Xu et al. 2009

Xu C, Guo L, Ping C-L, White DM. 2009. Chemical and isotopic characterization of size-fractionated organic matter from cryoturbated tundra soils, northern Alaska. Journal of Geophysical research 114, G03002.

These authors examined the isotopic composition and organic matter distribution in soil horizons and particle size fractions from two soils in Alaska, a moist acidic tundra and a moist non-acidic tundra. The organic matter quality and quantity in the deeper part of the active layer and down into the permafrost, material estimated to be between 3000 and 7000 years old indicated high susceptibility to microbial activity, that is, decomposition to CO2 and subsequent release to the atmosphere.

I read this paper as part of my background reading to understand the potential uses of a Picarro field-portable carbon isotope analyser; this paper includes a description of the δ13C values of organic matter throughout the soil / permafrost profiles of these Alaskan soils. These values, and the associated discussion of signatures of microbial activity, suggest it is quite possible to distinguish the source of CO2, permafrost-SOM, deep-SOM, shallow/autotrophic, based on the 13C content of effluxing CO2 and / or CO2 at various depths within a soil profile. Table 5 is particularly valuable in this regard.

Lin et al., 2009

Lin X, Wang S, Ma X, Xu G, Luo C, Li Y, Jiang G, Xie Z. 2009. Fluxes of CO₂, CH₄, and N₂O in an alpine meadow affected by yak excreta on the Qinghai-Tibetan plateau during summer grazing periods. Soil Biology and Biochemistry 41: 718-725.

I read this paper in an attempt to gain a better understanding of the methods used to compare greenhouse gas fluxes between ecosystems or treatments, and between gases, particularly the use of CO₂-equivalents when estimating total global warming potential contributions by ecosystems that may be simultaneously sources and sinks for the greenhouse gases CO₂, CH₄, and N₂O. In addition, this is one of a small number of studies I have been able to find that draw conclusions about global warming potentials based only on growing season measurements, rather than whole-year or growing season plus “cold season” (often what the tourist industry might call the shoulder seasons, spring and fall, though sometimes including winter as well).
These authors studied the effects of yak (Bos grunniens) excreta, dung and urine, on soil emissions of the three greenhouse gases. Excreta were hypothesized to increase GHG emissions because both are rich in nitrogen, especially inorganic forms of nitrogen such as urea, ammonia, and nitrate, contain sufficient water to stimulate microbial activity in dry soils, and, in the case of dung, are rich sources of labile organic carbon compounds and large microbial populations already present in the material. Furthermore, because production of CH₄ by grazing mammals is strongly linked to their digestive systems, fresh dung may contain considerable dissolved CH₄ that will be emitted quickly upon excretion.
The main results of this study were that while fresh dung did significantly shift a patch of meadow from a weak sink for CH₄ to a source, this difference was not sufficient to render the larger meadow area a net source because the spatial distribution of dung patches, as well as the duration of the CH₄ emission from dung, were relatively small. Urine application did not significantly increase CH₄ emission, which is surprising considering the high N concentration and rapid, large addition of water represented by urination by a yak; both factors are expected to increase methanogenesis.
Emissions of CO₂ were increased by dung, but not by urine when considering a longer, cumulative set of emissions. Interestingly, urine produced a significant pulse of CO₂ nearly immediately upon application to the soil, though whether this CO₂ is the result of hydrolysis of urea ((NH₂)₂CO + H₂O --- 2NH₃ + CO₂) or increased microbial respiration is not clear.
Emissions of N₂O were increased by both dung and urine application relative to untreated controls. However, the magnitude of the increase was less than predicted by IPCC (2001) guidelines for calculating the effects of grazing mammals on grasslands; those guidelines were based primarily on temperate low-altitude grasslands, not the high-altitude alpine meadows studied here. In general, patches of yak excreta accounted for an increase in N₂O emissions of up to about 10% compared to ungrazed and untreated control meadow, while total CO₂-equivalents emissions increased by about 1%, largely due to the small total areal extent of excreta patches.

Van Groenigen et al., 2005

Van Groenigen JW, Zwart KB, Harris D, van Kessel C. 2005. Vertical gradients of δ¹⁵N and δ¹⁸O in soil atmospheric N₂O – temporal dynamics in a sandy soil. Rapid Communications in Mass Spectrometry 19: 1289-1295.

These authors examined the production and consumption of N₂O in soil profiles using stable-isotope analysis. Enzymatic processes in the microbial nitrification and denitrification pathways all strongly fractionate isotopes of both N and O, with products of reactions significantly depleted in the heavier isotopes. This allows identification of regions of soil producing or consuming N₂O by distinguishing between local production and diffusion through the soil layer.

Similar to my own studies, gas concentrations were measured using diffusion wells, in this case probes buried in the soil and sampled by syringe; gas samples were measured by GC-IRMS. Internationally-recognized isotope samples for N₂O are not available, so standards for analysis were prepared from commonly available laboratory chemicals and abiotic chemical reactions to completion to avoid fractionation (e.g. reduction of N₂O to N₂ over copper at 600ºC).

The largest concentrations of N₂O were found at the deepest sampling position, 90cm. There was a negative logarithmic relationship between the δ¹⁵N value of N₂O in the soil and depth, consistent with a relatively large single source of N₂O at 90 cm and consumption shallower in the soil combined with vertical-upwards diffusion of N₂O; reduction of N₂O to N₂ enriches the remaining N₂O pool for ¹⁵N.

Miller et al. 2008

Miller MN, Zebarth BJ, Dandie CE, Burton DL, Goyer C, Trevors JT. 2008. Crop residue influence on denitrification, N2O emissions and denitrifier community abundance in soil. Soil Biology & Biochemistry 40: 2553-2562.

These authors conducted a factorial experiment using packed soil cores to examine the influence of varying levels of available carbon and nitrogen on the process of denitrification. The treatments consisted of addition of glucose at three levels and KNO3 at four levels in experiment 1, and additions of either red clover or barley straw crop residues with or without additional KNO3. They measured soil chemistry, including extractable organic carbon and NO3- concentration based on K2SO4 extractions, as well as N2O production, the molar ratio of N2O (N2O : (N2O + N2)), and a handful of bacterial genes by qPCR.

The experimental setup was very similar to what we used in SLSC 802 (Special Topics) in the fall of 2010; cylindrical soil cores with gas-exchange holes in the sides were filled with soil at 1 g cm-3 bulk density and a water content of 70% and placed in 1 L canning jars with lids fitted with a perforable septum for gas sampling. One important difference between this experiment and what we have done is that in this experiment, gas measurements were of total cumulative gas production, whereas we flushed each jar with ambient air after each sampling event. Presumably this difference will have important effects on the formation of anaerobic conditions and microbial consumption of N2O previously produced under less anoxic conditions.

Not surprisingly, minimal denitrification activity was found in treatments without added NO3-. Starting NO3- concentrations were 3 mg NO3--N kg-1 soil, and fell in all treatments without added NO3- to less than 1 mg NO3--N kg-1. Once this supply of readily available nitrate was used, it appears the bacteria ceased denitrification activity, or at least it was reduced.
The red clover had a much lower C:N ratio than the barley straw, 13:1 and 45:1, respectively, and more labile carbon. This difference appears to have driven the observed difference in denitrification activity, in a manner that reflects the results of the simple-C-source experiment 1. In general, more labile C and more available N leads to stronger denitrification activity and greater production of N2O; in sealed jars such as these, strong respiration under these conditions leads to anaerobic conditions and a fall in the molar ratio of N2O as nosZ-equipped microbes consume N2O as a terminal electron acceptor.

Extractable organic carbon (EOC) was a relatively poor predictor of denitrification, compared to respiration as measured by CO2 production. EOC is a measure of the instantaneous size of the pool of labile C, while respiration represents carbon that has already passed through a microbe’s metabolism. The distinction here may be between two different pools of carbon, as well as between an instantaneous snapshot measure and a series of measurements readily convertible to an estimate of the rate of a process.

In conclusion, these authors reiterate their finding that available C and available N (especially as NO3-) are strong predictors of denitrifying activity, across a range of C and N sources. I read this paper for the class SLSC 802 in the fall of 2010, but the portions describing denitrification physiology and especially the qPCR information will be generally useful to my other projects.

Delgado et al. 2010

Delgado JA, Del Grosso SJ, Ogle SM. 2010. 15N isotopic crop residue cycling studies and modeling suggest that IPCC methodologies to assess residue contributions to N2O-N emissions should be reevaluated. Nutrient Cycling in Agroecosystems 86: 383-390.

These authors reanalyzed two recent reviews of measuring nitrous oxide emissions from agricultural systems and used a model to simulate N2O emissions and NO3 leaching associated with cropping practices in Colorado and Iowa. In general, use of crop residue instead of or in addition to synthetic fertilizers significantly altered patterns of N loss, whereas IPCC recommendations assume no difference between these N sources in regards to N2O emissions. Microbial immobilization of nitrogen, particularly associated with residues with high C/N ratios, is a major factor in these differences, and these authors provide supporting arguments for their suggestion of revisions to IPCC recommendations and modeling.

Huang et al. 2004

Huang Y, Zou J, Zheng X, Wang Y, Xu X. 2004. Nitrous oxide emissions as influenced by amendment of plant residues with different C:N ratios. Soil Biology & Biochemistry 36: 973-981.

These authors examined the role of residue quality, in the form of C:N ratio and a range of crop residues, on N2O emissions from soils. They also measured CO2 emissions, and found strong correlations between organic-matter decomposition and respiration, and nitrogen cycling.

Gas fluxes of CO2 and N2O were highly correlated across all incubations. To ensure only respiration-derived CO2 was measured, the CO2 released by urea breakdown in urea-treated treatments was calculated and subtracted; respiration in the urea-only treatment was similar to that in the untreated controls. Both gas emissions were negatively correlated with residue C:N ratios. Finally, residue C:N ratios were negatively correlated with dissolved organic carbon concentrations.

Overall, higher C:N ratios in residues seem to result in slow decomposition of mainly recalcitrant organic matter, and low CO2 and N2O emissions. Addition of urea in conjunction with crop residues produces a range of N2O emissions depending on the C:N ratio of the residues.

This short paper may serve as a model for the work I will be doing in the special topics class in soil science, fall 2010.

Smith et al. 2003

Smith KA, Ball T, Conen F, Dobbie KE, Massheder J, Rey A. 2003. Exchange of greenhouse gases between soil and atmosphere: interactions of soil physical factors and biological processes. European Journal of Soil Science 54:779-791.

These authors present a broad review paper of the role of soil physical factors, mainly temperature and water-filled-pore-space, in controlling soil emissions of the greenhouse gases CO2, CH4, and N2O. The paper’s goal is stated to be to expose a variety of researchers to the links between soil physics and soil biology, as well as the importance of these fields to current research in many disciplines on global warming.

All three gases are produced and consumed in soil primarily by microorganisms, which respond to variation in soil physical parameters in different ways. In general, both temperature and WFPS impact GHG production. Higher temperatures almost always result in increased production of gases, though the Q10 values (measuring the magnitude of response to a change of 10º of temperature) vary widely in the literature for all three gases. The effect of WFPS is different, involving upper and lower bounds, though in the middle range increasing WFPS generally promotes increased gas production. Microbes are limited in their tolerance of dry conditions, such that gas production falls rapidly below some critical WFPS value; for CO2 this threshold is near 20%. WFPS is also indirectly important, through its effects on soil diffusivity. Higher WFPS as well as higher bulk density are associated with lessened CH4 oxidation, due to reduced diffusivity of both CH4 and atmospheric O2. Very high WFPS values are associated with reduction of N2O to N2, partly by limiting O2 supplies and creating larger anaerobic microsites, and partly by preventing the escape of N2O gas into rapid-diffusion pathways; it is trapped in the vicinity of microbes capable of using it as an electron acceptor.

There are other factors controlling net GHG emissions, such as the relationship between plant productivity and water table position, which will change the relationship between rates of soil organic matter oxidation to CO2 and the removal of CO2 from the atmosphere by plants; trees in particular can lower local water tables, increasing SOM oxidation while simultaneously consuming more CO2 than the previous wetland vegetation community.

I read this paper on the suggestion of my coworkers in the special topics class of fall 2010, but it applies well to the general area of my research. The reference list includes multiple interesting papers addressing particular specialties within this large topic.

Palmer et al. 2010

Palmer K, Drake HL, Horn MA. 2010. Association of novel and highly diverse acid-tolerant denitrifiers with N2O fluxes of an acidic fen. Applied and Enironmental Microbiology 76: 1125-1134.

These authors examined soils from an acidic fen in southern Germany, and discovered novel denitrifiers that are apparently adapted to local conditions and contribute to the cycling of nitrogen within the fen. Methods employed included measurement of soil parameters, microcosms to examine denitrification rates (both total denitrification and net production / consumption of N2O), cell counts of cultured organisms, and phylogenetic analysis using narG and nosZ sequences and RFLP.

Harding et al. 2001

Harding RJ, Gryning S-E, Halldin S, Lloyd CR. 2001. Progress in understanding of land surface/atmosphere exchanges at high latitudes. Theoretical and Applied Climatology 70: 5-18.

These authors review and discuss the implications of studies based in two international projects in northern Europe. WINTEX was a large study examining the effects of snow cover and long nights in winter on high-latitude ground-atmosphere exchange processes, while LAPP was an independent but complementary study examining most of the same processes in a range of high latitude sites during spring and summer.

Snow cover plays a major role in Arctic exchange processes. The high albedo of snow reflects much of the incident solar radiation, and insulates the frozen ground below, prolonging the period of snow cover to upwards of 9 months in the year in many places. Where vegetation is tall, such as in the boreal forest, the low solar angle reduces the effective net albedo of the landscape, allowing sunlight to warm the dark trees and speed springtime melting. This study mentions the importance of snow-surface aerodynamics, though it appears there is little solid information on this complex topic.

Snow melt is the major hydrological event of the year in much of the Arctic. The combination of frozen soils, very low evaporation rates, and often flat terrain means much of the Arctic is very wet or saturated while annual precipitation rates are consistent with arid or semi-arid conditions. These areas are the classic tundra systems, with abundant shallow lakes and ponds and very wet high-organic soils.

Differences in snow-surface dynamics and the timing of snowmelt create an extremely heterogeneous landscape, particularly in the vicinity of the northern treeline. There are often very large temperature and air-flow differences between patches of trees and adjacent lakes or clearings, which greatly complicate attempts to model the carbon dioxide emissions (for example) of such areas. Much of this paper is a series of evaluations of some of the models that have been applied to this region. In general, more sophisticated models that can take some of the extreme variability into account perform better than models that cannot account for differences in snow depth or insulating properties. However, this paper makes it clear that current modelling efforts still leave much to be desired in terms of predicting Arctic heat budgets and biological responses.

Water storage is also very difficult to model, and has large and variable impacts on other parts of the system. There appears to be large and unpredictable year-to-year variation in water storage and transport at the scale of catchments and basins, and the importance of soil water in controlling biological processes such as the decomposition of organic matter is large. Runoff matters, even on very gentle slopes.

This paper provides a useful overview of large-scale processes and attempts to understand these processes in the Arctic.

Philippot et al. 2002

Philippot L, Piutti S, Martin-Laurent F, Hallet S, Germon JC. 2002. Molecular analysis of the nitrate-reducing community from unplanted and maize-planted soils. Applied and Environmental Microbiology 68: 6121-6128.

These authors applied molecular techniques including PCR, RFLP, and sequencing to the study of soil bacteria relevant to crops. Dissimilatory nitrate reduction, the process that converts NO3- to NO2-, is widespread in prokaryotes, with the activity described in alpha, beta, and gamma Proteobacteria, gram-positive bacteria, and some archaea. There are two described enzymes that catalyze the reaction and provide energy to the organism; these authors focused on the membrane-bound protein, specifically one subunit that includes a distinctive set of components. Their approach was to design primers for a well-conserved region of the gene narG that amplify a 650bp region, and then subject the PCR product to cloning, RFLP analysis, and sequencing.

Community structure and diversity was compared between pots planted with maize versus unplanted controls. Maize (Zea mays) is a plant that facilitates gas diffusion in its roots under oxygen-stress soil conditions; this creates an aerobic region in the rhizosphere distinct from anaerobic conditions further from roots. While diversity, as measured by standard indices, did not differ between planted and unplanted soils, the structure of the communities did change, with numerous RFLP phylotypes found in only one or the other treatment. This suggests a role of rhizosphere conditions, likely involving both oxygen and root exudates, in selecting for particular groups of microorganisms.

Nitrate reduction occurs primarily or possibly only under aerobic conditions. The microbial cell gains energy from dissimilatory reduction of nitrate, and if it occurs in the rhizosphere, the plant may gain a readily-accessible form of nitrogen in the form of nitrite. Denitrification, the process that shuttles nitrogen atoms from nitrite to gaseous forms such as N2O or N2, can occur under a range of oxygen conditions, including aerobic, thus denitrifiers in the rhizosphere may compete with plant roots for nitrite. The fate of nitrite produced by dissimilatory nitrate reduction can also be to ammonium, though this appears to be rare in soil and more common in vertebrate guts and digested sludge, two environments typically lacking in oxygen.

This paper provides some molecular tools for my own studies of nitrogen dynamics in soils, especially the sequences of the degenerate primers. In addition, it provides some clarification of parts of the remarkably complex soil-nitrogen cycle.

Dandie et al. 2007

Dandie CE, Miller MN, Burton DL, Zebarth BJ, Trevors JT, Goyer C. 2007. Nitric oxide reductase-targeted real-time PCR quantification of denitrifier populations in soil. Applied and Environmental Microbiology 73: 4250-4258.

These authors examined the responses of two major components of the denitrifying bacteria fraction of soil bacteria to the addition of labile carbon (glucose) under denitrifying conditions. Denitrification is presented as a four-step process, with enzymes responsible for shuttling nitrate to N2 via nitrite, nitric oxide, and nitrous oxide. In this study, one of the enzymes responsible for the reduction of NO to N2O, cNOR, was examined using primers optimized for two different groups of denitrifying bacteria. This gene is found only in denitrifiers, unlike another enzyme, qNOR, found in many microorganisms and associated with detoxification, rather than utilization, of dangerous nitric oxide.

Primers for qPCR are presented in a table. Specific primers for the two variants of cNOR were developed in this study for use with SYBR green-based qPCR. 16s rRNA sequences were also studied, to examine the total population of soil bacteria; for these qPCR reactions, the TaqMan primers-plus-probe system was used, based on oligonucleotides published by Suzuki et al. 2000.

Two experiments were carried out. In the first, a preliminary experiment to establish the utility of qPCR in this area was based on inoculating soils with cultures of bacteria of known cell density, followed by qPCR evaluation of those soils. Under most conditions qPCR performed well, though at low cell densities of some genera of bacteria the signal was not distinguishable from the background noise also associated with sterilized soil. The second experiment forms the main body of work of this paper, and is an examination of the population dynamics of soil bacteria, divided into the hierarchical categories “denitrifiers” and “all bacteria”, under denitrifying conditions and with varying levels of added labile carbon in the form of glucose solutions in distilled water. In the second experiment, soil nitrate was maintained at a high level, to ensure sufficient raw material for detectable denitrification activity. As N2O accumulation was one of the measures of activity, nitrous oxide reductase activity that would reduce N2O to N2 was inhibited by maintaining an atmosphere of 10% acetylene in culture jars. Soils were maintained at 70% WFPS to encourage denitrification.

Total microbial biomass was also measured, using the CHCl3 fumigation-extraction technique. While cNOR sequences are almost certainly restricted to one copy per genome, 16s rRNA sequences may range in copy number up to 15 per genome, thus estimates of bacterial populations by qPCR of 16s may have a large error associated with it. Fumigation-extraction captures all carbon associated with cells, thus contributions by archaea and fungi will not be found by molecular methods such as 16s qPCR that are specific to bacteria. However, in this study, estimates of total bacterial population by the two methods were well correlated, with r2 = 0.69.

Denitrification occurred in this study. Soils treated with additional glucose showed greater depletion of nitrate, as expected when denitrifiers increase their activity in response to a food supply and conditions already favour denitrification. These authors provide two possible mechanisms, non-mutually-exclusive, that could lead to increased denitrification activity under added glucose. First, the population of denitrifiers could expand, through both additional cell replication and activation of dormant cells. This would increase the proportion of the bacterial population composed of denitrifiers. Second, the total population of soil organisms could increase, leading to increased respiration, a decrease in oxygenation, and establishment of anaerobic conditions more favourable for denitrification. This would not necessarily change the proportion of the population composed of denitrifiers. In this study, denitrifiers increased their proportion of the population as measured by comparative qPCR from less than 1% to about 2.4% of cell numbers.

This change in population components is central to the approach using qPCR advocated in this paper. As these authors state:

“Although absolute numbers may not be achievable, gross differences and changes in population size are still detectable. The differences observed between the two denitrifier populations studied are then real differences in the responses of these populations to the conditions tested.”

This general approach of examining relative changes in populations is applicable to a very wide array of studies of environmental microbiology, including my own planned studies in which the environmental factor under examination is biogeographical (i.e. latitude) and the functional diversity response is in terms of greenhouse gas cycling."

This paper is of great value to my studies. The qPCR methods are directly applicable, for example the primers presented here will be useful if I decide to examine multiple components of the denitrification pathway. The approach, as described above, is also useful. And the reference list is composed almost entirely of papers I am surprised I have not yet found in my literature searches.

Klotz and Stein 2008

Klotz MG, Stein LY. 2008. Nitrifier genomics and evolution of the nitrogen cycle. FEMS Microbiology Letters 278: 146-156.

These authors review the role of nitrifying microorganisms in the current nitrogen cycle, and their evolution and the emergence of biological nitrogen cycles in early Earth history. The current global nitrogen cycle has changed considerably in the past decades, due to the large increase in nitrogen in the cycle due to human activities. The early-Earth nitrogen cycle was probably mostly driven by abiotic processes. After the development of an oxygen-rich atmosphere, nitrogen cycling was almost entirely biotic, with most key processes driven largely or entirely by bacteria and archaea. In the last few decades, the anthropogenic abiotic processes of fertilizer production and fossil-fuel combustion combined with increased cultivation of N2-fixing crops, has transformed the global nitrogen cycle.

As presented in this paper, there are two lobes to the global nitrogen cycle. N2 gas in the atmosphere is fixed to NH3, by nitrogenase in bacteria and archaea, by the Haber-Bosch industrial process, and (in small quantities) by hydrothermal vents. The process of nitrification converts this ammonia to nitrite/nitrate. Nitrite/nitrate are returned to the atmosphere as N2 through denitrification, with production of N2O under weakly anaerobic conditions. The other lobe of the cycle is a “short circuit” that avoids the atmospheric N2 pool and cycles nitrite/nitrate back to ammonia through the processes of ammonification and through production and decomposition of organic matter containing nitrogen. There is another, minor short circuit, as “anammox”, anaerobic ammonia oxidation, returns ammonia to N2 directly.

This “mini review” focuses on the nitrification portion of the cycle. The first step, oxidation of ammonia to hydroxylamine (NH2OH) is carried out by ammonia oxidizing bacteria, abbreviated AOB. There are many acronyms in this paper, reflecting the many acronyms in the existing literature regarding global biogeochemical cycles. NOB are nitrite oxidizing bacteria, and they take the oxidized products of AOB, especially nitrite through to nitrate. Anammox bacteria, on the other hand, may run the same net process of NH3 to NO3- directly, without collaboration with other cells.

The discussion of the plausible evolutionary scenarios in this paper is interesting but not particularly relevant to my current research. This discussion focuses on the relative timing of major events, such as the emergence of nitrification, complete and incomplete denitrification, an oxygenated atmosphere, and nitrogen fixation. These factors interact with each other, creating conditions favourable or not to the evolution of each other and of possible detail shifts within.

The description of the role of hydroxylamine produced by early nitrifiers in stimulating evolution of metabolic pathways responsible for its detoxification initially reads as speculation, but a long and detailed description of the ways in which the various components of those metabolic detox enzymes and pathways function provides plenty of support for the arguments. One aspect of this discussion is that some enzymes are currently misclassified, and that very similar enzymes in different organisms have different names reflecting different ultimate functions rather than the usual (and preferred) enzyme naming scheme that reflects proximate function.

In the discussion concerned with anthropogenic climate change and nitrogen dynamics, especially in soils and ocean waters, interactions with methane are briefly considered. This is based on the observation that methanotrophs are often also ammonia-oxidizers, operating under a budget of consumption of both molecules that shifts as ammonia from fertilizer is added to the system.
Of greatest relevance to my current work is the section describing the gene ncyA. This encodes an enzyme (nitrosocyanin) involved in the pathway from ammonia to nitrite, and has only been found in AOB to date, as opposed to NOB, anammox, or heterotrophs; it seems to be involved in the chemistry of obligate chemolithotrophy as expressed by AOB. It seems likely the enzyme binds and reduces NO, a highly toxic intermediate in ammonia oxidation. The regulatory region adjacent to the gene is also suggestive of roles in this metabolic pathway, and regulation is linked to concentration of various nitrogen-with-oxygen compounds.

This review is very useful to my current research. This paper and the major references in it will be key to constructing a diagram of the complex nitrogen transformations occurring in soils, which will allow targeted hypothesis generation and testing regarding the communities and processes in the soils I am studying.

Kellman and Kavanaugh 2008

Kellman L, Kavanaugh K. 2008. Nitrous oxide dynamics in managed northern forest soil profiles: is production offset by consumption? Biogeochemistry 90: 115-128.

These authors measured surface flux and subsurface profiles of N2O at a number of paired sites in the managed forest of Nova Scotia. Half of the sites were clear-cut harvested three years before the study, the other half more than 50 years previously. Climate factors such as air temperatures and solar radiation were consistent across the study area. Fluxes and profiles were measured periodically through a 9-month snow-free period in 2005, from early March to late November.

Surface fluxes were measured by pulling samples into evacuated containers from chambers mounted on permanent collars. Similarly, profiles were measured by sampling from permanent probes buried in the walls of soil pits. Actual measurement of gas concentrations were in the laboratory using a gas chromatograph system.
The soil probes consist of 50cm PVC tubes, covered with a “water resistant porous membrane” (could they be using Gore-tex?) and buried in the walls of pits at depths of 0, 5, 20, and 35cm, with 0 at the mineral soil-organic layer interface. This provides a 50cm-long sampling space at four depths, replicated across 40 sites.

The relationship between profile N2O concentrations and surface flux was almost always non-significant. These authors attribute this lack of correlation to consumption of N2O in the soil profile. In contrast, the studies that have linked CO2 profiles to surface flux have relied on the (probably true) assumption that CO2 is not consumed in the soil, and moves through diffusion in a manner that can be predicted from soil physics. N2O profiles that include regions of consumption are complicated by the biological and chemical factors that control production and consumption, as well as movement. All of this leads to a disconnection between soil N2O cycling and surface-atmosphere exchange.

This paper should almost certainly be included in the introduction, methods, and/or discussion section(s) of my pits & probes manuscript. This is one of the few studies I have found that examined N2O in soil profiles; most others appear to focus on CO2 or in some cases the biogeochemistry of CH4.

Moffat et al. 1990

Moffat AJ, Johnston M, Wright JS. 1990. An improved probe for sampling soil atmospheres. Plant and Soil 121: 145-147.

These authors describe a new design of soil gas probe they produced to examine soil O2 levels, especially at landfills and other potentially polluted sites. The design is fairly complex, with moving parts. Essentially, the probe is driven into the soil to the desired depth, and then lifted slightly to create a void below the tip. The top of the probe is then rotated and tapped to open the tip, and a gas sample can be drawn from a tube on the side. These authors tested this probe by measuring O2 concentrations in a column of sand that had been flushed with pure N2; anoxic conditions were recorded in the deeper parts of this column.

Farías et al. 2009

Farías L, Fernández C, Faúndez J, Cornejo M, Alcaman ME. 2009. Chemolithoautotrophic production mediating the cycling of the greenhouse gases N2O and CH4 in an upwelling ecoystem. Biogeosciences 6: 3053-3069.

“Chemolithoautotrophy is the non-photosynthetic biological conversion of C1 molecules (usually CO2 or CH4) into organic matter."

These authors studied the chemolithoautrophic community in the south-eastern Pacific ocean, at a long-term research position on the small continental shelf of Chile. At this position, the water is about 90m deep, and is part of one of the largest and most productive upwelling regions of the world, where productivity is extremely high. For about 70% of the year, surface winds drive upwelling that brings nutrients such as NO3- up to the photic zone, allowing massive planktonic productivity. Some of these plankton are non-photosynthetic prokaryotic autotrophs, using inorganic molecules such as NH4+, NO2-, and HS- as electron donors to drive the energy-intensive process of fixing inorganic carbon, chiefly CO2, as organic matter. These organisms require oxygen as an electron acceptor, thus they are all aerobic organisms and do not thrive in anaerobic environments. Assimilation of CO2 in the dark is the diagnostic signal of the presence of these organisms. Some chemolithoautrophs use CH4 in addition to or instead of CO2, but always under aerobic conditions; anaerobic methanotrophs are outside the scope of this study, and have not been identified in this system.

Nitrous oxide accumulates in these waters, indicative of greater production than consumption. These authors state that N2O reduction can only occur through a single identified pathway, that of total denitrification that takes NO3- or NO2- all the way to N2, and only under extremely limited O2 conditions; they cite Elkins et al. (1978) and Farías et al. (2009). Variation in space and time in dissolved N2O patterns do suggest some consumption is occurring, but in general production outweighs consumption.

Kostina et al. 1994

Kostina NV, Stepanov AL, Umarov MM. 1994. Study of the complex of nitrous oxide-reducing microorganisms in the soil. Eurasian Soil Science 26: 81-87.

These authors extracted microorganisms from a range of soils apparently collected from various places in Russia and produced cultures of organisms capable of reducing nitrous oxide. The only nitrogen source in culture vials was N2O, and conditions were rendered anaerobic by flushing with argon gas.

Most cultures gradually lost their ability to reduce N2O, especially mixed-species cultures. A few pure strains were isolated that did not show this loss, and maintained high levels of activity in storage.

Pseudomonas spp. and Bacillus spp. contributed the vast majority of N2O-reducing activity in all soils, with other groups including Aeromonas spp., Micrococcus spp., Flavobacterium spp., Erwinia spp., and an organism identified as “similar to Corynebacterium” also showing some activity. Neither actinomycetes nor eukaryotes were found in any of the cultures capable of N2O-reduction, indicating this is a physiological process not possessed by these organisms.

This is an English translation of a paper that was probably originally in Russian: Pochvovedeniye 1993 25: 72-76.

Yu and Patrick 2004

Yu K, Patrick WH Jr. 2004. Redox window with minimum global warming potential contribution from rice soils. Soil Science Society of America Journal 68: 2086-2091.

These authors followed up a previous study (Yu and Patrick 2003) that discovered a critical range of soil redox potential (Eh) across a range of pH for rice-agriculture soils, by examining soil Eh in more detail. The critical range is based on minimizing total global warming potential of all 3 major greenhouse gases, in terms of CO2-equivalents; methane and nitrous oxide have much higher radiative forcing than does CO2, when looked at on a 100-year horizon.

The critical range, for all 8 soils studied, is between 180 and -150 mV, what might be considered “moderately reducing” for soils. CO2 production is modest in this range, though it is lower at more reducing conditions. CH4 production is nearly absent in this range, but is very large at redox conditions below -150 mV. N2O production is modest in this range as well, with much higher N2O production under oxidizing conditions (Eh > 180) due to strong nitrification activity.

This paper serves to support earlier ideas about the net effects of redox conditions on the production and consumption dynamics of these gases. The story with CO2 and CH4 is fairly simple, while N2O dynamics are more complicated because there are more pathways for both production and consumption of this gas.

Ettwig et al. 2009

Ettwig KF, van Alen T, van de Pas-Schoonen KT, Jetten MSM, Strous M. 2009. Enrichment and molecular detection of denitrifying methanotrophic bacteria of the NC10 phylum. Applied and Environmental Microbiology 75: 3656-3662.

These authors describe a series of experiments and procedures designed to investigate an enigmatic organism known as NC10, a bacteria in its own eponymous phylum that currently represents the only demonstrated case of biological reduction of nitrate coupled to oxidation of methane under anaerobic conditions. While anaerobic methane consumption has been observed in some archaea, it has not been found coupled to denitrification.

A laboratory culture eventually dominated by NC10 organisms of group a (a distinction within the phylum) was established, based on sediment collected from a eutrophic ditch draining agricultural land on the floodplain of the Rhine river in the Netherlands. This culture was grown and maintained under conditions in which the only carbon source was the sparge gas of CH4-CO2, and nitrogen was supplied with the mineral inputs as nitrate and nitrite, along with a wide range of other inorganic compounds and trace elements.

The major finding of this study was a wealth of knowledge of the basic characteristics of the NC10 organism, and confirmation that it does indeed oxidize methane under anaerobic conditions coupled to denitrification. This process is energetically favourable, and the theoretical stoichiometry matches the observed changes in chemical composition in these experiments, with the nitrite reduction to methane consumption ratio of 8:3.5, versus 8:3 based on mass balance calculations.

One of the surprising aspects of this organism is that its methane-oxidizing activity is completely inhibited by black butyl rubber, as is found in black rubber stoppers for serum vials and other glassware. Grey or red butyl rubber stoppers do not show such inhibition, and repeated boiling of black butyl rubber stoppers in HCl did not remove the inhibitory effects. Strictly anoxic conditions are not required for all aspects of working with this organism; brief exposure to atmospheric oxygen during liquid transfer, for example, did not inhibit methanotrophic activity.

Another strange feature of NC10 concerns its 16s rDNA sequences. General 16s primers do not amplify NC10 DNA. These authors developed new primers for the 16s region based on the DNA in their culture, which they were able to confirm as NC10 based on FISH observations. The new primers allowed them to work more easily with the NC10 DNA, which is not surprising, but the sequences of NC10 16s found did not differ in critical ways from the target regions of the general 16s primers. So, it is unknown why the general 16s primers do not work on NC10 DNA.
This paper was recommended to me and I would not likely have discovered it without this recommendation. I have results from the 2009 work at Alexandra Fjord that suggest simultaneous consumption of CH4 (oxidation) and N2O (reduction), and I did not know if these two processes might be linked in a single organism or within a system such as a symbiosis or food-chain as 2 halves of a redox couple.

My results may be more suggestive of an alternate situation. Rather than anaerobic oxidation of methane (weirdness) coupled to nitrate reduction, I may be looking for cases of reduction of nitrous oxide under aerobic conditions (weirdness) coupled to methane oxidation.

Firestone et al. 1980

Firestone MK, Firestone RB, Tiedje JM. 1980. Nitrous oxide from soil denitrification: Factors controlling its biological production. Science 208: 749-751.

These authors measured the faction of N2O in nitrogen gas outputs from soil slurries under a range of conditions of substrate and oxygen availability. Slurries were employed to avoid problems associated with diffusion of materials through a soil matrix, and the process of denitrification was studied using isotopic tracers, especially in the form of 13N in nitrate and other inputs.

The controls on the production of N2O from denitrification are the concentration of nitrite (NO2-) and the availability of oxygen (O2), with time-since-anoxic another important factor. Increasing nitrite increases N2O production and increasing NO3- does as well, but less strongly, suggesting the role of NO3- is indirect, and it is the NO2- produced from NO3- that matters. Aerobic conditions inhibit denitrification, rendering the entire pathway moot. The establishment of anaerobic conditions turns on denitrification, but in a stepwise process apparently related to protein synthesis. In a series of experiments, these authors found that in the initial period of anaerobiosis, N2 is the major output. Later, N2O production increases without an increase in its consumption, and N2O is the major output. Finally, N2O consumption catches up with production, and N2 is once again the major output. Adding O2 increases the proportion of total denitrification output that is N2O, but eventually O2 does inhibit denitrification completely.

Yates et al. 2007

Yates TT, Si BC, Farrell RE, Pennock DJ. 2007. Time, location, and scale dependence of soil nitrous oxide emissions, soil water, and temperature using wavelets, cross-wavelets, and wavelet coherency analysis. Journal of Geophysical Research 112, D09104.

These authors analyzed a dataset of soil parameters and N2O emission using three subtly-different wavelet-based statistical techniques. There were two main purposes to this study; first, to examine the predictive relationships (if any) between soil parameters such as water filled pore space (WFPS) or temperature and N2O emissions; second, to evaluate the utility of these 3 wavelet techniques in analyzing this type of data.

N2O emission data is characterized by high variance in space and time, and frequent extreme values. These characteristics make many sophisticated geospatial statistical techniques not suitable, and the high spatial and temporal autocorrelation of many soil parameters eliminates many other techniques. These authors describe these limitations and some of the techniques that have been employed, and settle on 3 varieties of wavelet analysis.

Wavelet techniques are related to Fourier-transforms, and they appear to be highly complex and sophisticated methods to transform data for analysis, rather than being analytical methods per se. A large fraction of this paper is concerned with detailed description of the parameters of the transformation, and the interpretation of the results. One of the key advantages of these techniques is they usually allow examination of data across a broad range of spatial scales, thus permitting identification of the spatial scale at which important soil processes occur. Beyond that, I did not understand much of this paper.

Besides the interpretation of the differences between the 3 wavelet techniques, which was quite frankly beyond my understanding, the main result of this study was that the soil parameters that can predict N2O emissions in this landscape vary through the season. Early, around snowmelt and soil thawing, soil temperature is predictive of emissions. Later in the season, temperature loses its usefulness, and individual landscape features may present WFPS as predictive, but not in a global sense. By mid-summer, the soil parameters measured in this study no longer bore any relationship to N2O emissions. This loss of predictive value shows how complex this system is, and shows how some modeling efforts need to change in order to improve estimates of landscape-scale N2O processes.

Besides demonstrating my ignorance of advanced geospatial statistical techniques, this paper is primarily useful to me for its clear introduction describing the basic controls on and processes of N2O production in soils. My previous understanding centred on the role of water in restricting O2 availability in soils leading to changes at both the community and cell-physiology levels and consequently N2O production patterns in space and time appears to be essentially correct, and is reinforced by the early introduction section of this paper and the references therein.