Nutrient addition as part of microbial enhanced oil recovery (MEOR) operations have important implications for more energy recovery from oil reservoirs, but very little is known about the in situ response of microorganisms after intervention. An analysis of two genes as biomarkers, mcrA encoding the key enzyme in methanogenesis and fthfs encoding the key enzyme in acetogenesis, was conducted during nutrient addition in oil reservoir. Clone library data showed that dominant mcrA sequences changed from acetoclastic (Methanosaetaceae) to CO2-reducing methanogens (Methanomicrobiales and Methanobacteriales), and the authentic acetogens affiliated to Firmicutes decreased after the intervention. Principal coordinates analysis (PCoA) and Jackknife environment clusters revealed evidence on the shift of the microbial community structure among the samples. Quantitative analysis of methanogens via qPCR showed that Methanobacteriales and Methanomicrobiales increased after nutrient addition, while acetoclastic methanogens (Methanosaetaceae) changed slightly. Nutrient treatment activated native CO2-reducing methanogens in oil reservoir. The high frequency of Methanobacteriales and Methanomicrobiales (CO2-reducers) after nutrient addition in this petroleum system suggested that CO2-reducing methanogenesis was involved in methane production. The nutrient addition could promote the methane production. The results will likely improve strategies of utilizing microorganisms in subsurface environments. (C) 2016, The Society for Biotechnology, Japan. All rights reserved.