A dark fermentation mixed bacterial population and its culture method and application

Abstract

The application belongs to the technical field of biological fermentation, and particularly relates to a dark fermentation mixed flora, a culture method thereof and application of the dark fermentation mixed flora in hydrogen production from waste oil reservoirs. In the application, a mixed flora capable of producing hydrogen by using waste oil reservoirs is disclosed, the mixed flora comprising Thermotoga hypogea, Thermotoga elfii, Thermus sp., Propionibacterium freudenreichii, Butyrivibrio fibrisolvens and Pseudomonas aeruginosa. The mixed flora can not only produce hydrogen by using crude oil as raw material, but also improve the flowability of the oil layer, improve the interface between the oil layer and the water layer, and crack heavy components in the crude oil, so that the oil layer can be fully utilized for fermentation hydrogen production, and the efficiency of hydrogen production by microorganisms using waste oil reservoirs is improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological fermentation, and particularly relates to a dark fermentation mixed flora, a culture method thereof, and application of the dark fermentation mixed flora in hydrogen production from abandoned oil reservoirs. BACKGROUND

[0002] The existing oil well exploitation process is generally divided into two stages. In the first stage, the pressure of the formation itself can be used to make the oil gush up. In the second stage, the water injection method is generally used to supplement the pressure of the formation, so that the oil can be lifted up by external force. After that, due to the increase in technical difficulty of lifting the oil to the bottom surface and the increase in exploitation cost, a large amount of unexploited oil remains in the deep formation, and the exploitation has to be abandoned to become an abandoned oil reservoir. At present, various ways to improve oil recovery have been proposed, such as polymer flooding, chemical flooding, thermal oil recovery, and microbial oil displacement.

[0003] Another way to utilize abandoned oil reservoirs is to use microorganisms to produce hydrogen from crude oil. After the second exploitation of the abandoned oil reservoir, the upper layer of the oil well includes the injected water layer and the rich oil layer below the water layer. After the microorganisms are injected into the water layer, the microorganisms can contact the oil layer and metabolize the hydrocarbon compounds to produce hydrogen. By subsequent separation and purification of the mixed gas of the oil well, hydrogen with high purity can be obtained. However, the hydrogen production efficiency of the microorganisms that can produce hydrogen from crude oil is relatively low, and the research on the microbial flora for producing hydrogen from crude oil is also relatively less. Therefore, there is an urgent need for a mixed flora that can improve the hydrogen production efficiency of the microorganisms for producing hydrogen from abandoned oil reservoirs by fermentation using crude oil as raw material. SUMMARY

[0004] To solve the above problems, a dark fermentation mixed flora for hydrogen production from abandoned oil reservoirs and a culture method thereof are provided. The dark fermentation mixed flora can not only produce hydrogen from crude oil, but also improve the flowability of the oil layer, improve the interface between the oil layer and the water layer, and crack the heavy components in the crude oil, so that the oil layer can be fully utilized for fermentation to produce hydrogen, and the hydrogen production efficiency of the microorganisms for producing hydrogen from abandoned oil reservoirs can be improved.

[0005] According to one aspect of the present application, a dark fermentation mixed flora for hydrogen production from abandoned oil reservoirs is provided, which comprises: Thermotoga hypogea, Thermotoga elfii, Thermus sp., Propionibacterium freudenreichii, Butyrivibrio fibrisolvens, and Pseudomonas aeruginosa.

[0006] Optionally, the mixed bacterial flora comprises: Thermotoga hypogea DSM-11164, Thermotoga elfii DSM-9442, Thermus sp. ATCC-27737, Propionibacterium freudenreichii CICC-10019, Butyrivibrio fibrisolvens DSM-3071 and Pseudomonas aeruginosa CICC-56144.

[0007] Optionally, the mixed bacterial flora is obtained by mixing the individual bacterial species after being separately cultured to the logarithmic growth phase.

[0008] Optionally, the inoculation amount of Thermotoga hypogea DSM-11164 is 5-15%, the inoculation amount of Thermotoga elfii DSM-9442 is 10-20%, and the inoculation amount of Thermus sp. ATCC-27737, Propionibacterium freudenreichii CICC-10019, Butyrivibrio fibrisolvens DSM-3071 and Pseudomonas aeruginosa CICC-56144 is 2-5%.

[0009] Optionally, the ratio of the inoculation amount of Thermotoga hypogea DSM-11164 to that of Thermotoga elfii DSM-9442 is 1:(2-3).

[0010] Optionally, the ratio of the inoculation amount of Thermotoga hypogea DSM-11164 to that of Thermotoga elfii DSM-9442 is 1:3.

[0011] Optionally, the ratio of the inoculation amount of Thermotoga hypogea DSM-11164 to that of Pseudomonas aeruginosa CICC-56144 is 1:0.8.

[0012] According to another aspect of the present application, a culture method of the mixed bacteria group for hydrogen production by dark fermentation of waste oil reservoir is provided, wherein each bacteria is separately cultured in dark to logarithmic growth phase, and then inoculated into mixed culture medium to obtain the mixed bacteria group for hydrogen production by dark fermentation of waste oil reservoir.

[0013] Optionally, in the separate dark culture, the culture temperature of Thermotoga hypogea, Thermotoga elfii and Thermus sp. is 70-75℃, and the culture temperature of other bacteria is 28-30℃, and the rotation speed is 100-300rpm, and the culture time is 2-4 days; in the mixed dark culture, the culture temperature is 28-30℃, the rotation speed is 100-300rpm, and the culture time is 2-4 days.

[0014] Optionally, in the separate dark culture, the culture medium of Thermotoga hypogea, Thermotoga elfii and Thermus sp. is TGY (Tryptone Glucose Yeast) medium; the culture medium of other bacteria in the separate dark culture and the mixed dark culture is TSB (Tryptic Soy Broth) medium.

[0015] According to another aspect of the present application, a mixed bacteria group for hydrogen production by dark fermentation of waste oil reservoir is provided.

[0016] The beneficial effects of the present application include but are not limited to:

[0017] 1. According to the mixed bacteria group for dark fermentation of the present application, Thermotoga hypogea and Thermotoga elfii can utilize crude oil for fermentation to produce hydrogen, and the mixed bacteria group formed by further culturing with Thermus sp., Propionibacterium freudenreichii, Butyrivibrio fibrisolvens and Pseudomonas aeruginosa can greatly improve the efficiency of producing hydrogen from crude oil compared with ordinary single bacteria or other mixed bacteria groups, and the culture and preparation process is simple and fast, which is very suitable for industrial production.

[0018] 2. The dark fermentation mixed bacteria colony according to the present application, wherein thermophilic bacteria and Pseudomonas aeruginosa are added, wherein the Pseudomonas aeruginosa can crack heavy components in crude oil to form light components, and the light components can be more easily contacted by hydrogen-producing bacteria in an experimental environment; in an actual production environment, the heavy crude oil layer is at the lower layer, and the water layer containing the bacteria colony is at the upper layer, the light components produced by cracking have low density, can react with the alkyl sulfate surfactant produced by the thermophilic bacteria, and can optimize the interface between the crude oil layer and the water layer by dispersing and emulsifying the crude oil layer, so that the light components produced by cracking can be more easily emulsified into the water layer, thereby shortening the contact path of the hydrogen-producing bacteria and the crude oil layer, promoting the contact effect of the hydrogen-producing bacteria and the crude oil layer, and greatly improving the hydrogen production efficiency of the mixed bacteria colony.

[0019] 3. The dark fermentation mixed bacteria colony according to the present application, further comprising Propionibacterium freudenreichii and Butyribacterium, which can fully utilize the organic acid metabolites in the water layer for fermentation and produce hydrogen, so that the mixed bacteria colony can not only utilize crude oil for hydrogen production, but also fully utilize the nutrients in the water layer for hydrogen production, thereby improving the hydrogen production efficiency, wherein the Propionibacterium freudenreichii can utilize the acid metabolites in the water layer to produce hydrogen and carbon dioxide, thereby improving the acidic environment of the water layer and providing more suitable growth conditions for other bacteria, thereby improving the hydrogen production efficiency of the mixed bacteria colony by improving the activity of each bacteria in the mixed bacteria colony.

[0020] 4. The dark fermentation mixed bacteria colony according to the present application, wherein the Propionibacterium freudenreichii can secrete various nutrients, which is beneficial to the growth of other bacteria in the mixed bacteria colony, and can also secrete various enzymes into the environment, thereby improving the metabolic level and growth rate of other bacteria in the bacteria colony, especially the extreme thermophiles and the E. thermophila, thereby enabling the two bacteria mainly responsible for hydrogen production from crude oil in the mixed bacteria colony to work at a higher activity, and through the synergistic cooperation of each bacteria in the mixed bacteria colony, the overall hydrogen production capacity of the mixed bacteria colony can be significantly improved.

[0021] 5. The dark fermentation mixed bacteria according to the present application, the Thermatogae and the Thermatogae can utilize the crude oil to produce hydrogen, wherein the Thermatogae has a stronger specificity and a higher efficiency of producing hydrogen by utilizing the light component alkane, and the Thermatogae can utilize various hydrocarbon components in the crude oil to produce hydrogen, but the efficiency of producing hydrogen is relatively low. Since the contact effect between the crude oil layer and the water layer is poor, the use of the two bacteria alone or the low or high proportion of the two bacteria will reduce the efficiency of producing hydrogen. When the inoculation amount of the Thermatogae is too large, the light component in the crude oil is limited and the contact effect between the oil layer and the water layer is poor, so that the mixed bacteria have a high hydrogen production rate in the early stage, but cannot fully utilize the crude oil substrate in the later stage. When the inoculation amount of the Thermatogae is too large, although the crude oil substrate can be fully utilized to produce hydrogen, the efficiency of producing hydrogen is relatively low. When the two bacteria are used in a specific proportion, the advantages of the two bacteria can be fully utilized, the crude oil substrate can be fully utilized, and the efficiency of producing hydrogen can be maximized.

[0022] 6. The dark fermentation mixed bacteria according to the present application, the Pseudomonas aeruginosa can crack the heavy component in the crude oil into the light component, and the Thermatogae can utilize the light component as the substrate to produce hydrogen fermentation, so that when there is a sufficient amount of Pseudomonas aeruginosa in the mixed bacteria, the hydrogen production capacity of the mixed bacteria can be promoted. However, if the inoculation amount ratio of the Thermatogae to the Pseudomonas aeruginosa is too low, that is, the content of the Pseudomonas aeruginosa in the mixed bacteria is too high, the number and activity of the Thermatogae and the Thermatogae will be affected. When the Pseudomonas aeruginosa and the Thermatogae are inoculated in a suitable inoculation amount ratio, the Pseudomonas aeruginosa will not affect the growth and hydrogen production of the Thermatogae and the Thermatogae due to too much, and can crack to produce light components that can meet the needs of the Thermatogae, thereby improving the overall hydrogen production efficiency of the mixed bacteria. DETAILED DESCRIPTION

[0023] The present application will be described in detail below with reference to the examples, but the present application is not limited to these examples.

[0024] In the examples of the present application, the raw materials and reagents are purchased through commercial channels unless otherwise specified.

[0025] Example 1

[0026] (1) Single dark culture

[0027] Thermotoga hypogea DSM-11164, Thermotoga elfii DSM-9442 and Thermus sp. ATCC-27737 activated seed liquid were inoculated in three groups of different TGY medium for single dark culture, culture conditions: temperature 73°C, rotation speed 100-300 rpm, culture time 3 days; Propionibacterium freudenreichii CICC-10019, Butyrivibrio fibrisolvens DSM-3071 and Pseudomonas aeruginosa CICC-56144 activated seed liquid were inoculated in three groups of different TSB medium for single dark culture, culture conditions: temperature 29°C, rotation speed 100-300 rpm, culture time 3 days.

[0028] (2) Mixed dark culture

[0029] The strains of each single dark culture in step (1) were inoculated in TSB medium for mixed dark culture, culture conditions: temperature 29°C, rotation speed 100-300 rpm, culture time 3 days, to obtain the dark fermentation mixed bacteria group. The inoculation amount of each strain was: Thermotoga hypogea DSM-11164 5%, Thermotoga elfii DSM-9442 15%, Thermus sp. ATCC-27737, Propionibacterium freudenreichii CICC-10019, Butyrivibrio fibrisolvens DSM-3071 and Pseudomonas aeruginosa CICC-56144 4%.

[0030] Example 2

[0031] (1) Single dark culture

[0032] Thermotoga hypogea DSM-11164, Thermotoga elfii DSM-9442 and Thermus sp. ATCC-27737 activated seed liquid were inoculated in three groups of different TGY medium for single dark culture, culture conditions: temperature 70℃, rotation speed 100-300 rpm, culture time 4 days; Propionibacterium freudenreichii CICC-10019, Butyrivibrio fibrisolvens DSM-3071 and Pseudomonas aeruginosa CICC-56144 activated seed liquid were inoculated in three groups of different TSB medium for single dark culture, culture conditions: temperature 28℃, rotation speed 100-300 rpm, culture time 4 days.

[0033] (2) Mixed dark culture

[0034] The strains of each single dark culture in step (1) were inoculated in TSB medium for mixed dark culture, culture conditions: temperature 28℃, rotation speed 100-300 rpm, culture time 4 days, to obtain the dark fermentation mixed bacteria group. The inoculation amount of each strain was: Thermotoga hypogea DSM-11164 was 10%, the inoculation amount of Thermotoga elfii DSM-9442 was 10%, the inoculation amount of Thermus sp. ATCC-27737, Propionibacterium freudenreichii CICC-10019, Butyrivibrio fibrisolvens DSM-3071 and Pseudomonas aeruginosa CICC-56144 was 5%.

[0035] Example 3

[0036] (1) Single dark culture

[0037] Thermotoga hypogea DSM-11164, Thermotoga elfii DSM-9442 and Thermus sp. ATCC-27737 activated seed liquid were inoculated in three groups of different TGY medium for single dark culture, culture conditions: temperature 75°C, rotation speed 100-300 rpm, culture time 2 days; Propionibacterium freudenreichii CICC-10019, Butyrivibrio fibrisolvens DSM-3071 and Pseudomonas aeruginosa CICC-56144 activated seed liquid were inoculated in three groups of different TSB medium for single dark culture, culture conditions: temperature 30°C, rotation speed 100-300 rpm, culture time 2 days.

[0038] (2) Mixed dark culture

[0039] The strains of each single dark culture in step (1) were inoculated in TSB medium for mixed dark culture, culture conditions: temperature 30°C, rotation speed 100-300 rpm, culture time 2 days, to obtain the dark fermentation mixed bacteria group. The inoculation amount of each strain was: Thermotoga hypogea DSM-11164 15%, Thermotoga elfii DSM-9442 20%, Thermus sp. ATCC-27737, Propionibacterium freudenreichii CICC-10019, Butyrivibrio fibrisolvens DSM-3071 and Pseudomonas aeruginosa CICC-56144 2%.

[0040] Example 4

[0041] (1) Single dark culture

[0042] The same as example 1.

[0043] (2) Mixed dark culture

[0044] The strains in each single dark culture in step (1) were inoculated into TSB medium for mixed dark culture, and the culture conditions were as follows: temperature 29°C, rotation speed 100-300 rpm, culture time 3 days, to obtain the mixed strain group for dark fermentation. The inoculation amount of each strain was as follows: 4% for Thermotoga hypogea DSM-11164, 16% for Thermotoga elfii DSM-9442, and 4% for each of Thermus sp. ATCC-27737, Propionibacterium freudenreichii CICC-10019, Butyrivibrio fibrisolvens DSM-3071, and Pseudomonas aeruginosa CICC-56144.

[0045] Example 5

[0046] (1) Single dark culture

[0047] The same as in Example 1.

[0048] (2) Mixed dark culture

[0049] The strains in each single dark culture in step (1) were inoculated into TSB medium for mixed dark culture, and the culture conditions were as follows: temperature 29°C, rotation speed 100-300 rpm, culture time 3 days, to obtain the mixed strain group for dark fermentation. The inoculation amount of each strain was as follows: 4% for Thermotoga hypogea DSM-11164, 16% for Thermotoga elfii DSM-9442, and 4% for each of Thermus sp. ATCC-27737, Propionibacterium freudenreichii CICC-10019, Butyrivibrio fibrisolvens DSM-3071, and Pseudomonas aeruginosa CICC-56144.

[0050] Example 6

[0051] (1) Single dark culture

[0052] The same as in Example 1.

[0053] (2) Mixed dark culture

[0054] The strains in each single dark culture in step (1) were inoculated into TSB medium for mixed dark culture, and the culture conditions were as follows: temperature 29°C, rotation speed 100-300 rpm, culture time 3 days, to obtain the mixed strain group for dark fermentation. The inoculation amount of each strain was as follows: 10% for Thermotoga hypogea DSM-11164, 10% for Thermotoga elfii DSM-9442, 4% for each of Thermus sp. ATCC-27737, Propionibacterium freudenreichii CICC-10019, Butyrivibrio fibrisolvens DSM-3071, and Pseudomonas aeruginosa CICC-56144.

[0055] Example 7

[0056] (1) Single dark culture

[0057] The same as in Example 1.

[0058] (2) Mixed dark culture

[0059] The strains in each single dark culture in step (1) were inoculated into TSB medium for mixed dark culture, and the culture conditions were as follows: temperature 29°C, rotation speed 100-300 rpm, culture time 3 days, to obtain the mixed strain group for dark fermentation. The inoculation amount of each strain was as follows: 10% for Thermotoga hypogea DSM-11164, 10% for Thermotoga elfii DSM-9442, 4% for each of Thermus sp. ATCC-27737, Propionibacterium freudenreichii CICC-10019, Butyrivibrio fibrisolvens DSM-3071, and Pseudomonas aeruginosa CICC-56144.

[0060] Example 8

[0061] (1) Single dark culture

[0062] The same as in Example 1.

[0063] (2) Mixed dark culture

[0064] The individual strains of each single dark culture in step (1) were inoculated into TSB culture medium for mixed dark culture, and the culture conditions were as follows: temperature 29°C, rotation speed 100-300 rpm, culture time 3 days, to obtain the dark fermentation mixed bacteria group. The inoculation amount of each strain was as follows: 5% of Thermotoga hypogea DSM-11164, 15% of Thermotoga elfii DSM-9442, 4% of Thermus sp. ATCC-27737, 4% of Propionibacterium freudenreichii CICC-10019, 4% of Butyrivibrio fibrisolvens DSM-3071, and 5% of Pseudomonas aeruginosa CICC-56144.

[0065] Comparative Example 1

[0066] (1) Single dark culture

[0067] Thermotoga hypogea DSM-11164 and Thermotoga elfii DSM-9442 activated seed solutions were inoculated into two groups of different TGY culture media for single dark culture, and the culture conditions were as follows: temperature 73°C, rotation speed 100-300 rpm, and culture time 3 days.

[0068] (2) Mixed dark culture

[0069] The two individual dark cultures in step (1) were inoculated into TSB culture medium for mixed dark culture, and the culture conditions were as follows: temperature 29°C, rotation speed 100-300 rpm, culture time 3 days, to obtain the dark fermentation mixed bacteria group. The inoculation amount of each strain was as follows: 5% of Thermotoga hypogea DSM-11164, and 15% of Thermotoga elfii DSM-9442.

[0070] Comparative Example 2

[0071] (1) Single dark culture

[0072] Thermotoga hypogea DSM-11164, Thermotoga elfii DSM-9442 and Thermus sp. ATCC-27737 activated seed liquid were inoculated in three groups of different TGY medium for single dark culture, the culture conditions were: temperature 73℃, rotation speed 100-300 rpm, culture time 3 days; Propionibacterium freudenreichii CICC-10019 and Butyrivibrio fibrisolvens DSM-3071 activated seed liquid were inoculated in two groups of different TSB medium for single dark culture, the culture conditions were: temperature 29℃, rotation speed 100-300 rpm, culture time 3 days.

[0073] (2) Mixed dark culture

[0074] The strains of each single dark culture in step (1) were inoculated in TSB medium for mixed dark culture, the culture conditions were: temperature 29℃, rotation speed 100-300 rpm, culture time 3 days, and the dark fermentation mixed bacterial community was obtained. The inoculation amount of each strain was: Thermotoga hypogea DSM-11164 was 5%, the inoculation amount of Thermotoga elfii DSM-9442 was 15%, and the inoculation amount of Thermus sp. ATCC-27737, Propionibacterium freudenreichii CICC-10019 and Butyrivibrio fibrisolvens DSM-3071 was 4%.

[0075] Comparative Example 3

[0076] (1) Single dark culture

[0077] Thermotoga hypogea DSM-11164 and Thermotoga elfii DSM-9442 activated seed liquid were inoculated in two groups of different TGY culture medium for separate dark culture, the culture conditions were: temperature 73℃, rotation speed 100-300 rpm, culture time 3 days; Propionibacterium freudenreichii CICC-10019, Butyrivibrio fibrisolvens DSM-3071 and Pseudomonas aeruginosa CICC-56144 activated seed liquid were inoculated in three groups of different TSB culture medium for separate dark culture, the culture conditions were: temperature 29℃, rotation speed 100-300 rpm, culture time 3 days.

[0078] (2) Mixed dark culture

[0079] The strains of each separate dark culture in step (1) were inoculated in TSB culture medium for mixed dark culture, the culture conditions were: temperature 29℃, rotation speed 100-300 rpm, culture time 3 days, and the dark fermentation mixed bacterial community was obtained. The inoculation amount of each strain was: Thermotoga hypogea DSM-11164 was 5%, the inoculation amount of Thermotoga elfii DSM-9442 was 15%, and the inoculation amount of Propionibacterium freudenreichii CICC-10019, Butyrivibrio fibrisolvens DSM-3071 and Pseudomonas aeruginosa CICC-56144 was 4%.

[0080] Comparative Example 4

[0081] (1) Separate dark culture

[0082] Thermotoga hypogea DSM-11164 and Thermotoga elfii DSM-9442 activated seed liquid were inoculated in two groups of different TGY culture medium for separate dark culture, the culture condition was: temperature 73℃, rotation speed 100-300 rpm, culture time 3 days; Propionibacterium freudenreichii CICC-10019 and Butyrivibrio fibrisolvens DSM-3071 activated seed liquid were inoculated in two groups of different TSB culture medium for separate dark culture, the culture condition was: temperature 29℃, rotation speed 100-300 rpm, culture time 3 days.

[0083] (2) Mixed dark culture

[0084] The strains of each separate dark culture in step (1) were inoculated in TSB culture medium for mixed dark culture, the culture condition was: temperature 29℃, rotation speed 100-300 rpm, culture time 3 days, to obtain the dark fermentation mixed bacteria group. The inoculation amount of each strain was: Thermotoga hypogea DSM-11164 was 5%, the inoculation amount of Thermotoga elfii DSM-9442 was 15%, and the inoculation amount of Propionibacterium freudenreichii CICC-10019 and Butyrivibrio fibrisolvens DSM-3071 was 4%.

[0085] Comparative Example 5

[0086] (1) Separate dark culture

[0087] Thermotoga hypogea DSM-11164, Thermotoga elfii DSM-9442 and Thermus sp. ATCC-27737 activated seed liquid were inoculated in three groups of different TGY culture medium for separate dark culture, the culture condition was: temperature 73℃, rotation speed 100-300 rpm, culture time 3 days; Pseudomonas aeruginosa CICC-56144 activated seed liquid was inoculated in TSB culture medium for separate dark culture, the culture condition was: temperature 29℃, rotation speed 100-300 rpm, culture time 3 days.

[0088] (2) Mixed dark culture

[0089] The strains of each single dark culture in step (1) were inoculated into TSB medium for mixed dark culture, and the culture conditions were as follows: temperature 29°C, rotation speed 100-300 rpm, culture time 3 days, to obtain the mixed strain of dark fermentation. The inoculation amount of each strain was as follows: 5% of Thermotoga hypogea DSM-11164, 15% of Thermotoga elfii DSM-9442, 4% of Thermus sp. ATCC-27737 and Pseudomonas aeruginosa CICC-56144.

[0090] Comparative Example 6

[0091] (1) Single dark culture

[0092] The same as Example 1.

[0093] (2) Mixed dark culture

[0094] The strains of each single dark culture in step (1) were inoculated into TSB medium for mixed dark culture, and the culture conditions were as follows: temperature 29°C, rotation speed 100-300 rpm, culture time 3 days, to obtain the mixed strain of dark fermentation. The inoculation amount of each strain was as follows: 5% of Thermotoga hypogea DSM-11164, 15% of Thermotoga elfii DSM-9442, 4% of Thermus sp. ATCC-27737 and Pseudomonas aeruginosa CICC-56144.

[0095] Comparative Example 7

[0096] (1) Single dark culture

[0097] Thermotoga hypogea DSM-11164, Thermotoga elfii DSM-9442 and Thermus sp. ATCC-27737 were inoculated into three groups of different TGY medium respectively for single dark culture, and the culture conditions were as follows: temperature 73°C, rotation speed 100-300 rpm, and culture time 3 days; Propionibacterium freudenreichii CICC-10019, Butyrivibrio fibrisolvens DSM-3071 and Serratia marcescens ATCC-13880 were inoculated into three groups of different TSB medium respectively for single dark culture, and the culture conditions were as follows: temperature 29°C, rotation speed 100-300 rpm, and culture time 3 days.

[0098] (2) Mixed dark culture

[0099] The strains in each single dark culture in step (1) were inoculated into TSB medium for mixed dark culture, and the culture conditions were as follows: temperature 29°C, rotation speed 100-300 rpm, and culture time 3 days, to obtain the dark fermentation mixed bacteria group. The inoculation amount of each strain was as follows: 5% for Thermotoga hypogea DSM-11164, 15% for Thermotoga elfii DSM-9442, and 4% for Thermus sp. ATCC-27737, Desulfotomaculum nigrificans DSM-122, Butyrivibrio fibrisolvens DSM-3071 and Serratia marcescens ATCC-13880.

[0100] Experimental Example 1

[0101] The dark fermentation mixed bacteria groups obtained in Examples 1-8 and Comparative Examples 1-7 above were inoculated into test medium containing crude oil respectively for testing of hydrogen production, and Tween 80 was added as a surfactant in each test to improve the contact effect of the mixed bacteria group with the crude oil in the medium. The contents of hydrogen and carbon dioxide in the fermentation gas were tested, and the average hydrogen production rate was calculated, and the results are shown in Table 1 below.

[0102] The test medium comprises: NH4CI 1.0 g / L, K2HPO4 0.3 g / L, KH2PO4 0.3 g / L, MgCI2·6H2O 0.2 g / L, CaCI2·2H2O 0.1 g / L, KCI 0.1 g / L, sodium acetate 0.5 g / L, yeast extract 2.0 g / L, proteose peptone 2.0 g / L, resazurin 0.0002 g / L, L-cysteine hydrochloride 0.5 g / L, glucose 3.0 g / L, Na2S 0.5 g / L, NaHCO3 2.0 g / L, trace element solution 10 ml, and 20 ml / L crude oil, wherein the trace element solution comprises: MnCI2·4H2O 6.4 g / L, CoCI2·6H2O 0.387 g / L, CuCI2H2O 0.035 g / L, Na2MoO4·2H2O 0.033 g / L, ZnCI2 0.027 g / L, FeCI2 1.062 g / L.

[0103] Table 1

[0104]

[0105] From the results of Examples 1, 4, 5, and 6, it can be seen from Table 1 that the ratio of the inoculation amounts of the two bacteria, i.e., T. extremus and T. edaphicus, is a main factor affecting the number of the two bacteria in the mixed bacteria group. When the ratio of the inoculation amounts of T. extremus and T. edaphicus is 1:3, the hydrogen production efficiency of the mixed bacteria group is the highest. This is because the two bacteria have different characteristics. T. extremus can only utilize the lighter components in the crude oil but has a high hydrogen production efficiency, while T. edaphicus has a relatively wide applicability but a relatively low hydrogen production efficiency. When the two bacteria are used in the above-mentioned ratio of 1:3, the highest hydrogen production efficiency can be obtained. From the comparison of the results of Examples 1, 7, and 8, it can be seen that the relationship between the inoculation amounts of P. aeruginosa and T. extremus affects the hydrogen production amount of the mixed bacteria group. If the ratio of the inoculation amounts of T. extremus and P. aeruginosa is too small, the number of P. aeruginosa in the mixed bacteria group is too large, which leads to a decrease in the activity of T. extremus and T. edaphicus due to the competition. When the inoculation amount of P. aeruginosa is too small, the light components produced by the lysis of P. aeruginosa are insufficient to supply T. extremus for hydrogen production fermentation, which also leads to a decrease in the hydrogen production rate of the mixed bacteria group. When the ratio of the inoculation amounts of T. extremus and P. aeruginosa is 1:0.8, the number of P. aeruginosa in the mixed bacteria group is appropriate, which neither affects the growth and activity of T. extremus and T. edaphicus nor causes the light components produced by the lysis of P. aeruginosa to be insufficient to meet the needs of T. extremus, so that the hydrogen production efficiency is the highest.

[0106] From the results of Example 1 and Comparative Examples 1, 2, 3, 4, 5, it can be seen that the hydrogen production efficiency is very low when only extreme thermophiles and E. fumarium are used as mixed bacteria, because the mixed bacteria in Example 1 can achieve better results after being mixed, the combination of thermophilic bacteria and P. aeruginosa can effectively improve the interface between oil layer and water layer, thereby greatly improving the efficiency of hydrogen production from crude oil, and P. freudenreichii and V. butyricum can not only make full use of the carbohydrates in the water layer for hydrogen production, but also reduce the acidic substances in the water layer to produce carbon dioxide, and can secrete enzymes and growth factors to promote the growth and reproduction of other bacteria, so that the mixed bacteria can significantly improve the hydrogen production efficiency after being mixed. From the results of Example 1 and Comparative Example 5, it can be seen that although the carbon dioxide content in Comparative Example 5 is small, the hydrogen content is relatively high, but the mixed bacteria fermentation hydrogen production efficiency is very low due to the acidic conditions of the fermentation environment, and the organic matter in the water layer cannot be fully utilized for auxiliary hydrogen production, so the overall hydrogen production rate is very low. From the results of Example 1 and Comparative Example 7, it can be seen that two other industrial bacteria commonly used in oil exploitation are used in Comparative Example 7, especially Serratia marcescens, which can also improve the flowability of the oil layer, but the hydrogen production efficiency of the mixed bacteria is much lower than that of Example 1.

[0107] From the results of Example 1 and Comparative Examples 2, 3, 4, it can be seen that in Comparative Examples 2, 3 and 4, the contact ability of extreme thermophiles and E. fumarium with crude oil decreases, the hydrogen production decreases, and the use of P. freudenreichii and V. butyricum to produce hydrogen and carbon dioxide from the organic matter in the water layer has little effect, which not only makes the hydrogen production rate significantly low, but also makes the carbon dioxide content in the produced gas too large, increasing the difficulty of hydrogen separation and purification in the later stage. From the results of Example 1 and Comparative Example 6, it can be seen that the inoculation amount of P. freudenreichii and V. butyricum is increased in the example, which increases the amount of carbon dioxide produced during fermentation, resulting in a lower hydrogen content in the mixed gas, and the increase in the inoculation amount of P. freudenreichii and V. butyricum also affects the hydrogen production efficiency of extreme thermophiles and E. fumarium, reducing the overall hydrogen production efficiency of the mixed bacteria.

[0108] The above is only an embodiment of the present application, and the protection scope of the present application is not limited by these specific embodiments, but is determined by the claims of the present application. Various modifications and changes can be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement, etc. within the technical idea and principle of the present application shall be included in the protection scope of the present application.

Claims

1. An application of a mixed microbial community in hydrogen production from waste oil reservoirs, characterized in that, The mixed bacteria are composed of the following bacteria: Extreme heat bacterium ( Thermotoga hypogea DSM-11164, Thermophyton floccosum ( Thermotoga elfii DSM-9442, thermophilic bacteria ( Thermus sp. ATCC-27737, Propionibacterium fischeri ( Propionibacterium freudenreichii CICC-10019, Vibrio butyricum ( Butyrivibrio fibrisolvens DSM-3071 and Pseudomonas aeruginosa ( Pseudomonas aeruginosa CICC-56144; The mixed microbial community is obtained by mixing and culturing various microbial species that have been cultured individually to the logarithmic growth phase. The culture method includes first culturing each microbial species individually in the dark to the logarithmic growth phase, and then inoculating them into a mixed culture medium for mixed dark culture to obtain the dark fermentation mixed microbial community for hydrogen production from waste oil reservoirs. The extreme heat bacillus ( Thermotoga hypogea The inoculum size for DSM-11164 is 5-15%, and the inoculum size for *Thermophyton floccosum* is... Thermotoga elfii The inoculum size for DSM-9442 is 10-20%, and the thermophilic bacteria ( Thermus sp. ATCC-27737, Propionibacterium fischeri ( Propionibacterium freudenreichii CICC-10019, Vibrio butyricum ( Butyrivibrio fiber-dissolving DSM-3071 and Pseudomonas aeruginosa ( Pseudomonas aeruginosa The inoculation dose of CICC-56144 is 2-5%.

2. The application according to claim 1, characterized in that, The extreme heat bacillus ( Thermotoga hypogea DSM-11164 and Thermophyton floccosum ( Thermotoga elfii The ratio of DSM-9442 inoculation dose is 1:(2~3).

3. The application according to claim 1, characterized in that, The extreme heat bacillus ( Thermotoga hypogea DSM-11164 and Thermophyton floccosum ( Thermotoga elfii The ratio of DSM-9442 inoculation doses was 1:

3.

4. The application according to claim 1, characterized in that, The culture method includes: in separate dark culture, *Extreme Heat Bacterium* (… Thermotoga hypogea Thermophyton floccosum ( ), Thermotoga elfii ) and thermophilic bacteria ( Thermae sp. The culture temperature for the first strain is 70-75℃, while the culture temperature for other strains is 28-30℃. The rotation speed is 100-300 rpm, and the culture time is 2-4 days. In the mixed dark culture, the culture temperature is 28-30℃, the rotation speed is 100-300 rpm, and the culture time is 2-4 days.

5. The application according to claim 4, characterized in that, The extreme heat bacillus (in the isolated dark culture) Thermotoga hypogea Thermophyton floccosum ( ), Thermotoga elfii ) and thermophilic bacteria ( Thermus sp. The culture medium for the bacteria was TGY (Tryptone Glucose Yeast); the culture medium for other bacteria in individual dark culture and for mixed dark culture was TSB (Tryptic Soy Broth).

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