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Massive emission of CO2 into atmosphere from consumption of carbon deposit is causing climate change. Researchers have applied metabolic engineering and synthetic biology techniques for improving CO2 fixation efficiency in many species. One solution might be the utilization of autotrophic bacteria, which have great potential to be engineered into microbial cell factories for CO2 fixation and the production of chemicals, independent of fossil resources. In this work, several pathways of Ralstonia eutropha H16 were modulated by manipulation of heterologous and endogenous genes related to fatty acid synthesis. The resulting strain B2(pCT, pFP) was able to produce 124.48mg/g (cell dry weight) free fatty acids with fructose as carbon source, a fourfold increase over the parent strain H16. To develop a truly autotrophic fermentation technique with H-2, CO2 and O-2 as substrates, we assembled a relatively safe, continuous, lab-scale gas fermentation system using micro-fermentation tanks, H-2 supplied by a hydrogen generator, and keeping the H-2 to O-2 ratio at 7:1. The system was equipped with a H-2 gas alarm, rid of heat sources and placed into a fume hood to further improve the safety. With this system, the best strain B2(pCT, pFP) produced 60.64mg free fatty acids per g biomass within 48h, growing in minimal medium supplemented with 9x10(3)mL/L/h hydrogen gas. Thus, an autotrophic fermentation technique to produce fatty acids was successfully established, which might inspire further research on autotrophic gas fermentation with a safe, lab-scale setup, and provides an alternative solution for environmental and energy problems.