394 Marine Microbiology 



The transformation of ammonia to nitrite has been shown to be 

 stoichiometric with the optimal reaction taking place between 

 pH 7.5 to 8.8 and at a substrate concentration of 3 to 5 X 10"^ 

 M (16, 26, 32). According to Hofman and Lees (22) the efficien- 

 cy with which the organism uses energy released during the 

 above reaction in order to assimilate CO- falls off as the concen- 

 trations of the reaction product ( NO2" ) and cell carbon increase 

 in the culture. This apparent decrease in free energy efficiency 

 has been attributed to an expenditure of energy in order to off- 

 set the tendency of the nitrite accumulating in the growth med- 

 ium to enter the cell. According to Engel and Alexander 

 (16), however, the resulting increase in nitrite concentration has 

 no apparent effect on substrate oxidation and cell proliferation 

 indicating that during the active period of cell growth, the free 

 energy efficiency of Nitrosomonas remains unchanged. 



Some 35 years ago, Kluyver and Donker (27) hypothesized 

 that the oxidation of ammonia takes place in three steps, each 

 consisting of two-electron changes: ammonia -> hydroxylamine 

 -^ hyponitrite -^ nitrite. The stoichiometric formation of nitrite 

 in the course of ammonia oxidation indicates that there is very 

 little, if any, accumulation of the intermediates during the re- 

 action (23). The use of suitable inhibitors which act selectively 

 on particular steps in the above suggested series of reactions has 

 proven to be a useful means of accumulating intermediates during 

 the reaction sequence. The first experimental evidence pointing 

 to hydroxylamine as an intermediate of ammonia oxidation was 

 demonstrated by Hofman and Lees (23) and has since been 

 confirmed by other workers (16-18, 24, 25, 34, 35). In the pres- 

 ence of 3 X 10-3 M hydrazine, the oxidation of ammonia by 

 Nitrosomonas cells leads to the accumulation of hydroxylamine 

 in the medium by apparently inhibiting the further metabolism 

 of hydroxylamine to nitrite. The observed effect of 10"^M 

 thiourea or allylthiourea in depressing the oxidation of ammonia 

 but not of hydroxylamine by Nitrosomonas also implicates hy- 

 droxylamine as an intenmediate in the nitrification process (21, 

 23). Moreover, Nitrosomonas cells, upon aging under refrigerated, 

 frozen or lyophilized conditions, lose their ammonia oxidizing 

 ability at a faster rate than that of hydroxylamine oxidation (9, 



