Denitrifying organisms are essential in removing fixed nitrogen pollutants from ecosystems (e.g. sewage sludge). They can be detrimental (e.g. for agricultural soil) and can also produce the greenhouse gas N2O (nitrous oxide). Therefore a more comprehensive understanding of this process has become increasingly important regarding its global environmental impact. Even though bacterial genome sequencing projects may reveal new data, to date the denitrification abilities and features in Gram-positive bacteria are still poorly studied and understood. The present review evaluates current knowledge on the denitrification trait in Gram-positive bacteria and addresses the likely existence of unknown denitrification genes. In addition, current molecular tools to study denitrification gene diversity in pure cultures and environmental samples seem to be highly biased, and additional novel approaches for the detection of denitrifying (Gram-positive) bacteria appear to be crucial in re-assessing the real diversity of denitrifiers.

Introduction

Denitrification is part of the global nitrogen cycle and allows respiration in low oxygen environments [1,2] by using nitrogenous compounds as terminal electron acceptors. It is a four-step modular process catalysed by different sequentially induced metalloproteins known as nitrate (NO3) reductase, nitrite (NO2) reductase, nitric oxide (NO) reductase and nitrous oxide (N2O) reductase (Table 1). Denitrification sensu stricto contains NO2 and NO respiration, converting fixed nitrogen into gaseous nitrogen that is lost into the atmosphere [3,4].

Table 1
The different stages of denitrification

The overall reaction is: NO3→NO2→NO→N2O→N2. Adapted from [4,10,19].

Reaction ΔG0′ Enzyme Gene encoding catalytic subunit 
NO3+2e+2H+→NO2+H2−161.1 kJ/mol Membrane-bound nitrate reductase: Nar narG 
  Periplasmic nitrate reductase: Nap napA 
NO2+e+2H+→NO+H2−76.2 kJ/mol Copper nitrite reductase: CuNiR nirK 
  Cd1 nitrite reductase: Cd1 NiR nirS 
NO+2e+2H+→N2O+H2−306.3 kJ/mol Cytochrome c NO reductase: cNOR cnorB 
  Quinol NO reductase: qNOR qnorB (norZ
  qCuANOR Gene not yet identified 
N2O+2e+2H+→N2+H2−339.5 kJ/mol NO reductase: N2OR nosZ 
Reaction ΔG0′ Enzyme Gene encoding catalytic subunit 
NO3+2e+2H+→NO2+H2−161.1 kJ/mol Membrane-bound nitrate reductase: Nar narG 
  Periplasmic nitrate reductase: Nap napA 
NO2+e+2H+→NO+H2−76.2 kJ/mol Copper nitrite reductase: CuNiR nirK 
  Cd1 nitrite reductase: Cd1 NiR nirS 
NO+2e+2H+→N2O+H2−306.3 kJ/mol Cytochrome c NO reductase: cNOR cnorB 
  Quinol NO reductase: qNOR qnorB (norZ
  qCuANOR Gene not yet identified 
N2O+2e+2H+→N2+H2−339.5 kJ/mol NO reductase: N2OR nosZ 

Since its discovery in 1886 by Gayon and Dupetit ([1], reviewed in [5]), a broad range of denitrifying bacteria and archaea, but also fungi [6] and foraminifers [7], have been described, and the genes and enzymes involved in the process have been thoroughly investigated. In the last two decades, however, in-depth biochemical and molecular denitrification research on bacteria has mainly focused on Gram-negative bacteria, while mostly overlooking Gram-positive denitrifiers. The aim of the present review is to summarize current knowledge on Gram-positive denitrification, investigate possible reasons why it has been underexplored and hypothesize on its ecological importance in the hope that future denitrification research will pay more attention to Gram-positive bacteria and other overlooked groups of micro-organisms.

Diversity of Gram-positive denitrifiers

Denitrification is a widespread trait among prokaryotes, and some authors have tried to record described denitrifying bacterial species. In 1981, Payne [2] listed a considerable number of genera that contain denitrifying members. One of these genera was the Gram-positive genus Bacillus, with denitrifying strains belonging to B. licheniformis, B. azotoformans and B. stearothermophilus (now Geobacillus stearothermophilus). A decade later, Zumft [8] compiled a list of almost 130 denitrifying bacterial species; however, the majority of them were Gram-negative species. Gram-positive denitrifying bacterial genera included in this survey are members of Bacillus, Brevibacillus, Geobacillus, Paenibacillus, Virgibacillus, Sporosarcina, Corynebacterium, Gemella, Jonesia, Propionibacterium and Tsukamurella. Shapleigh [5] also assembled a list of Gram-positive denitrifying species that were suggested to contain denitrifying strains. He added genera described by Shoun et al. [9] and the genus Frankia to Zumft's list [8]. In 2007, Philippot and co-workers [10] also included Paenibacillus terrae.

Similarly, Supplementary Table S1 (available at http://www.biochemsoctrans.org/bst/039/bst0390254add.htm) is an updated heuristic annotated list of strains that denitrify, or were once claimed to, how denitrification was assessed and the current status of our knowledge of their denitrification ability. Since Gram-positive bacteria, other than Bacillus, were classically considered as not containing true denitrifiers [3], it is no surprise that the majority of the publications on Gram-positive denitrifying taxa focuses on members of the Bacillales. For instance, Supplementary Table S1 contains several denitrifying strains belonging the genus Bacillus, such as strains of B. stearothermophilus (now Geobacillus stearothermophilus) [11], B. licheniformis [12], B. circulans and B. cereus [13], B. thermodenitrificans (now Geobacillus thermodenitrificans) [14] and strains that now belong to the genus Paenibacillus [15]. The Actinomycetales are a second group in which a wide variety of denitrifying bacteria has been found, with a large proportion of members of the genus Streptomyces [9,16]. In addition, a small number of non-spore-forming denitrifying Gram-positive bacteria have been reported as well, such as strains belonging to Corynebacterium. Besides a description of the denitrifying capacities of strains belonging to the Bacillales and Actinomycetales, Gram-positive denitrification reductases have also been described, e.g. in Bacillus halodenitrificans (now Virgibacillus halodenitrificans) [17], Bacillus firmus [18] and B. azotoformans [19] (Supplementary Table S1), but in general the denitrification trait, enzymes and genes in Gram-positive bacteria remain underexplored.

Why underexplored?

Phenotypic detection

Large numbers of novel taxa of cultured micro-organisms are described yearly, with an average of 616 novel taxa per year for the last decade (http://www.bacterio.cict.fr). Unfortunately, no mandatory minimum characterization of phenotypic traits is demanded for valid description of novel microbial taxa and, although ‘minimal standards’ are formulated as guidelines for many taxa, often no standardized protocols are recommended or required to describe new species. Hence, when dissimilatory nitrogen metabolism [including NO3 reduction, DNRA (dissimilatory nitrate reduction to ammonium) or denitrification] is investigated in novel species, phenotypes are often inaccurately deduced from miniaturized test panels using, for example, only a colorimetric NO3 reduction test. In addition, when the type strain is not a denitrifier, characterization of the dissimilatory nitrogen metabolism in other strains of the same species is often not considered, despite its strain-dependent nature [5]. For Gram-positive bacteria, this effect has also been enhanced by the general assumption that denitrification in Gram-positive bacteria is limited to some representatives of the genus Bacillus [3]. As a result, the actual prevalence of denitrification in described and cultivated bacteria is highly unknown and hence underestimated.

Another factor in the underestimation of the occurrence of denitrification in pure cultures is the ambiguous assessment of the ability of an organism to denitrify. Quick and easy tests, such as colorimetric NO3 and NO2 reduction tests, gas formation in Durham tubes and detection of a pH increase with a dye indicator in the growth medium [20], are seldom specific for the detection of denitrification, but are still widely applied. Currently, general consensus exists on the two main criteria for an organism to be named a true respiratory denitrifier [21], namely (i) nitrogen gases, principally N2O and N2, are products from NO3 and/or NO2 reduction, and (ii) the process is coupled to a significant growth yield increase. Using these criteria, denitrifiers can easily be distinguished from non-denitrifiers, in particular from those that express NO reductase as a protection against nitrosative stress [22]. In practice, NO3 is added to an inoculated growth medium and denitrification is tested through quantitative measurement of N2O and/or N2 after incubation. Often 10% acetylene is added to the headspace to block the reduction of N2O to N2, and N2O is measured, although this step can already be circumvented [23]. However, other processes such as DNRA or anammox can also generate N2O or N2 from NO3 or NO2 [3,24], whereas new denitrifiers, such as the recently described Methylomirabilis oxyfera [25], can follow alternative enzymatic pathways and may only produce known intermediates in trace amounts. Thus gas chromatographic analyses are also not incontestable as the sole investigative tool to demonstrate denitrification. It should also be mentioned that, independent of the method used, results will be dependent on the test conditions. For example, some bacteria can only start denitrification from NO2 and not from NO3 [8], although mostly only NO3 is tested as the electron acceptor. In addition, the use of other growth medium with different carbon sources or different pH and different incubation conditions, such as temperature, amount of oxygen etc., can influence the denitrifying ability of the tested organism [3]. As a result, many NO3-respiring NH4+-producing isolates have incorrectly been considered denitrifiers in routine testing [10], whereas true denitrifiers that start from NO2 have been overlooked. We do not believe that at present the current criteria for denitrifiers need updating, but correct interpretation of phenotypic observations to assess denitrification certainly deserves more attention.

Molecular detection

Although the issues mentioned above with phenotypic detection of denitrification are relevant for all bacteria and not solely for Gram-positive bacteria, molecular detection of the involved genes is more specifically a problem for Gram-positive denitrification.

Gene sequence analyses of key denitrification genes have been used to confirm isolates as denitrifiers [21] or, in culture-independent research, to monitor the influence of different physicochemical parameters on the abundance and diversity of denitrifying populations in situ. The majority of gene sequences available in the public databases encoding catalytic subunits of key denitrifying reductases, namely nirS, nirK, qnorB (quinol-dependent nitric oxide reductase B) and cnorB (cytochrome c-type nitric oxide reductase B) (Table 1), are retrieved from either Gram-negative or uncultured bacteria (Figure 1). As a result, all available molecular tools for nir and norB gene detection (e.g. described by Braker et al. [26], Hallin and Lindgren [27], Braker and Tiedje [28], Goregues et al. [29], Casciotti and Ward [30,31] and Flores-Mireles et al. [32]) only target Gram-negative bacteria. These primers have been used on denitrifying members of the genus Bacillus [33] and Paenibacillus [34], but without success.

Available Gram-positive nir and nor sequence data

Figure 1
Available Gram-positive nir and nor sequence data

Data collected from the National Center for Biotechnology Information Nucleotide database, September 2010 (http://www.ncbi.nlm.nih.gov/nucleotide/). Only a few nir and nor sequences of Gram-positive denitrifying bacteria are available. Genes annotated with the term nirS, nirK, cnorB or qnorB were included in the dataset. CFB group, Cytophaga/Flavobacterium/Bacteroides group.

Figure 1
Available Gram-positive nir and nor sequence data

Data collected from the National Center for Biotechnology Information Nucleotide database, September 2010 (http://www.ncbi.nlm.nih.gov/nucleotide/). Only a few nir and nor sequences of Gram-positive denitrifying bacteria are available. Genes annotated with the term nirS, nirK, cnorB or qnorB were included in the dataset. CFB group, Cytophaga/Flavobacterium/Bacteroides group.

When denitrification genes are occasionally detected in Gram-positive denitrifiers, their sequence phylogeny is very closely related to that of genes from Gram-negative denitrifiers [35]. This observation is in disagreement with the phylogeny of gene sequences from whole genome sequences. Jones et al. [36] showed that concatenated denitrification gene sequences from G. thermodenitrificans, one of the only confirmed Gram-positive denitrifiers of which the whole genome is currently available, were only distantly related to other known sequences. This sequence divergence obviously has consequences: nucleotide-based BLAST searches with a denitrification gene sequence of G. thermodenitrificans as the query will not find any homologous sequences; only the use of derived amino acid sequences will result in identification of the correct protein domain and retrieval of homologous sequences. Because of this, relevant denitrification gene sequences were probably overlooked, e.g. in metagenomic data, and therefore efforts are necessary to generate more whole genomes from Gram-positive or other confirmed denitrifiers [33], or even large fosmid libraries from the environment [37], to expand the knowledge on denitrification gene sequences that are not targeted by current primers. This information can then be used to design specific primers that target certain groups of gene sequences.

Lack of suitable primers and very divergent sequences can certainly explain the neglect of Gram-positive denitrifiers in the last two decades, in which research has been dominated by molecular analyses. However, another explanation could be the presence of new denitrification genes coding for unknown NO2 and NO reductases. This hypothesis is supported by the discoveries of the qCuANOR (quinol copper A NO reductase) enzyme purified from B. azotoformans [19], encoded by as yet unknown genes, the gNOR (g-type nitric oxide reductase) gene [38], encoding a hitherto unknown NO reductase in Sulfurimonas denitrificans, and the proposed presence of an NO dismutase catalysing the conversion of two NO compounds into N2 and O2 in a denitrifying aerobic methane oxidizer [25]. These discoveries suggest that the denitrification pathway is more redundant than previously thought. It seems that, despite intensive research for over many decades, our knowledge of denitrification is still incomplete and the existence of currently unknown new denitrification enzymes is very plausible.

Why important?

We have described above that denitrification may be a trait of many Gram-positive genera and we have tried to unravel why they are overlooked in current research. But is Gram-positive denitrification important from an ecological point of view? Unfortunately, molecular tools need to be available before its relevance, more specifically numerical importance and activity, in situ can be investigated. However, a range of culture-dependent denitrification studies have already isolated Gram-positive denitrifiers from different types of NOx (nitrogen oxides)-reducing ecosystems, although these reports are very limited. Main habitats of denitrifying bacteria are (i) sediments and the water column of aquatic systems, such as oceans, estuaries, rivers, lakes and lagoons, (ii) parts of wastewater treatment systems, such as activated sludge, and (iii) terrestrial systems, such as soil and the rhizosphere associated with it. Aquatic denitrifying Corynebacterium spp. and an Arthrobacter sp. have been isolated from around mangrove roots [32] and Bacillus spp. from lagoon sediments [29]. Gram-positive denitrifiers have also been isolated from wastewater treatment systems [3941], and Bacillus spp. are even essential for some specific wastewater treatments [42,43]. In terrestrial systems, denitrifying members of the genus Bacillus and Paenibacillus have already been found in rice soils and rice plant rhizospheres [11,44], bent grass and Bermuda grass [45], in soddy podzolic soil [13] and in a Korean night soil treatment system [46], whereas denitrifying Actinomycetes have already been isolated from agricultural soils [9,16]. In addition, microbial composition of terrestrial systems has been studied globally, in many types of soil and rhizospheres, and Gram-positive bacteria are found to be abundant in these soil ecosystems [16,47]. Since denitrification is a process of global and environmental concern, elucidating Gram-positive denitrification phenotypes, their associated enzymes and encoding genes, and their ecological contribution is pivotal for the further understanding and management of denitrifying ecosystems.

Concluding remarks

Denitrification is a trait of global ecological importance, in which Gram-positive bacteria could play a key role. Denitrification in all its aspects, from the phenotypic observation of nitrogen conversion to molecular detection of the encoding genes, has been underexplored in this large group of micro-organisms. The present review highlights the need for more stringent phenotypic characterization for the description of novel taxa, thus creating an elaborate and reliable inventory of the capacity to denitrify among cultivated and identified micro-organisms which are publically available for the generation of more whole genomes. In addition, failure of currently available molecular tools to detect denitrification genes in cultivated denitrifiers and the high divergence in phylogeny between PCR-amplified gene sequences and those retrieved from whole genome sequencing suggest incomplete and inaccurate in situ monitoring of the denitrification process. This underlines the necessity for new efforts in primer development and the need for other approaches to identify predominant denitrifying microbial taxa in environmental monitoring studies, as some researchers have already endeavoured [33,48,49]. Strengthened by recent discoveries, we also hypothesize that new hitherto unknown genes encode the denitrification process in common already cultivated bacteria.

Enzymology and Ecology of the Nitrogen Cycle: A Biochemical Society Focused Meeting held at University of Birmingham, U.K., 15–17 September 2010. Organized and Edited by Jeff Cole (University of Birmingham, U.K.), Rosa María Martínez-Espinosa (University of Alicante, Spain), David Richardson (University of East Anglia, Norwich, U.K.) and Nick Watmough (University of East Anglia, Norwich, U.K.).

Abbreviations

     
  • DNRA

    dissimilatory nitrate reduction to ammonium

Funding

This work was partially supported by the Geconcerteerde Onderzoeksactie (GOA) of Ghent University [grant number BOF09/GOA/005].

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Supplementary data