The objective of this study was to examine the effects of nitrate on methane production, important fermentation characteristics, Fibrobacter succinogenes, Ruminococcus albus, Ruminococcus flavefaciens, total bacteria, and methanogens using in vitro ruminal cultures. Potential adaptation of the above microbes and persistency of nitrate to mitigate CH(4) production were also evaluated. Methane production was reduced by 70% at 12 mu mol ml(-1) and nearly completely at >= 24 mu mol ml(-1) nitrate. Production of volatile fatty acids (VFAs) was affected to different extents at different nitrate concentrations. Over a series of six consecutive cultures receiving 12 mu mol ml(-1) nitrate, production of CH(4) and VFA did not change significantly. R. albus and R. flavefaciens seemed to adapt to nitrate, while F. succinogenes and methanogens did not. Nitrate may be used in achieving persistent mitigation of CH(4) production by ruminants. Crown Copyright (C) 2011 Published by Elsevier Ltd. All rights reserved.