阅读说明：本技术 一种厌氧微生物菌群降解γ-六六六的方法 (Method for degrading gamma-hexachloro cyclohexane by anaerobic microbial flora ) 是由 邹德勋 郑富文 仝婧婧 于 2021-05-18 设计创作，主要内容包括：一种厌氧微生物混合菌群降解γ-六六六的方法属于环境生物化学修复技术应用技术领域。该混合菌群从长期受六六六农药污染土壤中采用基础培养基多次传代富集驯化获得。该混合菌群由细菌和古菌组成,细菌包括Clostridium-sensu-stricto-7,Sedimentibacter,Lysinibacillus,Georgenia muralis,Anaerocolumna aminovalerica等菌株。古菌包括norank-d-Archaea,norank-o-Group-1.1c,Thermoplasmata,Bathyarchaeia,Methanothermobacter等菌株。本发明的微生物菌群能够以葡萄糖、乳酸、乙酸作为电子供体,有效地降解γ-六六六。微该混合菌群能够在添加乳酸的淹水条件下对实际的污染土壤起到良好的修复效果,γ-六六六的降解率可达近95％。(A method for degrading gamma-hexachloro cyclohexane by anaerobic microorganism mixed flora belongs to the technical field of application of environmental biochemical remediation technology. The mixed flora is obtained by multiple passage enrichment domestication of basic culture medium in soil polluted by hexachloro cyclohexane pesticide for a long time. The mixed flora is composed of bacteria and archaea, and the bacteria comprise strains of Clostridium _ sensu _ stricoto _7, Sementibacter, Lysinibacillus, Georgia muralis, Anaerocolmnavarias, and the like. The Archaea includes strains such as norank _ d _ Archaea, norank _ o _ Group _1.1c, thermoplasta, Bathyarcaeia, Methanotherobacter, etc. The microbial flora can effectively degrade gamma-hexachloro-cyclohexane by taking glucose, lactic acid and acetic acid as electron donors. The mixed flora can play a good role in repairing the actual polluted soil under the flooding condition of adding lactic acid, and the degradation rate of gamma-hexachloro cyclohexane can reach nearly 95%.)

1. A method for degrading gamma-hexachloro-cyclohexane by anaerobic microorganism mixed flora is characterized in that the flora comprises bacteria and archaea;

the bacteria comprise Clostridium _ sensu _ stricoto _7 with the abundance percentage of OTUs of 60-70%, Sedimiobacter of 8-15%, Lysinibacillus of 5-10%, Georgia mulalis of 5-10%, Anaerocolmma of 2-5%;

the Archaea comprises norak _ d _ Archaea with the abundance percentage of OTUs of 65-75%, norak _ o _ Group _1.1c of 5-10%, thermoplasamata of 3-8%, Batthyaroeia of 3-8% and Methanothermobacter of 3-8%.

2. The method of claim 1, wherein the bacteria comprise Clostridium sensu stricto 7 with 66% abundance of OTUs, Sedinibacter 12%, Lysinibacillus 9%, Georgia muralis 8%, and Anaerocolumnadorica 4%.

3. The method of claim 1, wherein said Archaea comprises norak _ d _ Archaea with 72% OTUs abundance, norak _ o _ Group _1.1c, thermoplasta 6%, bathyarchaea 6%, methanobacteria 5%.

4. The method according to claim 1, wherein the mixed population of anaerobic microorganisms degrades γ -hexa-in the water body using glucose, lactic acid, or acetic acid as an electron donor.

5. The method of claim 4, wherein the lactic acid concentration is 2-10 mM.

6. The method of claim 1, wherein the mixed population of anaerobic microorganisms is capable of degrading γ -hexa-lactic acid in flooded soils using lactic acid as an electron donor.

Technical Field

The invention relates to a method for removing gamma-hexachloro-cyclohexane in an organic chlorine pesticide difficult to degrade, in particular to a method for degrading gamma-hexachlorocyclohexane in an environment by utilizing microbial flora, wherein the method comprises a water environment and a soil environment, and belongs to the technical field of application of environmental biochemical remediation technology.

Background

Gamma-hexachloro (gamma-HCH) is a broad spectrum insecticide organochlorine pesticide that has been widely used in the past 70 years with increasing residual levels in soil and water environments, and has been listed in the stockholm convention persistent organic pollutants POPs in 2009 due to its persistent recalcitrance in the environment and its lipopexia and potential carcinogenicity as environmental endocrine disruptors. At present, researchers have screened many microbial pure cultures capable of anaerobically degrading gamma-HCH, which are mainly distributed in various genera such as Clostridium, Bacillus, Enterobacteriaceae, Desulfovibrio, and the like. However, single pure cultures often suffer from strong competition by indigenous microorganisms in actual field remediation resulting in reduced efficiency (Tomei and daugaulis, 2012). Mixed microbial populations tend to perform better than pure cultures. The mixed flora is taken as a biological colony with multiple coexisting bacteria, can decompose organic matters in the growth process, simultaneously degrade the organic matters in the environment by means of mutual symbiotic proliferation and synergistic metabolism among various microorganisms, and can activate other microorganisms with purification functions, so that a complex and stable microecological system (wool et al, 2010) is formed. Compared with a microbial pure culture, the gene of the microbial mixed flora enriched from the natural environment is more diverse, the metabolic pathways are more abundant, the metabolic function cross feeding (Zhang et al, 2020) can be enhanced, and the established stable survival relationship and the degradation performance are possessed, so the microbial mixed flora has more potential in the aspect of organic pollutant degradation.

Disclosure of Invention

The invention provides a mixed microbial flora with stable flora and good degradation performance, which aims at the treatment of gamma-hexachloro cyclohexane polluted site and the problem that general microbial pure culture is often weak in colonization ability in the environment, and is more suitable for repairing the polluted site.

The mixed microbial flora comprises bacteria and archaea.

The bacteria comprise Clostridium _ sensu _ stricoto _7 with the abundance percentage of OTUs of 60-70%, Sedimibacter of 8-15%, Lysinibacillus of 5-10%, Georgia mulalis of 5-10% and Anaerocolumna aminovalica of 2-5%.

The Archaea comprises norak _ d _ Archaea with the abundance percentage of OTUs of 65-75%, norak _ o _ Group _1.1c of 5-10%, thermoplasamata of 3-8%, Batthyaroeia of 3-8% and Methanothermobacter of 3-8%.

The bacteria include Clostridium _ sensu _ stricoto _7 with 66% OTUs abundance, Sedimentobacter 12%, Lysinibacillus 9%, Georgenia muralis 8%, and Anaerocolumnaderica 4%.

The Archaea comprises norak _ d _ Archaea with the abundance percentage of OTUs of 72%, norak _ o _ Group _1.1c of 9%, thermoplasamata of 6%, Bathyarcheia of 6% and Methanothermobacter of 5%.

The mixed microbial flora can take glucose, lactic acid and acetic acid as electron donors, and gamma-hexachloro cyclohexane as electron acceptors to effectively degrade 20mg/L gamma-hexachloro cyclohexane. Furthermore, lactic acid has better degradation effect as an electron donor mixed flora, and the concentration of the lactic acid is 2-10 mM. A soil remediation test in a laboratory shows that the mixed flora can efficiently degrade 20mg/kg of gamma-hexachloro cyclohexane in soil within 35 days, has a good remediation effect on the polluted soil, and has the potential of being applied to remediation of gamma-hexachloro cyclohexane polluted water and soil.

Drawings

FIG. 1 example 1 high throughput sequencing of mixed flora results in bacterial level composition.

FIG. 2 high throughput sequencing of mixed populations example 1 results in the archaea level composition.

FIG. 3 shows the degradation effect of the mixed flora on 20mg/L gamma-hexachloro cyclohexane under different electron donor conditions.

FIG. 4 shows the degradation effect of the mixed flora on 20mg/L gamma-hexachloro cyclohexane under different lactic acid concentrations.

FIG. 5 chromatogram of degradation of gamma-hexachloro-cyclohexane by mixed flora.

FIG. 6 shows the degradation effect of the mixed flora on 20mg/kg of gamma-hexachloro cyclohexane in soil.

Detailed Description

Example 1

The method for enriching the mixed flora of anaerobic microorganisms is characterized by comprising the following steps of:

(1) establishing deposit-free cultures (segment-free cultures): the microorganisms are from soil contaminated with sixty-six. 5g of the contaminated soil of hexachloro cyclohexane was inoculated into 50mL of anaerobic inorganic base medium, gamma-HCH (dissolved in acetone) was added to give a final concentration of 10mg/L, and 50mg/L of lactic acid was added as an electron donor. Displacing the gas so that the headspace is N₂The cells were cultured at 30 ℃ in a 150r/min incubator for 7 days. Then, 10% (v/v) of the soil suspension was transferred again to a fresh 50mL anaerobic inorganic base medium, and γ -hexachloro-cyclohexane (dissolved in acetone) was added to give a final concentration of 10mg/L, and the culture was carried out under the same conditions.

(2) Enrichment and domestication: the concentration of gamma-hexachloro cyclohexane in the culture medium is quantitatively detected by gas chromatography (GC-MS) periodically, and after the concentration is obviously reduced, the culture medium is transferred to a fresh inorganic salt culture medium again, and lactic acid and gamma-hexachloro cyclohexane are added. Continuously enriching and domesticating, gradually increasing the concentration of gamma-hexachloro cyclohexane to 50mg/L, maintaining the concentration of the pollutants, and continuously domesticating for multiple times to obtain the final anaerobic microorganism enrichment solution. And taking the culture solution with the same volume and coating the culture solution in an inorganic salt solid culture medium when the culture solution is transferred once, and comparing the colony characteristics and the number of strains on the plate every time.

The inorganic salt culture medium is prepared according to the following proportion: NaCl 1.00g/L, MgCl₂·6H₂O 0.50g/L，KH₂PO₄0.20g/L，NH₄Cl 0.30g/L，KCl 0.30g/L，CaCl₂·2H₂O 15mg/L，FeCl₂·4H₂O 15mg/L，CoCl₂·6H₂O 0.19mg/L，MnCl₂·4H₂O 0.1mg/L，ZnCl₂ 0.070mg/L，H₃BO₃ 0.006mg/L，Na₂MoO₄·2H₂O0.036 mg/L，NiCl₂·6H₂O 0.024mg/L，CuCl₂·2H₂O 0.002mg/L，Na₂WO₄·2H₂O 0.008mg/L，Sterilizing at 121 deg.C for 20min at pH of 7.2, and adding vitamin solution filtered with 0.22 μm filter membrane. The inorganic salt solid medium is prepared by additionally adding 1.5-2.0% of agar.

The method is adopted to obtain the mixed microbial flora, the mixed microbial flora comprises bacteria and archaea, and the high-throughput sequencing result belongs to the horizontal classification and is respectively shown in figures 1 and 2. The bacteria include Clostridium _ sensu _ stricoto _7 with 66% OTUs abundance, Sedimentobacter 12%, Lysinibacillus 9%, Georgenia muralis 8%, and Anaerocolumnaderica 4%.

The Archaea comprises norak _ d _ Archaea with the abundance percentage of OTUs of 72%, norak _ o _ Group _1.1c of 9%, thermoplasamata of 6%, Bathyarcheia of 6% and Methanothermobacter of 5%.

Example 2

The mixed bacterial population was inoculated into a sterile inorganic salt medium containing 20mg/L of γ -hexachloro cyclohexane in an inoculum size of 10% (v/v), cultured for 35 days at 30 ℃ in a 150r/min incubator with 6mM glucose, lactic acid and acetic acid as electron donors, and periodically sampled to determine the concentration of hexachloro cyclohexane in the medium, and the effect of the mixed bacterial population on the degradation of γ -hexachloro cyclohexane was examined, the results of which are shown in FIG. 3. The mixed flora can effectively degrade gamma-hexachloro cyclohexane in the culture medium, the final degradation rates of glucose, lactic acid and acetic acid groups are 70.11%, 88.00% and 86.92% respectively, and lactic acid has higher degradation rate as an electron donor.

Example 3

Among the three electron donors, the influence of the lactic acid concentration on the degradation performance of the mixed flora was examined, the lactic acid concentration was set to 2, 4, 6, 8, 10mM, respectively, the mixed flora was inoculated into a sterile inorganic salt medium containing 20mg/L of γ -hexachloro cyclohexane in an inoculum size of 10% (v/v), the medium was cultured at 30 ℃ for 35 days in a 150r/min incubator, and the concentration of hexachloro cyclohexane in the medium was measured by sampling periodically, and the results are shown in FIG. 4. The mixed flora has better degradation effect in the range of 6-10mM of lactic acid concentration, wherein 6mM is the optimal concentration. In the degradation process, tetrachlorocyclohexene (3,4,5,6-TeCCH) intermediate products are generated. FIG. 5 is a mass spectrum of a sample of mixed bacterial population cultured in mineral salt medium containing gamma-hexachloro cyclohexane for 7 days.

Example 4

Setting the mass-to-volume ratio of soil to water to be 1:2.5(g: mL), adding 10% (v/v) of mixed bacterial liquid, observing the repairing effect (M + Lac) of the mixed bacterial liquid on gamma-hexachloro-benzene with the concentration of 22.83mg/kg of actual polluted soil, taking the soil without adding lactic acid (M) and only adding deionized water (CK) as a control group, and culturing in an incubator at 30 ℃ and 150r/min, wherein the result is shown in FIG. 6. After flood repair for 63 days, the degradation rates of the M + Lac, the M and the CK groups can respectively reach 95.0%, 94.5% and 2.0%. The group only added with deionized water has extremely low degradation rate to gamma-hexachloro cyclohexane, the gamma-hexachlorocyclohexane can be effectively degraded by adding the mixed bacteria liquid, and the degradation rate can be improved in the early stage of repair by adding lactic acid.