254 Biochemical Society Transactions (2011) Volume 39, part 1 Denitrification in Gram-positive bacteria: an underexplored trait Ines Verbaendert*, Paul De Vos*†, Nico Boon‡ and Kim Heylen* 1 *Laboratory of Microbiology (LM-UGent), Department of Biochemistry and Microbiology, Ghent University, K.L. Ledeganckstraat 35, 9000 Gent, Belgium, †BCCM/LMG Bacteria Collection, Laboratory of Microbiology, Ghent University, K.L. Ledeganckstraat 35, 9000 Gent, Belgium, and ‡Laboratory of Microbial Ecology (LabMET), Ghent University, Coupure Links 653, 9000 Gent, Belgium Abstract 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 N 2 O (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 (NO 3 − ) reductase, nitrite (NO 2 − ) reductase, nitric oxide (NO) reductase and nitrous oxide (N 2 O) reductase (Table 1). Denitrification sensu stricto contains NO 2 − and NO respiration, converting fixed nitrogen into gaseous nitrogen that is lost into the atmosphere [3,4]. 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. Key words: denitrification, Gram-positive bacterium, molecular detection, nitrogen cycle. Abbreviations used: DNRA, dissimilatory nitrate reduction to ammonium. 1 To whom correspondence should be addressed (email Kim.Heylen@UGent.be). 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, Paenibacil- lus, 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 C  The Authors Journal compilation C  2011 Biochemical Society Biochem. Soc. Trans. (2011) 39, 254–258; doi:10.1042/BST0390254