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.

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.

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.

Harding et al. 2001

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

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

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

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

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

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

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

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.

Conrad 1999

Conrad R. 1999. Contribution of hydrogen to methane production and control of hydrogen concentrations in methanogenic soils and sediments. FEMS Microbiology Ecology 28: 193-202.

This author reviews the chemistry behind methane production by Archaea in anaerobic environments, focusing on the contribution of H2 rather than acetate to methanogenesis. The thermodynamics and kinetics of H2-driven CH4 production are distinct from those of acetate-driven, and the stoichiometry of the situation indicates that H2 should contribute 33% of the CH4 from a given ecosystem.

Methanogenesis in anaerobic sediments and soils is the end of a short chain of microbial interactions. First, organic matter is broken down by fermenting bacteria. The products of fermentation includes H2, and the other components such as alcohols and fatty acids, are further decomposed by syntrophic bacteria, also supplying some acetate to the environment. Finally, methanogens consume either H2 and CO2 or acetate (CH3CO2-) to produce methane.

There are many studies that show this expected pattern of methanogenesis, but many other that show either over- or under-representation of H2. Where H2 contributes less CH4 than expected, the most likely explanations involve sulfate reducers, microbes capable of outcompeting H2-consuming methanogens by more efficient use of H2 and faster population growth, based on the thermodynamics of the two guilds respective metabolisms. Such situations are common in marine and acidic freshwater sediments.

Where H2 contributes more CH4 than expected, including an Antarctic soil where H2 is the basis of 100% of CH4 production, the explanation is not as well established. The explanations that have been proposed, by this and other authors, include additional sinks of acetate such as scavenging by other organisms, additional sources of H2 including geological sources, or measurements of the system taken when it was far from equilibrium. The models of H2 and CH4 dynamics are mostly based on equilibrium conditions.

That addition of sulfate inhibits methanogenesis is well established. Competition explains this observation in sediments and soils where the biological community has had time to reach something like equilibrium, with methanogens outcompeted by sulfate reducers. However, addition of sulfate to sediment immediately and completely inhibits H2-driven CH4 production, which cannot be explained by ecosystem dynamics. A model involving a threshold H2 concentration, in which H2 levels lower than some critical level determined by the thermodynamics of the situation shut down that pathway, does explain these observations.

Lovley 1991

Lovley DR. 1991. Dissimilatory Fe(III) and Mn(IV) reduction. Microbiological Reviews 55: 259-287.

This author comprehensively reviews microbe-mediated reduction of iron and manganese in soils and sediments, in a long and detailed paper. Dissimilatory reduction is distinct from assimilatory reduction, in which metal ions are reduced when they are incorporated into cellular macromolecules such as enzymes and cofactors. Dissimilatory reduction is a process that ends with the accumulation of reduced metal outside the cell, and is responsible for the majority of iron and manganese reduction in sediments. While it has been observed in aerobic environments, such reduction occurs mainly in anaerobic conditions. Fe(III) in particular is most often reduced when it is the final electron acceptor in the anaerobic oxidation of organic molecules.

Microbes capable of dissimilatory metal reduction can be categorized in a number of ways. This author presents 5 categories, though I think there is considerable overlap between them, as in cases where one cell is able to reduce Fe(III) and metabolize a range of carbon sources. In the first category, reducing fermenting bacteria, the amount of Fe(III) reduced during metabolism is far less than the stoichiometry of the redox couple would suggest. This implies that Fe(III) is a minor electron acceptor during fermentation reactions, rather than the electron acceptor of choice or necessity for these organisms. Sulfur-oxidizing species in contrast do appear to reduce quantities of iron in line with stoichiometric predictions, but do not seem to gain significant energy from these reactions which occur under aerobic conditions on elemental sulfur. Hydrogen-oxidizing reducers appear to be abundant in anaerobic sediments, and seem to have high affinity for hydrogen gas; where Fe(III) is being reduced, hydrogen concentrations are low, and when hydrogen is added, Fe(III) reduction increases. These organisms need other material, such as simple organic molecules, to grow, as this reaction provides energy but little else. Organic-acid oxidizing reducers and aromatic-oxidizers are probably in many cases the same cells. As these molecules are sometimes the result of fermentation metabolisms, one possible food chain in anaerobic environments is fermentation, with some Fe(III) reduction, followed by greater levels of Fe(III) reduction linked to the decomposition of smaller organic molecules such as acetate. Such an ecological pairing is probably widespread, given the known abundance and diversity of fermenting species and the probable abundance of more aggressively iron-reducing species, many of which are probably Archaea rather than Bacteria.

There are three competing models for how Fe(III) is reduced in natural environments. The first is the enzymatic model, in which microbial cells employ membrane-bound or intracellular enzymes to transfer electrons to Fe(III) ions during the process of anaerobic oxidation of organic matter. The second is termed the redox model, and posits the majority of Fe(III) reduction is driven by equilibrium thermodynamics, with the relative levels of Fe(III) and Fe(II) in sediments controlled by abiotic factors such as temperature and pH. The third model is termed the direct-reduction model, in which some organic molecules react directly with Fe(III), without the intervention of cells or enzymes.

At first glance, the observation that Fe(III) reduction rates fall when microbes are removed from sediments supports the enzymatic model, but in fact all three models rely on microbial metabolisms. In the redox model, competing electron acceptors such as oxygen and nitrate are consumed by microbes, lowering their concentrations to levels too low to influence the transitions between Fe(III) and Fe(II). And the direct model relies on microbes releasing key organic molecules known to reduce Fe(III) in vitro. However, several other lines of evidence, such as the lack of spontaneous shifts in iron valency ratios in stored sediments, the large changes in pH associated with widely-used extraction methods, and the general rarity of rapidly-acting direct-reducing molecules all lead to the conclusion that while the other mechanisms may contribute some iron reducing activity in some situations, the overwhelming majority of Fe(III) reduction to Fe(II) occurring is driven by microbes and their enzymes.

This author spends nearly as much time discussing Mn(IV) reduction as Fe(III) reduction, but I have little interest in Mn chemistry at this time. However, in the discussion of the competing models of metal reduction, mention is made that Fe(II) in solution may reduce Mn(IV), removing accumulated Fe(II) from iron reduction and abiotically returning the iron to Fe(III) while generating Mn(II). This abiotic back-reaction closes the loop on cycling Fe(III).

Iron reduction is postulated as one of the first, if not the first, globally important metabolic pathway, with early Archaea using Fe(III) as their final electron acceptor in a generally reducing environment lacking free oxygen and nitrate. If Fe(III), a non-soluble, precipitating substance is the primary oxidizing agent, the redox environment of the Earth 2 billion years ago was upside-down compared to today: the surface and atmosphere was reducing, while buried and water-saturated sediments were oxidizing.

Freshwater swamps differ from aquatic and marine sediments in a number of ways, including a generally high organic content and plenty of sulfates. Under these conditions, both iron and manganese may cycle rapidly between reduced and oxidized forms, further reinforcing the idea that iron may cycle in a closed or nearly-closed loop between Fe(III) and Fe(II) in wet soils. Soils that periodically dry and become oxic, such as rice paddies, may also employ molecular oxygen in this cycling, with the formation of amorphous Fe(III) oxides during the dry season, and reduction of this iron during the wet season. These reactions would restrict methane production, at least during the early part of the wet season, because Fe(III) reduction diverts electrons away from methane production.

The physical structure of iron oxides in the environment has a large effect on rates of Fe(III) reduction and populations of microbes. More strongly crystalline forms, such as goethite and hematite, are not readily reduced, while amorphous forms are consumed rapidly. Presumably it is amorphous forms that accumulate when Fe(II) is oxidized to Fe(III) and precipitates from solution as an oxide, leading to a labile pool of iron distinct from the highly resistant pool of crystalline mineral iron.

When it is not back-oxidized rapidly, Fe(II) and Mn(II) accumulate in solution, and are subject to water movements, potentially removing them from the site of production. Dissolved, reduced metal can be problematic, as these ions are readily oxidized by atmospheric oxygen if water flows contact the atmosphere, and will precipitate as rusty powder in drinking water and irrigation infrastructure. This also suggests that Fe(II) will not accumulate in natural systems over long periods, and that measuring the amount of Fe(II) in a sample is not a good indicator of iron-reducing microbial populations outside of microcosm experiments.

Iron oxides tend to provide strong adsorption surfaces for many other substances, including phosphate and heavy metals. The release to solution and movement of these substances can be a major concern when iron oxides are reduced.

Banded iron formations in some sediments and rocks, in which magnetite is deposited, appear to be the result of dissimilatory iron reduction. Magnetite is a mixed-valence iron oxide that behaves magnetically; small, characteristically-shaped crystals of it are produced intracellularly by magnetotactic bacteria, but much larger amounts are produced by a range of iron-reducing bacteria. The associated organic matter, masses and crystal structures of banded iron formations strongly suggest ancient dissimilatory iron reduction coupled to the decomposition of organic matter.

There are a range of factors controlling iron and manganese reduction in natural environments. Metal reduction is decreased in the presence of alternate electron acceptors, especially oxygen and nitrate. Oxygen is a thermodynamically favourable electron acceptor compared to either Fe(III) or Mn(IV), and nitrate appears to inhibit Fe(III) reduction by lowering electron availability below necessary levels. From a biological perspective, aerobic bacteria can usually outcompete iron reducers, many of whom are obligate anaerobes and are killed or inhibited by the presence of oxygen.

As previously described, the form of metal available in the environment also has a major effect on metal reduction rates, with more strongly crystalline forms most resistant to chemical alteration by microorganisms. “Poorly-crystalline” forms, presumably including amorphous metal oxides, are the major source of oxidized metals for reducing cells. Thus, extraction and measurement procedures that involve only the least crystalline forms of Fe(III) oxides are a good measure of the iron available for reduction by microbes. The widely-used oxalate extraction method normally does not extract significant quantities of Fe(III) oxides bound in strongly crystalline forms, except when “catalytic quantities” of Fe(II) are also available, as when weakly crystalline mixed-valence minerals such as magnetite are present. In such systems, oxalate extraction will gather a larger fraction of the total dithionate-citrate extractable iron, even though much of the iron so extracted will be in a form not actually available to soil microbes.

This paper is an enormous review of iron metabolisms in soils and sediments. It did not actually answer my current question, about the cycling of Fe(III) in soils and how comparisons of amorphous (oxalate-extractable) and total (dithionate-extractable) can be used to support inferences about long-term redox status in soils. However, I do think I now have a better grasp of general soil iron and manganese conditions and chemical transformations.