高效固定CO2的极端微生物选育、基因差异性分析及增效技术研究

In order to solve the technical bottleneck problems for CO2 microbial fixation with high concentration in the extreme environment, we will breed efficient extreme environmental strains by means of membrane filtration, immobilization and gene engineering technologies, carry out the study on molecular biology identification of strains, analyze the genetic differences of CO2 tolerance and fixation force existed between the initial and mutated sera based on rbcl gene sequences, improve resistance genes of strains for CO2 ultimate tolerability, establish the reasonable bacterium group structure in order to realize coordination carbon fixation. ①Design a new training mode, separate microbial strains that can survive well in extreme environment with high CO2 concentration and high temperature and so on. ②Study the genetic structure and composition of strains, breed efficient target strains with high CO2 tolerability and fixation force and with the relative genetic stabiliy by means of the mutation breeding, PCR amplification and cloning and transcription technologies. ③Determinate rbcl gene sequences of strains, complete molecular biology identification of the strains, and explore the mechanism of carbon sequestration force and tolerance improvement of target strains from the view points of molecular systematics. ④Carry out the research of the genetic factors of CO2 toxicity, induce resistance genes with antagonist and detoxicification in the bodies of the target strains to improve their tolerance. ⑤Set up a reasonable bacterium group for collaborative fixing carbon in the common cultivation mode, improve the rate of carbon sequestration. ⑥Test culture characteristics of bacterium group to study the growth control mechanism, and establish an efficient biological system for CO2 fixatiom in extreme environment.

为解决极端环境中高浓度CO2微生物固定的技术瓶颈，利用膜过滤、固定化和基因工程等技术选育高效极端环境菌株，基于rbcl基因序列完成菌株的分子生物学鉴定、耐受性和固定力差异性原因分析，研究固碳株抗性基因以提高极限CO2耐受性，建立合理的菌群结构以实现协同固碳。①设计新型培养模式，分离耐高CO2浓度、高温等极端环境微生物，作为出发株；②研究其基因结构和组成，采用诱变育种、PCR扩增和克隆转录等技术培育具有高耐受性、固碳力的传代稳定的高效目标株；③测定菌株的rbcL基因序列并从系统学角度分析差异性，完成菌株的分子生物学鉴定，从分子学角度探寻目标株的固碳力和耐受性提高的机理；④研究目标株的抗CO2毒性遗传因子，诱导其体内的具有拮抗和解毒作用的抗性基因，提高耐受力；⑤采用共同培养方式建立合理菌群协同固碳，提高固碳率；⑥对菌群进行培养特性测试，研究其生长调控机制，建立高效固定极端环境CO2的生物体系。

本研究的目的是实现极端环境条件中CO2的高效固定。研究中分离和筛选出对极端环境适应性强且有较高CO2耐受性的初选株，研究其基因结构和组成，完成了影响固定CO2的内在因素及高CO2浓度诱导下的细胞蛋白组学分析，获得了提高耐受性和固碳力的有效途径。通过实时定量RT-PCR分析得到细胞内参与光合、与CCM代谢相关的特定基因所对应的转录丰度及途径。根据上述研究结果，通过定点诱变、PCR扩增和克隆转录等基因工程技术手段对出发株进行改造，获得了传代稳定的高效菌株，其中一株的最大CO2固定率达5.883gCO2/(L D)。目标菌株可耐受60%的CO2浓度，在100%浓度条件还能存活。借助基于rbcl基因的分子生物学方法进行了物种鉴定及基因差异性分析，从系统学角度探究了CO2耐受性和固定力提高的机理。在此基础上，从多角度研究提高藻株耐极端环境毒性和CO2固定力增效的途径，包括添加蛋白质合成抑制剂增加光合活性；通过碱液加入、低流速供气方式及高接种浓度等方式调控pH值以抵抗毒性；化学吸收与生物固定耦合的营养循环的半连续混合培养方式增加固碳效果等。设计了三种反应体系，对混合菌种进行较大规模的协同培养，在缩短生长周期、提高CO2固定力和利用率上都取得很好的效果。通过对数据进行过程模拟，验证了所设计的反应体系可以推广到任何尺度，为长期实验和实际应用提供依据。