A novel system integrating an in-situ and ex-situ power-to-gas (PtG) system was developed in the current study. A continuous stirred-tank reactor (CSTR) was operated using cattle manure as substrate at mesophilic temperature (37 °C ± 2 °C). The CH4 content in the biogas was upgraded to above 95% by H2 injection, which meets the highest criteria for grid injection without requiring CO2 removal. Furthermore, the bio-nature gas production was promoted by external CO2 and H2 injection. The volumetric methane production rate (VMPR) was significantly increased by 739% from 117.4 mL L−1·d−1 to 985 mL⋅L−1⋅d−1, which is higher than in other studies. Meanwhile, the volumetric biogas production rate (VBPR) was increased by 36.9% by H2 injection, increasing the conversion efficiency (82.56%) of the chemical oxygen demand (COD) to CH4. A significant increase in the specific methanogenic activity of dissolved hydrogen (SMA(Hdissolved)) and the enrichment in hydrogenotrophic methanogens (Methanobacterium) demonstrate that the CH4 production pathway was converted from acetoclastic methanogenesis (AM) pathway to hydrogenotrophic methanogenesis (HM) pathway. It is postulated that the change in proportion of different pathways of the CH4 production was caused by the strengthening of key enzymes (coenzyme F420 hydrogenase and coenzyme-B sulfoethylthiotransferase) by H2 injection. The integrated system represents a promising approach to achieve simultaneous CO2 emission reduction and bio-natural gas production.
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