目前，我国针对CO₂含量上升的问题提出“3060双碳目标”。现已开发出如碳捕获和储存（Carbon Capture and Storage，CCS）及碳捕获和利用（Carbon Capture and Utilization，CCU）等技术。煤矿采空区由于其存贮空间大、存储能力强等优势被认为具有巨大的CO₂封存潜力，且在采空区封存CO₂有助于降低煤自燃风险。但现有采空区CO₂封存技术仅能做到一次封存，且CO₂泄漏问题难以解决。对此，本文提出将微生物固碳技术应用于采空区中以解决上述问题。本实验的开展对后续拓展采空区微生物固碳技术具有重要的指导意义。

本文煤矿环境样品为固碳微生物提取源，通过理论与实验相结合的方法对分离驯化出的2种固碳微生物进行了生长周期测定、培养基和培养条件优化，模拟含煤环境对微生物的生长及固碳能力进行判断，并系统分析了固碳菌在含煤环境下的生长及CO₂固化特性。主要结论如下：

（1）在避光条件下成功分离出4种固碳微生物。采用富集、梯度稀释、涂布、划线、驯化等方法分离出单菌，经16S rDNA鉴定分别为铜绿假单胞菌、贝莱斯芽孢杆菌、嗜酸寡养单胞菌、溶酚假单胞菌。进一步研究发现，这些微生物在以CO₂气体为唯一碳源的无机液体培养基中生长周期有所差异，其中溶酚假单胞菌表现出了二次生长的特征，其余3种微生物在120 h内均进入衰亡期。

（2）采用单因素实验和响应面法对2种固碳菌进行了培养基优化。最优培养基组分和浓度如下：①铜绿假单胞菌为MnSO₄ 0.02 g/L、Na₂HPO₄ 1 g/L、KH₂PO₄ 1.8 g/L、MgSO₄ 0.3 g/L、CaCl₂ 0.05 g/L、NaCl 1 g/L、KNO₃ 0.896 g/L、FeCl₃ 0.0421 g/L、Na₂S₂O₃ 8.377 g/L、生物素5×10^-4 g/L；②溶酚假单胞菌为MnSO₄ 0.02 g/L、Na₂HPO₄ 1 g/L、KH₂PO₄ 1.8 g/L、MgSO₄ 0.5 g/L、CaCl₂ 0.05 g/L、NaCl 1 g/L、KNO₃ 0.94 g/L、FeCl₃ 0.03 g/L、Na₂S₂O₃ 12 g/L。

（3）针对不同培养条件对目标固碳菌的生长及CO₂固化能力进行了分析。发现两种固碳菌的固碳率在培养时间为48 h时存在最大值，CO₂固定率分别能达到98.13%和98.67%，即固定了57.28 mg和57.65 mg的CO₂，此时的平均单位菌液固碳速率分别为21.70 mg·L^-1·h^-1和21.84 mg·L^-1·h^-1。两种菌最适宜的pH为8、培养温度为35 ℃、接种量为15%、CO₂初始浓度为25%。

（4）模拟含煤环境实验发现固碳菌和煤粉可以进行协同固碳。添加1 g的煤粉时，2种固碳菌在48 h分别固定了44.71 mg和47.71 mg的CO₂，加入5 g煤粉时为20.58 mg和22.61 mg，均优于无菌对照组。表明添加煤粉后，该环境下固碳菌的生化反应和煤的吸附作用可以协同固定CO₂。

本文成功分离出了固碳微生物，并优化了培养基组成和培养条件，提高了固碳菌的生长活性，掌握了固碳菌在含煤环境下的CO₂固化特性。本实验的开展为采空区固碳技术和助力达成“双碳”目标提供了创新性的思路和解决方案，为后续研究打下坚实的理论基础与技术支撑。

At present, China has proposed the "3060 dual-carbon target" in response to the problem of rising CO₂ levels. Technologies such as Carbon Capture and Storage (CCS) and Carbon Capture and Utilization (CCU) have been developed. Coal mine goaf is considered to have huge potential for CO₂ storage due to large storage space and high storage capacity. And storing CO₂ in goaf helps reduce the risk of spontaneous combustion of coal. However, the existing goaf CO₂ sequestration technology can only achieve one-time sequestration, and the problem of CO₂ leakage is difficult to solve. In this paper, microbial carbon sequestration technology is proposed to be applied to the goaf to solve the above problems. This experiment is of great significance for the subsequent expansion of microbial carbon sequestration technology in Goaf.

In this paper, coal mine environmental samples were used as the extraction source of carbon-sequestering microorganisms. By combining theoretical and experimental methods, we measured the growth cycle, optimized the culture medium and culture conditions of the two carbon-sequestering microorganisms isolated and domesticated, and simulated the coal-containing environment to judge the growth and carbon sequestration ability of the microorganisms. The growth and CO₂ solidification characteristics of the carbon-sequestering microorganisms in the coal-containing environment were also systematically analyzed. The main conclusions are as follows:

(1) Four species of carbon-sequestering microorganisms were successfully isolated under light-avoidance conditions. Single bacteria were isolated by enrichment, gradient dilution, coating, scribing, and domestication. They were identified by 16S rDNA as Pseudomonas aeruginosa DSM 50071, Bacillus velezensis CBMB205, Stenotrophomonas acidaminiphila AMX 19, Pseudomonas phenolilytica RBPA9. Further studies revealed that these microorganisms differed in their growth cycles in an inorganic liquid medium with CO₂ gas as the sole carbon source. Among them, Pseudomonas phenolilytica showed secondary growth and the remaining three microorganisms entered into the decay phase within 120 h.

(2) The medium optimization of two carbon fixing bacteria was carried out by one-way experiment and response surface method. The optimal medium components and concentrations were as follows: ① Pseudomonas aeruginosa: MnSO₄ 0.02 g/L, Na₂HPO₄ 1 g/L, KH₂PO₄ 1.8 g/L, MgSO₄ 0.3 g/L, CaCl₂ 0.05 g/L, NaCl 1 g/L, KNO₃ 0.896 g/L, FeCl₃ 0.0421 g/L, Na₂S₂O₃ 8.377 g/L, D-Biotin 5×10^-4 g/L; ② Pseudomonas phenolilytica：MnSO₄ 0.02 g/L, Na₂HPO₄ 1 g/L, KH₂PO₄ 1.8 g/L, MgSO₄ 0.5 g/L, CaCl₂ 0.05 g/L, NaCl 1 g/L, KNO₃ 0.94 g/L, FeCl₃ 0.03 g/L, Na₂S₂O₃ 12 g/L.

(3) The growth and CO₂ fixation ability of the target carbon-fixing bacteria were analyzed under different culture conditions. It was found that the carbon fixation rates of the two types of carbon-fixing bacteria were maximized at the incubation time of 48 h. The CO₂ fixation rates reached 98.13% and 98.67%, respectively, while 57.28 mg and 57.65 mg of CO₂ were fixed. The average CO₂ fixation rates were 21.70 mg·L^-1·h^-1 and 21.84 mg·L^-1·h^-1. The optimal pH was 8, the incubation temperature was 35 ℃, the inoculum size was 15%, and the initial CO₂ concentration was 25%..

(4) The simulated coal-containing environment revealed that the carbon-sequestering bacteria and coal dust could carry out synergistic carbon sequestration. Two kinds of carbon-fixing bacteria fixed 44.71 mg and 47.71 mg of CO₂ in 48 h when adding 1 g of coal dust. The CO₂ fixation was 20.58 mg and 22.61 mg at 5 g adding amount. They were better than the sterile control group. The results showed that the biochemical reaction of the carbon-fixing bacteria and the adsorption of coal could fix CO₂ synergistically in this environment after the addition of coal powder.

In this paper, carbon-sequestering microorganisms were successfully isolated. The medium composition and culture conditions were optimized to improve the growth activity of the carbon sequestering bacteria, and the CO₂ curing characteristics of the carbon sequestering bacteria in the coal-containing environment were mastered. This experiment provides innovative ideas and solutions for the carbon sequestration technology in the air-mining area and helps achieve the "dual-carbon target". It lays a solid theoretical foundation and technical support for subsequent research.