General Microbiology - Schlegel H. 1987

Electron transport under anaerobic conditions
Denitrification and nitrate reduction

Phosphorylation coupled with electron transport during the respiration of aerobic organisms yields significantly more ATP than substrate-level phosphorylation during Fermentation. Therefore, it is hardly surprising that during biochemical evolution, a metabolic type emerged and was conserved in which hydrogen is transferred from an organic substrate to "bound oxygen." In this process, oxygen "carriers" can include nitrate, sulfate, carbonate, or Other Compounds, which are reduced by the substrate's hydrogen. The ability to transfer electrons to these compounds enables Bacteria to oxidize substrates in the absence of molecular oxygen and thereby extract more energy than is possible through fermentation.

Bacteria of this type possess an electron transport system and typically contain Cytochromes. Energy generation via electron transport-coupled phosphorylation involving the aforementioned "oxygen carriers" (or rather, terminal hydrogen acceptors) is fundamentally similar to respiration, where oxygen acts as the terminal hydrogen acceptor. However, because this process occurs under anaerobic conditions, it is referred to as Anaerobic respiration, with distinctions made between nitrate, sulfate, and carbonate respiration, and so on (Fig. 9.1). Bacteria capable of anaerobic respiration using inorganic hydrogen acceptors (nitrate, sulfate, carbonate) play a crucial role in both nature and human economic activity.

Microorganisms utilize nitrate for two main purposes. First, similar to most plants, many bacteria are able to extract nitrogen from it for the synthesis of nitrogen-containing cellular components. Such assimilatory nitrate reduction can take place under both aerobic and anaerobic conditions. Second, dissimilatory nitrate reduction, or "nitrate respiration," can also occur, where nitrate serves as the terminal hydrogen acceptor under anaerobic conditions. In both cases, nitrate is first reduced to nitrite with the aid of the molybdenum-containing enzyme nitrate reductase.

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Fig. 9.1. Processes of energy generation via Oxidative Phosphorylation under aerobic and anaerobic conditions ("aerobic respiration" and "anaerobic respiration").

Nitrate assimilation. During assimilation, nitrate is first reduced to nitrite and subsequently to ammonia; the latter is then used for the synthesis of Amino Acids and other nitrogen-containing cellular components. The first step is catalyzed by nitrate reductase; this enzyme (nitrate reductase B) is located in the Cytoplasm, and its synthesis is induced when nitrate serves as the sole nitrogen source in the nutrient medium. Nitrite is reduced to ammonia by nitrite reductase, a process that consumes 6 electrons. These electrons are supplied by NAD(P)H₂ (in Fungi and bacteria) or ferredoxin (in plants and certain bacteria). Nitrite reductase is a complex enzyme whose catalytic centers contain iron atoms—some incorporated into a heme group (siroheme) and others bound to sulfur atoms. The nitrite reductase reaction is similar to the transformations catalyzed by Nitrogenase and sulfite reductase, as the reduction of the substrate involves a 6-electron transfer without the release of any intermediates:

The formation of ammonium from nitrate is a reduction process that requires a substantial investment of reducing power:

Comparing Cell growth under aerobic conditions on media containing either nitrate or ammonium as the nitrogen source reveals that the former requires a greater expenditure of reducing power (hydrogen donor) than the reduction of ammonium (at equivalent biomass yields).

Nitrate respiration: denitrification. Denitrifying bacteria have the ability to reduce nitrate, via nitrite, to gaseous nitrous oxide (N₂O) and dinitrogen (N₂):

Thus, in the absence of oxygen, nitrate serves as the terminal hydrogen acceptor.

The ability to derive energy by utilizing nitrate as a terminal hydrogen acceptor with the formation of molecular nitrogen is widespread among bacteria. To date, this denitrification process has been detected exclusively in facultative aerobes; obligate anaerobes apparently lack denitrifying forms. Furthermore, these bacteria generally possess a complete Respiratory system, and the Synthesis of the Enzymes required for denitrification (membrane-bound nitrate reductase A and nitrite reductase) is induced only under anaerobic conditions (Fig. 9.2). In many denitrifiers, this induction occurs only in the presence of nitrate, although for some, the establishment of anaerobic conditions alone is sufficient. Many denitrifiers can grow using not only nitrate but also nitrite—and occasionally even nitrous oxide—as a hydrogen acceptor. This indicates that not only nitrate reductase A, but also dissimilatory nitrite reductase, is linked to their Respiratory Chain and participates in energy generation.

Fig. 9.2. Rate of molecular nitrogen formation in a cell suspension. N₂ is produced via the denitrification of nitrate under anaerobic conditions (acetate serves as the oxidized substrate). Bacteria were grown under various conditions: anaerobically in the presence of nitrate (I); aerobically in the presence of nitrate (II); aerobically in the absence of nitrate (III). (Van Olden E. In: Kluyver A.J., Van Niel C.B. The Microbe’s Contribution to Biology, Cambridge [Mass.]: Harvard Univ. Press, 1956.)

Enrichment culture of denitrifying bacteria. If soil or mud is inoculated into a medium containing hydrogen Donors, substrates for building cellular components, and nitrate, and the culture is incubated without access to air (see Table 6.3), various bacteria will develop: (a) in the presence of trace amounts of peptone and ethanol or propionic acid — Pseudomonas aeruginosa; (b) on glucose — Pseudomonas fluorescens; (c) on tartrate, succinate, or malate — Pseudomonas stutzeri; (d) on a medium with organic acids, alcohol, meat extract, and a high nitrate concentration (5–12% KNO₃) — Bacillus licheniformis; (e) in the presence of trace Yeast extract and H₂ as a hydrogen donor — Paracoccus denitrificans; (f) in the presence of sulfur or thiosulfate — Thiobacillus denitrificans. Because some denitrifiers use nitrate exclusively or predominantly as a hydrogen acceptor and are unable to reduce it to NH₄, it is necessary to Supplement the medium with a nitrogen source (peptone or an ammonium salt).

Soil nitrogen depletion due to denitrification. Temporary losses of nitrogen in localized soil areas are undoubtedly associated with The activity of denitrifying bacteria. This process becomes highly significant when anaerobic conditions develop in the soil, such as during waterlogging, particularly when organic fertilizers and nitrates are applied concurrently. In rice paddies, nitrate Fertilization can lead to adverse effects due to nitrite accumulation. Nitrite also accumulates in nitrate-containing wastewater subjected to insufficient aeration, and occasionally finds its way into drinking Water sources. The dependence of soil nitrogen depletion on aeration is related to the regulatory Features of the bacterial nitrate-reducing enzyme system. These enzymes are induced by nitrate only under anaerobic conditions (Fig. 9.2); molecular oxygen suppresses (represses) the synthesis of nitrate and nitrite reductases. If these enzymes have already been synthesized before the Cells come into contact with atmospheric oxygen, oxygen competes with nitrate for electrons supplied by the respiratory chain and also inhibits the function of the nitrate-reducing system.

The Role of denitrification in nature. Denitrification is the sole biological process capable of converting bound nitrogen into free N₂. From a global perspective, this process is critical for sustaining life on terrestrial landmasses. In normally aerated soils and aquatic environments, nitrate represents the end product of mineralization. Due to its high water solubility and weak binding to soil particles, nitrate ions would otherwise be leached from the soil and accumulate in seawater; the atmospheric concentration of molecular nitrogen would gradually decline, ultimately halting Plant Growth and terrestrial biomass production.

Nitrate reduction to nitrite via nitrate respiration. For a range of facultatively anaerobic bacteria (such as Enterobacter and Escherichia coli), nitrate can serve as the terminal hydrogen acceptor in the energy-yielding electron transport process. This type of "nitrate respiration" differs from denitrification in that only the initial step—namely, the reduction of nitrate to nitrite mediated by nitrate reductase A—is coupled to Electron Transport and energy transformation:

In this case, nitrite may accumulate in the culture medium, and no N₂ is produced.

Alternatively, nitrite can be reduced to ammonia via assimilatory nitrite reduction, followed by the release of NH₄ into the medium. This process is referred to as nitrate ammonification. The reduction of nitrite to ammonia does not provide The Cell with energy. Rather, it resembles a fermentative process in which nitrite acts as an exogenous electron acceptor. Consequently, nitrite reduction still confers a certain advantage: during glucose fermentation, a portion of the hydrogen atoms is diverted to reduce nitrite, resulting in a higher yield of acetate (see p. 264, reaction 3).

Methemoglobin formation resulting from nitrate accumulation in drinking water and food products. The consumption of nitrate-containing drinking water (frequently occurring during drought periods) and vegetables (such as "over-fertilized" spinach) can lead to illness due to the bacterial reduction of nitrate in canned goods or within the gastrointestinal tract. The microbial reduction of nitrate to nitrite, followed by the absorption of nitrite into the bloodstream, causes the formation of methemoglobin, in which oxygen is tightly (irreversibly) bound to Hemoglobin. As a result, erythrocytes partially lose their oxygen-carrying capacity, leading to cyanosis. This condition occurs exclusively in infants under 6 months of age. In such infants, nitrate-reducing bacteria apparently survive gastric passage and enter the intestine, where they reduce nitrate, proliferate, and accumulate nitrite. In older children and adults, these bacteria are destroyed by strongly acidic gastric juice, and nitrate ions are absorbed before they can undergo reduction under the favorable pH conditions found in the duodenum.