Pond et al., 2009

Pond, K.S., Wadhawan, S., Chiaromonte, F., Ananda, G., Chung, W.Y., Taylor, J., Nekrutenko, A., 2009. Windshield splatter analysis with the Galaxy metagenomic pipeline. Genome Research 19, 2144-2153.

These authors describe a novel software system that integrates several functions relevant for metagenomic analysis, generally defined as examining environmental samples of nucleic acids (typically DNA) without culturing the organisms present, and drawing inferences about the biological community (phylogenetic or functional) from the sequences. This is currently my favourite paper, for the quality of the writing, the density of information, the usefulness of the described methodology, and especially for the dataset they use as their demonstration of their system. I'm going to mostly use direct quotes from this paper, because there's no way I could say any of this better by paraphrasing.

The abstract starts with: 

How many species inhabit our immediate surroundings? A straightforward collection technique suitable for answering this question is known to anyone who has ever driven a car at highway speeds. 

The Introduction describes the existing resources available for metagenomic analyses, and how those resources can be expected to deal with prokaryotic and eukaryotic data. For example, while protein sequences are often employed in studies of prokaryotes (including the use of predicted protein sequences and open reading frames (ORFs) from DNA sequences), the small fraction of eukaryote genomes that codes for proteins makes such strategies less useful for investigating community composition of eukaryotes.

The authors undertook two voyages on sequential days in July of 2007, travelling from Pennsylvania to New Brunswick, in a minivan equiped with sticky tape on its bumper. They frequently refer to "windshield splatter", though this is slightly inaccurate, as the tape was affixed to the bumper of the vehicle, several decimeters closer to ground level than the windshield.

Jumping into the Results:

The most prominent difference between the two trips is in the number of reads identified with green plants (Viridiplantae): 10,242 in trip A versus 612 in trip B. It is unlikely that a two orders of magnitude difference reflects a genuine variation in species abundance of such a ubiquitous taxonomic group between the two trips. Because during each trip we collected two samples (left and right sides of the vehicle; see Methods) we were able to trace the majority (9317) of Viridiplantae reads to the left subsample. The most likely explanation for this overabundance is that a piece of plant material (e.g., a leaf or stem fragment) adhered to the collection surface. 

This illustrates a few of the striking differences between biology at the level of macroscopic organisms (e.g. most of botany, or the animals that a good naturalist would be expected to be familiar with) and microscopic, especially bacterial. A single leaf or stem fragment contains thousands to millions of cells in direct contact with each other in a dense 3-dimensional structure. Bacterial cells in the environment are often found in biofilms, which are typically a single cell layer or only a few cell layers thick, and cover a tiny area. Or they occur as individual cells, separated by multiple cell-length-equivalents from their neighbours. Also, identification to high taxonomic levels such as Order or Phylum is common in environmental microbiology, yet essentially unheard of for multicellular organisms - if it's big enough to see, it can be identified to Family or better by a person equiped with a readily-available guide. Yet they report a "green plant" - anything from roses to ginkos is included in that high-level taxon!

The list included unexpected entries such as the genus Homo even though the two trips were uneventful. Such matches are likely caused by road debris (which often includes roadkill) adhering to the collecting tape. Because few entries in NT and WGS databases are derived from, say, white-tailed deer (Odocoileus virginianus, a prevalent large mammal roadkill in the northeastern United States), reads truly representing this speces are more likely to match abundant human sequences. 

That first sentence, above, is probably my favourite sentence in the entire paper. "the two trips were uneventful." Just savour that, and ponder the meanings...

This is also another striking difference between metagenomics and related microbiological sampling and study strategies and how multicellular eukaryotes are most often studied. No ecologist would normally need to describe the probability of mistaking a sample derived from a white-tailed deer with that from a human, yet here, because of the way the databases used for comparison and identification are structured, consideration of roadkill rates (and roadside clean-up efforts, presumably) are required to refine the raw identifications derived from comparisons of DNA sequences. 

Existing tools for major steps in the environmental-sample-to-phylogeny experimental pipeline are difficult to use and make work together, thus: 

This is why our objective was to build a complete pipeline for homology-based taxonomic labeling of metagenomic reads that was self-contained and guided the user from data acquistion and QC, to database searches, and finally, actual metagenomic analyses. We demonstrate that the classification performance of our solution is on par with currently available applications...
Our second goal was to perform a eukaryotic metagenomic study on the organic matter collected on an automobile's windshield. Specifically, we were interested in addressing two questions: Can one identify eukaryotic taxa from random reads generated by the next-generation sequencing technology from environmental samples? and Is it possible to contrast species abundance between geographic locations? While this pilot analysis provides positive answers to both questions, it also raises important issues and limitations. 

I leave it to you to read this excellent paper and see the "issues and limitations" they describe.

And I *love* their methods: 

The front bumper of a 2006 Dodge Caravan ("The Wanderer") was divided at the license plate into "left" (passenger side) and "right" (driver side), and was taped with a double-sided carpet tape. On top of the carpet tape, a 3M 5414 Water Soluble Wave Solder Tape was affixed, exposing its sticky side. The tapes were applied on June 23, 2007, at 6 am EDT in State College, Pennsylvania, and removed in tubes containing Tris EDTA buffer at 12 pm EDT in Manchester, Connecticut. New tapes were again applied in Portland, Maine, at 5 pm EDT and removed in Moncton, New Brunswick, at 12 pm EDT the following day.

Note that they named the vehicle (with a pretty good name, in my opinion), and they describe "left" and "right" in opposition to the common standard among drivers - their description is based on a person standing in front of the vehicle, facing the windshield; their "left" is the vehicle's starboard side, and "right" is port. It's extremely unlikely "The Wanderer" is a right-hand-drive vehicle.

Their software is web-based and available at:  www.usegalaxy.org

Su et al. 2011

Su, C., Lei, L., Duan, Y., Zhang, K.-Q., Yang, J., 2011. Culture-independent methods for studying environmental microorganisms: methods, application, and perspective. Applied Microbiology and Biotechnology 93, 993-1003.

These authors provide a summary overview of the more recently-developed culture-independent methods and their use in studying microbial communities. Figure 1 shows the basics of each of several methods, that all start with the collection of an environmental sample (e.g. soil, water, mouth swab, etc.), and end with data analysis and evalution dependent on the hypotheses of the study.

(I'm not going to post Figure 1 here, I'm not interested in violating copyright)

In the introductory part of the review, these authors provide a list and a taxonomy of these methods.

PCR-based

·         DGGE/TGGE (denaturing/temperature gradient gel electrophoresis)

·         SSCP (Single-strand-conformation polymorphism)

·         RFLP (restriction fragment length polymorphism)

·         T-RFLP (terminal restriction fragment length polymorphism)

·         qPCR (quantitative PCR)

non-PCR-based

·         FISH (fluorescence in situ hybridization)

·         Microarray

·         Raman microspectroscopy
      NanoSIMS (nano-scale secondary ion mass spectrometry
     
     NGS (Next Generation Sequencing)  
        Pyrosequencing

The field of metagenomics is described apart from these methods, as a broad category of investigations of microorganisms in mixed, uncultured communities.

I found this paper a useful introduction to some of the terminology and methodology of environmental microbiology. At the moment, it seems unlikely I will be citing this paper directly, but its reference list will be useful, and I might want to re-read this in a few months, when I have gained some more familiarity with key concepts.

Lacelle et al. 2010

Lacelle D, Radtke K, Clark ID, Fisher D, Lauriol B, Utting N, Whyte LG. 2011. Geomicrobiology and occluded O2-CO2-Ar gas analyses provide evidence of microbial respiration in ancient terrestrial ground ice. Earth and Planetary Science Letters 306: 46-54.

These authors compared the gas composition of ice from massive ground ice bodies (e.g. 30 000-year-old buried snowbanks) to atmospheric gas concentrations and gas contents from glacial ice. They also cultivated some microorganisms collected from within the ice, and used some non-culture-dependent methods to examine the diversity of those organisms.

Loss of O2, as determined by comparison of the ratio of O2 to Ar in samples, as well as changes to the 13C-CO2 contents indicate heterotrophic microbial activity in the ice, most likely by organisms living in high-salt brine channels in cracks in the ice, using dissolved organic carbon as both energy and C source.

This paper provides an overview of methods for extracting and measuring the gases trapped in microscopic bubbles in permafrost and ground ice, as well as interesting findings about microbial activities.

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.

Davidson et al. 1991

Davidson EA, Hart SC, Shanks CA, Firestone MK. 1991. Measuring gross nitrogen mineralization, immobilization, and nitrification by 15N isotopic pool dilution in intact soil cores. Journal of Soil Science 42: 335-349.

These authors evaluated the use and limitations of the isotope-pool dilution technique when studying nitrogen dynamics in soil. Because addition of inorganic nitrogen compounds (NH4+, NO3-) can stimulate microbial activity in N-limited systems such as most soils, estimating the rate of these processes by tracking 15N through a system will almost certainly overestimate these rates. The isotope-pool dilution method, on the other hand, measures the dilution of enrichment in the nitrogen pool at the end of a particular process, relying on the assumption that additional product of metabolism will have negligible effects on the magnitude of that metabolism. In this study, immobilization of nitrogen was the main focus of investigation, comparing 15N isotope dilution in pools of either 15NH4+ or 15NO3-.

There are three key assumptions for the isotope-pool dilution method in this context. 1. Microorganisms do not discriminate between 15N and 14N; 2. rates of processes measured remain constant over the incubation period; 3. 15N assimilated during the incubation period is not remineralized. Previously, these assumptions had been evaluated for well-mixed soils, but not for unmixed field-collected soil samples. While fractionation by biological processes certainly does result in discrimination between isotopes of nitrogen, it is of negligible importance when injected solutions are very highly enriched and incubation periods are relatively short; in this case, injections were more than 90% 15N and incubations ran for 24 hours. Rates of measured processes will change if the population and / or activity of microorganisms changes, but again, over a 24-hour incubation period under controlled conditions this is unlikely. Highly enriched injections allow the use of small injection volumes, limiting the impact of nutrient enrichment. These authors were able to measure the remineralization of immobilized 15N, and estimated that between 1.0 and 1.6% of injected 15NH4+ appeared in the 15NO3- pool after 24 hours; they consider this an insignificant amount, but caution that longer incubations would almost certainly result in much more problematic amounts of remineralization.

This paper is clearly a major part of the basis of the project I am currently engaged in with Katherine from our 2009 field season at Alexandra Fjord. I probably should have read this paper long ago. The three main conclusions stated by these authors at the end of their paper I think can be quoted verbatim as justification for both why I (should have earlier) read this paper, and as a reminder to myself to include this paper in the methods & materials section of the eventual manuscript.

"Three points should be considered when applying the isotope dilution method.
1. Accurate estimation of both 14N and 15N initial pool sizes is important. Abiotic consumption of label, such as by clay fixation, can cause significant errors. A subset of intact cores may need to be destructively sampled directly after adding 15N for estimation of initial pool sizes.
2. Homogeneity of 15N enrichment throughout a soil sample is not possible, and perfectly uniform distribution of added label is not necessary. However, significant errors can arise from a bias in 15N distribution that is concurrent with a non-random
distribution of microbial processes. Distribution of label should, therefore, be as uniform as possible.
3. In situ gross immobilization rates may be overestimated by isotope dilution methods and underestimated by chloroform fumigation methods, depending on which (if any) kN factor is applied to the latter. Gross mineralization and gross nitrification estimates from isotope dilution are more reliable because these rates should not be affected by addition of 15N label in the form of the process products."

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.

Trinsoutrot et al. 2000

Trinsoutrot I, Recous S, Mary B, Nicolardot B. 2000. C and N fluxes of decomposing 13C and 15N Brassica napus L.: effects of residue composition and N content. Soil Biology and Biochemistry 32: 1717-1730.

These authors studied the decomposition process by soil microorganisms when isotope-labelled crop residues were added to soil. The crop used, oilseed rape Brassica napus (also known as canola) varies its nitrogen content of tissues, and the C:N ratio, depending on levels of N inputs by fertilization. This allows variation in input organic matter quality by manipulation of growing conditions; in this experiment, both carbon and nitrogen inputs to the plant included stable-isotope labels, in the form of 13C-CO2 and 15N-KNO3. Plant residues were added to soils and incubated for 168 days.

Initial C:N ratio and especially the labile-C fraction of organic-matter inputs are major controls of both the rate of decomposition and fate of matter through the system. Additionally, temperature, particle size of residues, and water content in the soil also strongly influence decomposition processes.

Here, N mineralization (the formation of NO3- and NH4+ pools in the soil from organic-N precursors) occurred in two phases. In the early phase, up to about 3 weeks, the N cycle resulted in net mineralization. Later, mineral N pools were depleted and N was immobilized, that is, incorporated into the tissues of microbial cells.

Carbon dioxide release during the experiment occurred through two pathways. The more direct route was rapid mineralization of organic matter, which I interpret as non-incorporation of organic matter by microbes, consuming such material but metabolizing it rapidly through respiration. The second, presumably slower route was through metabolization of material after incorporation into cells through respiration. Either way, the ultimate fate of much of the organic-C in the residues was release as CO2.

Differences in the N-content of residues affected decomposition rates early in the experiment, but by about 4 months the differences between high-N and low-N residues had evened out. Only a small fraction of labelled N from residues ended up in soil mineral-N pools; the majority was either immobilized into microbial cells or remained in recalcitrant organic matter fractions. Immobilization of unlabeled, SOM-derived N was enhanced by the addition of C through a substitution effect.

These authors conclude that 15N labelling was fraught with difficulties, and both under- and overestimated some pools and processes. However, the use of their model, named NCSOIL, improved their ability to trace the fate of added material through the system. This paper represents a study similar in some ways to our planned course activity in the special topics in soil science course, fall 2010.

Pennock et al. 1987

Pennock DJ, Zebarth BJ, De Jong E. 1987. Landform classification and soil distribution in hummocky terrain, Saskatchewan, Canada. Geoderma 40: 297-315.

These authors present a method of analysing irregular terrain for the purposes of examining important soil parameters such as soil depth and soil hydrology. Three variables (profile curvature, plan curvature, and gradient) can be calculated from a matrix of elevation data; taken together for a given point or area, these variables can be used to classify an area into one of seven landforms. These landforms are level (for summits or bottom lands), shoulders, backslopes, and footslopes; the three non-level forms come in “divergent” (convex plan) and “convergent” (concave plan) varieties. Shoulders have convex profiles, backslopes have flat profiles (i.e. constant gradient when looking up or down the slope), and footslopes are concave. Divergent landforms shed water laterally; convergent landforms tend to collect water. Where water collects and is moving slowly, rates of infiltration will be highest, and erosion will tend to deposit, rather than remove, material at these places.

The variables required to calculate profile and plan curvature and gradient are relatively easy to calculate from a matrix of elevation data, using an interpolating topographical software package and some differential calculus. The seven categories of slope elements can be estimated in the field by eye, making for a useful method for field studies.

This paper was on the recommended reading list for SLSC 834; in addition, the course instructor is Dr. Pennock, lead author of this study. The study site, near Hafford, Saskatchewan, is perhaps 1 to 1.5 hours drive away from Saskatoon, suggesting this area may be the destination of one of the day trips scheduled for the week of August 30, 2010.

Hayashi et al. 1998a

Hayashi M, van der Kamp G, Rudolph DL. 1998. Water and solute transfer between a prairie wetland and adjacent uplands, 1. Water balance. Journal of Hydrology 207: 42-55.

These authors report water balance data and analysis in the first of a pair of papers about the hydrology of wetlands in the prairie pothole region of central Saskatchewan. The second paper concerns dissolved material movements in the same example study system.

Through a series of measurements of water and water-related properties of soil as well as precipitation, total water balance and total water movements within the wetland were estimated. Direct precipitation and evaporation accounts for a small fraction of the total volume of water entering and leaving the pond in the middle of the hectare-scale wetland and surrounding catchment of uplands. Most of the water entering the pond is in the form of snowmelt during spring, and most of the water leaving the pond is in the form of horizontal groundwater flow, relatively shallowly (i.e. above a redox discontinuity at about 5-6m depth), driven strongly by evapotranspiration through the “willow ring” surrounding the pond and through crops (principally wheat during this experiment) on the uplands. Only a small amount of water is exported down to the aquifer; of a total input of 780mm into the pond, only about 3mm goes to recharging the underlying aquifer. Figure 3 provides definitions of the terminology, and Figure 12 the estimates of water balance.
This paper was on the suggested reading list for SLSC 834 in August 2010.

Christiansen 1979

Christiansen EA. 1979. The Wisconsinan deglaciation of southern Saskatchewan and adjacent areas. Canadian Journal of Earth Science 16: 913-938.

This author describes in considerable detail the process of deglaciation that occurred at the end of the last glacial maximum from about 17 000 years ago, as it occurs to Saskatchewan. Major geological features and patterns of melt-water drainage led to the inclusion of nearby parts of Alberta, Manitoba, Montana, and North Dakota in the analysis. Patterns during the deglaciation were identified by glacial landforms, many of which are apparent only from aerial photographs, with sample analysis in the lab including radio-carbon dating.

The period from 17 000 years to 10 000 years ago is divided into 9 phases corresponding to periods of rapid glacial retreat or temporary stasis or glacial advance. By 10 000 years ago the ice sheet that had covered nearly the entire province had retreated towards Hudson Bay and covered only a small part of the north of Saskatchewan. Glacial lakes formed from meltwater and from water flowing from the west (presumably sourced from glaciers in the Rocky mountains), sometimes reaching enormous sizes; these lakes were bordered by the glacier’s edge, and connected to each other via spillways that often carved large channels from the plains; the east-west valleys of southern Saskatchewan such as the Qu’Appelle valley are the remains of such spillways. Modern river systems such as the Saskatchewan River and the Churchill River formed during the glacial retreat, occupying low areas and spillway remnants.

The rate of deglaciation varied considerably over the studied 7 000 years, generally accelerating from about 150 m / yr to around 275 m / yr, though with frequent pauses, occasional re-advances, and variation across the glacial edge. The evidence from glacial lake-edge movements and depth patterns suggests the ice sheet melted fastest initially, but with the slowest retreat at that time indicating the sheet first thinned, and then retreated, especially around newly-uncovered Nunataks where the underlying land formed highlands.

This paper was on the suggested reading list for the course SLSC 834 in August 2010, but it is also personally interesting in describing Pleistocene events in areas I visit during Sunday drives across the center of the province.

Stark and Hart 1996

Stark JM, Hart SC. 1996. Diffusion technique for preparing salt solutions, Kjeldahl digests, and persulfate digests for nitrogen-15 analysis. Soil Science Society of America Journal 60: 1846-1855.

These authors evaluated the recently-developed Teflon-encased-acid-trap technique for collecting nitrogen from water samples for analysis, especially stable isotope (15N) analysis. Earlier acid trap techniques, which rely on the chemistry of ammonium/ammonia in solutions of varying pH, were based on suspended small vials containing acid solutions above a larger volume of sample solution at high pH. Ammonium present in the sample is converted to ammonia by high pH, and escapes solution into the gas phase. It is captured in acid solution; in a closed container with a separate acidic region, ammonium will migrate from the alkaline sample to the acid trap. Suspended trap designs are vulnerable to a number of problems, including vulnerability of the acid trap to contamination by alkaline solution and subsequent loss of captured ammonium. Teflon will pass gases but not liquids, and therefore provides an ideal barrier between the sample and the acid trap that will permit ammonia to enter the trap.

These authors conducted a set of six experiments to evaluate the utility of Teflon acid traps. First, blanks were evaluated, to determine the sensitivity of the technique to nitrogen derived from contamination in the various materials of the experiment (filter paper, Teflon, plastic bottles, etc.). There was some nitrogen detectable from these non-sample sources, but a correction factor could be constructed based on including a few blanks in sample sets. The second experiment examined the recovery of ammonium from 2M KCl solutions. These solutions are very similar to the solutions extracted from soils of Alexandra Fjord in 2009. As might be expected, incubation time correlated with recovery of ammonium; in addition, Teflon traps performed better over shorter incubations (up to about 6 days) than did suspended traps, though the difference disappeared at the longest (8 days) incubations.

The third experiment was of greatest interest to me. They examined the open-bottle time necessary to eliminate residual ammonium from samples used in experiment 2, and the incubation conditions to collect NO3- -derived nitrogen using Devarda’s alloy to reduce NO3- to NH4+. Residual ammonium left in solution at the end of the first reaction would contaminate NO3- examination and lead to overestimation of NO3- contents; this is especially true when the two forms of nitrogen have been previously enriched with 15N to different degrees. There is a trade-off between open bottle time (ranging in these experiments from one to five days) and recovery of NO3- from samples; long open-bottle times appear to lead to an unknown chemical change in samples, which these authors speculate, may involve sorption of atmospheric CO2. Long ammonium-collection times (i.e. experiment 2) do tend to collect the great majority of ammonium present, recovering on average more than 97% of ammonium from known-concentration samples. This suggests to me a long and potentially nitrate-losing open-bottle incubation is not necessary if ammonium collection has proceeded for at least six days. Agitation of samples, in these experiments often once per day, resulted in a strong improvement in nitrogen capture over at least the shorter (24-96 hour) incubations; our use of continuous agitation for seven days therefore seems likely to capture nearly all ammonium present in samples.

Experiments 4 through 6 were of little interest to me, as I am unfamiliar with Kjeldahl digests and persulfate digests and do not plan to use these techniques in my studies. From what I could glean based on my limited knowledge, the Teflon-acid-trap technique appears to work well with Kjeldahl digests and persulfate digests.

Overall, the Teflon-acid-trap technique performed very well, and had significant ease-of-use and contamination-avoiding advantages over earlier methods. One caution put forth by these authors is regarding the H2 gas evolved during Devarda’s alloy incubations; strong build up of gas can cause leaks, potentially allowing NH3 to escape and leading to underestimates of sample nitrate contents. They suggest storing bottles upside-down during such incubations, as leaks will then include sample solution, and leaking bottles can be easily identified.

Complete recovery of nitrogen was not possible on a routine basis using this technique, but 15N isotope ratios do not rely on 100% recovery if ammonium and / or nitrate concentrations have been measured by an independent method, such as the widely-used colorimetric techniques. The two isotopes of nitrogen do not migrate differently between alkaline sample and acid trap, thus even at relatively inefficient recovery rates, isotopic ratios are maintained. Blanks are important, but in general this technique is robust, reliable, and easy to conduct.

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.

Palmer et al. 2009

Palmer K, Drake HL, Horn MA. 2009. Genome-derived criteria for assigning environmental narG and nosZ sequences to operational taxonomic units of nitrate reducers. Applied and Environmental Microbiology 75: 5170-5174.

These authors compared the sequences of narG and nosZ genes to corresponding sequences of 16s rRNA genes, using in-silico analysis of sequences downloaded from GenBank. While similarities above 97% are commonly used for species- or genus-level taxonomic delineation for 16s sequences, this analysis found much lower threshold similarities for such delineation using the structural genes.

This paper is confusing to me. One part of the text appears to contradict itself, when the authors state that the Nar operon in Pseudomonas stutzeri A1501 is putatively alien in origin (i.e. recent horizontal transfer), then go on to state in the same paragraph that it appears unlikely that the Nar operon was horizontally transferred in any species. I may just be misunderstanding the meaning of the term “putatively alien” in regards to a bacterial gene sequence.

A greater puzzle is presented by the list of nosZ sequences. These authors downloaded 85 such sequences, where my own attempts to extract data from GenBank resulted in only 42 unique nosZ sequences. The list in a supplementary table includes several cases of multiple accessions of the same species but of different PD. The paper these clusters of PD-sequences are derived from is Dandie et al. (2007); a quick scan of this paper did not reveal what the distinction “PD” indicates.

Tuomivirta et al. 2009

Tuomivirta TT, Yrjälä K, Frize H. 2009. Quantitative PCR of pmoA using a novel reverse primer correlates with potential methane oxidation in Finnish Fen. Research in Microbiology 160: 751-756.

These authors developed novel primers for studying the methanotrophs of Finnish peatland fens. The existing primer pairs widely used to study methanotrophs, based on the gene pmoA, did not consistently amplify useful products in PCR using template DNA from these fens.

The novel primer pair was tested on 114 samples from two Finnish fens, representing the northern and southern peatlands of Finland. While A189f/A682r failed to provide useful products, the new primer A6821r in conjunction with A189f did produce strong bands. In qPCR, these primers produced results correlated with measured methane oxidation potential, further supporting their utility in these systems.

Schmidt et al. 2008

Schmidt SK, Reed SC, Nemergut DR, Grandy AS, Cleveland CC, Weintraub MN, Hill AW, Costellow EK, Meyer AF, Neff JC, Martin AM. 2008. The earliest stages of ecosystem succession in high-elevation (5000 metres above sea level), recently deglaciated soils. Proceedings of the Royal Society of London, Series B 275: 2793-2802.

These authors describe the microbial community and soil parameters of a chronosequence at the foreground of a receding glacier high in the Peruvian Andes. From a combination of aerial photography and previous work at this site, a series of sites of soils of increasing ages from zero to 79 years old was established. No surface plants, even lichens, are present on any of these new soils, and soil nutrient levels (carbon, nitrogen) are very low; the only organisms present are microorganisms.

Two previous hypotheses had been proposed to explain the dynamics of very early primary succession on new soil. Organic matter has been observed to accumulate slowly in new soils; the source of this material is either aeolian deposits (i.e. wind-borne plant detritus and pollen) or in-situ fixation of CO2 and N2. These are not mutually exclusive hypotheses, but the relative contributions of each are explored in this study.

The methods used here cover an extensive list of soil parameters. Three sets of soil samples were collected: for microbiological analysis, N-fixation measurement, and all other chemical analyses. The other chemical analyses include photosynthetic pigment extraction, soil total and mineral nitrogen, pyrolysis for identifying sources of carbon compounds (i.e. microbial-autotroph, microbial-heterotroph, plant), enzyme assays for common and informative microbial enzymes, and soil stability analysis of the resistance of these new soils to erosion forces such as water runoff.

These authors focused on the cyanobacterial fraction of the microbial community in this study; some details of other components of the biota are described in an earlier paper, Nemergut et al. (2007). Cyanobacteria are autotrophs also capable of fixing atmospheric nitrogen, thus they are ideal primary colonizers of new soil as they require little more than a source of moisture and air. Analysis of the community included the use of the P-test (Martin 2002); note that as in Nemergut et al. (2007), he is one of the authors of this study. The analytical approach is very similar to that employed in the earlier study, with a comparison of discovered sequences to published sequences from around the world. In this study, cyanobacterial sequences from zero and 4-year-old soils were similar to sequences from an extremely broad sample of habitats, including Antarctic lake ice, marine subseafloor sediments, urban aerosols, forest soils, and oil-polluted soils.

The soil chronosequence showed a clear pattern of stages of primary succession at every level of analysis. The soil microbial community became both more abundant and more diverse through time, soil nutrients increased, the chemical environment included increasing amounts and diversity of complex organic molecules, key enzyme pathways became established, and soil stability increased as soils aged. N-fixation showed a peak, with increasing N-fixation activity from the zero to 4-year-old soils (by two orders of magnitude), then declining by about half in the 79-year-old soils. This mirrors and precedes a widely-observed pattern in plant primary succession, in which nitrogen-fixing plants are among the first colonizers, but decline in abundance at later stages of succession. Enzyme and organic molecule patterns were consistent with a total absence of heterotrophs in the extremely young soils, increasing occurrence of organisms capable of decomposing plant matter in the 4-year-old soils, and a molecular ecology qualitatively similar to a mature plant-associated soil in the 79-year-old soil.

The list of procedures and level of detail of analysis in this paper is impressive. Many, though certainly not all, of these techniques will be models for my own work, especially in the summer of 2010. The molecular-diversity techniques pioneered by Martin (Martin 2002, Nemergut et al. 2007, this paper) as well as the techniques of analyzing soil pigments and soil nutrients are all very interesting.

Sørensen et al. 2006

Sørensen LI, Holmstrup M, Maraldo K, Christensen S, Christensen B. 2006. Soil fauna communities and microbial respiration in high Arctic tundra soils at Zackenberg, Northeast Greenland. Polar Biology 29: 189-195.

These authors sampled soil animals from three sites at Zackenberg station, Greenland, over three days in mid-summer. Two of the sites were considered mesic heath, with a mix of Cassiope tetragona and other High Arctic species of plants, while the third site was dominated by Dryas spp. and was considered dry heath; snow melts from the dry heath up to 20 days earlier than from the mesic heaths. Soil samples ranging down to about 6cm depth were collected, stored at 5ºC for up to two weeks, and analyzed by a range of methods in the laboratories in Europe.

Different groups of soil animals were extracted by varying methods. Soil microarthropods, a diverse group dominated by Collebola and Acari, were extracted by modified MacFadyen funnels into Benzoic acid. Enchytraeids and dipteran larvae were extracted in Baermann wet funnels with heating of the samples, into tap water. Protozoa were washed from soils in water and grown on media plates in the dark at 10ºC. Nematodes were collected by the Blender-Cotton wool method of Schouten and Arp (1991). Soil microbial respiration was measured in serum bottles, with the CO2 concentration in the headspace measured at zero, 5 and 25 hours, with a fully factorial design of nutrient amendments of C, N, and P. Soil pH and soil organic matter content, but not moisture content or other nutrient concentrations were determined using methods not clearly described, though presumably these procedures were similar to standard methods.

Once abundance and biomass data was collected, comparisons between plots were made using multivariate analysis and a software package named PRIMER 5.0. My understanding is the species counts were (log+1) transformed to reduce the influence of very abundant species, then analyzed using an approach similar to Principal Components Analysis. The result of this analysis was a clear difference between the dry heath and the two mesic heaths, while the two mesic heaths were not different from each other in parameter-space. A Bray-Curtis similarity matrix was also involved, though I’m not certain I understand how.

Different taxonomic groups were identified to different taxonomic levels; 19 species of Collembola and 7 species of Enchytraeids were found, for example, but Acari were identified to suborder (Cryptostigmata (oribatids), Prostigmata, Mesostigmata) and nematodes and protozoans were counted at those high taxonomic levels. While the two mesic heath sites were only marginally significantly different from each other, there was a clear increase in abundances in the dry heath site. For collembola at least, the dry heath site was also dominated by two highly abundant species, which differed from the majority of species in the mesic sites by being unpigmented and associated with sub-surface, rather than soil-surface, regions in the soil. The higher abundance of probably bacteria-eating nematodes at the dry heath strongly suggests higher turnover of microorganisms as well as generally higher biological activity from the higher populations of most soil animals.

These authors suggest higher organic matter decomposition rates at the dry heath, which seems reasonable given the higher animal populations there. However, their attribution of higher soil pH there to higher respiration levels seems like more of a stretch, absent supporting mineralogical and soil-nutrient data.

This paper provides an excellent example of the data that can be collected and analyzed from a brief but intensive study of soil invertebrates at a High Arctic site. In addition, meaningful information about differences in biodiversity between locations can be derived from studies of organisms not identified to fine taxonomic levels.