Cell Biology And Molecular Basis Of Denitrification Pdf
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This research investigated spatial-temporal variation in benthic bacterial community structure, rates of denitrification and dissimilatory nitrate reduction to ammonium DNRA processes and abundances of corresponding genes and transcripts at three sites—the estuary-head, mid-estuary and the estuary mouth EM along the nitrate gradient of the Colne estuary over an annual cycle. Denitrification rates declined down the estuary, while DNRA rates were higher at the estuary head and middle than the EM. In four out of the six 2-monthly time-points, rates of DNRA were greater than denitrification at each site.
- Cell biology and molecular basis of denitrification.
Cell biology and molecular basis of denitrification.
Pseudomonas aeruginosa is a metabolically flexible member of the Gammaproteobacteria. Under anaerobic conditions and the presence of nitrate, P.
This study focuses on understanding the influence of environmental conditions on bacterial denitrification performance, using a mathematical model of a metabolic network in P.
To our knowledge, this is the first mathematical model of denitrification for this bacterium. Analysis of the long-term behavior of the network under changing concentration levels of oxygen O 2 , nitrate NO 3 , and phosphate PO 4 suggests that PO 4 concentration strongly affects denitrification performance. The model provides three predictions on denitrification activity of P. One motivation for this study is to capture the effect of PO 4 on a denitrification metabolic network of P.
Simulating the microbial production of greenhouse gases in anaerobic aquatic systems such as Lake Erie allows a deeper understanding of the contributing environmental effects that will inform studies on, and remediation strategies for, other hypoxic sites worldwide.
Academic Editor: Christopher V. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The work conducted by the U. Department of Energy under Contract No. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist. Denitrification is a facultative anaerobic process in which nitrate is utilized as an alternative terminal electron receptor and dissimilatory nitrate is reduced to nitrogen gas via nitrogen oxides [ 1 — 3 ]. Since denitrification is one of the few pathways for producing atmospheric N 2 , it is a major component of the nitrogen cycle [ 4 ]. Denitrification occurs in several habitats such as soils, lakes, rivers and oceans [ 5 ].
Pseudomonas aeruginosa , a facultative ubiquitous, and metabolically flexible member of the Gammaproteobacteria, can perform complete denitrification under anaerobic conditions and the presence of nitrate. Complete denitrification consists of four sequential steps to reduce nitrate NO 3 to dinitrogen N 2 via nitrite NO 2 , nitric oxide NO , and nitrous oxide N 2 O , and each step of the pathway is catalyzed by denitrification enzymes such as nitrate reductase nar , nitrite reductase nir , nitric oxide reductase nor , and nitrous oxide reductase nos.
The identification and transcriptional control of denitrification genes encoding nar , nir , nor and nos has been largely established. Transcription is dependent on a hierarchy of the FNR-like Crp family transcription factors Anr and Dnr, the two-component system NarXL, and the CbbQ family protein NirQ [ 7 , 8 ], summarized in [ 4 ], allowing for experimental validation of N 2 O yield as environmental parameters change.
We have built a combined gene regulatory and metabolic network for the denitrification pathway in Pseudomonas aeruginosa PAO1, a well-studied denitrifier strain Fig. Environments such as Lake Erie experience seasonal periods of hypoxic conditions favorable for denitrification, and the endemic microbial community regulates expression of alternative respiratory pathways to adapt to low oxygen O 2 tension.
We are interested in using the model to investigate the effect of PO 4 on the denitrification performance of P. Although there are several studies on regulation of denitrification by kinetic mathematical modeling approaches e.
One of the challenges in building kinetic mathematical models of networks, such as systems of differential equations, is that many of the needed parameters are either not known or unmeasurable. Furthermore, for large networks, kinetic models are difficult to analyze mathematically. Therefore, we take a qualitative approach to model denitrification distinct from the quantitative denitrification models attempted previously.
We use a discrete model framework that provides coarse-grained information about the temporal biochemical output of the network in response to environmental conditions. This framework captures attractors and their biological correspondence, phenotypes yet it does not render any measurements of time or concentration.
In particular, we prefer a time-discrete and multi-state deterministic framework, Polynomial Dynamical System PDS [ 13 ], to model our denitrification network in Pseudomonas aeruginosa. Green solid arrows indicate upregulation and red dashed arrows indicate downregulation.
Our interest lies in perturbation of the external parameters O 2 , PO 4 , NO 3 and their effect on the long-term behavior of the network. The denitrification network consists of molecules, proteins and genes all of which can play an important role in the denitrification process in Pseudomonas aeruginosa.
The large gray rectangle represents the bacterial cell. The pathway begins with the phosphate-sensing two component regulatory system PhoRB [ 14 ]. PhoRB, the main PO 4 sensor activating the pho regulon, has been recently shown to be a regulator of PhoPQ transcription in the gammaproteobacterium Escherichia coli [ 15 ]. In light of the fact that Pseudomonas aeruginosa possesses a similar regulatory system to PhoRB in E.
Low oxygen O 2 tension, which is the major initial signal to turn on the denitrification pathway, can be sensed by Anr [ 1 ]. Under anaerobic conditions, Anr primarily promotes Dnr dissimilatory nitrate respiration regulator transcription [ 4 ].
The mechanism of inhibitory effect of PmrA on Dnr [ 17 ] is not known, so we assumed that the effect of Anr on Dnr can be reduced by PmrA. The main regulator of the system, Dnr, controls the expression of all denitrification genes nar , nir , nor , nos in the presence of NO [ 18 ].
Of particular note is the influence of the two-component system PhoPQ on PmrA expression and, subsequently, Dnr expression [ 17 ], suggesting that phosphorus P availability influences denitrification gene expression see Fig. This is particularly relevant, since linkages between anaerobic Fe III reduction and P release adsorbed to FeOOH in sediments have been recognized for many years [ 19 , 20 ], and recently documented in Lake Erie by stable isotope methods [ 21 ].
The actual mechanisms of the relationships in the denitrification network Fig. Thus, the network does not represent a biochemical reaction network, for instance, but rather captures the regulatory logic driving the network in a similar way that a circuit diagram explains the function of a circuit board.
In the network Fig. In the discrete setting that is used to model the denitrification network, each node e. Our interest lies in perturbation of the external parameters and their effect on the long-term behavior of the variables in the system.
Such values incorporate appropriate ranges of long-term nutrient and seasonal oxygen concentrations for Lake Erie [ 22 , 23 ]. The denitrification network is an open system; it exchanges molecules with the outside environment and responds to external stimuli [ 24 ]. The molecule NO 3 enters the bacterium and N 2 exits the system once the system is triggered by low O 2.
The model predicts the long-term behavior of the denitrification pathway under various environmental conditions and these predictions are either supported by the literature or validated by experimental results. There are two conditions that we did not focus on. The low NO 3 and low PO 4 condition and the low NO 3 and high PO 4 condition, while possible, are less likely in freshwaters based on a worldwide survey of lakes revealing that N:P stoichiometric ratios average above the ideal Redfield ratio of 16 [ 25 ].
However, a high P , high NO 3 condition can arise in lakes affected by agricultural nutrient inputs and deposition of P in sediments. The first condition low O 2 , low PO 4 and high NO 3 corresponds to the perfect condition for denitrification and the second condition low O 2 , high PO 4 and high NO 3 corresponds to the denitrification condition disrupted by PO 4 availability.
The remaining conditions can be labeled as aerobic conditions. These attractors indeed are steady states, each of which corresponds to one environmental condition.
This agrees with biology; Palsson highlighted that open systems eventually reach a homeostatic steady state and are in balance with their environment until the environmental conditions are perturbed [ 24 ]. Phenotypes, biological interpretations of the long-term behavior steady states , of the system under various environmental conditions can be found in Table 2. Based on the steady state analysis above, the Pseudomonas network model predicts that elevated PO 4 , hypothesized to increase under hypoxia, acts to modulate the transcriptional network to limit nos gene expression.
Thus, the physiological output under this condition will be an increased yield of N 2 O relative to N 2. While other studies have suggested linkages between N 2 O accumulation and factors such as nosZ vs. In an aquatic system, oxygen dissolves in water to be available to living aerobic organisms.
Hypoxia is the phenomenon of dissolved oxygen below 4 mgO 2 per liter. Common reasons for hypoxia include aerobic respiration of decaying algal biomass from bloom events. Such blooms are fueled by increased availability of N and P due to anthropogenic inputs such as agricultural runoff and industrial pollutants [ 33 ].
The linkage between high nutrient N , P loads and N losses N 2 and N 2 O through dissimilatory anaerobic processes was described recently [ 34 ]. Hypoxic low-oxygen areas, so-called dead zones, often occur in several large bodies of water affected by human activity, including Lake Erie, which is of particular interest. Establishing a better understanding of the nutrient cycling of Lake Erie has quite wide ranging socioeconomic impacts on its recreational area and economy, primarily fisheries.
Through denitrification, dead zones lead to microbial production of the greenhouse gas nitrous oxide N 2 O , which plays a crucial role in ozone layer depletion and climate change.
In addition to oxygen, the intersections of the nitrogen cycle with other geochemical cycles may be important factors influencing denitrification and nitrogen N sinks in aquatic systems. In particular, the increased availability of phosphorus P has been shown to dictate the rate of nitrogen removal in aquatic systems [ 34 ].
Indeed, the transcriptional regulatory network developed for P. The bacterium Pseudomonas aeruginosa is an example of an abundant microbe in aquatic systems [ 36 ], and analysis of Lake Erie metagenomic data sets reveals abundant pseudomonads capable of denitrification Unpublished data, DOE-JGI. This study describes a computational model of a denitrification network of this bacterium to capture the effect of PO 4 on its denitrification performance in order to shed light on greenhouse gas N 2 O accumulation during oxygen depletion.
Transcription is dependent on a hierarchy of the FNR-like Crp family transcription factors Anr and Dnr, the two-component system NarXL, and the CbbQ family protein NirQ [ 7 , 8 , 37 ], allowing for experimental measurement of N 2 O as external environmental parameters change. The model was constructed based on the pertinent biological literature.
Model predictions either agree with current published results or are validated by experimentation. The new biology that our model discovers is that PO 4 availability strongly affects the denitrification activity of P. The data presented here are the first to suggest a role for PO 4 in regulating the denitrification pathway in Pseudomonas aeruginosa.
Current efforts will be expanded to determine how PO 4 affects greenhouse gas N 2 O accumulation during denitrification in P.
According to the model, the activation of Dnr by Anr or the activation of nos in the presence of NO by Dnr can be prevented by high PO 4. These hypotheses will be tested utilizing quantitative reverse transcriptase PCR qRT-PCR to determine Dnr, norB nitric oxide reductase large subunit gene and nosZ encoding nitrous oxide reductase transcript levels in denitrifying cultures grown in increasing P.
Synergistic interactions between individual members of population of Pseudomonas aeruginosa may need to be taken into account and incorporated to the model. For instance, Toyofuku and his colleagues stated that denitrification performance of P. The model described here works well for cultured Pseudomonas , and the next step is to test natural complex microbial communities from different denitrification sites. The effects of PO 4 on N 2 O production will be tested in mesocosms of hypoxic Lake Erie water samples to see if the model described here predicts the community as a whole.
By testing the model on environmental samples in mesocosms from Lake Erie and elsewhere, the study can likely be applied broadly to other marine dead zones such as those that routinely occur in the Gulf of Mexico. Our network consists of two different sub-networks metabolic and gene regulatory and consequently different time scales.
From a discrete modeling perspective, this issue can be tackled or ignored only if the long-term behavior of the system is of interest. Due to inadequate information on the reaction rates, we do not focus on a stochastic framework. Even with a fully asynchronous update schedule, the attractors are preserved for each configuration of external parameters; however, this asynchronous update schedule requires more time steps to reach a steady state than a synchronous update schedule does.
Since an asynchronous update schedule provides us more on transient behavior of the system and we are interested in long-term behavior of the system, we prefer to use a deterministic framework with a synchronous update schedule, Polynomial Dynamical System PDS , which allows us to model regulatory networks over a finite field [ 13 ].
Definition 1 Let x 1 , x 2 , …, x n be variables which can take values in finite fields X 1 , X 2 , …, X n respectively. A Polynomial Dynamical System is a collection of n update functions. There are 14 variables, each of which is labeled for the mathematical formulation.
Table 3 indicates the variables, their discretization, update rules and the literature evidence that support these update rules.
Facultative anaerobic bacteria perform denitrification as a type of respiration that reduces oxidized forms of nitrogen in response to the oxidation of an electron donor such as organic matter. Denitrification can leak N 2 O, which is an ozone-depleting substance and a greenhouse gas that can have a considerable influence on global warming. The process is performed primarily by heterotrophic bacteria such as Paracoccus denitrificans and various pseudomonads ,  although autotrophic denitrifiers have also been identified e. Direct reduction from nitrate to ammonium , a process known as dissimilatory nitrate reduction to ammonium or DNRA ,  is also possible for organisms that have the nrf- gene. Other genes known in microorganisms which denitrify include nir nitrite reductase and nos nitrous oxide reductase among others;  organisms identified as having these genes include Alcaligenes faecalis , Alcaligenes xylosoxidans , many in the genus Pseudomonas , Bradyrhizobium japonicum , and Blastobacter denitrificans. Denitrification generally proceeds through some combination of the following half reactions, with the enzyme catalyzing the reaction in parentheses:. In nature, denitrification can take place in both terrestrial and marine ecosystems.
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Pseudomonas aeruginosa is a metabolically flexible member of the Gammaproteobacteria. Under anaerobic conditions and the presence of nitrate, P. This study focuses on understanding the influence of environmental conditions on bacterial denitrification performance, using a mathematical model of a metabolic network in P.
Skip to search form Skip to main content You are currently offline. Some features of the site may not work correctly. DOI: 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.
In this study, a novel nitrate reduction method using an electro-enzymatic system was investigated to treat an aqueous solution containing high concentrations of nitrate. This system was comprised of working electrodes and their counter electrodes, where enzymes for nitrate removal were located inside the microorganisms. A nitrate reduction mechanism for the electro-enzymatic system was proposed and a mathematical model was developed. The modeling results were compared to the experimental results and all the experimental results fit well with the model; thus, the derived mathematical model can be used for reactor control and effluent prediction. This is a preview of subscription content, access via your institution. Rent this article via DeepDyve. Keith, L.
B Corresponding author. Email: anqiang cqu. Environmental context. Industrial development has caused the release of hexavalent chromium and nitrates into the environment. Interactions of hexavalent chromium and nitrates with microorganisms are important both for understanding environmental behaviour and for treatment options. Bacterial removal of both chromium and nitrate was optimised in waters relevant to waste streams and the environment. An isolated strain of the bacterium Stenotrophomonas maltophilia strain W26, is shown to be capable of the simultaneous removal of nitrate and Cr VI under aerobic conditions.
PDF. Loading. SUMMARY. Denitrification is a distinct means of energy conservation, making.
The second step of the dissimilatory denitrification pathway in which nitrite NO 2 - is converted to nitric oxide NO is catalyzed by the enzyme nitrite reductase. Two distinct enzymes are found in nature that catalyze this reaction, and they contain different metal sites, either iron Fe , in the form of heme, or copper Cu Zumft, The Pseudomonas stutzeri P. In this assay, total nitrite reductase activity can be measured in whole cells using fumarate or some other carbon source as an electron source by measuring the disappearance of nitrite over time Thorgersen et al. Keywords : Nitrite reductase, Pseudomonas, Griess. Figure 1.
Metrics details. Microbial denitrification is not considered important in human-associated microbial communities. Accordingly, metabolic investigations of the microbial biofilm communities of human dental plaque have focused on aerobic respiration and acid fermentation of carbohydrates, even though it is known that the oral habitat is constantly exposed to nitrate NO 3 - concentrations in the millimolar range and that dental plaque houses bacteria that can reduce this NO 3 - to nitrite NO 2 -.
Tetrapyrroles pp Cite as. H eme d 1 is found only in a bacterial periplasmic enzyme, cytochrome cd 1 , that catalyses reduction of nitrite to nitric oxide. The unique features of the d 1 heme include saturation of two of the pyrrole rings and presence of two carbonyl groups. Reasons for the selection of the d 1 ring for an enzyme catalysing this reaction are discussed and related to model compound studies.
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