CN114314807A - A method for rapid domestication and enrichment of electrogenic bacteria - Google Patents

Abstract

The invention discloses a method for rapidly domesticating and enriching electricity-producing bacteria, comprising the following steps; Step 1: constructing a double-chamber microbial electrochemical system, using common suspended anaerobic fermentation microorganisms as seed sludge microorganisms, sodium acetate or sodium propionate As an electron donor, potassium ferricyanide is added to the cathode chamber as an electron acceptor, and the conductive carbon material carbon felt is placed as an electrode material in the anode chamber and the cathode chamber, respectively, to acclimate and enrich the electricity-producing bacteria; step 2 : By adding suspended biochar particles into the anode chamber, the enrichment speed of electricity-producing bacteria on the surface of the anode is accelerated, and by monitoring the electricity production rate, Coulomb efficiency, carbon Population structure of biofilm on felt surface, evaluating the effect of biochar particle addition on the rapid domestication and enrichment of electrogenic bacteria. The invention can efficiently and quickly use low-cost common anaerobic microorganisms as seed sludge to realize domestication and enrichment of electricity-producing bacteria.

Description

A method for rapid domestication and enrichment of electrogenic bacteria

technical field

The invention belongs to the technical field of sewage treatment, and in particular relates to a method for rapid domestication and enrichment of electricity-producing bacteria.

Background technique

The extracellular electron transfer behavior of microorganisms has important application value in the fields of environmental engineering technology such as water environment treatment, pollutant degradation, and soil remediation. However, how to use common microorganisms to rapidly domesticate and enrich electrogenic bacteria with extracellular electron transfer ability is one of the technical bottlenecks in the research and application of extracellular electron transfer behavior. Therefore, it is of great significance to develop a method for rapid domestication and enrichment of electrogenic bacteria using common anaerobic microorganisms.

At present, the methods for domesticating and culturing electrogenic bacteria mainly include culture medium domestication method and microbial electrochemical system culture method. ①The medium domestication culture method refers to the use of microbial purification technology, using ferric iron as an electron donor, domesticating and culturing electrogenic bacteria, and using LB medium for colony culture, purification and separation. The disadvantage is that the colony culture and purification and separation of this method need to be carried out in a sterile anaerobic incubator, which is difficult to operate and has high cost; (2) The microbial electrochemical system culture method is to construct a dual-chamber microbial electrochemical system, using glucose, Small molecular organics such as sodium acetate are electron donors, and electrogenic bacteria are enriched and cultured on the anode surface through a periodic domestication process.

SUMMARY OF THE INVENTION

In order to overcome the above technical problems, the purpose of the present invention is to provide a method for rapid domestication and enrichment of electricity-producing bacteria, which can efficiently and rapidly use low-cost common anaerobic microorganisms as seed sludge to achieve domestication and enrichment of electricity-producing bacteria.

In order to achieve the above object, the technical scheme adopted in the present invention is:

A method for rapidly domesticating and enriching electrogenic bacteria, comprising the following steps;

Step 1: Construct a two-chamber microbial electrochemical system, using 50 ml of suspended sludge from a fully mixed anaerobic fermentation system as seed sludge microorganisms, and 150 ml of sodium acetate or sodium propionate as electron donors into the anode chamber, Potassium ferricyanide is added to the cathode chamber as an electron acceptor, and the conductive carbon material carbon felt is placed as an electrode material in the anode chamber and the cathode chamber, respectively, to domesticate and enrich the electricity-producing bacteria;

Step 2: By adding biochar particles into the anode chamber, magnetic stirring at 200 rpm ensures that the biochar particles are evenly suspended in the device, so as to increase the contact area between the biochar and the suspended microorganisms, and accelerate the electricity-producing bacteria in the anode. The enrichment rate of the surface, by monitoring the electricity production rate of the system with and without the addition of biochar, the coulombic efficiency, and the population structure of the biofilm on the surface of the carbon felt, to evaluate the rapid domestication of electricity-producing bacteria by the addition of biochar particles enrichment boosting effect.

In the step 2, the source of biochar particles is apple wood chips, and the biochar is prepared by oxygen-limited pyrolysis. The apple wood chips are placed in a ceramic crucible, compacted with a ceramic pestle, covered with a lid, and placed in an ordinary muffle furnace, Control the heating rate to 12±1℃/min. When the temperature reaches 500℃, keep it for 2h. After the temperature of the muffle furnace returns to room temperature, take it out for use.

In the step 2, the particle size of the biochar particles is 2-5 mm, and the dosing concentration is 15 g/L.

In the step 1, the two-chamber microbial electrochemical system is made of glass material, the reaction volume of the anode chamber and the cathode chamber are both 200 ml, and the two chambers are separated by a cation exchange membrane, which is conducive to ion exchange. Make connections for 1 mm titanium wire.

The operating temperature of the dual-chamber microbial electrochemical system in the step 1 is room temperature (26±2°C).

In the step 1, the anode chamber and the cathode chamber of the dual-chamber microbial electrochemical system must be aerated with nitrogen with a purity of 99.9% at an aeration intensity of 1 L/min for 8 minutes before the reaction is carried out.

The mixing mode of the dual-chamber microbial electrochemical system in the step 1 is magnetic stirring, and the stirring is 200 rpm.

In the step 1, the dosage of sodium acetate or sodium propionate is 1500 mg/L, and the dosage is one-time dosage.

In the step 1, the microbial seed sludge comes from a completely mixed medium-temperature anaerobic fermentation system, which is used for the domestication and enrichment of electricity-producing bacteria. After the sludge is taken out, it is placed in an anaerobic serum bottle for cultivation. It can be used when the daily methane production is less than 5mL/day, and the concentration of sludge added to the anode chamber is 2.3g volatile solids/L.

In the step 1, 50mM/L of sodium bromoethane sulfonate (BES) is added to each anode chamber as a methanogenesis inhibitor to prevent the methanogens in the anaerobic seed sludge from metabolizing sodium propionate or sodium acetate Methane production.

The composition of the anode culture solution in the anode chamber for domestication and enrichment of electricity-producing bacteria in the step 1 is: ammonium chloride 500mg/L, potassium dihydrogen phosphate 200mg/L, sodium sulfate 40mg/L, potassium chloride 50mg/L L, aluminum chloride 0.5mg/L, calcium chloride 10mg/L, magnesium chloride 70mg/L, manganese chloride 0.8mg/L, cobalt chloride 1.2mg/L, nickel chloride 0.5mg/L, EDTA-sodium 3mg /L, ferrous sulfate 3.2mg/L, copper chloride 1.1mg/L, sodium manganate 0.1mg/L, zinc sulfate 3.2mg/L, boric acid 0.2mg/L.

In the step 1, the potassium ferricyanide in the cathode chamber uses ferric ions as electron acceptors, and the dosage is 50 mM/L.

Beneficial effects of the present invention:

The invention can realize the rapid domestication and enrichment of functional electrogenic bacteria;

Compared with the traditional method, the method uses the biochar green material prepared from solid waste, which is environmentally friendly and low in cost;

The method is simple to operate and easy to popularize and use.

Description of drawings

FIG. 1 is a schematic diagram of the microbial electrochemical experimental apparatus of the present invention.

Fig. 2 is a schematic diagram showing the change of the current density of each group during the reaction process of the present invention.

3 is a schematic diagram of the degradation of acetic acid and propionic acid and the change of the system coulombic efficiency during the reaction process of the present invention.

FIG. 4 is a schematic diagram of the enrichment and abundance of the electrogenic bacteria of the present invention on the carbon felt.

Detailed ways

The present invention will be described in further detail below in conjunction with the examples.

The system construction and operation steps of the present invention are as follows:

(1) Preparation of biochar particles: Place apple wood chips with a plane size of 1-3 square centimeters in a 200 ml ceramic crucible, fill the crucible with volume, and leave no gaps as much as possible to create an oxygen-deficient environment. Cover the crucible and place it in the muffle furnace, set the heating rate of the muffle furnace to 12 ± 1 °C/min, keep it at 500 °C for 2 hours, and then automatically close it. After the muffle furnace is cooled to room temperature, remove the crucible and obtain bio-charcoal. The biochar will be sieved with a stainless steel mesh to a particle size of 2-5 mm, stored in a plastic bag, placed in a dry place away from light, and used for later use.

(2) Source and pre-cultivation of microbial seed sludge: Take 500 ml of anaerobic sludge from the stable operation of the mesophilic anaerobic fermentation system, and pre-cultivate it in an anaerobic fermentation serum bottle for 10-15 days. After the daily methane production is less than 5mL/day, the sludge is stored in an anaerobic environment for use.

(3) Construction of the dual-chamber microbial electrochemical system: The dual-chamber microbial electrochemical system is shown in Figure 1, and its material is glass, and the working volume is 200 ml. The top of the glass bottle is sealed with a rubber stopper and a plastic bottle cap. There are two round holes on the side of each bottle, which are sealed with rubber stoppers and aluminum caps during the experiment. The junction between the anode chamber and the cathode chamber is separated by a cation exchange membrane, and is fixed and sealed with a water stop tape and a stainless steel water stop clip. The electrode materials used in this system are conductive carbon felts, each with an area of 6 cm ² . A titanium wire with a diameter of 1 mm is used for connection between the anode and the cathode.

(4) Group setting of the two-chamber microbial electrochemical system: according to the presence or absence of biochar particles and the type of electron donor, a total of acetic acid-biochar dosing group (Ac-BC), acetic acid-control group (Ac- CT), propionic acid-biochar addition group (Pr-BC), propionic acid-control group (Pr-CT).

(5) Start-up and operation of the dual-chamber microbial electrochemical system: 50mL of anaerobic sludge with a concentration of 2.3g/L and 150mL of microbial culture solution were added to the anode chamber of the system, and two groups of systems using acetic acid as the electron donor were added. Sodium acetate was added at a concentration of 1500 mg/L, and two groups of systems using propionic acid as electron donors were added with sodium propionate at a concentration of 1500 mg/L. Each group with biochar addition added biochar particles to the anode chamber at a concentration of 15g/L. Potassium ferricyanide was uniformly dosed in the cathode chamber, and ferric ions were used as electron acceptors at a concentration of 50mM/L.

(6) Determination of system operation indicators: During the operation of the system, regularly use a 5 ml plastic needle to take 2 ml of the mixture in the anode chamber, filter it with a filter membrane with a 0.45-micron pore size, and use a gas chromatograph (flame ionization detector) to measure The concentration of acetic acid or propionic acid, a 10Ω resistance is connected in series in the external circuit, the voltage across the resistance is measured in real time through a data collector and a computer, and the current intensity of the system is calculated using Ohm's law. At the end of the reaction, the microbial samples on the surface of the carbon felt were removed for high-throughput sequencing analysis.

(7) Analysis method of system operation results: Coulomb's law is used to calculate the electricity production efficiency of each group of microorganisms consuming electron donors. Based on the results of microbial high-throughput sequencing, the enrichment of typical electrogenic bacteria in each group was quantified at the family and genus levels, and the important role of biochar addition on the rapid domestication and enrichment of electrogenic bacteria was analyzed.

(8) Analysis of system operation results: Figure 1 shows the changes of current intensity in each group. As can be seen from Figure 2(a), when acetic acid was used as the electron acceptor, the overall current density of the Ac-BC group was significantly higher than that of the Ac-CT group, and the Ac-BC group reached the maximum current density of 1.6 A/m on the second day of operation. ² , while in the Ac-CT group, the current density of the control group reached the maximum value of 0.5A/m ² on the 4th day, which was 68.8% lower than that of the Ac-CT group. When acid was the electron acceptor, the overall current density of the Pr-BC group was significantly higher than that of the Pr-CT group. The Pr-BC group reached the maximum current density of 2.3 A/m ² on the third day of operation, while the Pr-CT group reached the peak current density. The time lag was 1.3A/m ² , which was 76.9% lower than that of the Pr-BC group. The coulombic efficiency calculation results in Figure 3 also show that the addition of biochar improves the coulombic efficiency of the system as a whole. Figure 4 shows the abundance of typical electrogenic bacteria Geobacteraceae (family level) in the biofilm on the electrode surface of each group after the reaction. 3.4-4.6% of the charcoal added group. Although differences in electron donors affected the enriched species of Geobacteraceae at the genus level, the addition of biochar significantly promoted the enrichment of typical electrogenic bacteria on the electrode surface.

Claims (10)

1. a method for rapid domestication and enrichment of electrogenic bacteria, is characterized in that, comprises the following steps; Step 1: Construct a two-chamber microbial electrochemical system, using 50 ml of suspended sludge from a fully mixed anaerobic fermentation system as seed sludge microorganisms, and 150 ml of sodium acetate or sodium propionate as electron donors into the anode chamber, Potassium ferricyanide is added to the cathode chamber as an electron acceptor, and the conductive carbon material carbon felt is placed as an electrode material in the anode chamber and the cathode chamber, respectively, to domesticate and enrich the electricity-producing bacteria; Step 2: By adding biochar particles into the anode chamber, magnetic stirring at 200 rpm ensures that the biochar particles are evenly suspended in the device, so as to increase the contact area between the biochar and the suspended microorganisms, and accelerate the electricity-producing bacteria in the anode. The enrichment rate of the surface, by monitoring the electricity production rate of the system with and without the addition of biochar, the coulombic efficiency, and the population structure of the biofilm on the surface of the carbon felt, to evaluate the rapid domestication of electricity-producing bacteria by the addition of biochar particles enrichment boosting effect. 2. a kind of method for rapid domestication and enrichment of electricity-producing bacteria according to claim 1, is characterized in that, in described step 2, the source of biochar particles is apple wood sawdust, and the biochar preparation method is oxygen-limited pyrolysis, and the The apple sawdust was placed in a ceramic crucible, compacted with a ceramic pestle, covered with a lid, and placed in a common muffle furnace. The temperature rise was controlled at 12±1°C/min. After returning to room temperature, take it out for use. 3 . The method for rapidly acclimating and enriching electricity-producing bacteria according to claim 1 , wherein in the step 2, the particle size of the biochar particles is 2-5 mm, and the dosing concentration is 15 g/L. 4 . 4. a kind of method for rapid domestication and enrichment of electricity-producing bacteria according to claim 1, is characterized in that, in the described step 1, the double-chamber microbial electrochemical system is made of glass material, and the reaction volumes of the anode chamber and the cathode chamber are 200 ml, the two chambers are separated by a cation exchange membrane, which is conducive to ion exchange, and the anode and the cathode are connected by a titanium wire with a diameter of 1 mm. 5. The method for rapidly acclimating and enriching electricity-producing bacteria according to claim 1, wherein in the step 1, the operating temperature of the dual-chamber microbial electrochemical system is room temperature (26±2°C); In the step 1, the anode chamber and the cathode chamber of the dual-chamber microbial electrochemical system must be aerated with nitrogen with a purity of 99.9% at an aeration intensity of 1 L/min for 8 minutes before the reaction is carried out. 6. a kind of method for rapid domestication and enrichment of electricity-producing bacteria according to claim 1, is characterized in that, in described step 1, the mixing mode of double-chamber microbial electrochemical system is magnetic stirring, and stirring is 200 rev/min; In the step 1, the dosage of sodium acetate or sodium propionate is 1500 mg/L, and the dosage is one-time dosage. 7. a kind of method for rapid domestication and enrichment of electricity-producing bacteria according to claim 1, is characterized in that, in described step 1, microbial seed sludge is derived from complete mixed type mesophilic anaerobic fermentation system, is used for electricity-producing bacteria domestication For enrichment, the sludge is taken out and then placed in an anaerobic serum bottle for cultivation. The purpose is to deplete the organic matter in the sludge mixture. When the daily methane production is less than 5mL/day, it can be used. The concentration of sludge added to the anode chamber is 2.3g volatile solids/L. 8. a kind of method for rapid domestication and enrichment of electricity-producing bacteria according to claim 1, is characterized in that, in described step 1, in each anode chamber, add the sodium bromoethane sulfonate (BES) of 50Mm/L as Methanogen inhibitor, used to prevent methanogens in anaerobic seed sludge from metabolizing sodium propionate or sodium acetate to produce methane. 9. the method for a kind of rapid domestication and enrichment of electricity-producing bacteria according to claim 1, is characterized in that, in described step 1, in the anode chamber, the anode culture liquid composition for domestication and enrichment of electricity-producing bacteria is composed of: chlorine Ammonium chloride 500mg/L, potassium dihydrogen phosphate 200mg/L, sodium sulfate 40mg/L, potassium chloride 50mg/L, aluminum chloride 0.5mg/L, calcium chloride 10mg/L, magnesium chloride 70mg/L, manganese chloride 0.8mg/L, cobalt chloride 1.2mg/L, nickel chloride 0.5mg/L, EDTA-sodium 3mg/L, ferrous sulfate 3.2mg/L, copper chloride 1.1mg/L, sodium manganate 0.1mg/ L, zinc sulfate 3.2mg/L, boric acid 0.2mg/L. 10. The method for rapidly acclimating and enriching electricity-producing bacteria according to claim 1, wherein the potassium ferricyanide in the cathode chamber in the step 1 uses ferric ion as an electron acceptor, and casts a The concentration is 50mM/L.

Images