WO2023039921A1 - Agent microbien composite pour la dégradation d'hydrocarbures pétroliers, son procédé de préparation et son utilisation - Google Patents

Agent microbien composite pour la dégradation d'hydrocarbures pétroliers, son procédé de préparation et son utilisation Download PDF

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Publication number
WO2023039921A1
WO2023039921A1 PCT/CN2021/119762 CN2021119762W WO2023039921A1 WO 2023039921 A1 WO2023039921 A1 WO 2023039921A1 CN 2021119762 W CN2021119762 W CN 2021119762W WO 2023039921 A1 WO2023039921 A1 WO 2023039921A1
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swh
petroleum
degrading
oil
soil
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PCT/CN2021/119762
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English (en)
Chinese (zh)
Inventor
王加宁
黄玉杰
霍乾伟
宋繁永
陈贯虹
张强
傅晓文
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山东省科学院生态研究所(山东省科学院中日友好生物技术研究中心)
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Publication of WO2023039921A1 publication Critical patent/WO2023039921A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
    • B09C—RECLAMATION OF CONTAMINATED SOIL
    • B09C1/00—Reclamation of contaminated soil
    • B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F11/00—Treatment of sludge; Devices therefor
    • C02F11/02—Biological treatment
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
    • C02F2103/10—Nature of the water, waste water, sewage or sludge to be treated from quarries or from mining activities

Definitions

  • the invention belongs to the technical field of microbes and soil remediation, and in particular relates to a petroleum hydrocarbon degrading composite microbial bacterial agent and its preparation method and application.
  • Soil oil pollution remediation is the main field of intensive research and development of environmental protection technologies in various countries in the world.
  • conventional methods at home and abroad include physical and chemical remediation, such as concentrated drying, solid-liquid separation, extraction and separation, Washing method, heat treatment and thermal desorption, chemical demulsification recovery method, etc.
  • physical and chemical remediation methods were mainly established for the recovery and treatment of crude oil in oil sludge in the early stage.
  • Physical and chemical remediation methods are difficult to avoid secondary pollution, so that the original habitat is destroyed, and it is difficult to be effective and applied to large-scale, medium-low concentration oil pollution control, and the cost is expensive. Therefore, the original physical and chemical methods need to be further improved, enhanced, optimized or supplemented/combined with biological methods.
  • Bioremediation usually uses endogenous or artificially added microorganisms or bacterial agents, repair plants and specific enzymes to strengthen/accelerate the degradation of petroleum hydrocarbons. Compared with physical and chemical methods, it is more environmentally friendly, and can minimize the damage to the soil ecosystem while removing pollutants, and is a sustainable restoration method.
  • the present invention provides a petroleum hydrocarbon degrading composite microbial agent and its preparation method and application.
  • the composite microbial agent can effectively degrade petroleum hydrocarbons in soil and oil sludge sand, and can be effectively applied biodegradation treatment of petroleum-contaminated soil.
  • a compound microbial bacterial agent for petroleum hydrocarbon degradation including Acinetobacter KJ-1 of preservation number CGMCC No.20664, Providencia rettii L1 of CGMCC No.16526, and Bacillus SWH-1 of GMCC No.1950 and Sphingobacterium SWH-2 of CGMCC No.1951.
  • the composite microbial agent is composed of a fermentation broth and a carrier peat soil, and the mass ratio of the fermentation broth to the peat soil is 1:5-15.
  • the effective number of live bacteria of Acinetobacter KJ-1 in the fermentation broth is 10 7 -10 9 /ml
  • the effective number of live bacteria of Providencia rettii L1 is 10 7 -10 9 /ml
  • the effective number of viable bacteria of Bacillus SWH-1 is 10 8 -10 9 /ml
  • the effective number of viable bacteria of Sphingobacterium SWH-2 is 10 7 -10 9 /ml.
  • the preparation method of the petroleum hydrocarbon degrading composite microbial bacterial agent is characterized in that it comprises the following steps:
  • step (3) The oil-degrading flora fermentation grade I seed liquid in step (2) is transferred to the fermentation medium again according to the volume ratio of 3-6%, and the air is cultivated until the total number of oil-degrading flora is 10 9 CFU/ ml or more, the oil-degrading flora fermentation grade II seed liquid is obtained, and the cultivation is expanded step by step, and the total number of oil-degrading flora is more than 10 9 CFU/ml;
  • step (2) Using peat soil as a carrier, mix the fermented bacteria liquid obtained in step (2) with peat soil, the mass ratio is 1:5-15, and the water content after drying is 10-15%, so as to obtain a petroleum hydrocarbon degradation compound Microbial agents.
  • the NB culture medium described in the step (1) contains 10 g of peptone, 3 g of beef powder, 5 g of sodium chloride, 18.0 g of agar, distilled water to 1000 mL, and a pH value of 7.0 to 7.4 per liter of culture medium;
  • the fermentation medium described in (2) and step (3) contains KH 2 PO 4 1g in every liter of fermentation medium, K 2 HPO 4 1g, MgSO 0.2g, CaCl 2 0.03g, FeCl 0.002g , Na 2 CO 3 0.2g, (NH 4 ) 2 SO 4 0.8g, nutrient broth 20g, the balance is water; the fermentation temperature is 28-37°C, the culture time is 18-24h, and the culture speed is 120 ⁇ 150rpm.
  • step (2) the effective activity of four bacterial strains of the oil-degrading bacteria Acinetobacter aviosa KJ-1, Providencia rettgeri L1, Bacillus SWH-1 and Sphingobacterium SWH-2 described in step (2)
  • the ratio of the number of bacteria is 1.5-2:1:1-2:2.
  • the petroleum pollutants are petroleum-contaminated soil or treated oily sludge.
  • the compound microbial bacterial agent is added to the soil twice, and the interval between the two additions is one week.
  • the petroleum hydrocarbon degradation composite microbial bacterial agent of the present invention can effectively degrade petroleum hydrocarbons in soil and oil sludge sand, can be effectively applied to the biodegradation treatment of petroleum-contaminated soil, and will not cause secondary pollution to the ecological environment of the soil.
  • the synergistic effect between Acinetobacter KJ-1, Providencia rettgeri L1, Bacillus SWH-1 and Sphingobacterium SWH-2 improves the degradation efficiency of final petroleum hydrocarbons; and the preparation process of solid bacterial agent is simple and easy The operation is low in cost, and has broad application prospects in the field of bioremediation of oil-contaminated soil and oil product leakage accident sites.
  • step (3) Using peat soil as the carrier of the oil-degrading bacterial agent, mix the fermentation liquid obtained in step (2) with the peat soil according to a mass ratio of 1:10, and dry it naturally in a cool place for 2 hours until the water content is 12 %, the final compound microbial bacterial agent for petroleum hydrocarbon degradation is obtained.
  • the 10L seed tank control fermentation parameters are: temperature 30°C, stirring speed 120r/min, ventilation rate 1:0.5-1, culture seed tank pressure 0.04-0.06Mpa, culture time is about 24h according to the growth condition;
  • the 500L fermenter control fermentation parameters are: temperature 30°C, stirring speed 120r/min, ventilation rate 1:0.6-1, culture seed tank pressure 0.04-0.06Mpa, culture time about 18-24h;
  • Fermentation medium composition Each liter of fermentation medium contains KH 2 PO 4 1g, K 2 HPO 4 1g, MgSO 4 0.2g, CaCl 2 0.03g, FeCl 3 0.002g, Na 2 CO 3 0.2g, (NH 4 ) 2 SO 4 0.8g, nutrient broth 20g, the balance is water;
  • the bacterial agent obtained in this example was stored at room temperature, and the detected bacteria were stored for 3, 6, and 9 months respectively, and the survival rate was calculated.
  • the results of the survival rate test are shown in Table 1.
  • Table 1 the petroleum-degrading bacterial agent prepared in Example 1 was stored at room temperature, and the survival rate reached 75% in three months, and the survival rate was 58% after half a year. Therefore, the survival period of the bacterial agent has reached the standard of biological bacterial agents;
  • Table 1 The survival rate of different storage periods of petroleum degrading bacterial agents
  • the preparation method of embodiment 1 petroleum hydrocarbon degradation composite microbial bacterial agent prepare respectively the composite bacterial agent (composite microbial agent 1) containing Providencia rettgeri L1, bacillus SWH-1 and Sphingobacterium SWH-2 , the composite bacterial agent (composite microbial agent 2) containing bacillus SWH-1 and Sphingobacterium SWH-2, the total number of viable bacteria is not much different from the composite bacterial agent of Example 1.
  • serial number condition parameter 1 As is - Keep moisture content at 20-25%, till tillage 2 As is - 2% bacterial agent - no water added, tilled 3 As it is - 2% bacterial agent - keep 20-25% water content, turn till 4 As it is - 2% bacterial agent - 2% peat - keep water content at 20-25%, tillage 5 As it is - 2% bacterial agent - 5% peat - keep water content at 20-25%, tillage 6 As it is - 2% bacterial agent - 5% straw - keep water content at 20-25%, plow 7 As it is - 2% bacterial agent - keep the water content at 30-35%, turn till .
  • the determination of oil content adopts the following method: add a certain amount of dichloromethane to the oil-contaminated soil, use a Soxhlet extractor to extract the remaining oil in the soil, add a sufficient amount of anhydrous sodium sulfate to the extract for dehydration, filter and spin Steam to make dichloromethane completely volatilize, use dichloromethane to constant volume, dilute to a certain multiple, use the gas chromatography internal standard method to measure the petroleum hydrocarbon content in the soil, calculate the petroleum content in the soil according to the standard curve, and use the following formula to calculate the petroleum degradation rate:
  • Oil degradation rate (%) (Wo-Wx)/Wo ⁇ 100%
  • Wo represents the oil content in the control soil
  • Wx represents the oil content in the treated soil
  • sample numbers 4, 5, and 6 had better effects, and the oil content decreased from the original 2.411% to 1.343%, 1.326% and 1.241%, and the degradation rates of petroleum hydrocarbons were 52.65%, 53.23% and 56.24% respectively .
  • the petroleum degradation rate was only 14.98%.
  • adding peat or straw can help ensure the permeability of the soil and promote the degradation of petroleum hydrocarbons. .
  • the petroleum hydrocarbon degrading composite microbial bacterial agent in Example 1 was taken to carry out the oil-contaminated soil remediation test, and the specific method was as follows: the petroleum-contaminated soil in the isolated island area of Dongying was taken, and the oil content of the soil was detected by gravimetric method, and the oil content of the soil was 6.729%.
  • Add 6.7g of ammonium nitrate and 1.8g of potassium dihydrogen phosphate to each kilogram of soil carry out sample treatment on oil-contaminated soil according to the parameters in Table 4, replenish water regularly, and plow, the water supply method is to calculate the water shortage after weighing, and spray when plowing Hydrate. As shown in Table 5 to the effective viable count determination result;
  • Immobilizing microorganisms can maintain the biological activity of microorganisms, and under suitable conditions, microorganisms can be rapidly multiplied in large quantities.
  • Different carriers have a greater impact on the effects of microorganisms.
  • different carrier additions also have an impact.
  • This experiment Using peat and straw as solid carriers, the effects of different carriers on their degradation rate were studied, and the results are shown in Table 9. Experiments have shown that straw is more conducive to microbial degradation of petroleum hydrocarbons when the addition amount is the same;
  • the composite bacterial agent in embodiment 1 is better than composite bacterial agent 1 and composite bacterial agent 2 to the degradation effect of petroleum hydrocarbon, further proves that live Acinetobacter KJ-1, Providencia rettgeri L1, Bacillus SWH- 1 and the synergy between Sphingobacterium SWH-2;

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  • Engineering & Computer Science (AREA)
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  • General Health & Medical Sciences (AREA)
  • Hydrology & Water Resources (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Medicinal Chemistry (AREA)
  • Water Supply & Treatment (AREA)
  • Soil Sciences (AREA)
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  • General Engineering & Computer Science (AREA)
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Abstract

L'invention concerne un agent microbien composite pour la dégradation d'hydrocarbures pétroliers, son procédé de préparation et son utilisation. L'agent microbien composite est composé d'Acinetobacter KJ-1 vivants et possédant le numéro de dépôt CGMCC n° 20664, de Providencia L1 possédant le numéro de dépôt CGMCC n° 16526, de Bacillus SWH-1 possédant le numéro de dépôt GMCC n° 1950, et de Sphingobacterium SWH-2 possédant le numéro de dépôt CGMCC n° 1951. L'agent microbien composite préparé pour la dégradation des hydrocarbures pétroliers peut dégrader efficacement les substances d'hydrocarbures pétroliers dans le sol et les boues de pétrole, peut être appliqué efficacement au traitement de biodégradation du sol contaminé par le pétrole, et n'induit pas de pollution secondaire sur l'environnement écologique du sol. En utilisant pleinement l'effet synergique des Acinetobacter KJ-1, Providencia L1, Bacillus SWH-1 et Sphingobacterium SWH-2 vivants, l'efficacité finale de la dégradation des hydrocarbures pétroliers est améliorée. L'agent microbien solide possède un processus de préparation simple, facile à mettre en œuvre et peu coûteux. Il présente de larges perspectives d'application dans le domaine de la biorémédiation des sols contaminés par le pétrole et des sites d'accidents dus à des fuites de produits pétroliers.
PCT/CN2021/119762 2021-09-17 2021-09-23 Agent microbien composite pour la dégradation d'hydrocarbures pétroliers, son procédé de préparation et son utilisation WO2023039921A1 (fr)

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CN202111090786.7A CN114644994A (zh) 2021-09-17 2021-09-17 一种石油烃降解复合微生物菌剂及其制备方法和应用
CN202111090786.7 2021-09-17

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Publication number Priority date Publication date Assignee Title
CN116769642A (zh) * 2023-05-04 2023-09-19 大连理工大学 一种石油降解微生物复合菌剂、复合菌剂培养基、制备方法和使用方法及用途

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