CN111607532A

CN111607532A - Method for driving denitrification of alcaligenes faecalis

Info

Publication number: CN111607532A
Application number: CN202010347167.0A
Authority: CN
Inventors: 刘星; 詹骥; 周顺桂
Original assignee: Fujian Agriculture and Forestry University
Current assignee: Fujian Agriculture and Forestry University
Priority date: 2020-04-28
Filing date: 2020-04-28
Publication date: 2020-09-01
Anticipated expiration: 2040-04-28
Also published as: CN111607532B

Abstract

The invention provides a method for driving denitrification of Alcaligenes faecalis, which comprises the steps of inoculating Alcaligenes faecalis bacteria and Geobacter bacteria in a low-nutrient medium to form a co-culture system in an anaerobic environment, and carrying out denitrification reaction on a system containing nitrate by adopting the co-culture system. The method can excite the Alcaligenes faecalis bacteria to carry out denitrification by electric driving force, and has wide application prospect.

Description

Method for driving denitrification of alcaligenes faecalis

Technical Field

The invention relates to a method for driving denitrification of alcaligenes faecalis, belonging to the technical field of microorganisms.

Background

Due to the increasing demand of human beings for grains, excessive chemical fertilizers are put into grain production activities. A large amount of unutilized fertilizer flows into a natural ecological system in the form of nitrate to cause the problems of water eutrophication and the like, and great damage is caused to the environment. Denitrification is the only way to mitigate these nitrate contaminations and has irreplaceable environmental value.

The complete denitrification is a process of completely reducing nitrate into nitrogen which is harmless to the environment by microorganisms, and has high environmental benefit. However, microorganisms that want to accomplish this need to have reductases and associated transporters necessary for each denitrification step. Because the variety of single microorganisms simultaneously possessing the reductase and the transporter is less, more microorganisms realize the denitrification process in a cooperative mode of mutualistic symbiosis in a biological community.

Alcaligenes faecalis is a facultative anaerobic bacterium with the ability to nitrify under aerobic conditions to produce NO₃ ^-、NO₂ ^-NO and N₂O and ability to undergo short-cut denitrification under anaerobic conditions, but alcaligenes, faecalis is considered to have NO ability to reduce NO under natural conditions due to lack of universal nitrate reductase₃ ^-The ability of the cell to perform.

Therefore, if a mutual-nutrition symbiotic system formed by microorganisms which are mutually symbiotic with Alcaligenes faecalis can be found for denitrification operation, the method has wide application prospect.

Disclosure of Invention

The invention provides a method for driving the denitrification of alcaligenes faecalis, which can effectively solve the problems.

The invention is realized by the following steps:

a method for driving denitrification of Alcaligenes faecalis comprises the steps of inoculating Alcaligenes faecalis bacteria and Geobacter in a low-nutrient medium to form a co-culture system in an anaerobic environment, and carrying out denitrification reaction on a system containing nitrate by adopting the co-culture system.

As a further improvement, the Alcaligenes faecalis strain is Alcaligenes faecalis DSM 30030.

As a further improvement, the Geobacter is at least one of Geobacter metalliriruducens GS15 and Geobacter sulfurruducens PCA.

As a further improvement, the inoculated Alcaligenes faecalis and Geobacter have an OD600nm value of 0.1-0.5, or a bacterial liquid in a logarithmic growth phase, and the inoculation amount is 10% by volume.

As a further improvement, the formula of the low-nutrient medium is NaHCO₃2.0-3.0g/L，NH₄Cl 0.20-0.30g/L，NaH₂PO4*H₂O 0.05-0.08g/L，KCl 0.05-0.15g/L，DL Vitamins 8-15ml/L，DLMinerals 8-12ml/L。

As a further improvement, acetate is also added to the co-cultivation system.

As a further improvement, the final concentration of acetate in the co-cultivation system is 15 mM.

As a further improvement, the co-culture is light-shielding culture, and the culture temperature is 28-32 ℃.

In a further improvement, the inoculation bacterial liquid of the Alcaligenes faecalis is bacterial liquid cultured in a low-nutrient medium added with 15mM acetate and 5mM nitrite, and the pH value of the medium is 6.5-7.5.

As a further improvement, the anaerobic environment is a strictly anaerobic environment.

The invention has the beneficial effects that:

the invention discloses a method for driving the denitrification of Alcaligenes faecalis, which can expand the types of methods for realizing complete denitrification by interaction of microorganisms, and takes geobacillus widely distributed in various anaerobic water environments as electric driving force to excite Alcaligenes faecalalis so as to realize the process of reducing nitrate to produce nitrogen and provide an idea for the research and treatment process of a new anaerobic denitrification mechanism.

The method has the advantages of simple operation, high efficiency, harmless product to the environment and the like, and the simple system construction process ensures that the method has the potential of popularization and application.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are required to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 shows the denitrification performance test and the related characterization of a Geobacter malmerides-Alcaligenes faecalis co-culture system. FIG. 1-A is a representation of the nitrate reduction performance of Geobacter metalrieducens, Alcaligenes faecalis and co-cultures thereof; FIG. 1-B is a representation of the nitrogen conversion in a Geobacter metalducedcens-Alcaligenes faecalis co-culture system; FIG. 1-C is an SEM image of a Geobacter metalglucens-Alcaligenes farecalis co-culture system; FIG. 1-D is a representation of the colony proportion of the Geobacter meterleducens-Alcaligenes farecalis co-culture system.

FIG. 2 shows the denitrification performance test and the related characterization of the Geobacter sulfureatedacens-Alcaligenes faecalis co-culture system. FIG. 2-A is a representation of the nitrate reduction performance of Geobacter sulfureateducens, Alcaligenes faecalis and co-cultures thereof; FIG. 2-B is a representation of the colony proportion of the Geobactillurreducens-Alcaligenes faecalis co-culture system.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be described clearly and completely with reference to the accompanying drawings of the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention. Thus, the following detailed description of the embodiments of the present invention, presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.

In the description of the present invention, the terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implying any number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the present invention, "a plurality" means two or more unless specifically defined otherwise.

The formula of the low nutrient medium (FWNN) of the invention is as follows:

FWNN formulations

The formula of the mixed liquid of DL Vitamins and DL Minerals is as follows:

DL Vitamins formula

DL Minerals formula

The Alcaligenes faecalis strain used in the invention is Alcaligenes faecalis DSM30030, which is purchased from China General Microbiological culture collection Center (CGMCC) and has the collection number of DSM 30030.

The Geobacter malmeriducens used in the invention are Geobacter malmeriducens GS15 or Geobacter sulfurducens PCA, which are purchased from American mode culture collection banks and have the deposit numbers of ATCC-53774 and ATCC-51573 respectively.

Example 1

(1) Alcaligenes faecalis and Geobacter metelliruducens GS15 were separately cultured to OD in FWNN medium supplemented with 15mM acetate and 5mM nitrite_600nmThe value is 0.3, and the pH value of the culture medium is 6.5-7.5.

(2) Dividing the culture system into 3 groups, inoculating Geobactermellirleducens GS15 cultured in the step (1) only in the first group, inoculating Alcaligenes faecalis bacteria cultured in the step (1) only in the second group, and inoculating Geobactermellirleducens GS15 cultured in the step (1) and the Alcaligenes faecalis bacteria for co-culture in the third group; the inoculation amount is 10% according to the volume ratio; the culture medium is FWNN culture medium with pH 7.0 and added with sodium acetate and sodium nitrate, the final concentration of the sodium nitrate is 5mM, and the final concentration of the sodium acetate is 15 mM;

(3) culturing at 30 deg.C in dark; and (3) testing the nitrate reduction and nitrogen conversion performance, the colony microscopic image and the population ratio of the three groups of culture systems.

The test method is as follows: an anaerobic flask having a total volume of 60ml was used as a culture vessel, 30ml of FWNN low nutrient medium supplemented with sodium nitrate and sodium acetate was added thereto, and a mixed gas (Ar: CO) was used₂80:20) inoculating the microorganism after removing oxygen and nitrogen. All experiments were set up in 3 replicate experimental groups.

Determination of nitrate, nitrite and ammonium salts. Filtering the culture solution with filter membrane with pore diameter of 0.22 μm, and respectively using dual-wavelength ultraviolet spectrophotometry [1 ]]N- (1-naphthyl) -ethylenediamine hydrochloride dyeing method [2 ]]And indophenol blue colorimetry [3]The concentrations of nitrate, nitrite and ammonium salt in the filtrate were determined. Determination of NO Using an ultraviolet Spectrophotometer (Shimadzu UV-2600, Japan)₃ ^-、NO₂ ^-And NH₄ ⁺。

Nitrous oxide and nitrogen gas were measured. Gas N was treated using a gas chromatograph (Agilent Technologies 7890B, USA) equipped with a peristaltic pump (Gilson Miniplus 3, Gilson, France) and three detectors (thermal conductivity detector TCD, electron capture detector ECD and flame ionization detector FID)₂O and N₂Is measured. According to a set program, a proper amount of headspace gas in an anaerobic culture bottle is sent to an instrument by a peristaltic pump for analysis, and high-purity helium gas is used for cleaning pipeline gas and calibrating a background value before each sample measurement. In order to ensure the balance of the air pressure in the anaerobic bottle, sterile mixed gas (Ar: CO) with the same volume needs to be supplemented into the bottle after each sample measurement₂＝80:20)。

All operations were performed under strictly anaerobic conditions.

The results show that neither g.metaleducens nor a.faecalis are able to reduce nitrate alone under low nutrient conditions, but their co-culture experimental groups reduced all 4.9mM of nitrate within 40 hours (fig. 1A); as can be seen in the results of monitoring the denitrification product of the system, the nitrate concentration decreased from 5.1mM to 0mM within 35 hours after inoculation, while the nitrogen concentration increased from 0.4mM to 2.5mM, and a small amount of ammonium salt (about 0.5mM) was consumed during colony growth (FIG. 1B); since no other nitrogen-containing species were detected in the system, it is considered that almost all of the nitrate nitrogen in the system was converted into nitrogen gas. Furthermore, the microcolonies of the co-culture system exhibited the phenomenon of colonization among microorganisms by massive pilous structures, suggesting that the establishment of this group of cross-breeding partners was related to the interspecies direct electron transfer mechanism in which geobacillus participates (FIG. 1C). The colony proportion data of Geobacter metelliruducens and Alcaligenes faecalis is 22.72%, and 77.28% reflects that A.faecalis accounts for a larger proportion of colonies in the system and dominates the colonies (FIG. 1D).

Example 2

(1) Alcaligenes faecalis and Geobacter metelliruducens PCA were separately cultured to OD in FWNN medium supplemented with 15mM acetate and 5mM nitrite_600nmThe value is 0.3, and the pH value of the culture medium is 6.5-7.5.

(2) Inoculating Geobacter metelliruducens PCA and Alcaligenes faecalis bacteria cultured in the step (1) into a culture medium for co-culture; the inoculation amount is 10% according to the volume ratio; the culture medium is FWNN culture medium with pH 7.0 and added with sodium acetate and sodium nitrate, the final concentration of the sodium nitrate is 5mM, and the final concentration of the sodium acetate is 15 mM;

(3) culturing at 30 deg.C in dark; the nitrate reduction and nitrogen conversion performance, colony microscopic image and population ratio of a Geobacter metallideuccus PCA and Alcaligenes faecalis bacteria co-culture system are tested. The test method was the same as in example 1.

All operations were performed under strictly anaerobic conditions.

The results showed that the Geobacter sulfureaters-Alcaligenes faecalis co-culture system achieved the reduction of the total 5mM nitrate in the system in about 64 hours, with a ratio of about 53.93% to 46.07%. (FIG. 2).

In conclusion, the invention comprises two core elements of the geobacillus Geobacter and the Alcaligenes faecalis. The method skillfully combines the interspecific mutualistic characteristic of the Geobacter Geobacter and the denitrification potential of the Alcaligenes faecalis, closely associates the Geobacter Geobacter and the Alcaligenes faecalis by a co-culture mode, breaks through the limitation of substrates together, creates a new denitrification mechanism in the category of microbial physiology, and realizes the rapid and harmless treatment of nitrate. The relative simplicity of the systems makes them easier to modify and improve in a modular fashion.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

[1]Norman R J,Stucki J W.The Determination of Nitrate and Nitrite inSoil Extracts by Ultraviolet Spectrophotometry[J].Soil Science Society ofAmerica Journal.1981,45(2):347-353.

[2]Dorich R A,Nelson D W.Direct Colorimetric Measurement of Ammoniumin Kcl Extracts of Soils[J].Soil Science Society of America Journal.1983,47(4):833-836.

[3]Norman R J,Edberg J C,Stucki J W.Determination of Nitrate in SoilExtracts by Dual-Wavelength Ultraviolet Spectrophotometry[J].Soil ScienceSociety of America Journal.1985,49(5):1182-1185.

Claims

1. A method for driving the denitrification of Alcaligenes faecalis is characterized in that Alcaligenes faecalis and Geobacter are inoculated in a low-nutrient medium to form a co-culture system in an anaerobic environment, and the co-culture system is adopted to carry out denitrification reaction on a system containing nitrate.

2. The method of driving the denitrification of Alcaligenes faecalis according to claim 1, wherein said Alcaligenes faecalis is DSM 30030.

3. The method for driving the denitrification of Alcaligenes faecalis according to claim 1, wherein the Geobacter bacteria is at least one of Geobacter metalliducens GS15 and Geobacter sulfurducens PCA.

4. The method for driving the denitrification of Alcaligenes faecalis according to claim 1, wherein the OD of said inoculated Alcaligenes faecalis and Geobacter bacteria_600nmThe value is 0.1-0.5, or the bacterial liquid in the logarithmic growth phase, and the inoculation amount is 10% by volume ratio.

5. The method for driving the denitrification of Alcaligenes faecalis according to claim 1, wherein the formulation of said low nutrient medium is NaHCO₃2.0-3.0g/L，NH₄Cl 0.20-0.30g/L，NaH₂PO₄*H₂O0.05-0.08g/L，KCl 0.05-0.15g/L，DL Vitamins 8-15ml/L，DL Minerals 8-12ml/L。

6. The method for driving the denitrification of Alcaligenes faecalis according to claim 1, wherein acetate is also added to the co-cultivation system.

7. The method for driving the denitrification of Alcaligenes faecalis according to claim 6, wherein the final concentration of acetate in the co-culture system is 15 mM.

8. The method for driving the denitrification of Alcaligenes faecalis according to claim 1, wherein the co-cultivation is a light-shielded cultivation and the cultivation temperature is 28-32 ℃.

9. The method for driving the denitrification of Alcaligenes faecalis according to claim 1, wherein the inoculated bacterial liquid of Alcaligenes faecalis is cultured in a low nutrient medium supplemented with 15mM acetate and 5mM nitrite, and the pH value of the medium is 6.5-7.5.

10. The method of driving the denitrification of Alcaligenes faecalis according to claim 1, wherein said anaerobic environment is a strictly anaerobic environment.