Microbial alliance for degrading acrylonitrile as well as preparation method and application of microbial alliance

Abstract

The invention discloses a microbial alliance for degrading acrylonitrile as well as a preparation method and application of the microbial alliance. The microbial alliance is composed of Burkholderia paragallinarum, Burkholderia zhejiangensis, Comamonas terrestris, Stenotrophomonas acidophilus and Trichoderma koningii. The ratio of the viable count of the burkholderia parakholderia to the viable count of the burkholderia zhejiang to the viable count of the comamonas terrestris to the viable count of the stenotrophomonas acidophilus to the viable count of the trichoderma koningii is 3: 1: 1: 1: 1. The microbial alliance has the advantages of stable flora structure, high acrylonitrile degradation efficiency, simple operation, low cost, no need of additional addition of other nutrients in the treatment process, and no secondary pollution, and belongs to an environment-friendly technology. The method has a wide application prospect in the field of treatment of acrylonitrile-containing wastewater (gas).

Description

Technical Field

[0001] The invention belongs to the technical field of microbial remediation and relates to a microbial consortium for degrading acrylonitrile and a preparation method and application thereof. Background Art

[0002] Acrylonitrile is the most important monomer in the production of the three major industrial synthetic materials. At the same time, acrylonitrile is also a toxic substance with carcinogenicity, mutagenicity and teratogenicity. With the annual growth of acrylonitrile demand and production in my country, the problem of acrylonitrile wastewater discharge is becoming more and more serious. If it is not effectively treated, it will cause serious pollution to the environment and seriously endanger human health. Acrylonitrile wastewater is a high-concentration organic wastewater that is difficult to degrade. How to effectively treat it has always been a problem for acrylonitrile companies.

[0003] Incineration, biological treatment and oxidation are usually used for acrylonitrile wastewater. Incineration is difficult to completely burn acrylonitrile wastewater, and a large amount of fuel needs to be added for combustion. The overall cost of this method is too high, the resource consumption is large, and the economic benefits are very low. Commonly used oxidation methods include ozone catalytic oxidation, electrocatalytic oxidation, photocatalysis, Fenton oxidation, etc., all of which are difficult to apply industrially due to secondary pollution and cost issues.

[0004] Compared with combustion and oxidation methods, biological treatment has the advantages of mild reaction conditions, low treatment energy consumption, and less secondary pollution. However, because the composition of acrylonitrile wastewater is complex and most pollutants are biologically toxic, they may have a greater impact on microorganisms, making the treatment effect unstable. Summary of the invention

[0005] An object of the present invention is to provide a microbial consortium for degrading acrylonitrile.

[0006] Another object of the present invention is to provide a method for preparing the microbial consortium.

[0007] Another object of the present invention is to provide the use of the microbial consortium in treating acrylonitrile-containing wastewater or acrylonitrile waste gas.

[0008] In order to achieve the above object, the present invention provides a microbial consortium for degrading acrylonitrile, characterized in that the microbial consortium is composed of Paraburkholderia sp., zhejiangensis sp.nov., Comamonas terrae., Stenotrophomonas acidaminiphila, and Trichoderma koningii, and the ratio of the number of live bacteria is 3:1:1:1:1.

[0009] The paraburkholderia, named as Paraburkholderia sp., with strain number NEAU-SY24, has been deposited in the China Center for Type Culture Collection in Wuhan, China, with the deposit number CCTCC AA2017029 and the deposit date of May 8, 2017.

[0010] Zhejiang Burkholderia, named Zhejiang Burkholderia (zhejiangensis sp.nov.), with strain number OP-1T, has been deposited in the China Center for Type Culture Collection in Wuhan, China, with the deposit number CCTCC AB2010354T and the deposit date of June 7, 2010.

[0011] The terrestrial Comamonas, named Comamonas terrae., with the strain number AS1P, has been deposited in the China Type Culture Collection in Wuhan, China, with the deposit number CCTCC AB 2020304 and the deposit date of November 23, 2020.

[0012] The acidophilic Stenotrophomonas, named as Stenotrophomonas acidaminiphila, with strain number X-2, has been deposited in the China Center for Type Culture Collection in Wuhan, China, with the deposit number CCTCC KB20081315 and the deposit date of March 13, 2007.

[0013] Trichoderma koningii, named as Trichoderma koningii, with strain number SMF2, has been deposited in the China Center for Type Culture Collection in Wuhan, China, with the deposit number CCTCC DF 20081034 and the deposit date September 9, 1981.

[0014] In another aspect, the present invention provides a method for preparing the microbial consortium, the method comprising:

[0015] Burkholderia parasitica, Burkholderia zhejiangensis, Comamonas terrestris, Oligotrophomonas acidophilus, and Trichoderma koningii were inoculated into sterilized LB liquid medium, and cultured in a shake flask at 25°C to 30°C in the dark until the late logarithmic growth period, and used as seed liquid respectively;

[0016] The seed liquid is inoculated into a sterilized nutrient fermentation medium at a volume ratio of 4-6%, and cultured at 25°C to 30°C until the OD600 of the bacterial liquid reaches 1.5 to obtain a fermentation liquid; preferably, the nutrient fermentation medium comprises 5.0 g / L yeast powder, 10.0 g / L sodium chloride, and 10.0 g / L peptone;

[0017] The fermentation broth is compounded, or further concentrated or dried as required to prepare a microbial consortium.

[0018] The microbial alliance of the present invention mainly utilizes the synergistic effect of five degradation bacteria to remove acrylonitrile organic matter, thereby providing an efficient, economical and environmentally friendly acrylonitrile wastewater and waste gas treatment method.

[0019] In a specific embodiment of the present invention, the microbial consortium for degrading acrylonitrile of the present invention is formed by uniformly mixing the fermentation broths of five bacterial species, namely, Burkholderia parasitica NEAU-SY24, Burkholderia zhejiangensis OP-1T, Comamonas terrestris AS1P, Oligotrophomonas acidophilus X-2, and Trichoderma koningii SMF2, with the ratio of the number of live bacteria being 3:1:1:1:1, and the OD600 in the bacterial agent reaches 1.5.

[0020] On the other hand, the present invention also provides the use of the microbial consortium in degrading acrylonitrile organic matter, including being used for degrading acrylonitrile in acrylonitrile-containing waste gas or wastewater in a biological filter.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) The present invention constructs a microbial consortium by artificially constructing a microbial consortium with fast film-forming speed, high degradation efficiency, stable bacterial community structure, and strong impact resistance. It can utilize acrylonitrile in waste gas or wastewater to meet its own needs, so as to achieve the purpose of degrading acrylonitrile without secondary pollution.

[0023] (2) The present invention obtains the microbial alliance in situ by sampling under actual working conditions, and uses bioinformatics to reveal its flora structure, and artificially constructs the microbial alliance based on this, so that the microbial alliance can adapt to actual working conditions, has high degradation efficiency, and has a simple process. The microorganisms have broad prospects for engineering applications. DETAILED DESCRIPTION

[0024] In order to enable those skilled in the art to better understand the technical solutions in this application, the present invention will be further described below in conjunction with specific embodiments. Obviously, the described embodiments are only part of the embodiments of this application, rather than all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of this application.

[0025] Example 1

[0026] The Bacillus subtilis is mixed evenly with the sterilized carrier material and then enriched and cultured to form a basement membrane, which is then placed in wastewater containing acrylonitrile and allowed to stand for two weeks to form a membrane to obtain a first bacterial community, which is then used as a research object to gradually increase the acrylonitrile content in the liquid phase, screen and domesticate to obtain a microbial consortium that can metabolize and degrade acrylonitrile. The specific carrier material can be, for example, wheat bran, zeolite powder, etc.

[0027] The specific screening and taming operation is carried out in the following steps:

[0028] (1) adding the first bacterial colony (inoculation amount 5%) to an inorganic salt medium (sterilized) containing acrylonitrile (0.25% by volume) as the sole carbon source, and culturing at 37° C. and 200 rpm for 5 days;

[0029] (2) transferring the above liquid culture medium to a new inorganic salt medium (sterilized) containing acrylonitrile (0.50% by volume) as the sole carbon source and culturing under the same conditions for 5 days;

[0030] (3) The above liquid culture medium was transferred to a new inorganic salt medium (sterilized) containing acrylonitrile (0.75% by volume) as the sole carbon source and cultured under the same conditions for 5 days;

[0031] (4) The above liquid culture medium was transferred to a new inorganic salt medium (sterilized) containing acrylonitrile (1.0% by volume) as the sole carbon source and cultured under the same conditions for 5 days;

[0032] (5) Streak culture on the plate, pick out single colonies and repeatedly streak culture to obtain multiple strains of pure and efficient degradation bacteria.

[0033] The specific components and contents of the above-mentioned inorganic salt culture medium are: K2HP04 0.5g / L, MgS04 0.5g / L, pH=7.2-7.6. The specific concentration of acrylonitrile in the culture medium is shown in the above steps; the purity of acrylonitrile used is chromatographically pure and is in solution state.

[0034] Take the culture fluid of 5 bacterial strains obtained according to the above steps, the bacteria are: NEAU-SY24 (paraburkholderia), OP-1T (burkholderia zekiensis), AS1P (comamonas terrestris), X-2 (acidophilic oligotrophic monocytogenes), SMF2 (Trichoderma koningii), centrifuge at 3000 rpm for 10 minutes, discard the supernatant, add distilled water to the precipitate to resuspend, centrifuge at 3000 rpm for 10 minutes, repeat 3 times, and obtain 5 pure bacterial strains. Transfer the single strain to the sterilized LB liquid culture medium, shake the flask at 25°C to 30°C in the dark, 150r / min for 16-48h, until the late logarithmic growth period, and use them as seed liquid respectively.

[0035] The seed liquid (inoculation amount 5%) of the above 5 strains of bacteria were respectively added to the sterilized LB liquid culture medium, and the culture was activated by shaking at 25°C to 30°C and 150 r / min for 16-48 hours until the OD600 of the bacterial solution reached 1.5; then the above culture solution was centrifuged, resuspended with physiological saline to prepare a bacterial solution OD600=1.5, and the live bacterial counts of NEAU-SY24 (paraburkholderia), OP-1T (burkholderia zeae), AS1P (comamonas terrestris), X-2 (acidophilic oligotrophomonas) and SMF2 (Trichoderma koningii) were proportioned in a ratio of 3:1:1:1:1 to obtain a mixed bacterial solution, namely the microbial alliance.

[0036] The composition and content of the LB liquid culture medium are preferably: 5.0 g / L yeast powder, 10.0 g / L sodium chloride, 10.0 g / L peptone, pH = 7.2-7.6.

[0037] The mixed bacterial solution (inoculation amount 5%) was added to the inorganic salt culture medium (sterilized) containing acrylonitrile wastewater, and cultured at 37°C and 150 r / min on a shaker for 180 h;

[0038] The specific components and contents of the above-mentioned inorganic salt culture medium are: K2HP04 0.5g / L, MgS04 0.5g / L, pH=7.2-7.6, and the acrylonitrile concentration in the wastewater is 71mg / L.

[0039] According to the purge and trap / gas chromatography method for the determination of acrylonitrile and acrolein in water quality of the Ministry of Ecology and Environment of the People's Republic of China (standard number: HJ 806-2016), it was determined that the degradation and removal rate of acrylonitrile reached 98.6%.

[0040] Example 2

[0041] In this embodiment, the composition and content of the LB liquid culture medium are preferably: 5.0 g / L yeast powder, 10.0 g / L sodium chloride, 10.0 g / L peptone, pH = 7.2-7.6.

[0042] Step 1: Activate the microbial alliance members NEAU-SY24 (Burkholderia parasitica), OP-1T (Burkholderia zekiensis), AS1P (Comamonas terrestris), X-2 (Oligomonas acidophilus) and SMF2 (Trichoderma koningii) on LB solid medium for 16-48 hours, transfer the single strains to sterilized LB liquid medium, and culture them in a shake flask at 25°C to 30°C in the dark at 150 rpm for 16-48 hours until the late logarithmic growth period, and use them as initial seed liquid respectively;

[0043] Step 2: The initial seed liquid of the above five bacterial strains is placed in a sterilized LB liquid medium at an inoculation rate of 4-6%, and fermented at 25°C-30°C and 150rpm until the OD600 of the bacterial liquid reaches 1.5 to obtain a fermentation liquid;

[0044] Step 3: centrifuge the bacterial solution obtained in step 2, resuspend it with physiological saline to prepare a bacterial solution OD600 = 1.5, and mix the live bacterial counts of NEAU-SY24 (Burkholderia parasitica), OP-1T (Burkholderia zekiensis), AS1P (Comamonas terrestris), X-2 (Acidophilus oligotrophomonas) and SMF2 (Trichoderma koningii) in a ratio of 3:1:1:1:1 to obtain a mixed bacterial solution, i.e., a microbial alliance;

[0045] The microbial consortium (4-6% by volume) was added to the biofilter filled with fillers, and then the biofilter was flooded with wastewater containing acrylonitrile to carry out aeration and biofilm formation operation for two weeks;

[0046] Step 4: After step 3, it was observed that obvious biofilm was formed on the filler. Inorganic salt culture medium was used as the circulating fluid. The culture medium composition was: K2HP04 0.5g / L, MgS04 0.5g / L. Acrylonitrile waste gas was mixed with air as the carrier gas. The acrylonitrile concentration in the carrier gas was 30.0mg / m 3 The biofilter was operated continuously for one week at 27°C, with a gas residence time of 50s, a circulating liquid flow rate of 10L / min, and a circulating liquid pH controlled at 6.0-8.0. The acrylonitrile concentration in the gas phase at the inlet and outlet of the device was determined according to the gas chromatography method for the determination of acrylonitrile in exhaust gas from stationary pollution sources of the Ministry of Ecology and Environment of the People's Republic of China (Standard No.: HJ / T 37-1999). It was determined that the degradation and removal rate of acrylonitrile in the gas phase reached 98.3%.

Claims

1. A microbial consortium for degrading acrylonitrile, the microbial consortium consisting of Paraburkholderia sp., zhejiangensis sp.nov., Comamonas terrae., Stenotrophomonas acidaminiphila, and Trichoderma koningii; In the microbial consortium, the ratio of the number of live bacteria of the paraburkholderia, the zhejiang Burkholderia, the terrestrial Comamonas, the acidophilic oligotrophomonas and the Trichoderma koningii is 3:1:1:1:1; in, The Burkholderia parakholderiae is the Burkholderia parakholderiae with the deposit number of CCTCC AA 2017029; The Burkholderia zhejiangensis is the Burkholderia zhejiangensis with a deposit number of CCTCC AB 2010354T; The soil Comamonas is the soil Comamonas with a deposit number of CCTCC AB 2020304; The acidophilic oligotrophic mononas is the acidophilic oligotrophic mononas with the deposit number of CCTCC KB 20081315; The Trichoderma koningii is the Trichoderma koningii with a preservation number of CCTCC DF 20081034.

2. The method for preparing the microbial consortium according to claim 1, comprising: (1) Burkholderia parasitica, Burkholderia zhejiangensis, Comamonas terrestris, Oligotrophomonas acidophilus, and Trichoderma koningii were inoculated into sterilized LB liquid culture medium, and cultured in a shake flask at 25° C. to 30° C. in the dark until the late logarithmic growth period, and the culture solutions were used as seed solutions; (2) inoculating the above seed solution into a sterilized nutrient fermentation medium at a volume ratio of 4-6%, and culturing at 25° C. to 30° C. until the OD600 of the bacterial solution reaches 1.5 to obtain a fermentation solution; (3) compounding the fermentation broth, or further concentrating or drying it as needed to obtain a microbial consortium; Wherein, the nutrient fermentation medium includes 5.0 g / L yeast powder, 10.0 g / L sodium chloride, and 10.0 g / L peptone.

3. The method according to claim 1, characterized in that: Before the implementation of step (1), Bacillus subtilis is placed in wastewater containing acrylonitrile to form a static film to obtain a first bacterial population, the first bacterial population is continuously transferred to 0.25%, 0.50%, 0.75% and 1.0% volume of an inorganic salt culture medium (sterilized) with acrylonitrile as the sole carbon source, and cultured at 37°C and 200 r / min in a shaking incubator for 5 days, streaking on a plate, picking a single colony and repeatedly streaking to obtain a culture solution of the Burkholderia parasitica, Burkholderia zhejiangensis, Comamonas terrestris, Oligotrophomonas acidophilus or Trichoderma koningii, and the culture solution is centrifuged to obtain pure bacterial cells; The specific components and contents of the inorganic salt culture medium are: K2HP04 0.5 g / L, MgS04 0.5 g / L, pH=7.2-7.

6.

4. The method according to claim 2 or 3, characterized in that: The process of compounding the fermentation liquid in step (3) is as follows: the fermentation liquid obtained in step (2) is centrifuged, resuspended with physiological saline to prepare a bacterial liquid OD600=1.5, and the ratio of the live bacteria count of the Burkholderia parasitica, Burkholderia zhejiangensis, Comamonas terrestris, Oligotrophomonas acidophilus and Trichoderma koningii is 3:1:1:1:1 to obtain a mixed bacterial liquid, namely the microbial alliance.

5. Use of the microbial consortium according to claim 1 in degrading acrylonitrile organic matter.

6. The use according to claim 5, characterized in that Including being used for degrading acrylonitrile in waste gas or wastewater containing acrylonitrile in a biological filter.

7. A method for degrading acrylonitrile organic matter, characterized in that: The method comprises: inoculating the microbial consortium described in claim 1 into a sample to be processed containing acrylonitrile organic matter, and culturing for a period of time, thereby achieving degradation of the acrylonitrile organic matter.