Denitrification is a distinct means of energy conservation, making use of N oxides as terminal electron acceptors for cellular bioenergetics under anaerobic, microaerophilic, and occasionally aerobic conditions. The process is an essential branch of the global N cycle, reversing dinitrogen fixation, and is associated with chemolithotrophic, phototrophic, diazotrophic, or organotrophic metabolism but generally not with obligately anaerobic life. Discovered more than a century ago and believed to be exclusively a bacterial trait, denitrification has now been found in halophilic and hyperthermophilic archaea and in the mitochondria of fungi, raising evolutionarily intriguing vistas. Important advances in the biochemical characterization of denitrification and the underlying genetics have been achieved with Pseudomonas stutzeri, Pseudomonas aeruginosa, Paracoccus denitrificans, Ralstonia eutropha, and Rhodobacter sphaeroides. Pseudomonads represent one of the largest assemblies of the denitrifying bacteria within a single genus, favoring their use as model organisms. Around 50 genes are required within a single bacterium to encode the core structures of the denitrification apparatus. Much of the denitrification process of gram-negative bacteria has been found confined to the periplasm, whereas the topology and enzymology of the gram-positive bacteria are less well established. The activation and enzymatic transformation of N oxides is based on the redox chemistry of Fe, Cu, and Mo. Biochemical breakthroughs have included the X-ray structures of the two types of respiratory nitrite reductases and the isolation of the novel enzymes nitric oxide reductase and nitrous oxide reductase, as well as their structural characterization by indirect spectroscopic means. This revealed unexpected relationships among denitrification enzymes and respiratory oxygen reductases. Denitrification is intimately related to fundamental cellular processes that include primary and secondary transport, protein translocation, cytochrome c biogenesis, anaerobic gene regulation, metalloprotein assembly, and the biosynthesis of the cofactors molybdopterin and heme D1. An important class of regulators for the anaerobic expression of the denitrification apparatus are transcription factors of the greater FNR family. Nitrate and nitric oxide, in addition to being respiratory substrates, have been identified as signaling molecules for the induction of distinct N oxide-metabolizing enzymes.

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Cell biology and molecular basis of denitrification.

Currently available microbiological techniques are not designed to deal with very slowly growing microorganisms. The enrichment and study of such organisms demands a novel experimental approach. In the present investigation, the sequencing batch reactor (SBR) was applied and optimized for the enrichment and quantitative study of a very slowly growing microbial community which oxidizes ammonium anaerobically. The SBR was shown to be a powerful experimental set-up with the following strong points: (1) efficient biomass retention, (2) a homogeneous distribution of substrates, products and biomass aggregates over the reactor, (3) reliable operation for more than 1 year, and (4) stable conditions under substrate-limiting conditions. Together, these points made possible for the first time the determination of several important physiological parameters such as the biomass yield (0.066 ± 0.01 C-mol/mol ammonium), the maximum specific ammonium consumption rate (45 ± 5 nmol/mg protein/min) and the maximum specific growth rate (0.0027 · h−1, doubling time 11 days). In addition, the persisting stable and strongly selective conditions of the SBR led to a high degree of enrichment (74% of the desired microorganism). This study has demonstrated that the SBR is a powerful tool compared to other techniques used in the past. We suggest that the SBR could be used for the enrichment and quantitative study of a large number of slowly growing microorganisms that are currently out of reach for microbiological research.

The sequencing batch reactor as a powerful tool for the study of slowly growing anaerobic ammonium-oxidizing microorganisms

Nitrous oxide emission during wastewater treatment.

Straw return has been widely recommended as an environmentally friendly practice to manage carbon (C) sequestration in agricultural ecosystems. However, the overall trend and magnitude of changes in soil C in response to straw return remain uncertain. In this meta-analysis, we calculated the response ratios of soil organic C (SOC) concentrations, greenhouse gases (GHGs) emission, nutrient contents and other important soil properties to straw addition in 176 published field studies. Our results indicated that straw return significantly increased SOC concentration by 12.8 ± 0.4% on average, with a 27.4 ± 1.4% to 56.6 ± 1.8% increase in soil active C fraction. CO2 emission increased in both upland (27.8 ± 2.0%) and paddy systems (51.0 ± 2.0%), while CH4 emission increased by 110.7 ± 1.2% only in rice paddies. N2O emission has declined by 15.2 ± 1.1% in paddy soils but increased by 8.3 ± 2.5% in upland soils. Responses of macro-aggregates and crop yield to straw return showed positively linear with increasing SOC concentration. Straw-C input rate and clay content significantly affected the response of SOC. A significant positive relationship was found between annual SOC sequestered and duration, suggesting that soil C saturation would occur after 12 years under straw return. Overall, straw return was an effective means to improve SOC accumulation, soil quality, and crop yield. Straw return-induced improvement of soil nutrient availability may favor crop growth, which can in turn increase ecosystem C input. Meanwhile, the analysis on net global warming potential (GWP) balance suggested that straw return increased C sink in upland soils but increased C source in paddy soils due to enhanced CH4 emission. Our meta-analysis suggested that future agro-ecosystem models and cropland management should differentiate the effects of straw return on ecosystem C budget in upland and paddy soils.

Effects of straw carbon input on carbon dynamics in agricultural soils: a meta-analysis.

Microbial ecology of denitrification in biological wastewater treatment.

Nitrous oxide can be a harmful by-product in nitrogen removal from wastewater. Since wastewater treatment systems operate under different aeration regimens, the influence of different oxygen concentrations and oxygen fluctuations on denitrification was studied. Continuous cultures of Alcaligenes faecalis TUD produced N2O under anaerobic as well as aerobic conditions. Below a dissolved oxygen concentration of 5% air saturation, the relatively highest N2O production was observed. Under these conditions, significant activities of nitrite reductase could be measured. After transition from aerobic to anaerobic conditions, there was insufficient nitrite reductase present to sustain growth and the culture began to wash out. After 20 h, nitrite reductase became detectable and the culture started to recover. Nitrous oxide reductase became measurable only after 27 h, suggesting sequential induction of the denitrification reductases, causing the transient accumulation of N2O. After transition from anaerobic conditions to aerobic conditions, nitrite reduction continued (at a lower rate) for several hours. N2O reduction appeared to stop immediately after the switch, indicating inhibition of nitrous oxide reductase, resulting in high N2O emissions (maximum, 1.4 mmol liter-1 h-1). The nitrite reductase was not inactivated by oxygen, but its synthesis was repressed. A half-life of 16 to 22 h for nitrite reductase under these conditions was calculated. In a dynamic aerobic-anaerobic culture of A. faecalis, a semisteady state in which most of the N2O production took place after the transition from anaerobic to aerobic conditions was obtained. The nitrite consumption rate in this culture was equal to that in an anaerobic culture (0.95 and 0.92 mmol liter-1 h-1, respectively), but the production of N2O was higher in the dynamic culture (28 and 26% of nitrite consumption, respectively).

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N.G. Grobben

Papers

Nitrous oxide production by Alcaligenes faecalis under transient and dynamic aerobic and anaerobic conditions