Trace metals play an important role in the growth of methanogens and methane formation. In order to understand the requirement of trace metals for methanogens, the accurate measurement of trace metals inside cells of methanogens was required. In this study, acclimated-methanogens were cultured in batch feed substrate gas (H-2/CO2) at 37degreesC. The balance and uptake of trace metals for cell of acclimated-methanogens were investigated by using inductively coupled plasma mass spectrometer (ICP-MS) and scanning probe microscopy. Cells of the acclimated-methanogens were harvested in the early stationary growth phases and separated by centrifugation and filtration using cellulose membrane under anaerobic condition. In order to remove trace metal ions on the surface of the cells, it was washed with different liquids-distilled water, physiological salt solution, phosphate buffer and EDTA mixture liquid, in different experiments. By using scanning probe microscopy, we found that cell lysis occurred and thus transitional trace metals severely lost when cells were washed with distilled water, while the minimum washing which effect cell lysis was obtained by using phosphate buffer. Washed cells were broken by wet digestion method and analyzed for the content of trace metals by using ICP-MS. The major elements contained in the methanogens cell were obtained, and it was clear that, for conventional method, the shortage of Fe will be created when methanogens grew up to about 1.32 g l(-1), and Zn, Cu, Ni and Co contents will be lacked when methanogens grow up to 1.13, 0.12, 4.8 and 30 g l(-1), respectively. In conclusion, to achieve a high productivity of methane fermentation, Fe, Cu, Ni and Zn contents in the fermentation liquid should be greatly increased. (C) 2003 Elsevier Ltd. All rights reserved.