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.

Lacelle et al. 2008

Lacelle D, Juneau V, Pellerin A, Lauriol B, Clark ID. 2008. Weathering regime and geochemical conditions in a polar desert environment, Haughton impact structure region, Devon Island, Canada. Canadian Journal of Earth Science 45: 1139-1157.

These authors examined the soils and waters near the Haughton crater on Devon Island, to determine the importance of chemical and mechanical weathering in this polar desert environment. They examined dissolved material in streams, lakes, snow, and groundwaters, and the size distribution, shape, and chemical composition of particles from soils in several different local landforms and parent materials.

Despite low temperatures, low precipitation, and very low vegetation presence, significant chemical weathering was found. Signs of chemical weathering, rather than mechanical, include rounded surfaces and pits in sand particles and the concentrations of Ca2+, Mg2+, and HCO3- in waters. Signs of mechanical weathering were also found, including sharp fracture lines in particles.

A gradient of increasing chemical weathering and decreasing mechanical weathering was found from the surface to the permafrost table. Thermal buffering reduces the frequency of frost-driven forces and thermal expansion from daily at the surface to annually at the permafrost table. The permafrost acts as a barrier to water movement, creating relatively wet condtions at depth that allow aqueous chemistry including the dissolution of dolomite to proceed.

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.