing the organic pollutants and inorganic nutrient solution with pH of 4-8 into the bioreactor to perform degradation reaction so as to degrade the organic pollutants.
6. The use according to claim 5, wherein the bioreactor is a biotrickling filter; the organic pollutants are introduced from the bottom of the tower, and the inorganic nutrient solution is sprayed downwards from the top of the tower.
7. Use according to claim 4 or 5, characterized in that:
the pH value of the inorganic culture medium or the inorganic nutrient solution is 7.0-8.0; and/or
The temperature of the degradation reaction is 20-35 ℃.
8. The use according to any one of claims 3 to 5, wherein the preparation method of the bacterial liquid containing pseudomonas comprises the following steps:
1) Slant culture: inoculating the pseudomonas into a slant LB solid culture medium, and culturing for 24-36 hours at the temperature of 30-35 ℃ to obtain slant thalli;
2) And (3) performing expansion culture: picking the obtained in step 1) with an inoculating loopInoculating the slant thallus into LB liquid culture medium, culturing at 30-35 deg.C for 24-36 h to obtain OD 600 Bacterial liquid of =0.1 to 0.2, namely bacterial liquid containing the pseudomonas.
9. Use according to claim 4 or 5, characterized in that:
the inorganic salt culture medium or the inorganic nutrient solution comprises the following components: na (Na) 2 HPO 4 ·12H 2 O 4.0～5.0g/L、KH 2 PO 4 0.8～1.2g/L，(NH 4 ) 2 SO 4 2.3～2.8g/L，MgSO 4 ·7H 2 O0.18-0.23 g/L, anhydrous CaCl 2 0.022-0.24 g/L, 0.8-1.2 mL/L of trace element mother liquor and deionized water as solvent;
the trace element mother solution comprises the following components: feSO 4 ·7H 2 O 0.8～1.2g/L、CuSO 4 ·5H 2 O 0.015～0.025g/L、H 3 BO 3 0.013～0.015g/L、MnSO 4 ·4H 2 O 0.08～0.13g/L、ZnSO 4 ·7H 2 O 0.08～0.13g/L、Na 2 MoO 4 ·2H 2 O 0.015～0.025g/L、CoCl 2 ·6H 2 O0.015-0.025 g/L, and deionized water as solvent.
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