"Aerobic Methanotrophy and Nitrification: Processes

Aerobic Methanotrophy and Nitrification: Processes and Connections

Advanced article Article Contents . Introduction . Methanotrophs . Ammonia Oxidisers . Linkages between Methane and Ammonia Oxidisers . Conclusion . Acknowledgements

Lisa Y Stein, University of Alberta, Edmonton, Alberta, Canada Reál Roy, University of Victoria, Victoria, British Columbia, Canada Peter F Dunfield, University of Calgary, Calgary, Alberta, Canada

Online posting date: 16th April 2012

This article is dedicated to the memory of Roger Knowles.

Ammonia and methane are structurally similar molecules. Not surprisingly therefore, microorganisms that use methane as a sole energy source (methanotrophs) and microorganisms that use ammonia as a sole energy source (ammonia oxidisers or nitrifiers) share many similarities. They have several key enzymes in common, most especially the ammonia monooxygenase/particulate methane monooxygenase enzyme family. The two groups are proposed to have a common evolutionary history. They occupy similar ecological niches, and compete for nitrogen. Enzymatically, nitrifiers are capable of methane oxidation, and methanotrophs are capable of nitrification. Microbial ecologists have attempted to find specific inhibitors for either group in order to study their respective roles in the environment. The contribution of ammonia oxidisers to methanotrophy in natural systems appears to be very minor, however methanotrophs may sometimes have important roles in the nitrogen cycle.

single oxygen atom from O2 into the respective molecules via a monooxygenase enzyme, yielding methanol (CH3OH) from CH4 and hydroxylamine (NH2OH) or possibly alternate N-oxides from NH3 (Figure 1). Energy released in the monooxygenation is not conserved, in fact the microbes expend reducing power to reduce the second oxygen atom from O2. However, methanol and hydroxylamine are further oxidised in processes coupled to energy generation. See also: Chemolithotrophy; Nitrification; The Metabolism of Anammox Similarities between bacterial methanotrophs and nitrifiers have long been evident. Both grow obligately on their particular substrate (NH3 for nitrifiers and CH4 for methanotrophs), but are capable of cooxidising the alternative substrate. Both contain intracellular membrane systems, inhabit oxic/anoxic interface environments, rely on monooxygenase reactions catalysed by ammonia NO ? b

a

Introduction

NH3

Methane (CH4) and ammonia (NH3) are highly reduced molecules and therefore suitable growth substrates for microbes. They can be oxidised either aerobically or anaerobically to yield energy. The anaerobic processes are only recently yielding up their secrets, particularly methane oxidation coupled to sulfate-reduction and ammonia oxidation coupled to nitrite-reduction. However, the focus of this review is on aerobic processes. The first step in aerobic methane or ammonia oxidation is the introduction of a eLS subject area: Microbiology How to cite: Stein, Lisa Y; Roy, Reál; and Dunfield, Peter F (April 2012) Aerobic Methanotrophy and Nitrification: Processes and Connections. In: eLS. John Wiley & Sons, Ltd: Chichester. DOI: 10.1002/9780470015902.a0022213

CH4

a a a

HNO? NH2OH CH3OH

? d c

N2 O

c

NO2−

? b e

HCHO

f

h

CHOOH

g

CO2

i

Cell biomass Figure 1 Overlapping pathways for ammonia and methane oxidation. Black lines denote bacterial pathways; grey lines denote putative pathways for ammonia-oxidising thaumarchaea. Enzymes catalysing each process are: a, ammonia/methane monooxygenase; b, hydroxylamine oxidoreductase; c, nitrite reductase; d, nitric oxide reductase; e, methanol dehydrogenase; f, formaldehyde dehydrogenase; g, formate dehydrogenase; h, enzymes of the serine or ribulose monophosphate pathway and i, enzymes of the Calvin–Benson–Bassham cycle. The question marks in the thaumarchaeal pathway denote uncertainty regarding the intermediate produced by ammonia monooxygenase, the enzyme that oxidises this intermediate to nitrite, and enzymes that form NO/N2O from the intermediate of ammonia oxidation or from reduction of nitrite as measured by Santoro et al. (2011). The dashed line denotes a possible role of NO in ammonia-oxidation (Schleper and Nicol, 2010).

eLS & 2012, John Wiley & Sons, Ltd. www.els.net

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monooxygenase (AMO) or methane monooxygenase (MMO), possess hydroxylamine oxidoreductase systems and require copper. Both cooxidise a range of other substrates and are inhibited by many of the same compounds. In a seminal review from 1989, ‘Physiology, biochemistry, and specific inhibitors of CH4, NH4+ and CO oxidation by methanotrophs and nitrifiers’, Be´dard and Knowles reviewed some of the early work into these microorganisms, particularly stressing the physiological, biochemical and ecological parallels between them. Our knowledge has expanded considerably since 1989. In the following section we will briefly introduce these interesting groups of microorganisms and update what is now known about the relationships between them.

Methanotrophs Several properties were once considered to unify the methanotrophs: they were all proteobacteria that possessed a particulate methane monooxygenase enzyme (pMMO), fixed carbon from formaldehyde produced as an intermediate of methane oxidation, and were obligate organisms unable to grow on substances containing a C–C bond (Hanson and Hanson, 1996). All of these generalities now have exceptions.

Systematics and ecology Aerobic methanotrophs occur in the phylum Verrucomicrobia, the phylum-level candidate division NC10 and in the Alphaproteobacteria and Gammaproteobacteria classes of the phylum Proteobacteria (Table 1). The most recently discovered methanotrophic groups are those two outside the Proteobacteria. Neither has been validly named, although the genus ‘Methylacidiphilum’ has been proposed for the Verrucomicrobia methanotrophs and ‘Methylomirabilis’ for the NC10 methanotrophs. Both differ from proteobacterial methanotrophs in their ecological and physiological properties, but not in the key steps of methane oxidation. Methylacidiphilum is a thermoacidiphile (optima 608C and pH 2) that occupies geothermal environments and uses pMMO for methane oxidation (Op den Camp et al., 2009). Methylomirabilis is, paradoxically, an anaerobe that carries out aerobic methanotrophy. It produces O2 via the dismutation of nitric oxide (NO) (Ettwig et al., 2010), and uses this to oxidise methane. Setting aside this rather bizarre source of O2, the actual oxidation of methane appears to be the same as other methanotrophs, via pMMO. In early studies, the known (proteobacterial) methanotrophs were placed into three groups: Type I, Type II and Type X (Table 1). This grouping was based primarily on differences in membrane lipids, carbon fixation pathways, the forms of MMO present and geometric arrangement of intracellular membranes. Types I and X were later shown to be gammaproteobacteria and Type-II alphaproteobacteria. For a more detailed description of the 2

distinguishing characteristics of these groups see an excellent early review by Hanson and Hanson (1996). This naming convention was popular with many microbial ecologists, especially where simplification into a few ecological groups aided interpretation of community patterns. Many early studies attempted to define ecological differences between the Types I, II and X methanotrophs, but these generalisations have usually proven to be site-specific. For example, it was initially considered that Type I’s should predominate over Type II’s under methane limitation because of their more efficient C-fixation pathway (Ribulose monophosphate cycle versus Serine cycle) (Hanson and Hanson, 1996). However, while this held true in early competition studies, further studies have shown that many cultured Type II’s have a lower methane threshold for survival than Type I’s, and that the most methane-poor habitats (oxic upland soils receiving methane only from the atmosphere), support a mix of alphaproteobacterial and/or gammaproteobacterial methanotroph species depending on soil pH (Dunfield, 2006). A further problem with the Type I–II–X system is that exceptions continue to be discovered: Type-II methanotrophs possessing ‘signature’ Type I and X membrane lipids (Dedysh et al., 2007) and vice versa (Heyer et al., 2005); alphaproteobacteria like Methylocapsa and Methylocella that do not have membrane arrangements characteristic of the classical Type II methanotrophs Methylosinus and Methylocystis (Table 1); gammaproteobacteria genera Methylothermus and Methylohalobius that may not be monophyletic with the Type I+X group (Heyer et al., 2005); and of course Methyacidiphilum and Methylomirabilis, which clearly deserve new categories. The Type I–II–X system is becoming more obfuscating than useful, and a species-based or family-based system is now more practical. The major families are outlined in Table 1. The ecology of aerobic methanotrophs has been reviewed in detail elsewhere (Hanson and Hanson, 1996; Dunfield, 2006; Conrad, 2007; Op den Camp et al., 2009; Semrau et al., 2010). Different species have adapted to wide ranges of environmental temperature, pH and salinity. They usually inhabit oxic/anoxic interfaces of methanogenic environments such as rice paddies, peat bogs and lake sediments, where they act as a filter to remove some proportion (10–90%) of the methane produced before it can reach the atmosphere (Laanbroek, 2010). The oxic/anoxic interfacial areas include surface sediments as well as rhizospheres of aerenchymous plants (Laanbroek, 2010). Intriguing but still largely unknown methanotrophs also live in oxic upland soils that are rarely methanogenic, particularly native forests. These oxidise the trace level of methane in the atmosphere (presently near 1.8 ppmv). They are usually called ‘high-affinity’ methanotrophs, based on the observation that the apparent affinity to methane observed in these soils is several orders of magnitude higher than that measured in pure cultures of methanotrophs. It is still not known how these organisms survive on such a minimal methane source – they may be true oligotrophs or they may be capable of oxidising other substrates besides


Table 1 Family level classification of methanotrophs Phylum and class

Proteobacteria (Gammaproteobacteria)

Proteobacteria (Gammaproteobacteria)

Proteobacteria (Alphaproteobacteria)

‘Methylothermaceae’ Methylohalobiusa and Methylothermusa

II Methylocystaceae Methylocystis and Methylosinus

Verrucomicrobia

Beijerinckiaceae Methylocella Methylocapsa and Methyloferula

NC10 ‘Methylomirabilis’

‘Methylacidiphilaceae’ ‘Methylacidiphilum’

Type II: membrane stacks along the cell periphery, parallel to the cell envelope

Methylocapsa: Type III membrane stack parallel to the long axis Methylocella and Methyloferula: cytoplasmic membrane invaginations or vesicles

?

Carboxysome-like structures or vesicular membranes

Includes the most thermophilic and halophilic methanotrophs known

Mesophilic, neutrophilic to mildly acidophilic

Includes the most coldadapted methanotrophs known, mild acidophiles

Mesophilic and neutrophilic

Includes the most acidophilic methanotrophs known, mesophiles to thermophiles

Ribulose monophosphate pathway and Calvin– Benson–Bassham cycle

Ribulose monophosphate pathway

Serine cycle

Serine cycle, Calvin– Benson–Bassham cycle

Calvin–Benson– Bassham cycle

Calvin–Benson–Bassham cycle

43–65 +/2 + +

58–63 2 + +

60–67 +/2 + +/2

60–63 +/2 +/2 +/2

59 2 + ?

41–46 2 + +

I and X Methylococcaceae Methylococcus (X), Methylocaldum (X), Methylobacter, Methylomicrobium, Methylomonas, Methylosarcina, Methylosoma, Crenothrixa, Clonothrixa and Methylosphaera

Internal membranes or compartments

Type I: membrane bundles perpendicular to the cell envelope

Type I: membrane bundles perpendicular to the cell envelope

Environmental tolererances

Diverse: includes thermophiles and halophiles

Carbon fixation pathway

G+C mol% sMMO pMMO Obligate methylotrophs

+, all species positive; 2, all species negative; +/2, some species positive. The family Crenotrichaceae is validated, but is phylogenetically a subset of the Methylococcaceae.



NC10

Type Family Genera

a

Proteobacteria (Alphaproteobacteria)

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methane (Dunfield, 2006). See also: Methanotrophy in Extreme Environments; Wetlands Methanotrophs have an obvious role in regulating atmospheric methane, which is the second most important greenhouse gas and has a global forcing potential 25 times worse than CO2 per molecule. Consequently, much of the research on these bacteria aims to understand how their activity is regulated, and how anthropogenic activities may alter their role in the global methane budget. For example, it has been noted that agricultural/silvicultural practices, particularly fertilisation, reduce the activity of methanotrophs and thereby affect methane fluxes (Bodelier and Laanbroek, 2004; Dunfield, 2006). The use of methanotrophs in landfill cover soils to reduce methane emissions is a good example of how the bacteria can be utilised practically (Semrau et al., 2010). See also: Climate Change and Biogeochemical Impacts

formaldehyde to CO2 via formate involves alternate pathways that incorporate the C1-carrying coenzymes tetrahydromethanopterin (H4MPT) and/or tetrahydrofolate (H4F). Formaldehyde and formate are fed into one of two carbon fixation pathways – either the ribulose monophosphate (RuMP) or Serine cycle. Some methanotrophs can also operate a Calvin–Benson–Bassham cycle to fix atmospheric CO2, and this may in fact be the sole carbon source for the verrucomicrobial methane oxidisers (Op den Camp et al., 2009; Khadem et al., 2011). Formaldehyde is highly toxic and its concentration must be kept low. The pathways of oxidation from formaldehyde must therefore be carefully regulated to balance energy production, carbon fixation and formaldehyde toxicity.

Physiology

Nitrification, the aerobic oxidation of ammonia to nitrate, can be separated into two processes: ammonia oxidation to nitrite and nitrite oxidation to nitrate. Ammoniaoxidising microorganisms grow chemolithotrophically (i.e. in the absence of organic molecules) using ammonia as a sole energy source. Below is a brief summary of the ammonia-oxidation process. See also: Chemolithotrophy; Nitrification

It is beyond the scope of this introduction to describe the biochemistry of methanotrophs in detail, the reader is referred to detailed reviews (Hakemian and Rosenzweig, 2007; Chistoserdova et al., 2009; Semrau et al., 2010). MMO exists in two phylogenetically unrelated forms: a soluble form (sMMO) and a membrane-bound form (pMMO). Both enzymes perform the same fundamental reaction, but with different cellular localisations, sources of reductant, kinetics (notably, the pMMO has a higher affinity to methane) and cooxidation profiles for competitive substrates (the sMMO appears to cooxidise a broader substrate spectrum). The pMMO is universal to all known methanotrophs with the exception of Methylocella spp. and Methyloferula spp. The sMMO is present as an additional system in many species, and as the only MMO in Methylocella and Methyloferula. The latter two genera, which are members of the Beijerinckiaceae family, are in many ways outliers among methanotrophs. They are the only methanotrophs that lack extensive internal membrane systems in which pMMO is bound, and Methylocella are unusually versatile in their energy metabolism (Dedysh et al., 2005). The discovery of metabolism of 2-C and 3-C compounds in Methylocella overturned the axiom that methanotrophs were all obligately methylotrophic. Now several other strains of methanotrophs have also been demonstrated to not be obligate, but among these Methylocella has the broadest substrate range (Dedysh et al., 2005; Belova et al., 2011; Dunfield et al., 2010). The biochemistry of methane oxidation beyond the initial monooxygenase step follows a modular design (Chistoserdova et al., 2009). Different methanotroph species mix-and-match different combinations of available modules for the steps e–i in Figure 1. The oxidation of methanol to formaldehyde is carried out by methanol dehydrogenase, usually a pyrroloquinoline quinone-dependent alcohol dehydrogenase of the methanol/ethanol family, which may take several forms in terms of tertiary subunit structure (Chistoserdova et al., 2009). The oxidation of 4

Ammonia Oxidisers

Systematics and ecology Ammonia-oxidising microorganisms perform the ratelimiting step of nitrification, the oxidation of ammonia to nitrite. This capability is shared by some bacteria and archaea. Chemolithotrophic ammonia-oxidising bacteria occur within the proteobacterial classes Betaproteobacteria and Gammaproteobacteria. Chemolithotrophic ammoniaoxidising archaea belong to the Thaumarchaeota phylum. Ammonia-oxidisers, both bacteria and archaea, are usually found in close partnerships with nitrite-consuming microorganisms, either the aerobic nitrite oxidising bacteria or anaerobic ammonia-oxidising (anammox) bacteria, both of which depend on nitrite produced by ammonia oxidisers. Nitrite-oxidising bacteria oxidise nitrite to nitrate, whereas anammox bacteria consume both ammonia and nitrite to produce dinitrogen. See also: Nitrification; The Metabolism of Anammox The discovery of the ammonia-oxidising thaumarchaea came after 100 years of study of bacterial ammoniaoxidation. Cultivation of the first thaumarchaeon, Nitrosopumilus maritimus, was accomplished in 2005 (Ko¨nneke et al., 2005) and this isolate has since been shown to have a much higher affinity for ammonium than bacterial ammonia-oxidisers (Martens-Habbena et al., 2009). Growth of ammonia-oxidising thaumarchaea at very low ammonium concentrations differentiates their niche from that of most ammonia-oxidising bacteria. Furthermore, thaumarchaea appear to dominate the ammonia-oxidising community in acidic soils (below pH 5) where they can acquire ammonium from organic matter via mineralisation



rather than from inorganic forms (Yao et al., 2011). Preference for organic versus inorganic ammonium is thus another potential marker that may discriminate thaumarchaeal from bacterial ammonia-oxidisers in soils. Another differentiating feature was discovered following cultivation of the first soil thaumarchaeon, Nitrososphaera viennensis, whereby pyruvate was required for optimal growth and carbon assimilation (Tourna et al., 2011). Although heterotrophic carbon assimilation does occur in some bacterial ammonia oxidisers, organic carbon is not a widely used media additive to enhance growth of chemolithotrophic ammonia-oxidising bacteria (Stein, 2011). Accumulating evidence suggests that ammonia-oxidising thaumarchaea are responsible for the majority of ammonia oxidation on Earth, particularly in marine environments and acid soils (Schleper and Nicol, 2010). This is perhaps due to nitrogen limitation in most environments, in addition to predominance of organic over inorganic forms of nitrogen. Conversely, in N-saturated ecosystems, the ammonia-oxidising bacteria tend to be more predominant, suggesting once again that ammonia load as well as source are critical factors that differentiate the niches of the two ammonia-oxidising groups (Schleper and Nicol, 2010; Stein, 2011). The role of ammonia-oxidising microorganisms in the global N-cycle has received a large amount of media attention as scientists and policy makers have recognised the impact of anthropogenic inputs of reactive-N to Earth’s terrestrial, aquatic and marine environments (Canfield et al., 2010). Some of these effects, aside from human health costs, include nitrate pollution leading to eutrophication and coastal hypoxia, and atmospheric release of nitrogen oxides (NO and N2O) that contribute to global warming and destruction of stratospheric ozone. Although it is clear that ammonia-oxidising bacteria are major sources of deleterious N-oxides, particularly in environments receiving inorganic N inputs, it remains unclear whether ammonia-oxidising thaumarchaea are also major sources. Recently, it was shown that ammonia-oxidising thaumarchaea in marine systems can produce N2O at very low levels (Santoro et al., 2011). However, considering their relative abundance, this source could be significant globally. However, whether soil thaumarchaea are similarly active in producing N2O remains an unanswered question. See also: Biogeochemical Cycles; Climate Change and Biogeochemical Impacts; Nitrogen Budgets

Physiology Both ammonia-oxidising bacteria and thaumarchaea can grow autotrophically; the bacteria fix CO2 via the Calvin– Benson–Bassham cycle, whereas the thaumarchaea use the 3-hydroxyprotionate-4-hydroxybutyrate cycle. As mentioned above, a thaumarchaeon isolated from soils benefits from addition of simple organic molecules such as pyruvate (Tourna et al., 2011), and ammonia-oxidising thaumarchaea in acidic soils appear to acquire ammonia from mineralised organic material instead of from inorganic sources (Schleper and Nicol, 2010). Both of these observations suggest a greater inclination of ammonia-oxidising

thaumarchaea towards a mixotrophic, or even heterotrophic, lifestyle than their bacterial counterparts. The pathway for ammonia-oxidation in both bacteria and thaumarchaea is shown in Figure 1. Note that as of this writing, the ammonia-oxidising pathway of the thaumarchaea remains highly speculative. As additional ammonia-oxidising thaumarchaea and bacteria are isolated, genome-sequenced and physiologically examined, we are better understanding how they differ from one another. The genes encoding AMO are not syntenous; bacterial operons are in the order of amoCAB and are coregulated, whereas the amo genes in thaumarchaea are usually separated from each other by one or more other genes (Schleper and Nicol, 2010). However, both bacterial and archaeal AMO enzymes are inhibited by acetylene, suggesting similar structural features and catalytic mechanisms (Offre et al., 2009). Perhaps the most surprising difference between the two groups is the inability to detect a hydroxylamine intermediate formed from the monooxygenation of ammonium in the thaumarchaea. It is possible that because thaumarchaea respond to such low levels of ammonia, our methods for detecting transient intermediates of the ammonia oxidation pathway are simply too insensitive. The isotopic signature of nitrous oxide produced by marine thaumarchaea is similar to that of nitrous oxide produced directly from hydroxylamine oxidation by ammonia-oxidising bacteria, suggesting that the pathway is identical between the two microbial groups (Santoro et al., 2011). Adding to the mystery, there are no gene homologues of iron-containing enzymes in available thaumarchaeal genomes, including those for hydroxylamine oxidoreductase and associated cytochromes (Schleper and Nicol, 2010). In ammonia-oxidising bacteria, the gene cluster encoding hydroxylamine oxidoreductase includes genes for the two cytochrome c enzymes that relay electrons to the quinone pool for generation of proton-motive force and for activating AMO (Klotz and Stein, 2008). This hydroxylamine-ubiquinone redox module (HURM) has its evolutionary origins from the anaerobic ammonia-oxidisers, or anammox bacteria (Klotz and Stein, 2008). Although HURM cannot be reconstructed from genomic or physiological information from Nitrosopumilus maritimus, a hypothesis is that ammonia-oxidising thaumarchaea use copper enzymes to relay electrons from the oxidation of an as-yet-to-be-determined N-oxide intermediate (most likely hydroxylamine or nitroxyl) to membrane-bound electron carriers for generation of a proton motive force (Schleper and Nicol, 2010).

Linkages between Methane and Ammonia Oxidisers Taxonomy Ammonia oxidisers belong to the archaeal phylum Thaumarchaeota and the Betaproteobacteria and Gammaproteobacteria classes of the phylum Proteobacteria.


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Methanotrophs belong to the phyla Verrucomicrobia and Candidate Division NC10, and to the Alphaproteobacteria and Gammaproteobacteria classes of the Proteobacteria. At the class level, the only group known to include both methanotrophs and ammonia oxidisers is therefore the Gammaproteobacteria, but they occur in distinct phylogenetic families even in this case. Of course, given the lack of knowledge about most bacteria in nature, it is conceivable that we will yet find ammonia oxidisers in the Verrucomicrobia or NC10 phyla, or methanotrophs amongst the Thaumarchaeota. However, at the moment it appears that ammonia-oxidising clades are separated from methanotrophic clades by deep evolutionary branches – a fact that makes their fundamental similarities more intriguing. As yet, there is no clear evidence of recent lateral transfer of the key monooxygenase genes conferring either methanotrophic or ammonia-oxidising phenotypes from one species to another, although other genetic systems related to the processes show multiple apparent transfers and deletions.

Evolution of monooxygenases Obligate, chemolithotrophic ammonia oxidation is performed by microorganisms that possess AMO. Methanotrophy requires one form of an MMO, either pMMO or Gammaproteobacteria (Crenothrix) pmoA DQ295902, DQ295899 and AF264136

sMMO. In 1989, Be´dard and Knowles noted that ‘Because of its inhibitor profile and membrane location, ammonia monooxygenase is thought to _ closely resemble the particulate MMO’. It was later discovered that the enzymes did not merely resemble each other, they were evolved from a single ancestral enzyme. AMO and pMMO both belong to the copper-containing membrane-bound monooxygenase superfamily (CuMMO). The sMMO belongs to the soluble di-iron monooxygenase family of enzymes and is evolutionarily separate from pMMO/AMO. The respective genes encoding pMMO and AMO (usually pmoCAB or amoCAB) can be easily aligned, demonstrating that they have evolved from common ancestral genes. The pmoA/amoA gene has been most extensively sequenced, and a phylogeny is shown in Figure 2. The major taxonomic groups based on 16S ribosomal ribonucleic acid (rRNA) sequence analysis are also distinguishable based on amoA/pmoA, indicating limited horizontal transfer of these key genes. One exception may be the gammaproteobacterium Crenothrix. The branching points between the major groups are poorly resolved and tend to differ with the phylogenetic inference method as well as the pmo/amo gene used (Op den Camp et al., 2009; Tavormina et al., 2011). The Thaumarchaeota usually form the most distant branch and a molecular clock logic would place the ancestor on this branch. On the other hand, the ability to detoxify and metabolise the products of monooxygenation (esp.

Candidate division NC10 pmoA FP565575

Betaproteobacteria amoA AB079055, U76552, DQ008439, AF037108, AF032438, U31655, U89833, AF042170 and AF042171 50

54 63

Thaumarchaeota amoA DQ085098, DQ534815, DQ278583, DQ278576, DQ278580, DQ278555,

Gammaproteobacteria pmoA and amoA 77

DQ534874, DQ500963 and DQ278503

Verrucomicrobia pmoA EU223862, EF591086, EU223859 and FJ462791

0.2

69

57

EU223855, EF591087,

Alphaproteobacteria pmoA AM410177, AJ431387, AY662377, AY662385, EU723751, AY500133, AJ278727, AJ459018, AJ459003, AJ458998, AJ459008, AJ431388, AM283546, EU647260 and FN433469

Gammaproteobacteria pxmA EU722431, EU722432 and EU722433

AJ581836, AY354041, AY550734, AY354039, AY829010, U89304, AF508901, AF533666, AF307139, NC_002977, AF153344, AF150795, U31653, AY945762, AY007285, EF623678, AB275418, AF047705, AB453964, AF510077, AF016982, EU071096, U31654, AY007286, AB176705, AY195660 and AB302948

Figure 2 Phylogenetic tree of partial (495 nucleotides) pmoA/amoA gene sequences of major groups of ammonia oxidisers and methanotrophs. The accession numbers of the sequences used to create the tree are shown. The tree was constructed with TREE_PUZZLE, a quartet maximum-likelihood method, using a Schoeniger–von Hasseler distance calculation (Schmidt et al., 2002). Support values for major nodes are given, and multifurcations are drawn when the support for a bifurcation is 550%. The bar represents 0.2 changes per nucleotoide position.

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hydroxylamine) should have evolved before connecting these pathways to a CuMMO (Klotz and Stein, 2008). These detox systems appear to have evolved in ancestors of the Planctomycetes (anammox bacteria) and Proteobacteria, as extant relatives maintain these systems. This in turn suggests that the genes encoding the ancestral CuMMO may have evolved first in anaerobic proteobacteria and later been transferred to the verrucomicrobia and thaumarchaea (Klotz and Stein, 2008; Tavormina et al., 2011). Additional members of the CuMMO superfamily, particularly from anaerobic microorganisms, will be required to verify its evolutionary history. At present the only clear conclusion is that a single CuMMO ancestral enzyme has evolved specialised capacity for oxidising either ammonia or methane in particular phylogenetic groups.

Hydroxylamine The CuMMOs of methanotrophs and bacterial ammonia oxidisers are similar enough to one another that methanotrophs are excellent nitrifiers and generate hydroxylamine as a product of ammonia monooxygenation (Be´dard and Knowles, 1989; Nyerges and Stein, 2009). Hydroxylamine is an extremely toxic molecule and must be transformed or removed quickly to prevent cellular damage. A physiological study of four cultivated methanotrophs showed significant differences in their capacity for ammonia oxidation and hydroxylamine detoxification as well as their resistance to high ammonium or nitrite levels (Nyerges and Stein, 2009). Some methanotrophs have homologues to hydroxylamine oxidoreductase like that found in bacterial ammonia-oxidisers, which are highly expressed in response to ammonium (Campbell et al., 2011). These hao-encoding methanotrophs exhibit high rates of ammonia conversion to nitrite relative to methanotrophs that lack hao gene homologues (Nyerges and Stein, 2009; Campbell et al., 2011). Some methanotrophs that lack hao genes encode a hydroxylamine reductase (hybrid cluster protein; prismane protein) that is speculated to reduce hydroxylamine back to ammonium. For methanotrophs, pathways for handling hydroxylamine do not closely follow vertical phylogenetic lines of descent (as modelled with 16S rRNA), suggesting multiple lateral gene transfer events and deletions. Methanotrophs that encode hydroxylamine oxidoreductase have high nitrifying activities and tend to be resistant to nitrite, whereas those with different detoxification strategies are poor nitrifiers with high sensitivity to nitrite. Such physiological differences may explain why some lineages of methanotrophic bacteria are more successful in N-saturated ecosystems than others.

Ammonium inhibition/stimulation of methanotrophs A consequence of the oxidation of ammonia by pMMO and sMMO is often an inhibitory effect of ammonium fertilisation on methanotrophic activity in soil (reviewed in

Bodelier and Laanbroek, 2004; Dunfield, 2006). The degree of inhibition depends on the concentration of fertiliser and undoubtedly also on the metabolic features of the particular methanotroph species present (e.g. the presence of hydroxylamine oxidoreductase). The inhibition can be a short-term substrate competition whereby ammonia blocks access of methane to the active site of MMO, but it can also be a long-term effect of toxic ammonia oxidation products, particularly hydroxylamine and nitrite. It is suspected that the decline of methane oxidation activity in agricultural soils compared to uncultivated soils is due to long-term inhibitory effects of fertiliser nitrogen on methanotroph populations, caused by excessive nitrification activity in the native methanotrophs. On the other hand, it is also clear that methanotrophic bacteria, like all bacteria, require nitrogen for growth, and therefore fertilisation of soils can stimulate methane oxidation activity in many cases (Bodelier and Laanbroek, 2004). The genetic potential of particular methanotrophs to deal with ammonium and its oxidation products is probably a key determinant of whether effects are stimulatory or inhibitory, although clear data to this effect in situ is lacking. In N-saturated ecosystems, the methanotroph community composition does tend to shift with fertilisation (e.g. Noll et al., 2008), although correlation of specific methanotrophic populations with their nitrification/ denitrification gene markers has yet to be demonstrated.

Denitrification Ammonia-oxidising bacteria and thaumarchaea contain homologues to the copper-containing nitrite reductase, NirK (Schleper and Nicol, 2010). The primary physiological role of NirK in Nitrosomonas europaea is to assist in the efficient conversion of hydroxylamine to nitrite, perhaps acting as an electron sink to prevent the accumulation of hydroxylamine (Stein, 2011). The product of nitrite reduction is the toxic molecule NO, which is reduced to N2O by nitric oxide reductase (Nitrosomonas europaea encodes multiple enzymes with this activity). During ammonia oxidation, no greater than 10% of NH3-N is ever converted to N2O. The majority is converted to nitrite to complete the energy-generating pathway (Stein, 2011). Nitrite reduction to N2O by NirK and nitric oxide reductase is a minor pathway that appears to be involved in electron flow, but not as its own central metabolic or energy-generating process. Conversely, in metatranscriptome studies, abundances of thaumarchaeal amoA and nirK transcripts are often matched, suggesting that both gene products are essential to the ammonia oxidation process in these organisms. The common presence of NirK in both ammonia-oxidising bacteria and thaumarchaea suggests a linkage of nitrite reduction to nitrification, rather than solely to respiratory denitrification as has long been believed. Although this partial denitrification activity is probably a side-reaction of ammonia-oxidisers, it contributes significantly to the atmospheric N2O reservoir (Wrage et al., 2001).


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Many methanotrophic bacteria also encode partial denitrification pathways and have the ability to reduce nitrate and/or nitrite to N2O via nitrate, nitrite, and nitric oxide reductases (Nyerges et al., 2010; Campbell et al., 2011). These enzymes may also serve a combined function of hydroxylamine detoxification and respiration. In one experiment, two methanotrophic strains, one with high tolerance to ammonium and one with high tolerance to nitrite, were cultured together (Nyerges et al., 2010). The nitrite-tolerant strain was generally more competitive and produced a larger amount of N2O with concomitant nitrite loss. This experiment suggested that methanotrophs with denitrifying capacity may outcompete other methanotrophs in ecosystems with high N-loading as they have capacity to withstand nitrosative stress and may even derive a growth benefit in the presence of both oxygen and nitrite. Finally, the discovery of oxygenic methanotrophs that denitrify NO2 2 (Ettwig et al., 2010) clearly raises new possibilities for linking the nitrogen and methane cycles in the environment. These organisms apparently do not produce N2O, but dismutate NO produced by nitrite reductase directly to O2 and N2. The O2 is then used to fuel pMMO under anoxic environmental conditions. A better understanding of how these organisms contribute to nitrogen and methane turnover in nature will require further research.

Specific inhibitors If methanotrophs and nitrifiers can use each others’ substrates, an obvious question arises: How much of the methane oxidation observed in the environment is in fact caused by nitrifiers? And conversely, how much of the nitrification and N2O flux is actually caused by methanotrophs? Key tools to answer such questions are specific metabolic inhibitors. An ideal metabolic inhibitor should inhibit only the target organisms. Unfortunately, because of their physiological similarities, most inhibitors of methane oxidation are also inhibitors of nitrification and therefore do not strictly meet this criterion. Some of the better-known inhibitors of methanotrophs and nitrifiers are nitrapyrin, allylthiourea, picolinic acid, acetylene, potassium cyanide and chlorocolinic acid. They can be useful to eliminate both processes, but are of little use where the goal of a study is to separate the ecological impacts of methanotrophs versus nitrifiers. Most of the early attempts to develop differential inhibitors for methanotrophs and nitrifiers were reviewed in Be´dard and Knowles (1989). Dicyandiamide (NH2CNHNHCN) and picolinic acid each showed a small differential inhibition on methane oxidation versus nitrification, but the differences were too small to make them useful in the field. Subsequently the discovery of diallylsulfide (CH2CHCH2)2S as a suicide substrate for AMO (Juliette et al., 1993) provided the basis to evaluate it as a differential inhibitor. Although methane oxidation in a lake sediment was two orders of magnitude less susceptible 8

to inhibition by diallylsulfide than was nitrification, its poor water solubility proved a serious limitation (Roy and Knowles, 1995). Dimethyl sulfoxide (DMSO) can be used as a solvent but care has to be taken to measure the inhibitory effect of DMSO. Studies using pure cultures of methanotrophic bacteria showed little inhibition by DMSO alone, but inhibition of methane oxidation in a forest soil was shown by Saari and Martikainen (2001). This work in fact showed that DMSO may have potential as a differential inhibitor in some sites. Methylfluoride (CH3F) was reported as a differential inhibitor between methane oxidation and methane production (Oremland and Culbertson, 1992). This gas is highly soluble in water. However, subsequent work showed that CH3F also inhibited other processes. Methanogens, especially those growing acetotrophically, were also susceptible to inhibition by CH3F (Frenzel and Bosse, 1996). In addition, it inhibited nitrification and N2O production in soil, lake sediment, and cultures of Nitrosomonas europaea and Nitrosococcus oceanus (Miller et al., 1993). Bodelier and Frenzel (1999) found that at a CH4/CH3F/ NH4+ molar ratio of 0.1:1:18 in rice field soil, CH3F selectively inhibited methanotrophs over nitrifiers. The molar ratios are critical for a differential effect as the substrates act competitively on the enzyme active sites. Use of all the above inhibitors is complicated because none is truly specific. Effects are often ecosystem-specific and experiments require considerable optimisation to select proper concentrations where a differential inhibition is evident. However, the recent discovery that phenylacetylene acts as a selective inhibitor of AMO in some nitrifiers is promising. Initial studies with phenylacetylene demonstrated that a small amount affected AMO activity in Nitrosomonas europaea, but not the AMO in Nitrosococcus oceanus nor pMMO in seven methanotrophs (Lontoh et al., 2000). In a landfill cover soil, it was found that phenylacetylene suppressed the synthesis of bacterial and archaeal amoA, but not pmoA of methanotrophs (Lee et al., 2009). The authors discussed the likelihood that Ser168 and/or Val/Met176 in amoC control the substrate range of the AMO since they are the only two amino acid substituted for Asp168 and Glu176 in pmoC of methanotrophic bacteria (Lee et al., 2009). This inhibitor seems promising for future study, and may even be useful in field application to reduce N2O emissions from landfill soil when fertiliser is added to stimulate methane oxidation (Im et al., 2011).

Methanotrophs as nitrifiers? There is good evidence that, in rare situations, methanotrophs do contribute to soil nitrification, particularly in the production of N2O. Nitrification by aerobic methane-oxidising bacteria is dependant on methane since they cannot grow on NH3. Freshwater sediments and flooded soils with accumulated organic matter have a large oxygen demand and limited O2 diffusivity, so organic matter is converted largely to methane by methanogens. These environments provide ideal conditions for aerobic methanotrophic



bacteria to grow at oxic/anoxic interfaces (Section ‘Systematics and Ecology’). The same environments also promote the production of NH+ 4 and support nitrifiers at the same oxic/anoxic interfaces. Therefore, competitive substrate cooxidation is a distinct possibility. Megraw and Knowles (1989) observed methanedependent nitrification in a humisol that was enriched with methane. Although not a natural system, this demonstrated that when methanotrophic populations are highly enriched in a system they might account for nitrification. Bodelier and Frenzel (1999) extended this to a natural system highly enriched in methanotrophs, a ricefield soil. Using a combination of CH3F and C2H2 to specifically inhibit nitrifiers and methanotrophs in rice field soil, they demonstrated that nitrifying bacteria made no measurable contribution to methane consumption, but methanotrophs made an appreciable (perhaps as much as 100%) contribution to nitrification. Some more recent studies of methane oxidation and N2O emission in soils using stable isotopes and specific inhibitors provide further evidence of the role of methanotrophic bacteria in nitrification. Under controlled laboratory conditions, a silt loam soil at a 60% water-filled spore space exposed to 13C–CH4 and varying concentrations of 15 NH4 15NO3 showed possible N2O production from methanotrophic bacteria (Acton and Baggs, 2011). The emission of N2O by a landfill cover soil also appeared primarily due to methanotrophic bacteria, based on methane-dependent N2O flux and on isotopic ratios of methanotroph fatty acids (Mandernack et al., 2000). The importance of methanotrophic nitrification to N2O emissions from landfill cover soils was further supported in two recent studies (Lee et al., 2009; Im et al., 2011). Phenylacetylene was used as a specific inhibitor of the AMO with little effect on the pMMO. It was found to suppress N2O production and activity of bacterial and thaumarchaeal nitrifiers, but not the gamaproteobacterial (Type I) methanotrophs (Im et al., 2011). The authors suggested that N2O in this system was produced by both archaeal nitrifiers and alphaproteobacterial (Type II) methanotrophs. N2O emissions could be reduced by a combination of phenylacetylene (to inhibit archaeal nitrifiers) and fertilisation (to allow gammaproteobacterial methanotrophs to predominate over the alphaproteobacteria). Sutka et al. (2003) noted that the 15N isotopomeric composition of N2O formed by hydroxylamine oxidation in a methanotroph (Methylococcus capsulatus) was similar to that measured in soils and oceans, whereas that formed by either hydroxylamine oxidation or denitrification in a nitrifying bacterium (Nitrosomonas europaea) was lower. Although the basis for comparison in this study is based on too few organisms, and did not include thaumarchaea, the results are at least suggestive of a role for methanotrophs in global N2O production. Another possible candidate for N2O emission is the thaumarchaea, which (compared to nitrifying bacteria) also produce N2O with a 15N isotopomeric composition closer to that estimated for the oceanic source (Santoro et al., 2011).

It has been suggested that the discrepancy between the average d15 NN2 O and d18 ON2 O values of atmospheric sources from soil and oceans and the isotopic composition of tropospheric N2O may be explained by the contribution of methanotrophic bacteria in methanerich habitats. A recent study tested this hypothesis by measuring the values of d15N and d18O of N2O produced during the cooxidation of NH4+ by Methylosinus trichosporium and Methylomonas methanica (Mandernack et al., 2009). This study concluded that methanotrophs in high-methane habitats may be an important source of 18O-enriched N2O to the atmosphere and explain the difference between 18O-enriched tropospheric N2O and more 18O-depleted sources of N2O from soils and oceans.

Nitrifiers as methanotrophs? On the other hand, there is no clear evidence for ammonia oxidisers acting as a methane sink in the environment. Bacterial ammonia-oxidisers such as Nitrosomonas europaea and Nitrosococcus oceanus can oxidise methane (Be´dard and Knowles, 1989). Although it was demonstrated experimentally that Nitrosococcus oceanus can completely oxidise CH4 to CO2 and incorporate C from this oxidation (Be´dard and Knowles, 1989), few subsequent studies have looked at the contribution of ammonia-oxidising bacteria to methane oxidation in nature. These few studies have largely delivered negative results. A study of methane oxidation in a littoral sediment suggested that nitrifiers were unlikely to be important in this process (Bosse et al., 1993). Calculations in multiple studies indicated that populations of nitrifying bacteria would need to be several orders of magnitude higher than observed populations in order to account for measured methane oxidation rates in soils and sediments (reviewed in Dunfield, 2006). Experiments using specific inhibitors of methanotrophy versus nitrification fail to show any contribution of nitrifiers to methane oxidation (Roy and Knowles, 1995; Bodelier and Frenzel, 1999). The cooxidation of methane by nitrifying bacteria almost certainly occurs, but the rate is so small compared to the rate of methane oxidation by methanotrophs that it is never more than a tiny fraction of the total methane oxidation. The maximum specific CH4 oxidation rate in nitrifying bacteria is lower than the minimum rate in any methanotroph, and their affinity to methane is generally lower than the affinity of methanotrophs for methane (Be´dard and Knowles, 1989). However, a big unknown is the potential role of the nitrifying thaumarchaea. The rate of methane cooxidation by thaumarchaea is not yet known. If they have a higher affinity to ammonia than the nitrifying bacteria, it is likely that they also have a higher affinity to methane, and their rates of methane cooxidation could be much higher. The discovery of these archaea may provide incentive to look again at the potential of nitrifiers to oxidise methane in natural systems.


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Conclusion Since Be´dard and Knowles reviewed the early work into methanotrophic and nitrifying bacteria in 1989, our knowledge of the diversity of these groups has greatly expanded, opening up many new avenues of enquiry. Particularly intriguing are the discoveries of nitrifying thaumarchaea and nonproteobacterial methanotrophs. Although these organisms all have monooxygenase enzymes, they appear to have different enzymatic mechanisms for dealing with methanol and hydroxylamine. The rapidly expanding database of genome sequences of diverse methanotrophic and nitrifying isolates is providing glimpses of additional metabolic diversity that await further testing and validation. These discoveries have required a reconsideration of the relative roles and interactions of methanotrophs and nitrifiers in nature.

Acknowledgements This paper was supported by the Natural Sciences and Engineering Research Council of Canada Discovery Grant Program, as well as by Genome Alberta/Genome Canada (Hydrocarbon Metagenomics).

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Schmidt HA, Strimmer K, Vingron M and Haeseler A (2002) TREE-PUZZLE: maximum likelihood phylogenetic analysis using quartets and parallel computing. Bioinformatics 18: 502– 504. Semrau JD, DiSpirito AA and Yoon S (2010) Methanotrophs and copper. FEMS Microbiology Reviews 34: 496–531. Stein LY (2011) Heterotrophic nitrification and nitrifier denitrification. In: Ward BB, Arp DJ and Klotz MG (eds) Nitrification, pp. 95–114. Washington, DC: ASM Press. Sutka RL, Ostrom NE, Ostrom PH et al. (2003) Nitrogen isotopomer site preference of N2O produced by Nitrosomonas europeae and Methyloccus capsulatus Bath. Rapid Communication in Mass Spectrometry 17: 738–745. Tavormina PL, Orphan VJ, Kalyuzhnaya MG et al. (2011) A novel family of functional operons encoding methane/ammonia monooxygenase-related proteins in gammaproteobacterial methanotrophs. Environmental Microbiology Reports 3: 91–100. Tourna M, Stieglmeier M, Spang A et al. (2011) Nitrososphaera viennensis, an ammonia oxidizing archaeon from soil. Proceedings of the National Academy of Sciences of the USA 108: 8420–8425. Wrage N, Velthof GL, van Beusichem ML and Oenema O (2001) Role of nitrifier denitrification in the production of nitrous oxide. Soil Biology and Biochemistry 33: 1723–1732. Yao H, Gao Y, Nicol GW et al. (2011) Links between ammonia oxidizer community structure, abundance, and nitrification potential in acidic soils. Applied and Environmental Microbiology 77: 4618–4625.

Further Reading Arp DJ and Stein LY (2003) Metabolism of inorganic N compounds by ammonia-oxidizing bacteria. Critical Reviews in Biochemistry and Molecular Biology 38: 471–495. Chistoserdova L, Vorholt JA and Lidstrom ME (2005) A genomic view of methane oxidation by aerobic bacteria and anaerobic archaea. Genome Biology 6: 208. Dalton H (2005) The Leeuwenhoek Lecture 2000. The natural and unnatural history of methane-oxidising bacteria. Philosophical Transactions of the Royal Society B 360: 1207–1222. Dedysh SN and Dunfield PF (2010) Facultative methanotrophs. In: Timmis KN (ed.) Handbook of Hydrocarbon and Lipid Microbiology, pp. 1967–1976. Berlin: Springer-Verlag. Jetten MSM, Niftrik L, Strous M et al. (2009) Biochemistry and molecular biology of anammox bacteria. Critical Reviews in Biochemistry and Molecular Biology 44: 65–84. Klotz MG and Stein LY (eds) (2011) Research on Nitrification and Related Processes, Part B. San Diego, CA: Elsevier. Rosenzweig A and Ragsdale SW (eds) (2011) Methods in Methane Metabolism, Part B: Methanotrophy. Methods in Enzymology, vol. 495. London: Academic Press. Scheutz C, Kjeldsen P, Bogner JE et al. (2009) Microbial methane oxidation processes and technologies for mitigation of landfill gas emissions. Waste Management and Research 27: 409–455. Trotsenko YA and Murrell JC (2008) Metabolic aspects of aerobic obligate methanotrophy. Advances in Applied Microbiology 63: 183–229. Ward BB, Arp DJ and Klotz MG (eds) (2011) Nitrification. Washington, DC: ASM Press.


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