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.

Liptzin 2006

Liptzin D. 2006. A banded vegetation pattern in a High Arctic community on Axel Heiberg Island, Nunavut, Canada. Arctic, Antarctic, and Alpine Research 38: 216-223.

This author attempted to explain the observation of banded vegetation on a slope that lacked the usual factors that generate such patterns. In temperate and tropical locations, banded vegetation, also known as “tiger stripes”, forms on shallow slopes in dry areas with a consistent direction of water flow. Plants at a position on the slope increase water retention and facilitate further colonization by plants. Similarly, some locations experience consistent wind direction carrying sea spray that kills trees at some positions. In cold environments, patterned ground from cryoturbation on shallow slopes can also lead to banded vegetation. However, the study site in this paper lacks all of these features, including cryoturbation despite the presence of permafrost within 50cm at most locations.

Some aspect of soil properties is the obvious explanatory hypothesis, which this author explores after describing the transects measuring plant diversity and the soil pits used to examine soil properties. In general, features that would normally be expected to influence plant diversity and abundance such as soil moisture or exchangeable cation levels, had no significant impact in the various statistical tests employed in this study. However, soil type did have some effect, as a few species of plants were found only on sandy soil, and nitrogen levels were negatively correlated with species richness.

The discussion section of this paper is an excellent example of a chain of logical reasoning working through a series of potential explanations. While this paper is interesting, it’s only relevant to my own studies in a narrow area around potential starting points in looking for explanations for whatever patterns I may find in my biogeography studies in 2010. However, this paper seems remarkably suitable as an introduction to the basics of modern soil science research, and may be relevant to my not-quite-mothballed interest in an undergraduate course about the current state of the scientific literature.

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.

Li et al. 2009

Li X-R, Du B, Fu H-X, Wang R-F, Shi J-H, Wang Y, Jetten MSM, Quan ZX. 2009. The bacterial diversity in an anaerobic ammonium-oxidizing (anammox) reactor community. Systematic and Applied Microbiology 32: 278-289.

These authors studied the bacterial community that developed inside a bioreactor running on sewage sludge under anaerobic conditions. Like Lim et al. (2008), the main focus of this study was in the applications of ammonia-oxidizing bacteria (AOB) to water treatment facilities. The expected chemistry of anaerobic ammonia oxidation catalyzed by microorganisms (“anammox”) includes the use of nitrite as the electron acceptor in a near-one-to-one ratio with the consumption of ammonia or ammonium. The energy derived from this process is used by the cell to fix CO2, thus making these organisms autotrophs. This alters the underlying stoichiometry slightly, as some nitrite is diverted to CO2 fixation rather than ammonia oxidation.

The study of anaerobic AOB is still quite new, with the five described genera of such organisms all named with “Candidatus” prefixes, indicating recent species descriptions. All are in one group (taxonomic level unknown), the Brocadiales, within the phylum Planctomycetes. Aerobic AOB are in other groups, and include some species within the genus Nitrosomonas in the Beta-Proteobacteria that are capable of limited ammonia-oxidizing activity under anaerobic conditions, and can apparently survive long periods without oxygen.

These authors did not develop novel primers for PCR or qPCR in this study. Instead, they used published primer sets; I gather they did not use the TaqMan double-dye system for qPCR, as no mention of probes is made. The target genomic sequences were portions of the 16s rRNA gene, using E. coli as a standard. Oddly, the overall procedure included normal PCR, followed by cloning and insertion into plasmids, followed by qPCR of plasmid DNA containing the 16s sequences. It is unclear to me exactly why this was done, though later in the paper there are a few sequence-based phylogenetic trees that might have been based on sequences derived from this cloning procedure. In any case, the qPCR did provide informative results regarding the composition of bacterial groups within the reactor.

Of the sequences identified, the great majority were unlike cultivated organisms, highlighting the utility of these techniques in studying environmental samples. AnAOB produced approximately 16% of sequences, with aerobic AOB less than 1%. Non-AOB in three phyla constituted the majority of sequences, including 38% Chlorobi, 21% Chloroflexi, and 7% Bacteriodetes. These are filamentous heterotrophic bacteria, and appear to be closely associated with the granules that formed in the reactor solution after a few months. These authors suggest further research on the ecophysiology of these groups to answer questions regarding energy and material cycles within these systems.

In addition to 16s sequences, the hzo locus was also studied. This is a gene that produces an enzyme that catalyzes the oxidation of hydrazine (rocket fuel; N2H4) to N2 gas. No mention is made of the possibilities for N2O production or consumption in this process. The gene is restricted to AnAOB only, or at least that is the inference based on the observation that hydrazine is a unique intermediate molecule of the anammox process.

This study provides a useful example of the combination of qPCR and molecular-phylogenetic approaches in studying a microbiological system. Applied together, the two approaches allow the extraction of useful information regarding taxonomic diversity, both richness and evenness, among functional groups of organisms.

Lim et al. 2008

Lim J, Do H, Shin SG, Hwang S. 2008. Primer and probe sets for group-specific quantification of the genera Nitrosomonas and Nitrosospira using real-time PCR. Biotechnology and Bioengineering 99: 1374-1383.

These authors developed precise primer and probe sets for TaqMan-based quantitative PCR to examine ammonia-oxidizing bacteria (AOB) associated with wastewater treatment facilities. These molecular tools have very low rates of false-positive and false-negative errors associated with them, and will be useful primarily to work on improving the nitrogen-removal capacity of wastewater treatment. However, AOB are nearly ubiquitous, such that these molecular tools will also be useful to a wide variety of less-directly-applied studies.

These authors purchased eight strains of nitrifying AOB commonly found in water treatment plants, eight non-nitrifying bacteria also commonly found in such plants, and collected, identified, and purified seven strains of nitrifying bacteria directly from a pair of water treatment plants in operation. Using published sequences of the 16s rRNA genes of these organisms, sets of primers and probes were constructed.

The development and evaluation of these primer/probe sets followed two basic procedures: first, “in silico” evaluation of potential primer and probe binding sites, and calculation of potential mismatches in various combinations. For example, a set developed for one organism may also be highly likely to amplify a related organism, reducing specificity of the assay. Second, sets were optimized for PCR conditions and trialed with the often-variable real sequences derived from culture collections or field samples. Iterating between these two processes allowed a series of final best-fit sequences to emerge, that have high specificity and low failure rates.

As a template for developing very good molecular tools, this paper provides some excellent advice regarding qPCR primer development. I will be using a different system that does not include probes, simplifying some steps of this process, but the basic pattern of invention, computer evaluation, wet-lab evaluation, further computer evaluation, and refinement will still be useful.

Himmelheber et al. 2009

Himmelheber DW, Thomas SH, Löffler FE, Taillefert M, Hughes JB. 2009. Microbial colonization of an in situ sediment cap and correlation to stratified redox zones. Environmental Science & Technology 43: 66-74.

These authors previously studied the changes in geochemistry associated with the common practice of adding a sediment cap to cover contaminated sediments at the sediment-water interface. Such caps are commonly clean sand, with the underlying idea being the layer of sand provides a transport barrier to various contaminants moving through the system by diffusion. Sediment geochemistry, like soils, includes layers of redox conditions generated by both biotic and abiotic factors. These zones of chemical conditions migrate upwards when a sediment cap is added; not surprising considering the effect the cap has on diffusion of oxygen and other chemicals important for redox considerations.

This study shows that the microbial populations also migrate upwards when a cap is added. The primary concern here seems to be the effect this population shift may have on the transport and decontamination of such pollutants as are often found in the river-bottom sediments of the eastern USA. The primary effect is likely positive: populations of bacteria and archaea in sediments will metabolize, mineralize, and generally detoxify most compounds moving up from the sediments to the cap. A few classes of contaminants, however, may not be decontaminated and it is possible their transport and release into the water column may be accelerated by these microbes.

There are two key parts of the methods of this paper that interest me. First, the microbial populations were analyzed by a range of techniques including real-time quantitative PCR (qPCR). The procedure of primer design, evaluation, and data interpretation looks very similar to what I will be attempting with my own samples. Second, diversity estimates for the various strata within the sediments, derived from qPCR data, includes the use of the statistical technique Canonical Correspondence Analysis. This allows direct testing of hypotheses regarding the relationship between environmental parameters, in this case depth below surface, and estimates of biodiversity such as the Shannon-Weiner index.

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.