Nitrobacter would grow in a similar medium containing sodium nitrite instead of ammonium sulphate. There being no organic carbon in these media, the organisms had no source of carbon for their nutrition, except the CO₂ of the air, or possibly that of bicarbonate in solution. It therefore followed that the organisms must obtain their carbon supply from one of these sources. Unlike green plants, the nitrous and nitric organisms are able to carry on this carbon assimilation in the dark, and must therefore obtain the energy needed for the process from some chemical reaction. The only sources of energy in Winogradsky’s solutions were the nitrogen compounds, and it consequently followed that the organisms must derive their energy supply by the oxidation of ammonia and nitrite respectively. The release of energy obtained by these two reactions has been calculated by Orla-Jensen to be as follows:-- (NH₄)₂CO₃ + 3O₂ = 2HNO₂ + CO₂ + 3H₂O + 148 Cals. KNO₂ + O = KNO₃ + 22 Cals. The exact process by which ammonium carbonate is converted into nitrite is not at present known. The two groups of organisms are extremely selective in their source of energy. The nitrous organisms can derive their energy only by the oxidation of ammonia to nitrite, and the nitric organisms only by the oxidation of nitrite to nitrate. In culture media they are, indeed, inhibited by soluble organic compounds such as sugars. Under natural conditions, however, they appear to be less sensitive, since ammonium carbonate is readily nitrified in substrata rich in organic matter. The rapid nitrification that takes place during the purification of sewage is an example of this. The conditions in culture, with regard to aeration and the removal of metabolic products from the neighbourhood of the organisms, are very different from those in the soil, and perhaps account for the discrepancies found. The oxidation of ammonium carbonate by nitrosomonas results in the formation of nitrous acid. The organisms are very sensitive to acidity, and can only operate if the nitrous acid produced is neutralised by an available base. In normal soils calcium carbonate supplies this base, and in acid soils the formation of nitrite is, as a rule, increased by the addition of lime, or of calcium or magnesium carbonate. There is evidence that in the absence of calcium carbonate, other compounds can be used as a base. It was found by Hopkins and Whiting[32] that in culture solution the nitrifying organisms could use insoluble rock phosphate as a base, producing therefrom the soluble acid phosphate. There is evidence, however, that in ordinary soil containing calcium carbonate very little solution of phosphate takes place in this way. The further oxidation of nitrite to nitrate by _Nitrobacter_ does not produce acid, and requires no further neutralising base.