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.

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.

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.

Smith et al. 2003

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

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

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

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

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

Palmer et al. 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.

Martin and Rygiewicz 2005

Martin KJ, Rygiewicz PT. 2005. Fungal-specific PCR primers developed for analysis of the ITS region of environmental DNA extracts. BMC Microbiology 5:28.

These authors designed new primers for PCR and related molecular biology investigations of soil fungi, especially mycorrhizae. These are very diverse organisms, and, because of the commonalities between fungal and plant DNA, studies of fungal samples closely entwined with plant tissue as in root-associated mycorrhizae can be very complex. The new primers were designed to produce a range of PCR products suitable for techniques such as qPCR, Length-Heterogeneity PCR (LH-PCR) and T-RFLP analyses.

The primers as a suite were designed around a nested approach, with new outer primers amplifying a long DNA sequence of approximately 1000bp, and later primer pairs amplifying regions within that long sequence. Most inner primer pairs generate products of approximately 500bp length.

These authors also used a different DNA extraction method, based on xanthogenate and Tween (X/T) that involves little to no tissue grinding, compared to the standard method based on CTAB. The X/T method preferentially extracts fungal DNA from cells on the outside of particles, for example fungal cells not penetrating plant roots. This reduces the amount of plant DNA and associated plant-derived compounds in resulting extracts. Combining the two techniques may allow for some interesting studies of fungal micro-ecology.

This paper’s novel primers should be more specific and more useful to my own qPCR studies of Arctic soil microbes, including soil fungi. The methods section detailing some of the decisions made and considerations involved in primer design will also be useful.

Philippot et al. 2002

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

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

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

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

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

Siciliano et al. 2007

Siciliano SD, Ma W, Powell S. 2007. Evaluation of quantitative polymerase chain reaction to assess nosZ gene prevalence in mixed microbial communities. Canadian Journal of Microbiology 53: 636-642.

These authors examined the usefulness of qPCR in studying populations of soil bacteria, especially denitrifiers using the gene nosZ that codes for nitrous oxide reductase. This enzyme catalyzes the final reaction in the process of denitrification, converting N2O to N2. Normally, it is expressed only in severely anaerobic conditions, as it allows the use of N2O as the terminal electron acceptor during metabolism.

There are a number of factors that control the efficiency of PCR in quantitative PCR applications. The efficiency is a major component of the calculations that allow qPCR to estimate gene copy numbers in samples and thus to be used to examine population dynamics of non-culturable microorganisms from environmental samples. Of particular importance is consistency of efficiency between the amplification of the standard DNA template and the amplification of all templates in the unknown samples. Variation between the standard and the unknowns can lead to severe under- or over-estimation of target populations, while variation in efficiency between different templates within the unknown samples can lead to misestimations of relative proportions of organisms.

These authors evaluated the efficiency of qPCR in a range of experimental templates, and in a range of combinations simulating mixed populations. Little variance in efficiency was found, and this variance was not associated with genetic distance from a reference organism. The experimental design did not allow a direct examination of the influence of the geographical differences in the sources of the test sequences (Arctic, temperate-grassland, Antarctic), but this lack of association with the reference organism does indicate low or no variation among PCR efficiencies associated with some other variable.

The influence of varying PCR efficiencies among templates within a sample becomes less severe as the number of different templates rises. In a typical soil sample with perhaps 1000 different templates, no one template can utterly dominate amplification by outcompeting for primers, thus the resulting mix of amplicons at the end of 40 rounds of PCR will most likely be representative of the population mixture in the environment.

This paper is of obvious high utility to my own work, not least because the individual machine used to perform qPCR is the same individual machine that I will be using. For this and other reasons, this paper was suggested to me, repeatedly. Future reference to this paper, when I am developing my methods and when I am writing up the next paper or two, seems likely.

Dandie et al. 2007

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

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

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

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

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

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

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

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

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

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

Klotz and Stein 2008

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

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

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

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

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

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

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

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

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.