The Biochemistry of Nitrifying Microorganisms

Abstract

SUMMARY 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. 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. 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. 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. 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. 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. 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 bacteri...

Bacterial Cytochromes: II. Functional Aspects

“…using organic electron donors as well as molecular hydrogen, and which reduce CO2 to methane and hydrocarbons, using both organic and inorganic sources of electrons (e.g., M ethanobacter sp.). The energ y-coupling systems which invariably involve cytochromes re quire little comment as they have been reviewed most competently recently (3,4,6,7,11). We confine our discussion in this section to some further, if limited, extension of this previously presented material and add some com ments on new findings.…”

“…Taken together with the simultaneous publication in the same volume of the review on energy-coupling mechanisms by H. D. Peck, Jr. (3), and other excellent discussions (4)(5)(6)(7)(8)(9)(10)(11), there is ample justification for the decision to limit space available to consideration of general perspectives with attention confined to those special topics for which up-dating is neces sary, or which require further amplification. As stated (1), our effort is presented in two parts.…”

“…In whatever manner the electron trans port chain is constituted, coupled phosphate esterifi cation is generally de monstrable and can be localized in steps involving cytochrome components. The overall pattern suggested (54) and documented in numerous research es (3,11) is generation of ATP by the coupled passage of reducing equiv alents to the terminal acceptor via a more or less complete cytochrome sys tem. In systems such as ammonia and nitrite oxidizers which generate re ducing equivalents at oxidation potentials too high to function in pyridine nucleotide reduct ion, A TP-as generated by oxidative phosphorylation-is used to effect reversed electron transport (3,11,54).…”

Partial nitrification—operational parameters and microorganisms involved

“…All researches along the partial nitrification or nitritation line explore the inhibitory factors (for example free NH 3 or toxic compounds) that have a selective inhibition on the nitrite oxidizers and a minimal affect on the ammonia oxidizers, or the competitive factors (temperature, dissolved oxygen (DO) which do not inhibit nitrite oxidizers but can give a competitive advantage to ammonia oxidizers over the nitrite oxidizers. Some of the works investigating nitrite build-up during nitrification were performed by Wallace and Nicholas (1969), Aleem (1970), Painter (1970), Focht and Chang (1975), Anthonisen (1976), Sharma and Ahlert (1977), Suthersan and Ganczarczyk (1986), Jayamohan and Ohgaki (1988), Hanaki and Wantawin (1990) and Philips et al (2002) which focused on a number of factors, such as the free ammonia (FA) concentration, pH, temperature, dissolved oxygen (DO) concentration and inhibitory compounds. The ways to accomplish the aim have been described by many authors, but the general conclusion is not clear and easy.…”

NAD+ - and NADP+-Dependent Glutamate Dehydrogenases in Nitrobacter agilis

“…Nitrobacter agilis ATCC 14123 was grown in 40 1 batches at 28 "C for 5 d with vigorous aeration in an inorganic medium as described by Aleem (1968) and Wallace & Nicholas (1969). Cells were harvested at 4 "C and 30000g in a Sorvall RC-2B centrifuge fitted with a continuous-flow rotor, with a flow Abbreviation : GDH, glutamate dehydrogenase.…”