Effects of carbon, nitrogen, and electron acceptor availability on anaerobic N2-fixation in a beech forest soil

The effects of different energy sources and inhibitors on the nitrogenase activity of a beech forest soil of north-east Bavaria (Germany) were determined using the acetylene-reduction method. Simple sugars, cellobiose and starch greatly stimulated nitrogenase activity under anaerobic, but not under aerobic, conditions; of the substrates tested, cellobiose yielded the highest anaerobic activity. In contrast, organic acids had no appreciable stimulating effect. Glucose-stimulated anaerobic nitrogenase activity was linked to the formation of butyrate (implicating the involvement of clostridia) and to the apparent growth of anaerobic N2-fixing microorganisms. The N2-fixing microorganisms culturable under anaerobic conditions in unamended soil approximated 105 cells g−1 dry wt soil and increased three orders of magnitude after 2 d of anaerobic incubation with glucose. In contrast, no N2-fixing microorganisms were culturable under aerobic conditions. Inhibition of anaerobic nitrogenase activity by supplemental ammonium and nitrate occurred in both unamended and glucose-amended soils. Nitrate was sequentially transformed to N2O and N2, with minor amounts also being reduced to ammonium. Thus, nitrate appeared to have an indirect inhibitory effect on N2-fixation, the reduction of nitrate being more competitive for available reductant than N2-fixation. This competitive effect was not observed with sulfate. These results suggest that in situ N2-fixation in the forest soil examined may be regulated in part by the availability of specific reductant sources and the presence of the competing reductant sink nitrate.