CN109456894B - Enrichment culture and rapid separation method for microorganisms with electricity generation and sulfur oxidation functions in water sediments - Google Patents
Enrichment culture and rapid separation method for microorganisms with electricity generation and sulfur oxidation functions in water sediments Download PDFInfo
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- CN109456894B CN109456894B CN201811197822.8A CN201811197822A CN109456894B CN 109456894 B CN109456894 B CN 109456894B CN 201811197822 A CN201811197822 A CN 201811197822A CN 109456894 B CN109456894 B CN 109456894B
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- 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
- C12N1/02—Separating microorganisms from their culture media
Abstract
The invention discloses a method for enriching, culturing and quickly separating microbes with an electricity generation and sulfur oxidation function in water sediments, which comprises the following steps: uniformly mixing and sieving water body sediments rich in organic matters, filling a beaker filled with ferrous sulfide with the sieved sediments, putting the beaker into a wide-mouth vessel filled with water, enabling the water level to be 5-20 cm higher than the beaker, and keeping the dissolved oxygen concentration of the overlying water body to be 6-7 mg/L and the ambient temperature to be 20-30 ℃; after 10-15 days of operation, collecting sediments with the surface layer of 1-6 cm; and (3) sieving the collected sediments in a wide-mouth vessel filled with filter sterilization water, and collecting small bacterium masses in a sieve to obtain the microorganism with the function of producing the electricity and the sulfur oxidation. The method has the advantages of simple operation, low cost, obvious enrichment culture and separation effects on the electrogenic sulfur oxidation functional microorganisms, and capability of effectively solving the problem of difficult culture of the electrogenic sulfur oxidation functional microorganisms.
Description
Technical Field
The invention relates to the technical field of environmental microbiology, element biological geochemical cycle and environmental bioengineering, in particular to a method for enriching, culturing and quickly separating microorganisms with an electricity generation and sulfur oxidation function in water sediments.
Background
With the development of economic society and the acceleration of social industrialization process, pollutants such as organic matters, sulfate, heavy metals and the like enter a water body through runoff and sedimentation and are accumulated in a large amount in sediments. Because polluted water sediments usually have lower oxidation-reduction potential, sulfate is easily reduced into sulfide by organic matters under the action of sulfate reducing bacteria, and the sulfide is further combined with heavy metals in the sediments to form black metal sulfide. This is also the main reason for the black and odorous phenomenon of the sediment in the water body.
In recent years, with the development and application of microbial physiological ecology and electrochemical analysis detection means, more and more research evidences indicate that Cable bacteria (Cable bacteria) exist in water body sediments. The long-line electroactive microorganisms mediate a direct-current electric field for electron transfer from bottom to top. This process is also known as Electrogenic sulfur oxidation (e-SOx). The process of the produced electricity sulfur oxidation mediated by long linear electroactive microorganisms such as cable bacteria is a key link of the electron transfer between the sediment and an overlying water interface, couples the reduction process of electron donor substances (organic matters, sulfides and the like) in the sediment and an overlying electron acceptor (oxygen and nitrate), and plays an important role in the in-situ repair of the sediment of the black and odorous water body. However, at present, there is still no method for pure culture of long linear electroactive microorganisms for mediating electrogenesis sulfur oxidation in water body sediments, and little understanding is available about functional activity characteristics of cable bacteria and the like in water body sediments for mediating electrogenesis sulfur oxidation. Therefore, researching and establishing the enrichment culture and rapid separation method of the long linear electroactive microorganisms for mediating the electrogenesis sulfur oxidation process in the water body sediments is an important prerequisite for revealing the active characteristics of the long linear electroactive microorganisms for mediating the electrogenesis sulfur oxidation function and accelerating the in-situ repair of the black and odorous water body sediments.
Disclosure of Invention
The invention aims to provide a method for enriching, culturing and quickly separating the electrogenesis sulfur oxidation function microorganisms in water sediments, which has the advantages of simple operation, low cost and obvious effects of enriching, culturing and separating the electrogenesis sulfur oxidation function microorganisms and can effectively solve the problem of difficult culture of the electrogenesis sulfur oxidation function microorganisms.
The invention is realized by the following technical scheme:
a method for enriching, culturing and quickly separating microbes with an electricity generation and sulfur oxidation function in water sediments comprises the following steps:
(1) putting ferrous sulfide into a cup, filling water body sediments rich in organic matters into the ferrous sulfide in the cup, putting the cup into a wide-mouth vessel filled with water, enabling the water level to be higher than the opening of the cup, carrying out aeration culture in the wide-mouth vessel, and collecting sediments on the surface layer in the cup, namely sediments enriched in microorganisms with the electricity generation and sulfur oxidation functions;
(2) in the sterile water, the sediment of the microorganism which is enriched to have the function of producing the oxysulfide of electricity is screened, and the small bacterial colony in the screen is collected.
Preferably, the sieve is a sieve with a diameter of 0.1-0.5 mm.
Preferably, the method for enriching, culturing and rapidly separating the microorganisms with the function of generating the oxysulfide of the electricity generated in the sediments of the water body comprises the following specific steps:
(1) uniformly mixing water body sediments rich in organic matters, sieving the water body sediments with a sieve with the aperture of 0.1-0.5 mm, and filling the sediments with the particle size of less than 0.1-0.5 mm after sieving into a cup containing ferrous sulfide;
(2) putting the cup in the step (1) into a wide-mouth vessel filled with water, enabling the water level to be 5-20 cm higher than the opening of the beaker, aerating the wide-mouth vessel by adopting an inflator pump, keeping the dissolved oxygen concentration of the overlying water body at 6-7 mg/L and the ambient temperature at 20-30 ℃, and collecting sediments with the surface layer of 1-6 cm after continuously running for 10-15 days;
(3) and (3) in a vessel containing filter sterilization water, taking 1-6 cm sediment on the surface layer obtained in the step (2), passing through a sieve with the pore diameter of 0.1-0.5 mm, and collecting small bacterial colonies in the sieve to obtain the electricity generation sulfur oxidation functional microorganisms.
Preferably, the ferrous sulfide in the step (1) accounts for 1/3-2/3 of the volume of the cup.
Preferably, the aperture of the sieve is 0.35mm, the dissolved oxygen concentration of the overlying water body is 7mg/L, the ambient temperature is 24 +/-1 ℃, the running time is 15 days, and the sediment with the 1-6 cm collecting surface layer is the sediment with the 2cm collecting surface layer.
Preferably, the water contained in the wide-mouth vessel in the step (2) is raw water of a water body system for digging water body sediments, artificially prepared simulated raw water or deionized water.
More preferably, the water contained in the wide-mouth vessel in the step (2) is raw water of a water body system for digging out water body sediments.
Preferably, the filter sterilization water in the step (3) is obtained by filtering raw water of a water system for digging water sediments, artificially prepared simulated raw water or deionized water with a 0.22 μm bacterial filter membrane.
More preferably, the filter sterilized water in the step (3) is obtained by filtering raw water of a water system for digging out water body sediments by using a 0.22 μm bacterial filter membrane.
Compared with the prior art, the invention has the advantages that:
the method has the advantages of simple operation, low cost and obvious enrichment culture and separation effect on the microorganisms with the electricity generation and sulfur oxidation functions, can effectively solve the problem of difficult culture of the microorganisms with the electricity generation and sulfur oxidation functions, and provides important method means and experimental materials for deeply researching the electricity generation and sulfur oxidation functions of the microorganisms and establishing the in-situ repair technology of the black and odorous water body sediments based on the in-situ regulation and control of the functional microorganisms.
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1 enrichment culture and isolation of Electricity-producing Sulfur Oxidation functional microorganisms in Black-odor river sediment from Foshan City of Guangdong province
Taking a black-odor river sediment with 5300.08-6129.32 mg/kg of sulfide content in Fushan City in Guangdong province, uniformly mixing, sieving by a sieve with the aperture of 0.35mm, and filling a beaker filled with ferrous sulfide accounting for 1/3 volume of the sediment with the particle diameter smaller than 0.35mm after sieving. Putting the beaker into a rectangular plastic box filled with the original river gushing water, and enabling the water level to be 10cm higher than the opening of the beaker; aerating into the wide-mouth vessel by adopting an aeration (oxygen) pump to ensure that the dissolved oxygen concentration of the overlying water body is kept at 7mg/L, the environmental temperature is 24 +/-1 ℃, and measuring the dissolved oxygen, pH, ORP and hydrogen sulfide concentrations of the sediments at different depths by adopting a microelectrode every day; after 15 days of continuous operation, the sulphide content of the deposit was less than 50 mg/kg. In a rectangular plastic box filled with the river gushing water filtered and sterilized by a bacterial filter membrane of 0.22 mu m, 2cm of sediment on the surface layer in the beaker is taken to pass through a sieve with the aperture of 0.35mm, and the small bacterial clusters in the sieve are collected. The weight of the collected small colonies is about 12000 times of the weight of the collected small colonies from the original sediment sample (black-smelling river sediment) which is not subjected to enrichment culture.
Example 2 enrichment culture and isolation of microorganisms capable of producing oxysulfide Oxidation by Electricity in lake sediment eutrophicated in Guangzhou, Guangdong province
Eutrophic lake sediment with the sulfide content of 1203.02-2129.38 mg/kg in Guangzhou city, Guangdong province is taken, evenly mixed and sieved by a sieve with the aperture of 0.50mm, and the sediment with the particle diameter smaller than 0.50mm is filled in a beaker filled with ferrous sulfide accounting for 1/2 volume. Putting the beaker into a rectangular glass jar filled with the original lake water, and enabling the water level to be 15cm higher than the opening of the beaker; aerating into the wide-mouth vessel by adopting an aeration (oxygen) pump to keep the dissolved oxygen concentration of the overlying water body at about 6mg/L, keeping the environmental temperature at 25 +/-1 ℃, and measuring the dissolved oxygen, pH, ORP and hydrogen sulfide concentrations of the sediments at different depths by adopting a microelectrode every day; after 15 days of continuous operation, the sulphide content of the deposit was less than 30 mg/kg. In a rectangular glass jar filled with 0.22 μm bacterial filter membrane for filtration and sterilization, 3cm of sediment on the surface layer in the beaker is taken to pass through a sieve with the aperture of 0.50mm, and the small bacterial clusters in the sieve are collected. The weight of the collected small bacterial colony is about 6800 times of that of the small bacterial colony collected from the original sediment sample (eutrophic lake sediment) which is not subjected to enrichment culture and has the same weight.
Example 3 enrichment culture and isolation of Electricity-producing Sulfur Oxidation functional microorganisms in sediments of certain reservoir of Whitman, Guangdong province
Taking reservoir sediments with the sulfide content of 201.08-539.05 mg/kg in Guangzhou city, Guangdong province, uniformly mixing, sieving by a sieve with the aperture of 0.1mm, and filling a beaker filled with ferrous sulfide accounting for 2/3 volume with the sediments with the particle diameter smaller than 0.1mm after sieving. Putting the beaker into a rectangular glass jar filled with raw reservoir water, and enabling the water level to be 20cm higher than the opening of the beaker; aerating the wide-mouth vessel by adopting an aeration (oxygen) pump to keep the dissolved oxygen concentration of the overlying water body at 7mg/L, keeping the environmental temperature at 28 +/-1 ℃, and measuring the dissolved oxygen, pH, ORP and hydrogen sulfide concentrations of the sediments at different depths by adopting a microelectrode every day; after 10 days of continuous operation, the sulphide content in the deposit was less than 20 mg/kg. In a rectangular glass jar filled with raw reservoir water filtered and sterilized by a 0.22 mu m bacterial filter membrane, 2cm of sediment on the surface layer in a beaker is taken to pass through a sieve with the aperture of 0.1mm, and small bacterial colonies in the sieve are collected. The weight of the collected small clumps was about 9800 times that of the small clumps collected from the same weight of the original sediment sample (reservoir sediment) without enrichment culture.
The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.
Claims (5)
1. A method for enriching, culturing and quickly separating microorganisms with an electricity generation and sulfur oxidation function in water sediments is characterized by comprising the following steps:
(1) uniformly mixing water body sediments rich in organic matters, sieving the water body sediments with a sieve with the aperture of 0.1-0.5 mm, and filling the sediments with the particle size of less than 0.1-0.5 mm after sieving into a cup containing ferrous sulfide;
(2) putting the cup in the step (1) into a wide-mouth vessel filled with water, enabling the water level to be 10-20 cm higher than the opening of the beaker, aerating the wide-mouth vessel by adopting an inflator pump, keeping the dissolved oxygen concentration of the overlying water body at 6-7 mg/L and the ambient temperature at 20-30 ℃, and collecting sediments with the surface layer of 1-6 cm after continuously running for 10-15 days;
(3) in a vessel containing filter sterilization water, taking 1-6 cm sediment on the surface layer obtained in the step (2), passing through a sieve with the aperture of 0.1-0.5 mm, and collecting small bacterial colonies in the sieve to obtain an electrogenesis sulfur oxidation functional microorganism;
the ferrous sulfide in the step (1) accounts for 1/3-2/3 of the volume of the cup;
the microorganism with the function of generating the electric sulfur oxidation is cable bacteria.
2. The method for enrichment culture and rapid separation of microorganisms with electrogenesis sulfur oxidation function in water sediments according to claim 1, wherein the aperture of the sieve is 0.35mm, the dissolved oxygen concentration of the overlying water is 7mg/L, the ambient temperature is 24 ± 1 ℃, the operation time is 15 days, and the sediments on the collection surface layer with the length of 1-6 cm are the sediments on the collection surface layer with the length of 2 cm.
3. The method for enrichment culture and rapid separation of microorganisms with electricity generation and sulfur oxidation functions in water sediments as claimed in claim 1, wherein the water contained in the wide-mouth vessel in the step (2) is raw water of a water system for digging up the water sediments.
4. The method for enrichment culture and rapid separation of microorganisms with electricity generation and sulfur oxidation functions in water sediments according to claim 3, wherein the water contained in the wide-mouth vessel in the step (2) is raw water of a water system for digging sediments.
5. The method for enrichment culture and rapid separation of microorganisms with electricity generation and sulfur oxidation functions in water sediments as claimed in claim 1, wherein the filter sterilized water of step (3) is obtained by filtering raw water of water system with dug water sediments with 0.22 μm bacterial filter membrane.
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| EP4077224A1 (en) * | 2020-01-13 | 2022-10-26 | OASE GmbH | Method for conditioning water, sediments and/or sludges |
| CN113371847B (en) * | 2021-06-11 | 2022-07-19 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Method for obtaining a large number of cable bacteria and application of cable bacteria in removing sulfides in black and odorous sediments |
| CN116239276A (en) * | 2023-01-04 | 2023-06-09 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Method for inhibiting discharge of nitrous oxide in wetland sediments by utilizing cable bacteria |
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