CN111440704A - Convenient method and device for efficiently enriching N-DAMO bacteria - Google Patents
Convenient method and device for efficiently enriching N-DAMO bacteria Download PDFInfo
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- CN111440704A CN111440704A CN202010191876.4A CN202010191876A CN111440704A CN 111440704 A CN111440704 A CN 111440704A CN 202010191876 A CN202010191876 A CN 202010191876A CN 111440704 A CN111440704 A CN 111440704A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
- C12M29/06—Nozzles; Sprayers; Spargers; Diffusers
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
- C12M29/20—Degassing; Venting; Bubble traps
Abstract
The invention discloses a convenient method and a convenient device for efficiently enriching N-DAMO bacteria, belonging to the field of anaerobic methane oxidation microorganism enrichment. The invention solves the problems of slow growth and metabolism, difficult enrichment culture and the like of enriched N-DAMO bacteria. The simple anaerobic sequencing batch reactor provided by the invention comprises a main reactor glass bottle, a water exchange pipe, an air inlet pipe and an air pressure balance pipe which are arranged on a sealed bottle plug, a methane air supply system connected with the air inlet pipe and a water exchange system connected with the water exchange pipe. And (3) adding a microbial sample to be cultured into the reactor, adding a liquid basic culture medium and nitrite, filling methane, performing closed culture, periodically replacing the supernatant with a fresh culture medium, and continuously culturing to finally obtain an enrichment culture of the N-DAMO bacteria. The invention provides a method and a device for efficiently enriching and culturing N-DAMO bacteria, which are convenient and quick and are beneficial to acquisition and utilization of N-DAMO bacteria resources.
Description
Technical Field
The invention relates to a convenient method and a convenient device for efficiently enriching N-DAMO bacteria, belonging to the field of anaerobic methane oxidation microorganism enrichment.
Background
In the biological treatment of waste waterMore or less having CH4While protecting the aqueous environment, the production and release of (b) increases the content of atmospheric greenhouse gases. On the other hand, due to the shortage of organic carbon source, the conventional biological treatment technology of nitrification and denitrification wastewater is difficult to realize economic and efficient denitrification and even difficult to discharge after reaching the standard.
The existing Nitrite type Anaerobic methane oxidation (N-DAMO) can solve the problem of CH4The emission reduction and the insufficient carbon source for biological denitrification of the wastewater. The N-DAMO process is with CH4Reduction of nitrite NO as an electron donor2 -Generation of nitrogen gas N2. The reaction equation is as follows: 3CH4+8NO2 -+8H+→3CO2+4N2+10H2OΔG0'＝-928kJ mol-1. The process is widely applied to natural ecological environments such as farmlands, wetlands, rivers and lakes and the like, and is an important link of the carbon-nitrogen circulation of the biosphere. However, the N-DAMO bacteria are autotrophic microorganisms, slow in growth and metabolism, difficult in enrichment culture and difficult to develop engineering application and practice. And the existing enrichment culture research on N-DAMO bacteria mostly focuses on reactor improvement to increase CH4The absorption utilization ratio of (1). However, most of the methods have the defects of complex culture system, high operation and management cost and the like, and are not enough to support the requirements of research and development of N-DAMO strain resources by scientific research and engineering application technologies. Therefore, the method and the device for rapidly, simply and conveniently enriching the N-DAMO bacteria are provided, the N-DAMO bacterial culture is rapidly, simply and conveniently obtained, and a foundation is necessary for obtaining N-DAMO strain resources, researching theories and researching engineering application technologies.
Disclosure of Invention
The invention provides a convenient method and a device for efficiently enriching N-DAMO bacteria, aiming at solving the problems of slow growth and metabolism, difficulty in enrichment culture and the like of the existing enriched N-DAMO bacteria.
The technical scheme of the invention is as follows:
a convenient device for efficiently enriching N-DAMO bacteria comprises a reaction system, a methane gas supply system and a water changing system, wherein the reaction system is respectively communicated with the methane gas supply system and the water changing system;
the reaction system comprises a reactor glass bottle 9, an air pressure balance tube 7 and an air bag 8, wherein one end of the air pressure balance tube 7 penetrates through a sealing bottle stopper of the reactor glass bottle 9 and is inserted into the reactor glass bottle 9, the other end of the air pressure balance tube 7 is communicated with the air bag 8, and a stop valve is arranged on the air pressure balance tube 7;
the methane gas supply system comprises a methane gas bottle 14, a ventilation pipe 1 and an aeration head 10, wherein one end of the ventilation pipe 1 is communicated with the methane gas bottle 14, the other end of the ventilation pipe 1 penetrates through a sealing bottle plug of a reactor glass bottle 9 to be connected with the aeration head 10 arranged in the reactor glass bottle 9, and the ventilation pipe 1 is provided with a stop valve;
trade the water system include stock solution bottle 12, trade water pipe 3, peristaltic pump 5, atmospheric pressure balance pipe 7 and air pocket 8, the sealed bottle plug that reactor glass bottle 9 was passed to the one end of trading water pipe 3 inserts inside the ware glass bottle 9, trade the water pipe 3 other end and be connected with peristaltic pump 5's hose one end, it stops the valve to be equipped with on trading water pipe 3, the sealed bottle plug that the other end of peristaltic pump 5 hose passed stock solution bottle 12 inserts inside stock solution bottle 12, the sealed bottle plug that stock solution bottle 12 was passed to the one end of atmospheric pressure balance pipe 7 inserts inside stock solution bottle 12, the other end and the air pocket 8 intercommunication of atmospheric pressure balance pipe 7, be equipped with the valve that stops on atmospheric pressure balance pipe 7.
Further defined, the reactor vial 9 is a jar having a volume of 1L.
Further, the air pressure balance tube 7 is inserted into the bottle for a distance of 0.5 cm.
The method for efficiently enriching the N-DAMO bacteria by using the device comprises the following specific operation processes:
the first stage is as follows: adding a liquid basic culture medium, nitrite and wetland soil into a reactor glass bottle 9, opening a stop valve on an air exchange tube 1, filling methane, closing the stop valve after the filling, placing the air exchange tube in an air bath oscillator for closed culture, and performing a second stage after 24 hours of culture;
and a second stage: opening a stop valve on a peristaltic pump 5 and a water changing pipe 3, discharging supernatant in a reactor glass bottle 9 into a liquid storage bottle 12, pouring liquid in the liquid storage bottle 12, injecting a liquid basic culture medium added with nitrite into the liquid storage bottle 12, changing the flow direction of the liquid in a hose of the peristaltic pump 5, ensuring that the distance between the pipe orifice of the water changing pipe 3 and the bottom of the reactor glass bottle 9 is 2cm, injecting a fresh liquid basic culture medium in the liquid storage bottle 12 into the reactor glass bottle 9, closing the stop valves on the peristaltic pump 5 and the water changing pipe 3 after the liquid basic culture medium is changed, opening the stop valve on a ventilation pipe 1 to fill methane, closing the stop valve on the ventilation pipe 1 after the gas filling is finished, and continuously placing the culture medium in an air bath oscillator for closed culture;
and a third stage: the second phase was repeated up to day 60.
Further defined, 0.25g KHCO per liter of liquid basal medium3，0.30g CaCl2·2H2O，0.05g KH2PO4，0.20g MgSO4·3H2O, 0.5m L acidic trace element liquid and 0.2m L alkaline trace element liquid.
Further defined, each liter of the acidic trace element liquid comprises: 2.085g FeSO4·3H2O，0.120g CoCl2·6H2O，0.320g CuSO4，0.014g H3BO3，0.068g ZnSO4·3H2O，0.500g MnCl2·4H2O，0.095gNiCl2·6H2O and 8.75M L HCl, with a HCl concentration of 12M.
Further limited, each liter of the alkaline microelement liquid comprises: 0.063g SeO2，0.050g Na2WO4·2H2O，0.242g Na2MoO4And 0.400g NaOH.
Further defined, the nitrite salt is NaNO2Adding NaNO2The concentration of the resulting solution in the liquid basal medium was 1 mmol/L.
Further limiting, the time duration of each second stage is 24 hours, wherein the discharge time of the supernatant in the reactor glass bottle 9 is 15 minutes, the time for injecting the fresh liquid basic culture medium added with the nitrite is 10 minutes, the time for supplementing methane gas is 5 minutes, and the closed culture time in the air bath oscillator is 19.5 hours.
Further limiting, the sealed culture conditions in the reactor glass bottle 9 are: the temperature is 30 + -1 deg.C, pH is 7.0-7.5, and air pressure in bottle is 101 kPa.
The invention has the following beneficial effects:
(1) the N-DAMO bacterial culture medium provided by the invention is more beneficial to enriching N-DAMO flora;
(2) the method can quickly and effectively enrich the N-DAMO flora, and provides method guidance for resource acquisition and engineering application of the N-DAMO bacterial strains;
(3) the device reactor and the matching device provided by the invention have the advantages of cheap and easily-obtained components, simple and convenient assembly and contribution to engineering application.
Drawings
FIG. 1 is a schematic view of an apparatus for enriching N-DAMO bacteria according to the present invention.
In the figure, 1-a ventilation pipe, 3-a water changing pipe, 5-a peristaltic pump, 7-an air pressure balancing pipe, 8-an air bag, 9-a reactor glass bottle, 9, 10-an aeration head, 12-a liquid storage bottle and 14-a methane gas bottle.
Detailed Description
The experimental procedures used in the following examples are conventional unless otherwise specified. The materials, reagents, methods and apparatus used, unless otherwise specified, are conventional in the art and are commercially available to those skilled in the art.
Detailed description of the preferred embodiment 1
The device enriched for N-DAMO bacteria was assembled as shown in FIG. 1.
The first stage is that 100g of wetland soil with the depth of 40-60cm in the natural protection area is taken and placed in a reactor glass bottle 9, 0.6L of liquid basic culture medium is added, and NaNO is added2Making its concentration in liquid basic culture medium 1 mmol/L, adjusting pH to 7.0-7.5 with 1 mol/L HCl, opening the stop valve of ventilation tube 1, charging high-purity (99.99%) methane gas, closing the stop valve of ventilation tube 1 after 5min, sealing the whole system, placing in an air bath oscillator, and adjusting methane partial pressure (P) at 30 deg.CCH4) Continuously culturing under the condition of 101kPa for 24 hours, wherein NO is added2 -The reaction is ended and the precipitate is left to stand.
Second stageOpening the peristaltic pump 5 and the check valve on the water changing pipe 3, discharging the supernatant of 0.4L in the glass bottle 9 of the reactor into the liquid storage bottle 12, pouring the liquid in the liquid storage bottle 12, adding NaNO2The liquid basic culture medium is filled into the liquid storage bottle 12, wherein the NaNO is2The content is 1 mmol/L, the flow direction of the liquid in the hose of the peristaltic pump 5 is changed, the distance between the pipe orifice of the water changing pipe 3 and the bottom of the reactor glass bottle 9 is ensured to be 2cm, the fresh liquid basic culture medium in the liquid storage bottle 12 is injected into the reactor glass bottle 9, the volume of the input liquid basic culture medium is 0.4L, after the liquid basic culture medium is changed, the peristaltic pump 5 and the check valve on the water changing pipe 3 are closed, and the device is continuously placed in an air bath oscillator for closed culture
And a third stage: the second phase was repeated up to day 60.
Wherein each liter of the liquid basal medium comprises 0.25g KHCO3，0.30g CaCl2·2H2O，0.05gKH2PO4，0.20g MgSO4·3H2O, 0.5m L acidic trace element liquid and 0.2m L alkaline trace element liquid, wherein each liter of acidic trace element liquid contains 2.085g FeSO4·3H2O，0.120g CoCl2·6H2O，0.320g CuSO4，0.014g H3BO3，0.068g ZnSO4·3H2O，0.500g MnCl2·4H2O，0.095g NiCl2·6H2O and 8.75m L HCl, wherein the HCl concentration is 12M, and each liter of alkaline microelement liquid contains 0.063g SeO2，0.050g Na2WO4·2H2O，0.242g Na2MoO4And 0.400g NaOH.
Each second stage was 24 hours in duration, so the entire N-DAMO bacterial enrichment process of embodiment 1 was 60 days in duration, and the enriched culture was obtained in reactor vial 9. For NO in enrichment culture2 -The content is detected, and NO is found in the enrichment culture2 -The removal efficiency is as high as 99%, which shows that the enrichment of N-DAMO flora by using the device and the method of the invention is successful, and the N-DAMO flora has excellent N-DAMO metabolic activity.
Detailed description of the preferred embodiment 2
Examination of environmental suitability of N-DAMO bacteria tested under different pH, temperature, NaC L concentration conditions.
First, unlike the first embodiment, the liquid basal medium was enriched with N-DAMO under different pH conditions (pH of the liquid basal medium was adjusted using 1 mol/L HCl solution or 1 mol/L NaOH solution), pH gradients were examined to be 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0, comparing to show that the enrichment of N-DAMO bacteria was more favored at pH 7-8, and N-DAMO bacteria exhibited good metabolic activity.
Scheme II: unlike the first embodiment, the N-DAMO is enriched under different temperature conditions. The temperature gradients were examined at 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃ and 45 ℃. The comparison shows that the temperature is 30-40 ℃, which is more beneficial to the enrichment of N-DAMO bacteria, and the N-DAMO bacteria show good metabolic activity.
Third, different from the first embodiment, NaCl is added into the liquid basic culture, and the concentration of NaCl in the liquid basic culture solution is 0 g/L, 1.0 g/L, 2.5 g/L, 5.0 g/L, 10.0 g/L and 15.0 g/L respectively.
Detailed description of preferred embodiments 3
Growth factors (vitamins, heme and betaine) can further enhance the metabolic growth capacity of N-DAMO bacteria.
In a fourth embodiment, different from the first embodiment, the vitamin solution is added into the liquid basic culture, and the concentrations of the vitamin solution in the liquid basic culture solution are respectively 0m L/L, 0.01m L/L, 0.02m L2/L, 0.03m L/L, 0.05m L/L, 0.07m L/L and 0.10m L/L. compared with the first embodiment, the addition of the vitamin solution can obviously improve the growth metabolic activity of DAMO bacteria, and when the vitamin solution is 0.02m L/L, the enrichment of N-DAMO bacteria is more favorable, and the N-DAMO bacteria show good metabolic activity, wherein 2.00g of biotin, 5.00g of thiamine hydrochloride, 5.00g of g D-Ca-pantothenic acid, 5.00g of lipoic acid, 2.00g of riboflavin, 0.00g of riboflavin, 0.10g of B-ba-5, 3600 g of vitamin and 3600 g of vitamin B27.00 g and 3600 g of p-aminobenzoic acid.
Fifth, different from the first embodiment, heme is added into the liquid foundation culture, and the concentrations of heme in the liquid foundation culture solution are respectively 0 mug/L, 2 mug/L, 4 mug/L, 6 mug/L, 10 mug/L, 15 mug/L, 20 mug/L and 30 mug/L.
Sixth, different from the first embodiment, betaine is added into the liquid foundation culture, and the concentrations of the betaine in the liquid foundation culture solution are respectively 0 mg/L, 0.1 mg/L, 0.2 mg/L, 0.3 mg/L, 0.5 mg/L, 0.7 mg/L and 1.0 mg/L.
Claims (10)
1. A convenient device for efficiently enriching N-DAMO bacteria is characterized by comprising a reaction system, a methane gas supply system and a water changing system, wherein the reaction system is respectively communicated with the methane gas supply system and the water changing system;
the reaction system comprises a reactor glass bottle (9), an air pressure balance pipe (7) and an air bag (8), wherein one end of the air pressure balance pipe (7) penetrates through a sealing bottle stopper of the reactor glass bottle (9) and is inserted into the reactor glass bottle (9), the other end of the air pressure balance pipe (7) is communicated with the air bag (8), and a stop valve is arranged on the air pressure balance pipe (7);
the methane gas supply system comprises a methane gas bottle (14), a ventilation pipe (1) and an aeration head (10), wherein one end of the ventilation pipe (1) is communicated with the methane gas bottle (14), the other end of the ventilation pipe (1) penetrates through a sealing bottle plug of a reactor glass bottle (9) to be connected with the aeration head (10) arranged in the reactor glass bottle (9), and a middle stop valve is arranged on the ventilation pipe (1);
trade water system include liquid storage bottle (12), trade water pipe (3), peristaltic pump (5), atmospheric pressure balance tube (7) and air pocket (8), the sealed bottle plug that the one end of trading water pipe (3) passed reactor glass bottle (9) inserts inside reactor glass bottle (9), trade the hose one end of water pipe (3) other end and peristaltic pump (5) and be connected, be equipped with the stop valve on trading water pipe (3), the other end of peristaltic pump (5) hose passes the sealed bottle plug of liquid storage bottle (12) and inserts inside liquid storage bottle (12), the one end of atmospheric pressure balance tube (7) is passed the sealed bottle plug of liquid storage bottle (12) and is inserted inside liquid storage bottle (12), the other end and air pocket (8) the intercommunication of atmospheric pressure balance tube (7), be equipped with the stop valve on atmospheric pressure balance tube (7).
2. The portable apparatus for the efficient enrichment of N-DAMO bacteria according to claim 1, wherein said reactor vial (9) is a 1L capacity jar.
3. The portable apparatus for highly efficient enrichment of N-DAMO bacteria according to claim 1, wherein said pressure equalization tube (7) is inserted into the flask at a distance of 0.5 cm.
4. The method for the efficient enrichment of N-DAMO bacteria using the apparatus of claim 1, wherein the specific procedures of the method are as follows:
the first stage is as follows: adding a liquid basic culture medium, nitrite and wetland soil into a reactor glass bottle (9), opening a stop valve on an air exchange tube (1) to fill methane, closing the stop valve after the completion of the inflation, placing the air exchange tube in an air bath oscillator for closed culture, and entering a second stage after the culture is carried out for 24 hours;
and a second stage: opening the peristaltic pump (5) and the stop valve on the water changing pipe (3), discharging the supernatant in the glass bottle (9) of the reactor into the liquid storage bottle (12), pouring the liquid in the liquid storage bottle (12), the liquid basic culture medium added with the nitrite is injected into a liquid storage bottle (12), the flowing direction of the liquid in a hose of a peristaltic pump (5) is changed, and the distance between the pipe orifice of the water changing pipe (3) and the bottom of the reactor glass bottle (9) is ensured to be 2cm, the fresh liquid basal culture medium in the liquid storage bottle (12) is injected into the reactor glass bottle (9), after the liquid basic culture medium is replaced, the peristaltic pump (5) and the stop valve on the water changing pipe (3) are closed, then opening a stop valve on the ventilation pipe (1) to fill methane, closing the stop valve on the ventilation pipe (1) after the filling is finished, and continuously placing the ventilation pipe in an air bath oscillator for closed culture;
and a third stage: the second phase was repeated up to day 60.
5. The method for the efficient enrichment of N-DAMO bacteria according to claim 4, wherein 0.25g KHCO per liter of the liquid basal medium is included3，0.30g CaCl2·2H2O，0.05g KH2PO4，0.20g MgSO4·3H2O, 0.5m L acidic trace element liquid and 0.2m L alkaline trace element liquid.
6. The method of claim 5 wherein said acidic trace element liquid comprises, per liter: 2.085g FeSO4·3H2O，0.120g CoCl2·6H2O，0.320g CuSO4，0.014gH3BO3，0.068g ZnSO4·3H2O，0.500g MnCl2·4H2O，0.095g NiCl2·6H2O and 8.75M L HCl, with a HCl concentration of 12M.
7. The method of claim 5 wherein said basic trace element liquid comprises, per liter: 0.063g SeO2，0.050g Na2WO4·2H2O，0.242g Na2MoO4And 0.400g NaOH.
8. The method of claim 4 wherein the nitrite salt is NaNO2，NaNO2The concentration in the liquid basal medium was 1 mmol/L.
9. The method for the efficient enrichment of N-DAMO bacteria according to claim 4, wherein the duration of each of said second phases is 24 hours, wherein the time for the supernatant to drain out of the reactor flask (9) is 15min, the time for the fresh liquid basal medium supplemented with nitrite is 10min, the time for the methane gas to be supplemented is 5min, and the time for the closed cultivation in the air bath shaker is 19.5 hours.
10. The method for the efficient enrichment of N-DAMO bacteria according to claim 4, characterized in that the closed culture conditions in the reactor vial (9) are: the temperature is 30 + -1 deg.C, pH is 7.0-7.5, and air pressure in bottle is 101 kPa.
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Application publication date: 20200724 |