THE BIOCHEMISTRY OF NITRIFYING MICROORGANISMS

• 1 Biological nitrification is mediated primarily by two genera of bacteria, Nitrosomonas and its marine form Nitrosocystis, oxidizing ammonia to nitrite, and Nitrobacter, converting nitrite into nitrate. These are chemoautotrophic organisms since they usually derive their energy for growth by oxidizing these inorganic nitrogen compounds and their carbon from carbon dioxide, carbonates or bicarbonates.
• 2 The morphology and structure of these Gram-negative bacteria studied by electron microscopy show numerous intracellular membranes reminiscent of those in photosynthetic bacteria and blue-green algae. These structures may therefore be associated with the production of ATP.
• 3 The bacteria are difficult to grow in pure cultures in sufficient amounts for biochemical work since their generation time is around 10 hr. and the yields are only about one hundredth of those obtained with heterotrophic bacteria. Thus in continuous cultures great care must be taken to avoid ‘wash-out’ of the cells. Since Nitrosomonas and Nitrosocystis produce copious amounts of nitrous acid, which would eventually retard growth, pH stat units are used to titrate the cultures continuously with a solution of sodium carbonate, to hold the pH around 7–8.
• 4 The respiratory chain which is associated with cell membranes, contains flavin, quinones and many cytochromes linking to oxygen as a terminal acceptor. In Nitro-somonas-Nitrosocytis hydroxylamine is oxidized by the electron transfer chain and in Nitrobacter nitrous acid is utilized. The ammonia-oxidizing system, which in Nitrosomonas probably resides near the cell surface, does not appear to survive cell breakage. During the oxidation of hydroxylamine and nitrous acid by the respiratory chains, a phosphorylation occurs but the P/O ratios around 0–30 are low. There is little energy reserve material in the cells, possibly β-hydroxybutyrate and some metaphosphates and as soon as the oxidative processes are impaired the cells cease dividing.
• 5 Chemoautotrophic bacteria have a novel way of producing reduced nicotinamide adenine dinucleotide (NADH). This involves a reversal of electron flow from reduced cytochrome c to nicotinamide adenine dinucleotide (NAD) that is energy-dependent, thus requiring adenosine triphosphate.
• 6 Reductase enzymes, nitrate, nitrite and hydroxylamine reductases in Nitrobacter and nitrite and hydroxylamine reductases in Nitrosomonas, have been described. They appear to be readily extracted in soluble form and are probably assimilatory enzymes since ¹⁶N labelled nitrate, nitrite and hydroxylamine respectively in Nitrobacter and the last two in Nitrosomonas are readily incorporated into cell nitrogen. It has been suggested that a particulate nitrate reductase in Nitrobacter is coupled to the synthesis of adenosine triphosphate but adequate experimental evidence for this concept has not been produced.
• 7 Some recent observations with Nitrobacter suggest that it grows on acetate, deriving all its energy and carbon skeletons from this source but the mean generation time for the bacterium is unchanged. Under these conditions the carbon dioxide fixing enzymes of the pentose pathway are suppressed. This then is a case of facultative chemoautotrophy but there is no increase in the biosynthesis of the TCA enzymes. Whether this is a widespread phenomenon in other chemoautotrophic bacteria remains to be established. If this does prove to be the case it would aid their survival in a variety of habitats and extend their distribution in soils and seas.
• 8 The carbon dioxide fixing enzymes of the pentose pathway are found in the soluble parts of the cells. The major route is via the carboxydismutase system with only a small incorporation via the phosphoenolpyruvate carboxylase enzyme. Enzymes of the tricarboxylic acid cycle have low activities compared with those in heterotrophs and this overall slow metabolism, rather than the lack of a specific enzyme such as NADH oxidase, may well account for the slow growth of these bacteria. Although there is very active glutamic dehydrogenase in Nitrosomonas that utilizes ammonia, the enzyme has a very small activity in Nitrobacter. This poses a problem of the route of incorporation of nitrite nitrogen into cell nitrogen in the latter bacterium.
• 9 A few heterotrophic fungi have been described which oxidize ammonia to nitrate but their activity is small compared with that of the nitrifying bacteria.
• 10 It is concluded that the nitrifying bacteria which have many novel biochemical features not met with in other organisms merit further study.