Biomethane Generation Mechanism Based On Anaerobic Digestion Under Coal Reservoirs Conditions And Microbial Electrolytic Cell

China has been exploring and developing coalbed methane（CBM）for more than 40years.However,the commercial development of CBM needs to be further improved,especially for deep CBM development,which is in urgent need of new stimulation techniques.Additionally,the proposal of China’s dual carbon goal urgently requires CCUS technology that can be commercialized.The birth of coalbed gas bioengineering（CGB）provides an effective way to increase coalbed methane production and reduce carbon dioxide emission.Microbe is the cheapest"labor force"in nature,as long as there is a suitable environment and enough nutrients,it will conscientiously"work".This cheap labor force is also the core of CGB to achieve coal biogasification and CO₂ methanation.Previous the field tests involved injecting the nutrient solution required for microbial gas production into the coal seam to generate biogas through anaerobic digestion.However,combined with the coal reservoir reconstruction technology,there are few reports on the engineering test of injecting bio-fracturing fluid containing high efficiency bacteria into coal reservoir through large-scale hydraulic fracturing.Also,related theories and technologies still need to be systematically studied.For example,how microorganisms perform anaerobic digestion of coal in the in situ environment of coal reservoirs conditions to produce methane,and the mechanism of biogas generation in this high-pressure environment is not clear.The mechanism of CBM stimulation and CO₂ reduction under the synergistic effect of microbes-mediated CO₂ in coal reservoirs still needs further investigation.Microbial electrochemistry has been shown to be effective in promoting methane production from anaerobic digestion of coal under atmospheric pressure conditions,whether the same can promote biomethane production under simulated in situ coal reservoirs conditions needs to be further investigated.In this paper,the mechanism of biomethane generation under in situ coal reservoirs conditions is investigated by combining indoor experiments with theoretical analysis,guided by theories of CBM geology,microbial electrochemistry and molecular biology.Furthermore,the promotion mechanism of microbial electrolysis cell（MEC）for biomethane production under in situ coal reservoirs conditions was investigated.Finally,the study explores the mechanisms of CBM stimulation and carbon reduction potential under the synergistic effect of microbes-mediated CO₂ in coal reservoirs,leading to several main conclusions:（1）The evolution of gas-solid-liquid-bacteria in anaerobic digestion systems with the participation of supercritical CO₂（SY）and gaseous CO₂（GY）was investigated by using self-developed anaerobic digestion device simulating in situ conditions of coal reservoirs.The results indicate that the stressing effect of supercritical CO₂ on the microbial community in SY system will be gradually lifted with the anaerobic digestion,and the diversity and abundance will be gradually restored.Supercritical CO₂ acts as a hydrolytic microbe in the early stages of anaerobic digestion and works in combination with Paraclostridium and Trichococcus adapted to the environment to ensure proper methane production from anaerobic digestion.The electron donors provided by supercritical CO₂ extracts,CO₂,and small molecule acids and alcohols provide a good buffer for archaea to cope with extreme environments,making the recovery period of archaeal communities shorter than that of bacterial communities.The biomethane production of SY system was 13.24 m L/g,of which the biotransformation of CO₂ reached10.94 m L/g.The stressing effect of gaseous CO₂ on the microbial community in the GY system was weaker than that of supercritical CO₂,and the bacterial and archaeal communities were able to resume hydrolysis and acid-producing fermentation quickly,with a final biomethane production of 8.02 m L/g,of which the biotransformation of CO₂reached 4.48 m L/g.Biomethane in SY and GY systems is derived from microbial degradation of the substrate coal on the one hand,and from CO₂ on the other hand,with small molecule organic acids and alcohols providing electrons for the methanation of CO₂.Paraclostridium and Trichococcus were active throughout the anaerobic digestion process in SY and GY systems,and they both belonged to Gram-positive bacteria with tolerance to extreme environments,which not only ensured the normal hydrolysis stage in SY and GY systems,but also confirmed that the hydrolysis of coal accompanied the whole anaerobic digestion process.（2）The effect of MEC on gas-solid-liquid-bacteria in SY and GY systems and the electrochemical characteristics of their anode biofilms were studied using the self-developed microbial electrolysis cell and anaerobic digestion coupled system（MEC-AD）under coal reservoirs conditions.The results showed that the hydrolysis of coal in the MEC-SY system was further improved based on the supercritical CO₂ extraction,and the MEC attenuated the stressing effect of supercritical CO₂ to a certain extent to protect the bacteria community.27%of bacteria on anode biofilm adapted to the environment survived and these microorganisms adapt to their environment by secreting polysaccharides and lipids for self-protection.Meanwhile,Geobacter and Methanosaeta on anode biofilms facilitate CO₂ reduction by establishing direct interspecies electron transfer（DIET）.The biomethane yield in the MEC-SY system was enhanced by 14.35%compared to the SY system with a yield of 15.14 m L/g.The bioconversion of CO₂ in the MEC-SY system increased by 18.64%compared to the SY system,with a bioconversion of 12.98 m L/g.In the MEC-GY system,on the one hand,MEC stabilizes the activity of microbial communities,on the other hand MEC strengthens the synergy between electroactive microorganisms and hydrolysis and acid-producing fermentation bacteria in the system,thus promoting the hydrolysis of coal.The biomethane yield in the MEC-GY system increased by 22.94%compared to the GY system,with a yield of 9.86 m L/g,where the bioconversion of CO₂ increased by 89.28%compared to the GY system,with a conversion of 8.48 m L/g.In addition,the percentage of live bacteria on the MEC-GY anode biofilm was 51.85%higher than that on the MEC-SY anode biofilm,and the uniform distribution of biofilms and the content of proteins that play a role in electron transfer in 56%of them.This promotes the efficiency of electron transfer between biofilm and electrode and determines the higher improvement of the MEC-GY system compared to the MEC-SY system.（3）SY,GY,MEC-SY,MEC-GY systems were subjected to comparative analysis based on metagenomics and LC-MS metabolomics techniques.Multivariate statistics and differential analysis were carried out from the aspects of the number of species stimulated by different pressures,different atmospheres and external electric fields,the abundance of genes involved in related enzymes in key metabolic pathways,and extracellular metabolites.The results showed that the microorganisms in the high-pressure environment participated by CO₂ showed negative effects on the relevant metabolism and signal transduction of the cell wall and cell membrane.This leads to the need for microorganisms to make corresponding adaptations and adjustments in the process of participating in the microbial degradation of coal,prolonging the cycle of coal anaerobic digestion.To further clarify the effects of pressure,gas composition and CO₂ phase state on the bacterial species and metabolism,the inert gas was used as a control to conduct research.The results show that the gas atmosphere is more likely to affect microorganisms than pressure at the same pressure,and the phase state of CO₂ is also a key factor affecting the number of the microbial community.The external field promoted the synthesis of the enzymes involved in cytochrome c,NADH dehydrogenase,succinate-Q reductase,and quinone-related enzymes on the one hand,thus enhancing extracellular electron transfer.On the other hand,the anodic biofilm in both the MEC-SY and MEC-GY systems promoted biomethane production in the form of DIET.In addition,the functional genes of the microbial community after the stimulation of the applied electric field were strengthened with relatively vigorous metabolism,which also provided ideas and microbes sources for the CGB.From the differential metabolite statistics,different phases of CO₂ have a greater impact on the differential metabolites of the AD system,and the combined effect of supercritical CO₂ extraction and microorganisms is irreplaceable by other means and is engineeringly implementable.In addition,the MEC has a higher improvement space for the GY system than the SY system,while being able to compensate for the lack of supercritical CO₂ extraction on anaerobic digestion metabolites.（4）This study proposes a mechanism for coalbed methane（CBM）stimulation through the synergistic effect of microbes-mediated CO₂ in coal reservoirs.In shallow coal reservoirs,CBM stimulation through the biogas enhancement,reservoir pressure enhancement and microbial reservoir modification involving anaerobic digestion of CO₂.In the middle coal reservoir,the combined action of supercritical CO₂ extraction and microorganisms can stimulate CBM through the biogas enhancement,reservoir pressure enhancement and microbial reservoir dual modification.Meanwhile,CO₂ microbial methanation achieves both CBM stimulation and carbon negative reduction in shallow and central coal reservoirs.Modification of coal reservoirs directly by supercritical CO₂extraction at depth to achieve CBM stimulation.Therefore,with the participation of microorganisms,the combination of CO₂ and CBM development achieves both increased CBM stimulation and low-negative carbon emission reduction.Ultimately,this study can be summarized as“four transformations”,the combination of microorganisms and CO₂transforms coal liquefaction from a single microbial hydrolysis to two pathways:microbial hydrolysis and supercritical CO₂ extraction.The carbon source of methane is converted from coal to a dual carbon source of coal and CO₂.CBM stimulation from a single enhancement in biogas to reservoir modification and biogas enhancement.CBM development is transformed from a single low-carbon energy acquisition to a dual purpose of low-carbon energy acquisition and CCUS.There are 85 figures,74 tables and 232 references in this thesis.