Method for improving electricity production, electrochemically active microorganism abundance and biomass of a microbial fuel cell by simulating microgravity

Abstract

The application discloses a method for improving electricity generation, electrochemically active microorganism abundance and biomass of a microbial fuel cell under simulated microgravity. The microbial fuel cell is a novel electrochemical device for generating electricity by using electrochemically active microorganisms, and has a good application prospect in the space field. However, the feasibility of the microbial fuel cell applied to the space field has not been reported. A key scientific problem is how the unique microgravity condition in the space field affects the microbial fuel cell and the electrochemically active microorganism, which is not clear. In order to solve the problem, the application discloses a method for improving electricity generation, electrochemically active microorganism abundance and biomass of a microbial fuel cell under simulated microgravity. Under the simulated microgravity condition, the total biomass of the microbial fuel cell is increased, and the abundance of the electrochemically active microorganism is improved, so that the biomass of the electrochemically active microorganism is increased, and the electricity generation of the microbial fuel cell is increased. The application provides a theoretical basis for the feasibility of the microbial fuel cell applied to the space field.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of space biology, and particularly relates to a method for improving the electricity generation, electrochemically active microorganism abundance and biomass of microorganisms by simulating microgravity. TECHNICAL BACKGROUND

[0002] A microbial fuel cell (MFC) is a novel electrochemical device that utilizes electrochemically active bacteria (EAB) to catalyze polarization reactions. EAB has a unique extracellular electron transfer capability, can oxidize organic matter and transfer the electrons generated by the oxidation of organic matter to the extracellular, forming an output current. Based on this feature, MFCs can be applied to the field of wastewater treatment, which can degrade organic matter in wastewater while converting chemical energy in organic matter into electrical energy, achieving simultaneous wastewater treatment and energy recovery. Compared with existing wastewater treatment technologies, MFCs are expected to overcome the bottleneck of high energy consumption in wastewater treatment. Previous studies have shown that MFCs can effectively degrade domestic wastewater and industrial wastewater while stably generating electricity. Electricity generation can be applied to lighting, mobile phone charging and sensor power supply. Moreover, scaled-up MFCs have also been constructed and applied to pilot-scale and long-term wastewater treatment studies, which have also shown good wastewater treatment efficiency and stable energy recovery capacity, all of which indicate that MFCs have good application prospects in the field of wastewater treatment.

[0003] In fact, the research on EAB and MFC originated from the field of space. In 1921, American scientists first discovered that certain microorganisms could produce electrical signals under certain conditions, and researchers named these microorganisms with electrochemical activity as EAB. Since then, the unique electricity generation characteristics of EAB have attracted the attention of researchers at the U.S. National Aeronautics and Space Administration (NASA). Researchers at NASA proposed using EAB as an emergency power supply to improve the reliability of manned space missions. In 1984, researchers at NASA successfully manufactured a MFC for space applications, with the main fuel being astronaut excrement such as urine. However, due to the lack of research on the electricity generation and biological mechanism of EAB at that time, the electricity generation performance of the MFC was extremely low, making it difficult to be truly applied in the space field.

[0004] However, MFC and EAB research has become a hot spot, and has rapidly developed in the civil field. After decades of research, the mechanism of EAB electricity generation has been basically elucidated, the electricity generation efficiency of MFC has increased by several hundred times, and the commercialization and application of MFC have also rapidly developed. For example, Plant-e in the Netherlands uses EAB in the rhizosphere of plants to use organic matter in the soil as fuel to power mobile phones, street lamps and other equipment. At the same time, researchers at the University of the West of England have constructed a urine MFC to convert urine into electrical energy, which can provide lighting for the LED lights in the toilet. These basic research accumulation and technological progress provide a good foundation for the application of MFC in space.

[0005] MFC and EAB have good application prospects in the space field. MFC is expected to be applied to space wastewater treatment and water reuse. Astronauts will generate liquid and solid waste during long-term on-orbit stay in space stations, and urine is the main liquid waste. Urine treatment and water reuse can improve the closure and reliability of the space life support system, meet the needs of electrolytic water and astronaut drinking water. However, urine treatment technology is an internationally recognized problem, and the main space urine treatment technology is distillation. The distillation method has high water recovery rate for treating urine, but it relies on high temperature and high pressure reaction conditions, and the energy consumption is extremely high. MFC can generate electricity using urine as raw material, and can generate electricity while treating urine, which is expected to solve the problem of high energy consumption of existing space urine treatment.

[0006] However, due to the limited resources of space experiments and space load experiment projects, the feasibility of MFC application in space has not been reported. In fact, compared with the ground environment, the unique microgravity environment in space may affect the performance of MFC. This is because MFC is often a mixed culture system, which contains not only EAB with electrochemical activity, but also microorganisms without electrochemical activity. Microgravity can directly affect the expression of microbial genes and proteins, the synthesis of metabolic products, and thus change the growth and metabolism of microorganisms, community evolution, and morphological structure. Therefore, the types, proportions and biomass of the two types of microorganisms (EAB and microorganisms without electrochemical activity) in MFC under microgravity conditions may change, thereby affecting the performance of MFC. SUMMARY

[0007] To solve this problem, the present application reports a method for improving MFC electricity generation, electrochemically active microbial abundance and biomass under simulated microgravity, which specifically comprises:

[0008] The method for improving the electricity generation, electrochemically active microorganism abundance and biomass of a microbial fuel cell under simulated microgravity conditions, characterized in that: a microbial fuel cell for mixed culture is constructed and started under 1g gravity conditions, and after the microbial fuel cell is started, the microbial fuel cell is placed under simulated microgravity conditions for operation, under the simulated microgravity conditions, the electricity generation of the microbial fuel cell is increased; the principle of the method is that: the microbial fuel cell for mixed culture contains electrochemically active microorganisms and non-electrochemically active microorganisms, under the simulated microgravity conditions, the total biomass of the microbial fuel cell is increased, and the abundance of the electrochemically active microorganisms is increased, so that the biomass of the electrochemically active microorganisms is increased, resulting in the increased electricity generation of the microbial fuel cell.

[0009] The microbial fuel cell comprises a biological anode and a chemical cathode, wherein the biological anode is derived from mixed bacteria in a natural anaerobic environment, and the electron acceptor of the chemical cathode is potassium ferricyanide.

[0010] The simulated microgravity conditions are realized by a three-dimensional rotator, the rotation speed of the three-dimensional rotator is 0.3-15 RPM, the rotation is 9-degree-of-freedom rotation, and the center gravity vector cumulative value is 0.

[0011] The electrochemically active microorganisms at the genus level include Geobacter, and the non-electrochemically active microorganisms include Rhodobacter, Nitratireductor, Arcobacter, Dysgonomonas, Azonexus, Lentimicrobium, Sphaerochaeta, Cellulomonas, Desulfovibrio and Acidovorax.

[0012] The electrochemically active microorganisms at the species level include Geobacter sulfurreducens, Geobacter sp.DSM9736, Geobacter pickeringii, Geobacter uraniireducens, Geobacter sp.OR-1 and Geobacter metallireducens, and the non-electrochemically active microorganisms include Bacteroidia bacterium 44-10, Azonexus hydrophilus, Lentimicrobium saccharophilum, Sphaerochaeta pleomorpha and Sphaerochaeta globosa.

[0013] Compared with the prior art, the present application discloses for the first time the effect of simulated microgravity on MFC electricity generation, and reveals the biological mechanism of simulated microgravity for improving MFC electricity generation, i.e. simulated microgravity can improve the total biomass of MFC and the proportion of EAB in the mixed bacteria, resulting in the increase of EAB biomass in MFC and further the increase of electricity generation. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 The photos of the microbial fuel cell in Example 1 of the present application and the running under simulated microgravity

[0015] Figure 2 The effect of simulated microgravity on MFC electricity generation in Example 1 of the present application

[0016] Figure 3 The effect of simulated microgravity on the internal resistance and composition of MFC in Example 1 of the present application

[0017] Figure 4 The effect of simulated microgravity on the biomass and three-dimensional structure of biofilm of MFC in Example 1 of the present application

[0018] Figure 5 The effect of simulated microgravity on the abundance of electrochemically active microorganisms in MFC in Example 1 of the present application DETAILED DESCRIPTION

[0019] Four groups of MFCs (MFCs 1-4) were constructed, each group of MFCs having the same size, and the physical diagram is shown in Fig. 1a. The anode chamber and the cathode chamber of the MFCs are square, and the inside is a hollow cylinder. The working volume of the anode chamber and the cathode chamber is about 28 mL, and they are separated by a proton exchange membrane. The anode of the MFC is a piece of 2 cm*2 cm carbon cloth, and the cathode is a piece of 2 cm*2 cm carbon paper. Each MFC was inoculated with the effluent of the seed MFC anode which had been stably running for a long time in the laboratory, and the inoculation ratio was 50%. The MFC was fed with an anode liquid containing 10 mM sodium acetate, while the cathode liquid contained 10 mM potassium ferricyanide. The external resistance of the MFC was 1000 Ω, and when the external voltage dropped to <10 mV, the fresh anode liquid was completely replaced. Each liter of anode liquid contained 0.82 g NaAc, 5.85 g NaCl, 0.13 g KCl, 0.31 g NH4Cl, 6.08 g NaH2PO4·2H2O, 21.83 g Na2HPO4·12H2O, 12.50 mL of mineral solution and 5.00 mL of vitamin solution. Each liter of cathode liquid contained 3.29 g K3[Fe(CN)6], 5.85 g NaCl, 6.08 g NaH2PO4·2H2O and 21.83 g Na2HPO4·12H2O.

[0020] After inoculation, all four MFC groups were run at room temperature (approximately 16-23°C). Once each MFC group had started, it was randomly divided into two groups: the Exp group (MFCs1-2) and the Control group (MFCs3-4). First, under normal gravity conditions (1g), both the Exp group (MFCs1-2) and the Control group (MFCs3-4) were run for one cycle, and the voltage of each MFC group for one cycle was collected. Then, the Exp group was placed on a three-dimensional rotator to simulate microgravity conditions (μg) and run... Figure 1 (b) The Control group continued to operate under 1g conditions, and each group's MFC ran for 6 cycles, with voltage data collected for each MFC during the 6 cycles. During the 6 operating cycles, except for necessary fluid changes, the Exp group operated under μg conditions.

[0021] Figure 2 The electricity generation of each group of MFCs under 1g and μg conditions is shown. For example... Figure 2 As shown in Figure a, the power generation of the Exp and Control groups under 1g conditions is basically similar, with peak voltages of 542.5mV and 527.6mV, respectively, and power generation of 1.06c and 0.99c per cycle, respectively. When the Exp group runs for the first cycle under μg conditions, the power generation of the Exp and Control groups remains similar, with the peak voltage of the Exp group (618mV) slightly higher than that of the Control group (562.6mV), but the power generation of the two MFC groups is basically the same, at 1.15c and 1.16c, respectively. Interestingly, as the Exp group runs under μg conditions, the difference in power generation between the two MFC groups becomes more obvious. Specifically, after the Exp group runs for 6 cycles under μg conditions, the peak voltage of the Exp group reaches 649mV, while that of the Control group is only 560mV. In this cycle, the power generation of the Exp group reaches 1.63c, while that of the Control group is only 1.13c. Summarizing these results, it can be found that the power generation of the Exp group is basically similar to that of the Control group under 1g conditions, while the power generation under μg conditions is significantly higher than that of the Control group, indicating that simulated microgravity improves the power generation of MFC.

[0022] After 6 cycles, the polarization curves and electrochemical impedance spectroscopy (EIS) of each group of MFCs were tested. The polarization curves were measured by the variable resistance method. First, each group of MFCs was replaced with fresh medium and left open for 3 h. Then, each group of MFCs was connected to a variable resistance box, and the external resistance was gradually reduced from 90,000 Ω to 10 Ω. The step size was 10,000 Ω from 90,000 Ω to 10,000 Ω, 2,000 Ω from 10,000 Ω to 1,000 Ω, 200 Ω from 1,000 Ω to 100 Ω, and 10 Ω from 100 Ω to 10 Ω. Each external resistance value was maintained for 5 min, and the output voltage after 5 min was recorded. The electrochemical impedance spectroscopy (EIS) of each group of MFCs was measured using a two-electrode system with a perturbation voltage of 10 mV and a scan range of 10 KHz to 100 mHz. The analysis method was based on previous literature, and the fitting model was Rs + Ra | Qa + Rc | Qc, where Rs was the ohmic resistance, Ra and Rc were the charge transfer resistances of the anode and cathode, and Qa and Qc represented the double-layer capacitances of the anode and cathode.

[0023] Figure 3 a-b shows the results of the polarization curve test of each group of MFCs. As shown in Figure 3 b, after 6 cycles of simulated microgravity conditions, the maximum power density of the Exp group reached 0.50 mW, while the maximum power density of the Control group was only 0.31 mW. This indicates that the maximum power generation performance of MFCs running under simulated microgravity conditions is 1.61 times that of MFCs running under 1 g conditions. The higher power generation of MFCs is due to the lower internal resistance. By testing EIS and fitting, it can be found that. Compared with the Exp group and the Control group, Rs and Rc did not show significant differences. However, the Ra of the Exp group was only 6,065.8 Ω, while the Ra of the Control group reached 9,863.9 Ω. Ra represents the charge transfer resistance of the anode. This indicates that under simulated microgravity conditions, the MFC anode has higher electrochemical activity and lower charge transfer resistance, resulting in higher power generation performance. Since the MFC anode is a biological electrode, the electrochemical activity and charge transfer resistance are directly related to the biomass and EAB abundance of the anode microorganisms. Therefore, it can be reasonably guessed that simulated microgravity conditions may affect the biomass and abundance of EAB in the MFC anode, and further analysis is needed to determine the specific effects.

[0024] After the polarization curve and electrochemical impedance spectroscopy tests, the anodes of each group of MFCs were divided into three parts, one part was used for confocal laser scanning microscope (CLSM) observation, one part was used for metagenomics test, and one part was used for metaproteomics test. CLSM observation includes the following process: the anode sample is first dyed with live / dead bacteria staining kit for 5 min, then washed with 100 mM PBS for 3 times to remove excess dye, and finally sliced and observed with laser confocal scanning microscope. The excitation / absorption wavelengths of the two fluorescent dyes SYTO9 and PI are 480 nm / 530 nm and 488 nm / 660 nm respectively. At least 3 random observation points are selected for each sample, and Z-stack mode is used for scanning, and the scanning results are reconstructed in three dimensions. Figure 4 The results of CLSM observation are shown in Figures 2a and 2b, Figure 4 a and 4b are the Exp group and the Control group respectively. Compared with Figure 4 As can be seen from Figures 2a and 2b, the Exp group has a higher fluorescence intensity, and the mean fluorescence intensity (Mean FI) is 1.21 times that of the Control group, which indicates that the simulated microgravity condition has increased the biomass to a certain extent. At the same time, it can be seen that the 3D metabolic structure volume (Volume) of the Exp group is significantly lower than that of the Control group, indicating that the thickness of the biofilm under simulated microgravity conditions is thinner. Therefore, it can be concluded that the simulated microgravity condition slightly increases the biomass of the MFC and significantly changes the structure of the MFC anode biofilm. Considering that the power generation of the MFC under simulated microgravity conditions is greatly improved, while the biomass is only slightly increased, it can be reasonably speculated that the increase in power generation of the MFC under simulated microgravity conditions is not only due to the increase in biomass, but also possibly due to changes in the community structure.

[0025] The metagenomics test mainly includes: DNA extraction, library construction, on-machine sequencing, raw data quality control, data assembly after quality control, microbial species annotation and gene function annotation. Figure 5 c shows the Detrended correspondence analysis (DCA) dimensionality reduction analysis of the elimination trend of 4 MFC samples at the Unigene level. It can be seen that there are obvious differences in the metagenomics results of the two groups of samples, indicating that the types of microorganisms and functional genes under simulated microgravity conditions have changed significantly. Figure 5aThe analysis at genus level showed that the most abundant genus in both Exp and Control groups was Geobacter. Geobacter is a widely reported EAB and is also the most common EAB in MFC, which is considered as the core microorganism to maintain electricity production in MFC. It is worth noting that the abundance of Geobacter was significantly different in the two groups of MFC, which was only 34.5% in the Control group and reached 55.7% in the Exp group, indicating that the abundance of EAB was significantly increased under simulated microgravity conditions. In addition to Geobacter, other types of microorganisms were also present in both groups of MFC, including Rhodobacter, Nitratireductor, Arcobacter, Dysgonomonas, Azonexus, Lentimicrobium, Sphaerochaeta, Cellulomonas, Desulfovibrio and Acidovorax. Since Geobacter can directly utilize sodium acetate in the anolyte to produce electricity, the end product is CO2, so it does not need to cooperate with other microorganisms to produce electricity. At the same time, there is no research report that the microorganisms of these genera can directly produce electricity, so these microorganisms are considered to be microorganisms without electrochemical activity, and the abundance of these microorganisms under simulated microgravity conditions has decreased.

[0026] Figure 5bThe results of each group of MFCs at the species level are shown. Among the groups of MFCs, Geobacter species mainly include Geobacter sulfurreducens, Geobacter sp. DSM 9736, Geobacter pickeringii, Geobacter uraniireducens, Geobacter sp. OR-1 and Geobacter metallireducens, Geobacter soil. By comparing the Exp group and the Control group, it can be found that all species of Geobacter are enriched under simulated microgravity conditions, except for Geobacter soil. In particular, Geobacter sp. OR-1, which had an abundance of only 6.0% in the Control group, reached 26.3% in the Exp group. At the same time, other microorganisms that have not been reported to have electrochemical activity, including Bacteroidia bacterium 44-10, Azonexus hydrophilus, Lentimicrobium saccharophilum, Sphaerochaeta pleomorpha and Sphaerochaeta globosa, significantly reduced in abundance. Therefore, it can be concluded that simulated microgravity conditions significantly increase the abundance of EAB in MFCs, which is the main reason for the improvement of MFC power generation performance under simulated microgravity conditions.

Claims

1. A method for simulating microgravity to improve the abundance and biomass of microbial fuel cells in terms of power generation and electrochemical activity, characterized in that: A microbial fuel cell with mixed bacterial culture was constructed and started under 1g gravity conditions. After the microbial fuel cell was started, it was placed under simulated microgravity conditions. Under simulated microgravity conditions, the power generation of the microbial fuel cell increased. The principle of the method is that the microbial fuel cell with mixed bacterial culture contains electrochemically active and non-electrochemically active microorganisms. Under simulated microgravity conditions, the total biomass of the microbial fuel cell increases, and the abundance of electrochemically active microorganisms increases. Therefore, the increased biomass of electrochemically active microorganisms leads to increased power generation of the microbial fuel cell. The simulated microgravity conditions are achieved by a three-dimensional rotator with a rotation speed of 0.3-15 RPM, a 9-degree-of-freedom rotation, and a central gravity vector cumulative value of 0.

2. The method for improving the abundance and biomass of microbial microorganisms in a microbial fuel cell by simulating microgravity as described in claim 1, wherein the microbial fuel cell includes a bioanode and a chemical cathode, the bioanode is a mixed culture from a natural anaerobic environment, and the electron acceptor of the chemical cathode is potassium ferricyanide.

3. The method for improving the power generation, electrochemically active microbial abundance, and biomass of microbial fuel cells by simulating microgravity as described in claim 1, wherein the electrochemically active microorganisms include Geobacter at the genus level, and the non-electrochemically active microorganisms include Rhodobacter, Nitratireductor, Arcobacter, Dysgonomonas, Azonexus, Lentimicrobium, Sphaerochaeta, Cellulomonas, Desulfovibrio, and Acidovorax.

4. The method for determining the electricity generation, electrochemically active microbial abundance, and biomass of microbial fuel cells as described in claim 1, wherein the electrochemically active microorganisms include, at the species level, *Geobacter sulfurreducens*, *Geobacter sp. DSM9736*, *Geobacter pickeringii*, *Geobacter uraniireducens*, *Geobacter sp. OR-1*, and *Geobacter metallireducens*, and the non-electrochemically active microorganisms include *Bacteroidia bacterium 44-10*, *Azonexus hydrophilus*, *Lentimicrobium saccharophilum*, *Sphaerochaeta pleomorpha*, and *Sphaerochaeta globosa*.

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