Screening method of microorganisms capable of relieving odor of kitchen waste

Abstract

The invention particularly discloses a screening method of microorganisms capable of relieving odor of kitchen waste. The method comprises the following steps: acquiring a sample for enrichment culture; separating and purifying on a specific selective culture medium which respectively takes degradation of amines, hydrogen sulfide and skatole as targets to obtain a single colony; and finally, extracting genome DNA of the strain, amplifying the 16S rRNA gene of the strain, and carrying out sequence alignment, so that the strain is accurately identified. By designing and applying the selective culture medium aiming at the specific odor-causing substances, efficient and directional screening of the target functional strains is realized. According to the method disclosed by the invention, the acinetobacter wuelsii, the acinetobacter baumannii and the bacillus licheniformis are successfully screened and identified, and the strains can be used for effectively degrading main odor-causing substances in the kitchen waste and have a good application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbiological technology, and in particular, to a method for screening functional microorganisms, and more particularly to a method for isolating and identifying microbial strains capable of degrading specific odor-causing substances in kitchen waste. BACKGROUND

[0002] Kitchen waste is an important component of municipal solid waste, which is characterized by high water content and diverse organic matter content, and is extremely prone to decomposition under the action of microorganisms. During this decomposition process, various organic matters in kitchen waste, such as proteins, sulfur-containing amino acids and heterocyclic compounds, will degrade to produce volatile malodorous substances, such as amines represented by trimethylamine, hydrogen sulfide and skatole, etc. These substances not only cause serious environmental odor pollution, but also may pose a threat to public health.

[0003] In order to handle kitchen waste and control the odor problem caused by it, there are various technical methods in the prior art. The traditional treatment methods mainly include sanitary landfill and incineration. However, this traditional sanitary landfill method occupies a large amount of land resources, and the leachate and methane generated under anaerobic conditions will cause secondary pollution to the soil and atmosphere. Although the incineration method can realize the reduction of waste, it has high requirements for the classification and dehydration of waste before incineration, and may produce substances harmful to human body and environment such as dioxin during the combustion process, and the equipment investment and operation cost are also relatively high. In addition, there are also some treatment methods, such as physical adsorption method and chemical deodorant method, which are usually only suitable for treating low-concentration waste gas and are not suitable for kitchen waste which is a continuous and high-concentration odor source.

[0004] Compared with the above-mentioned traditional treatment methods, using microorganisms to degrade kitchen waste is an environmentally friendly and resource-potential treatment method. Through the metabolic activity of specific microorganisms, odor-causing organic matter can be converted into harmless substances, solving the odor problem from the source. However, the prerequisite for efficient microbial treatment is to obtain functional strains that can specifically degrade target odor-causing substances.

[0005] Currently, there are still deficiencies in the screening method for obtaining such functional strains. A part of the existing technologies uses random isolation or conventional enrichment culture for screening, which lacks clear screening pressure and direction, resulting in low screening efficiency and difficulty in efficiently obtaining target strains from complex microbial communities. Another part of the technology realizes the importance of screening, but lacks sufficient pertinence in the design of screening medium, and fails to effectively distinguish and enrich microorganisms capable of degrading different types of odor-causing substances such as amines, sulfur compounds or skatole. In addition, the traditional strain identification method based on morphology and physiological and biochemical indicators has limited accuracy and is prone to misjudgment. Therefore, there is still a need in the art for a technical solution that can efficiently and directionally isolate specific functional microorganisms and accurately identify them, in order to meet the urgent needs of harmless and resourceful treatment of kitchen waste. SUMMARY

[0006] In order to overcome the deficiencies in the prior art, the present application provides a screening method for microorganisms that can alleviate the odor of kitchen waste, aiming to screen microbial strains that can efficiently degrade odor-causing substances such as amines, hydrogen sulfide and skatole in kitchen waste, and provide new technical means for biological treatment and resource utilization of kitchen waste.

[0007] In order to achieve the above purpose, the present application designs a screening method for microorganisms that can alleviate the odor of kitchen waste, which specifically comprises the following steps: Strain enrichment: The soil sample is taken into the culture medium and shaken for 0.5-96 h for strain enrichment.

[0008] Preferably, the soil sample is from kitchen waste. During shaking culture, a container filled with glass beads and inorganic salt liquid is used, and the culture temperature is 30℃.

[0009] Strain preliminary screening: The enriched bacterial liquid is diluted and spread on the microbial screening medium, and incubated at 10-65℃ until colonies appear.

[0010] Preferably, 0.1-1 mL of the enriched bacterial liquid is added to a sterilized test tube containing 0.9-9 mL of sterile water, mixed, and sequentially gradient diluted to 10 -1 ~10 -8 -6 , 10 -7 and 10 -8 3 gradients of 0.05-1 mL are taken, and spread on solid culture medium plates.

[0011] Preferably, the culture medium is divided into three types, which are: ​i) The culture medium for degrading amine is as follows: trimethylamine hydrochloride 0.001-10 g / L, KH2PO4 1.0-10 g / L, K2HPO4 2.6-15 g / L, MgSO4•7H2O 0.2-8 g / L, NH4Cl 0.2-10 g / L, KCl 0.25-12 g / L, 0.01-5% yeast extract, deionized water 1 L, and the pH value is 7.2; and the solid culture medium is added with agar 20 g / L, and the rest of the components are the same as above.

[0012] ii) The culture medium for degrading hydrogen sulfide is as follows: Na2S2O3•H2O 1-20 g / L, KNO3 0.5-7.0 g / L, KH2PO4 0.05-10 g / L, NaHCO3 0.05-8 g / L, MgCl2•6H2O 0.002-1 g / L, FeSO4•7H2O 0.01-3 g / L, NH4Cl 0.05-10 g / L; and the solid culture medium is added with agar 20 g / L, and the rest of the components are the same as above.

[0013] iii) The culture medium for degrading skatole is as follows: skatole 0.01-5 g / L, KH2PO4 0.002-3 g / L, K2HPO4 0.05-8.5 g / L, MgSO4 0.05-10 g / L, NH2SO4 0.5-8 g / L, KCl 0.001-6 g / L, deionized water 1 L, and the pH value is 7.2; and the solid culture medium is added with agar 20 g / L, and the rest of the components are the same as above.

[0014] Strain separation and purification: single colonies are picked and inoculated into fresh screening culture medium, and repeated subculture is carried out until single colonies with stable and consistent morphology are separated.

[0015] Strain preservation: the obtained single colonies are transferred to slant culture medium, and after good growth, are preserved in a 4°C refrigerator, and meanwhile, single colonies are picked and inoculated into culture medium, and the culture conditions are as follows: 10-65°C, 10-1000 rpm, 1-240 h.

[0016] Gene sequencing and identification: The genomic DNA of the bacteria in the bacterial liquid sample is extracted as a template, and the 16S rRNA gene fragment is amplified by PCR, and after PCR amplification and purification, sequencing is performed.

[0017] Preferably, the specific conditions for PCR amplification of the 16S rDNA gene fragment are as follows: pure water 22 μL, Easy Taq PCR super Mix 25 μL, and 1 μL of each of the forward primer, the reverse primer and the template, wherein: the nucleotide sequence of the forward primer 16SF is: TACGGYTACCTTGTTACGAC; Reverse primer nucleotide sequence 16SR is: AGAGTTTGATCMTGGCTCAG; The PCR amplification reaction program is: 95℃ for 5min; 95℃ for 30s, 52℃ for 30s, 72℃ for 135s, cycle 30 times; and then 72℃ for 5min.

[0018] Strain identification: The sequencing results in step (5) are subjected to sequence BLAST comparison in the NCBI database, and the colony identification of the screened microorganism is carried out in combination with the colony map, the microorganism strains degrading amines, hydrogen sulfide and skatole in kitchen waste are screened, and the Acinetobacter ursingii, Acinetobacter baumannii and Bacillus licheniformis with the NCBI numbers of MH368423.1, FJ907197.1 and MT642946.1 are obtained.

[0019] The microorganism screening method provided by the application solves the problems of low screening efficiency and lack of pertinence in the prior art by introducing a highly targeted screening strategy. The core of the technical concept of the application is to design and apply three different selective media, and the main or only nutrient source for the growth of microorganisms is the main odor-causing substance in kitchen waste, i.e. amines, the precursor of hydrogen sulfide and skatole. This design exerts precise and strong selective pressure on the culture environment, so that only microorganisms with the ability to degrade specific odor-causing substances can obtain growth advantage and be enriched in the culture medium, while the growth of other microorganisms is effectively inhibited. This substrate-based screening strategy directly realizes the directional enrichment and efficient separation of target functional strains, which is significantly different from the random screening method of the prior art.

[0020] The integrity of the technical solution of the application is also reflected in the accurate verification of the screening results. After obtaining the purified single colony by selective culture, the application further uses molecular biology means for strain identification. By extracting the genomic DNA of the strain and amplifying and sequencing the 16S rRNA gene sequence which is the gold standard in bacterial taxonomy, and then comparing the sequencing results with the public database, the screened strains can be accurately identified to the species level from the molecular level. This method overcomes the ambiguity and uncertainty brought by the dependence of traditional identification methods on morphology and physiological and biochemical indicators, ensures the accuracy of the identity of the strains obtained by the final screening, and lays a reliable technical foundation for subsequent application.

[0021] Therefore, by implementing the screening method of the present application, functional strains capable of degrading various key odor-causing substances in kitchen waste can be effectively isolated and obtained from complex environmental samples. The present scheme not only provides an efficient and reliable methodology, but also obtains effective strains identified as Acinetobacter uerisii, Acinetobacter baumannii and Bacillus licheniformis through the method. These strains have been confirmed to exhibit degradation capability for amines, hydrogen sulfide and skatole, etc., and can be used as effective microbial resources, directly applied to the biological treatment process of kitchen waste, to reduce the generation of malodorous gases from the source, and have clear industrial application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 is a picture of a single microbial colony screened in Example 1.

[0023] Figure 2 is a Venn diagram of community composition analysis of part of the representative microbial samples screened by different selective media in Example 1 of the present application. DETAILED DESCRIPTION

[0024] The preferred embodiments of the present application are described below in conjunction with the accompanying drawings, and it should be understood that the preferred embodiments described herein are only used to illustrate and explain the present application, and are not used to limit the present application.

[0025] Example 1.

[0026] As shown in Figure 1 , Figure 2 , the present embodiment describes the screening method and identification steps of the microorganisms for relieving the odor of kitchen waste as follows: (1) 20 g of kitchen soil sample was taken into a sterilized inorganic salt liquid medium containing an appropriate amount of glass beads (250 mL flask, 100 mL culture solution) and cultured at 10°C for 0.5 h on a shaker for bacterial enrichment; (2) 0.1 mL of the enriched bacterial solution was added to a sterilized test tube containing 0.9 mL of sterile water, mixed well, and sequentially diluted to 10 -1 ~10 -8 , -6 , -7 , -8 3 gradients, 0.2 mL of each gradient was diluted and inoculated on the microbial screening medium, and incubated at 10°C, with a total of 3 parallel incubations until colonies appeared; (3) single colonies capable of growing on the screening medium were picked and inoculated into fresh screening medium, and the same operation was repeated 2-3 times until single colonies with consistent morphology were isolated; (4) the obtained single colony is inoculated on a slant medium, and after good growth, is stored in a refrigerator at 4°C, and meanwhile, a single colony is picked and inoculated in a culture medium, and the culture conditions are as follows: temperature: 10°C; rotation speed: 10 rpm; and culture time: 10 h; (5) the genomic DNA of the bacteria in the bacterial liquid sample is extracted as a template, and a 16S rRNA gene fragment is amplified by PCR, and after PCR amplification and purification, sequencing is performed; The specific conditions for PCR amplification of the 16S rRNA gene fragment are as follows: 22 μL of pure water, 25 μL of Easy Taq PCR super Mix, 1 μL of each of the forward primer and the reverse primer, and 1 μL of the template; The nucleotide sequence of the forward primer 16SF is: TACGGYTACCTTGTTACGAC. The nucleotide sequence of the reverse primer 16SR is: AGAGTTTGATCMTGGCTCAG. The PCR amplification reaction program is as follows: 95°C for 5 min; 95°C for 30 s, 52°C for 30 s, and 72°C for 135 s; a total of 30 cycles; and 72°C for 5 min.

[0027] To further verify the targeting and selectivity of the screening method, high-throughput sequencing is performed on the microbial samples obtained by screening through different selective media, to analyze the differences in the community composition, and the results are shown in Figure 2 In Figure 2 , the samples S11, S12 and S13 are derived from amine-degrading medium; the samples S21, S22 and S23 are derived from hydrogen sulfide-degrading medium; and the samples S31, S32 and S33 are derived from skatole-degrading medium. Figure 2 Each petal-shaped set in Figure 2 represents an independent microbial sample, and the internal numbers represent the number of unique operational taxonomic units (OTUs) in the sample, and the number in the central white area represents the number of core OTUs common to all analyzed samples. From Figure 2 , it can be clearly seen that the number of unique OTUs of the samples derived from different selective media is significantly different. For example, the number of unique OTUs of the sample s.11 derived from amine-degrading medium is 131, the number of unique OTUs of the sample s.23 derived from hydrogen sulfide-degrading medium is 199, and the number of unique OTUs of the sample s.33 derived from skatole-degrading medium is 69. These data objectively show that the three different selective media used in this embodiment each enrich a group of species to form a unique microbial community, which directly proves that the screening strategy proposed in the present application has high targeting and selectivity.

[0028] (6) The sequence BLAST comparison by NCBI data confirms that the screened microorganisms are Acinetobacter ursingii, Acinetobacter baumannii and Bacillus licheniformis; The culture medium of step (2) is three kinds, respectively: i) The culture medium raw material for degrading amines: trimethylamine hydrochloride 0.001-10 g / L, KH2PO4 1.0-10 g / L, K2HPO4 2.6-15 g / L, MgSO4•7H2O 0.2-8 g / L, NH4Cl 0.2-10 g / L, KCl 0.25-12 g / L, 0.01-5% yeast extract, deionized water 1 L, pH value is 7.2; solid culture medium adds agar 20 g / L, and the rest of the ingredients are the same as above.

[0029] ii) The culture medium raw material for degrading hydrogen sulfide: Na2S2O3•H2O 1-20 g / L, KNO3 0.5-7.0 g / L, KH2PO4 0.05-10 g / L, NaHCO3 0.05-8 g / L, MgCl2•6H2O 0.002-1 g / L, FeSO4•7H2O 0.01-3 g / L, NH4Cl 0.05-10 g / L; solid culture medium adds agar 20 g / L, and the rest of the ingredients are the same as above.

[0030] iii) The culture medium raw material for degrading skatole: skatole 0.01-5 g / L, KH2PO4 0.002-3 g / L, K2HPO4 0.05-8.5 g / L, MgSO4 0.05-10 g / L, NH2SO4 0.5-8 g / L, KCl 0.001-6 g / L, deionized water 1 L, pH value is 7.2; solid culture medium adds agar 20 g / L, and the rest of the ingredients are the same as above.

[0031] Example 2 The screening method and identification steps of the microorganism for relieving the odor of kitchen waste are as follows: (1) Take 20 g of kitchen waste sample and put it into a sterilized inorganic salt liquid culture medium containing an appropriate amount of glass beads (250 mL flask, 100 mL culture solution) and culture at 65°C for 96 h on a shaking table for bacterial enrichment; (2) Take 0.1 mL of the enriched bacterial solution and add it to a sterilized test tube containing 0.9 mL of sterile water, mix well, and sequentially dilute to 10 -1 ~10 -8 -6 ​、10 -7 、10 -8 3 Gradient each 0.2 mL dilution coating on microbial screening medium, constant temperature culture at 65℃, a total of 3 parallel, until the colony appears; (3) picking up single colonies that can grow on the screening medium, inoculating into fresh screening medium, repeatedly performing the same operation 2~3 times, until the morphologically stable single colonies are isolated; (4) the obtained single colonies are transferred to the slant medium, and after good growth, they are stored in a refrigerator at 4℃, and single colonies are picked up and inoculated in the medium, and the culture conditions are: temperature: 65℃; rotation speed: 1000 rpm; culture time: 96 h; (5) extracting the genomic DNA of the bacteria in the bacterial liquid sample as a template, PCR amplifying the 16S rRNA gene fragment, and sequencing after PCR amplification and purification; The specific conditions for PCR amplifying the 16S rRNA gene fragment are as follows: 22 μL of pure water, 25 μL of Easy Taq PCR super Mix, 1 μL of each of the forward and reverse primers and the template; The forward primer nucleotide sequence 16SF is: TACGGYTACCTTGTTACGAC; The reverse primer nucleotide sequence 16SR is: AGAGTTTGATCMTGGCTCAG; The PCR amplification reaction program is: 95℃ for 5 min; 95℃ for 30 s, 52℃ for 30 s, 72℃ for 135 s; a total of 30 cycles; 72℃ for 5 min.

[0032] (6) confirming that the screened microorganisms are Acinetobacter ursingii, Acinetobacter baumannii and Bacillus licheniformis through sequence BLAST comparison of NCBI data; Among them, the medium of step (3) is 3, respectively: i) the culture medium for degrading amines: trimethylamine hydrochloride 0.001-10 g / L, KH2PO4 1.0-10 g / L, K2HPO4 2.6-15 g / L, MgSO4•7H2O 0.2-8 g / L, NH4Cl 0.2-10 g / L, KCl 0.25-12 g / L, 0.01-5% yeast extract, deionized water 1 L, pH value is 7.2; solid medium adds agar 20 g / L, the rest of the ingredients are the same as above.

[0033] ii) Culture medium raw materials for degrading hydrogen sulfide: Na2S2O3-H2O 1-20 g / L, KNO3 0.5-7.0 g / L, KH2PO4 0.05-10 g / L, NaHCO3 0.05-8 g / L, MgCl2-6H2O 0.002-1 g / L, FeSO4-7H2O 0.01-3 g / L, NH4Cl 0.05-10 g / L; solid culture medium adds agar 20 g / L, and the rest of the ingredients are the same as above.

[0034] iii) Culture medium raw materials for degrading skatole: skatole 0.01-5 g / L, KH2PO4 0.002-3 g / L, K2HPO4 0.05-8.5 g / L, MgSO4 0.05-10 g / L, NH2SO4 0.5-8 g / L, KCl 0.001-6 g / L, deionized water 1 L, pH value is 7.2; solid culture medium adds agar 20 g / L, and the rest of the ingredients are the same as above.

[0035] The above results show that the screening method proposed in the present application has good applicability and stability under different culture parameter combinations, and can reliably screen out microbial strains with target degradation function.

Claims

1. A method for screening microorganisms capable of alleviating odor of food waste, characterized by, The method comprises the following steps: The soil sample is placed in a culture medium for shaking culture for 0.5-96 h to enrich the bacteria; The diluted sample is coated on a microbial screening culture medium and incubated at a constant temperature of 10-65 DEG C until colonies appear; A single colony is picked and inoculated into fresh screening culture medium, and the subculture is repeated until a single colony with stable and consistent morphology is isolated; The obtained single colony is transferred to a slant culture medium, and after good growth, it is stored in a refrigerator at 4 DEG C; meanwhile, a single colony is picked and inoculated into a culture medium, and the culture conditions are as follows: 10-65 DEG C, 10-1000 rpm, and 1-240 h; The genomic DNA of the bacteria in the bacterial liquid sample is extracted as a template, and a 16S rRNA gene fragment is amplified by PCR; after PCR amplification and purification, sequencing is performed; The sequencing results in step (5) are subjected to sequence BLAST comparison, and the colony map is combined to identify the colonies of the microorganism to be screened, so that the microorganism strain capable of degrading amines, hydrogen sulfide and skatole in kitchen waste is screened.

2. The screening method according to claim 1, characterized in that: The culture medium in step (2) is as follows: i) for degrading amines, the culture medium raw materials are as follows: trimethylamine hydrochloride 0.001-10 g / L, KH2PO4 1.0-10 g / L, K2HPO4 2.6-15 g / L, MgSO4•7H2O 0.2-8 g / L, NH4Cl 0.2-10 g / L, KCl 0.25-12 g / L, 0.01-5% yeast extract, deionized water 1 L, and the pH value is 7.2; for solid culture medium, agar 20 g / L is added, and the remaining components are the same as above; ii) for degrading hydrogen sulfide, the culture medium raw materials are as follows: Na2S2O3•H2O 1-20 g / L, KNO3 0.5-7.0 g / L, KH2PO4 0.05-10 g / L, NaHCO3 0.05-8 g / L, MgCl2•6H2O 0.002-1 g / L, FeSO4•7H2O 0.01-3 g / L, NH4Cl 0.05-10 g / L; for solid culture medium, agar 20 g / L is added, and the remaining components are the same as above; iii) for degrading skatole, the culture medium raw materials are as follows: skatole 0.01-5 g / L, KH2PO4 0.002-3 g / L, K2HPO4 0.05-8.5 g / L, MgSO4 0.05-10 g / L, NH2SO4 0.5-8 g / L, KCl 0.001-6 g / L, deionized water 1 L, and the pH value is 7.2; for solid culture medium, agar 20 g / L is added, and the remaining components are the same as above.

3. The screening method of claim 1, wherein: The dilution of step (2) is to add 0.1-1 mL of the enriched culture liquid into a sterilized test tube containing 0.9-9 mL of sterile water, mix well, and then dilute by gradient into 10 -1 ~10 -8 -6 -7 -8 3 gradients of 0.05-1 mL, respectively, and then spread on solid culture medium plates.​​​ 4. The screening method of claim 1, wherein: In step (5), the specific conditions for PCR amplification of the 16S rDNA gene fragment are as follows: 22 μL of pure water, 25 μL of Easy Taq PCR super Mix, 1 μL of each of the forward primer, the reverse primer and the template, wherein the nucleotide sequence of the forward primer 16SF is TACGGYTACCTTGTTACGAC; the nucleotide sequence of the reverse primer 16SR is AGAGTTTGATCMTGGCTCAG; PCR amplification reaction program: 95℃ 5min; 95℃ 30s, 52℃ 30s, 72℃ 135s, cycle 30 times; then 72℃ 5min.

5. The screening method of claim 1, wherein: The step (6) is comparing and screening through the NCBI database to obtain Acinetobacter ursingii with NCBI numbers of MH368423.1, FJ907197.1 and MT642946.1, Acinetobacter baumannii and Bacillus licheniformis.

6. The screening method of claim 1, wherein: The step (1) is that the soil sample is obtained from kitchen waste.

7. The screening method of claim 1, wherein: In the step (1), the container filled with glass beads and inorganic salt liquid is used in the shock culture, and the culture temperature is 30 DEG C.

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