Nitrification and denitrification as a source for NO and NO2 production in high-strength wastewater

Most studies of bacterial denitrification have used nitrate (NO 3؊ ) as the first electron acceptor, whereas relatively less is understood about nitrite (NO 2 ؊ ) denitrification. We isolated novel bacteria that proliferated in the presence of high levels of NO 2 ؊ (72 mM T proliferated even in the presence of higher levels of NO 2 ؊ (100 mM), and both were considerably more resistant to acidic NO 2 ؊ than were the other strains noted above. These results indicated that O. anthropi YD50.2 is a novel denitrifier that has evolved reactive nitrogen oxide tolerance mechanisms.Environmental bacteria maintain the global nitrogen cycle by metabolizing organic and inorganic nitrogen compounds. Denitrification is critical for maintenance of the global nitrogen cycle, through which nitrate (NO 3 Ϫ ) or nitrite (NO 2 Ϫ ) is reduced to gaseous nitrogen forms such as N 2 and nitrous oxide (N 2 O) (19, 47). Decades of investigations into denitrifying bacteria have revealed their ecological impact (9), their molecular mechanisms of denitrification (13,25,47), and the industrial importance of removing nitrogenous contaminants from wastewater (31, 36). Bacterial denitrification is considered to comprise four successive reduction steps, each of which is catalyzed by NO 3 Ϫ reductase (Nar), NO 2 Ϫ reductase (Nir), nitric oxide (NO) reductase (Nor), and N 2 O reductase (Nos). The reaction of each enzyme is linked to the electron transport chain on the cellular membrane and accompanies oxidative phosphorylation, implying that bacterial denitrification is of as much physiological significance as anaerobic respiration (25, 47). Most denitrifying bacteria are facultative anaerobes and respire with oxygen under aerobic conditions. Because denitrification is induced in the absence of oxygen, it is considered an alternative mechanism of energy conservation that has evolved as an adaptation to anaerobic circumstances (13, 47).Nitrite and NO are hazardous to bacteria, since they generate highly reactive nitrogen species (RNS) under physiological conditions and damage cellular DNA, lipid, and proteins (28,37). Denitrifying bacteria are thought to be threatened by RNS since they reduce NO 3 Ϫ to generate NO 2 Ϫ and NO as denitrifying intermediates. Furthermore, denitrifying bacteria often inhabit environments where they are exposed to NO 2 Ϫ and NO and hence high levels of RNS. Recent reports suggest that pathogenic bacteria invading animal tissues are attacked by NO generated by macrophages (12). Such bacteria involve denitrifiers, and some of them, for example, Neisseria meningitidis (1) and Pseudomonas aeruginosa, acquire resistance to NO by producing Nor (44). The utilization (reduction) of NO by Brucella increases the survival of infected mice (2). These examples suggest that production of a denitrifying mechanism affects bacterial survival of threats from both endogenous and extracellular RNS. However, the mechanism of RNS tolerance induced by denitrifying bacteria is not fully understood.Ubiquitous gram-negative Ochrobactrum strains ar...

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confidence: 99%

Novel Denitrifying Bacterium Ochrobactrum anthropi YD50.2 Tolerates High Levels of Reactive Nitrogen Oxides

Doi

Takaya

Takizawa

2009

“…Analysis has shown that broiler litter contains potentially valuable plant nutrients, including nearly 30% crude protein and high levels of minerals and some heavy metals (36). For this reason, litter is commonly used as a fertilizer but environmental concerns, such as nutrient, nitrate, and phosphate runoff to streams, ponds, and groundwater have limited its acceptability (53,54). Another disadvantage is the tendency for ammonia and other volatile gases to escape into the atmosphere, resulting in unpleasant odors.…”

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confidence: 99%

Evaluation of Broiler Litter with Reference to the Microbial Composition as Assessed by Using 16S rRNA and Functional Gene Markers

Lu¹,

Sánchez

Hofacre

et al. 2003

185

160

Very little is known about the microbial composition of animal bedding wastes, including poultry litter, and what is known has been deduced from standard culture methods, by which some fastidious organisms that exist in the environment may not be detected. We evaluated the bacterial composition of poultry litter by using a combination of culture and molecular detection. Total aerobic bacteria in poultry litter were detected by culture at 10 9 CFU/g of material. Enteric bacteria such as Enterococcus spp. and coliforms composed 0.1 and 0.01%, respectively, of the total aerobic cultivatable bacteria in poultry litter; no Salmonella strains were detected by culture. In order to characterize the most abundant bacterial groups, we sequenced 16S ribosomal DNA (rDNA) genes amplified by PCR with microbial community DNA isolated from poultry litter as the template. From the 16S rDNA library, 31 genera were identified. Twelve families or groups were identified with lactobacilli and Salinococcus spp. forming the most abundant groups. In fact, 82% of the total sequences were identified as gram-positive bacteria with 62% of total belonging to low G؉C gram-positive groups. In addition to detection of 16S rDNA sequences associated with the expected fecal bacteria present in manure, we detected many bacterial sequences for organisms, such as Globicatella sulfidofaciens, Corynebacterium ammoniagenes, Corynebacterium urealyticum, Clostridium aminovalericum, Arthrobacter sp., and Denitrobacter permanens, that may be involved in the degradation of wood and cycling of nitrogen and sulfur. Several sequences were identified in the library for bacteria associated with disease in humans and poultry such as clostridia, staphylococci, and Bordetella spp. However, specific PCR targeting other human and veterinary pathogens did not detect the presence of Salmonella, pathogenic Escherichia coli, Campylobacter spp., Yersinia spp., Listeria spp., or toxigenic staphylococci. PCR and DNA hybridization revealed the presence of class 1 integrons with gene cassettes that specify resistance to aminoglycosides and chloramphenicol. Only from understanding the microbial community of animal wastes such as poultry litter can we manage animal disease and limit the impact of animal waste on the environment and human and animal health.

“…The experiments described here demonstrate that denitrifiers are a likely source of the NO that a bacterium such as B. subtilis might encounter in its natural environment. The finding that coincubation with A. cycloclastes was as effective at inducing hmp expression as that with 2.4.3 suggests that NO-inducible stimulons respond to the nM concentrations of NO typically found in the environment (33,37). In addition, this result also dem- onstrates that biologically produced NO is as good or better at inducing expression of genes in the NO stimulon than the NO-generating compounds used in most studies (Fig.…”

Section: Discussionmentioning

confidence: 70%

“…The most reactive intermediate is NO, and several in vitro studies demonstrate that the denitrifiers Paracoccus denitrificans, Pseudomonas stutzeri, and Achromobacter cycloclastes maintain steady-state NO concentrations in the low nM range when respiring either nitrate or nitrite (14,15). Studies with denitrifying wastewater and soil samples also measured NO concentrations at low nM levels (33,37). This efficient reduction of NO is most likely due to the high substrate affinity and catalytic activity of the NO reductase expressed by denitrifiers (15,17,34,44).…”

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confidence: 99%

Assessing the Impact of Denitrifier-Produced Nitric Oxide on Other Bacteria

Choi

Naal

Moore

et al. 2006

A series of experiments was undertaken to learn more about the impact on other bacteria of nitric oxide (NO) produced during denitrification. The denitrifier Rhodobacter sphaeroides 2.4.3 was chosen as a denitrifier for these experiments. To learn more about NO production by this bacterium, NO levels during denitrification were measured by using differential mass spectrometry. This revealed that NO levels produced during nitrate respiration by this bacterium were in the low M range. This concentration of NO is higher than that previously measured in denitrifiers, including Achromobacter cycloclastes and Paracoccus denitrificans. Therefore, both 2.4.3 and A. cycloclastes were used in this work to compare the effects of various NO levels on nondenitrifying bacteria. By use of bacterial overlays, it was found that the NO generated by A. cycloclastes and 2.4.3 cells during denitrification inhibited the growth of both Bacillus subtilis and R. sphaeroides 2.4.1 but that R. sphaeroides 2.4.3 caused larger zones of inhibition in the overlays than A. cycloclastes. Both R. sphaeroides 2.4.3 and A. cycloclastes induced the expression of the NO stress response gene hmp in B. subtilis. Taken together, these results indicate that there is variability in the NO concentrations produced by denitrifiers, but, irrespective of the NO levels produced, microbes in the surrounding environment were responsive to the NO produced during denitrification.During complete denitrification, nitrate is sequentially reduced to dinitrogen gas through the generation of the intermediates nitrite, nitric oxide (NO), and nitrous oxide (N 2 O) (44). Each reductive step is catalyzed by a separate nitrogen oxide reductase. The concerted activity of the four nitrogen oxide reductases typically maintains a steady flow of intermediates (15,44). It is important that bottlenecks be avoided, since some of these intermediates are reactive. The most reactive intermediate is NO, and several in vitro studies demonstrate that the denitrifiers Paracoccus denitrificans, Pseudomonas stutzeri, and Achromobacter cycloclastes maintain steady-state NO concentrations in the low nM range when respiring either nitrate or nitrite (14,15). Studies with denitrifying wastewater and soil samples also measured NO concentrations at low nM levels (33, 37). This efficient reduction of NO is most likely due to the high substrate affinity and catalytic activity of the NO reductase expressed by denitrifiers (15,17,34,44).NO is a free radical that can potentially interact with an array of targets in both prokaryotic and eukaryotic cells (25). When exposed to NO, many organisms induce a set of genes whose products provide protection against the deleterious effects of NO (32). The variety of genes induced subsequent to NO exposure indicates that NO can impact a range of cell functions and that most organisms have specific mechanisms for mitigating NO toxicity. Even though NO is an obligate intermediate during denitrification, the impact of the NO produced during denitrification on other m...