Yu K, Patrick WH Jr. 2004. Redox window with minimum global warming potential contribution from rice soils. Soil Science Society of America Journal 68: 2086-2091.
These authors followed up a previous study (Yu and Patrick 2003) that discovered a critical range of soil redox potential (Eh) across a range of pH for rice-agriculture soils, by examining soil Eh in more detail. The critical range is based on minimizing total global warming potential of all 3 major greenhouse gases, in terms of CO2-equivalents; methane and nitrous oxide have much higher radiative forcing than does CO2, when looked at on a 100-year horizon.
The critical range, for all 8 soils studied, is between 180 and -150 mV, what might be considered “moderately reducing” for soils. CO2 production is modest in this range, though it is lower at more reducing conditions. CH4 production is nearly absent in this range, but is very large at redox conditions below -150 mV. N2O production is modest in this range as well, with much higher N2O production under oxidizing conditions (Eh > 180) due to strong nitrification activity.
This paper serves to support earlier ideas about the net effects of redox conditions on the production and consumption dynamics of these gases. The story with CO2 and CH4 is fairly simple, while N2O dynamics are more complicated because there are more pathways for both production and consumption of this gas.
Thursday, January 28, 2010
Ettwig et al. 2009
Ettwig KF, van Alen T, van de Pas-Schoonen KT, Jetten MSM, Strous M. 2009. Enrichment and molecular detection of denitrifying methanotrophic bacteria of the NC10 phylum. Applied and Environmental Microbiology 75: 3656-3662.
These authors describe a series of experiments and procedures designed to investigate an enigmatic organism known as NC10, a bacteria in its own eponymous phylum that currently represents the only demonstrated case of biological reduction of nitrate coupled to oxidation of methane under anaerobic conditions. While anaerobic methane consumption has been observed in some archaea, it has not been found coupled to denitrification.
A laboratory culture eventually dominated by NC10 organisms of group a (a distinction within the phylum) was established, based on sediment collected from a eutrophic ditch draining agricultural land on the floodplain of the Rhine river in the Netherlands. This culture was grown and maintained under conditions in which the only carbon source was the sparge gas of CH4-CO2, and nitrogen was supplied with the mineral inputs as nitrate and nitrite, along with a wide range of other inorganic compounds and trace elements.
The major finding of this study was a wealth of knowledge of the basic characteristics of the NC10 organism, and confirmation that it does indeed oxidize methane under anaerobic conditions coupled to denitrification. This process is energetically favourable, and the theoretical stoichiometry matches the observed changes in chemical composition in these experiments, with the nitrite reduction to methane consumption ratio of 8:3.5, versus 8:3 based on mass balance calculations.
One of the surprising aspects of this organism is that its methane-oxidizing activity is completely inhibited by black butyl rubber, as is found in black rubber stoppers for serum vials and other glassware. Grey or red butyl rubber stoppers do not show such inhibition, and repeated boiling of black butyl rubber stoppers in HCl did not remove the inhibitory effects. Strictly anoxic conditions are not required for all aspects of working with this organism; brief exposure to atmospheric oxygen during liquid transfer, for example, did not inhibit methanotrophic activity.
Another strange feature of NC10 concerns its 16s rDNA sequences. General 16s primers do not amplify NC10 DNA. These authors developed new primers for the 16s region based on the DNA in their culture, which they were able to confirm as NC10 based on FISH observations. The new primers allowed them to work more easily with the NC10 DNA, which is not surprising, but the sequences of NC10 16s found did not differ in critical ways from the target regions of the general 16s primers. So, it is unknown why the general 16s primers do not work on NC10 DNA.
This paper was recommended to me and I would not likely have discovered it without this recommendation. I have results from the 2009 work at Alexandra Fjord that suggest simultaneous consumption of CH4 (oxidation) and N2O (reduction), and I did not know if these two processes might be linked in a single organism or within a system such as a symbiosis or food-chain as 2 halves of a redox couple.
My results may be more suggestive of an alternate situation. Rather than anaerobic oxidation of methane (weirdness) coupled to nitrate reduction, I may be looking for cases of reduction of nitrous oxide under aerobic conditions (weirdness) coupled to methane oxidation.
These authors describe a series of experiments and procedures designed to investigate an enigmatic organism known as NC10, a bacteria in its own eponymous phylum that currently represents the only demonstrated case of biological reduction of nitrate coupled to oxidation of methane under anaerobic conditions. While anaerobic methane consumption has been observed in some archaea, it has not been found coupled to denitrification.
A laboratory culture eventually dominated by NC10 organisms of group a (a distinction within the phylum) was established, based on sediment collected from a eutrophic ditch draining agricultural land on the floodplain of the Rhine river in the Netherlands. This culture was grown and maintained under conditions in which the only carbon source was the sparge gas of CH4-CO2, and nitrogen was supplied with the mineral inputs as nitrate and nitrite, along with a wide range of other inorganic compounds and trace elements.
The major finding of this study was a wealth of knowledge of the basic characteristics of the NC10 organism, and confirmation that it does indeed oxidize methane under anaerobic conditions coupled to denitrification. This process is energetically favourable, and the theoretical stoichiometry matches the observed changes in chemical composition in these experiments, with the nitrite reduction to methane consumption ratio of 8:3.5, versus 8:3 based on mass balance calculations.
One of the surprising aspects of this organism is that its methane-oxidizing activity is completely inhibited by black butyl rubber, as is found in black rubber stoppers for serum vials and other glassware. Grey or red butyl rubber stoppers do not show such inhibition, and repeated boiling of black butyl rubber stoppers in HCl did not remove the inhibitory effects. Strictly anoxic conditions are not required for all aspects of working with this organism; brief exposure to atmospheric oxygen during liquid transfer, for example, did not inhibit methanotrophic activity.
Another strange feature of NC10 concerns its 16s rDNA sequences. General 16s primers do not amplify NC10 DNA. These authors developed new primers for the 16s region based on the DNA in their culture, which they were able to confirm as NC10 based on FISH observations. The new primers allowed them to work more easily with the NC10 DNA, which is not surprising, but the sequences of NC10 16s found did not differ in critical ways from the target regions of the general 16s primers. So, it is unknown why the general 16s primers do not work on NC10 DNA.
This paper was recommended to me and I would not likely have discovered it without this recommendation. I have results from the 2009 work at Alexandra Fjord that suggest simultaneous consumption of CH4 (oxidation) and N2O (reduction), and I did not know if these two processes might be linked in a single organism or within a system such as a symbiosis or food-chain as 2 halves of a redox couple.
My results may be more suggestive of an alternate situation. Rather than anaerobic oxidation of methane (weirdness) coupled to nitrate reduction, I may be looking for cases of reduction of nitrous oxide under aerobic conditions (weirdness) coupled to methane oxidation.
Firestone et al. 1980
Firestone MK, Firestone RB, Tiedje JM. 1980. Nitrous oxide from soil denitrification: Factors controlling its biological production. Science 208: 749-751.
These authors measured the faction of N2O in nitrogen gas outputs from soil slurries under a range of conditions of substrate and oxygen availability. Slurries were employed to avoid problems associated with diffusion of materials through a soil matrix, and the process of denitrification was studied using isotopic tracers, especially in the form of 13N in nitrate and other inputs.
The controls on the production of N2O from denitrification are the concentration of nitrite (NO2-) and the availability of oxygen (O2), with time-since-anoxic another important factor. Increasing nitrite increases N2O production and increasing NO3- does as well, but less strongly, suggesting the role of NO3- is indirect, and it is the NO2- produced from NO3- that matters. Aerobic conditions inhibit denitrification, rendering the entire pathway moot. The establishment of anaerobic conditions turns on denitrification, but in a stepwise process apparently related to protein synthesis. In a series of experiments, these authors found that in the initial period of anaerobiosis, N2 is the major output. Later, N2O production increases without an increase in its consumption, and N2O is the major output. Finally, N2O consumption catches up with production, and N2 is once again the major output. Adding O2 increases the proportion of total denitrification output that is N2O, but eventually O2 does inhibit denitrification completely.
These authors measured the faction of N2O in nitrogen gas outputs from soil slurries under a range of conditions of substrate and oxygen availability. Slurries were employed to avoid problems associated with diffusion of materials through a soil matrix, and the process of denitrification was studied using isotopic tracers, especially in the form of 13N in nitrate and other inputs.
The controls on the production of N2O from denitrification are the concentration of nitrite (NO2-) and the availability of oxygen (O2), with time-since-anoxic another important factor. Increasing nitrite increases N2O production and increasing NO3- does as well, but less strongly, suggesting the role of NO3- is indirect, and it is the NO2- produced from NO3- that matters. Aerobic conditions inhibit denitrification, rendering the entire pathway moot. The establishment of anaerobic conditions turns on denitrification, but in a stepwise process apparently related to protein synthesis. In a series of experiments, these authors found that in the initial period of anaerobiosis, N2 is the major output. Later, N2O production increases without an increase in its consumption, and N2O is the major output. Finally, N2O consumption catches up with production, and N2 is once again the major output. Adding O2 increases the proportion of total denitrification output that is N2O, but eventually O2 does inhibit denitrification completely.
Patrick and DeLaune 1972
Patrick WH Jr., DeLaunce RD. 1972. Characterization of the oxidized and reduced zones in flooded soil. Soil Science Society of America Proceedings 36: 573-576.
These authors measured the thickness of the oxidized layer in flooded soils. For Eh measurements, a platinum electrode was pushed down through the soil at a rate of 2mm/hour, sufficiently slowly for the electrode tip to reach near-equilibrium conditions as it descended. Concentrations of reduced and oxidized forms of Manganese, Iron, Sulfur, and Nitrogen were also measured.
Within a few days of submergence, soils showed a clear redox-potential profile as measured by the platinum electrode, with the oxidized layer above the reduced layer, and a transition from above 200 mV to below 200 mV across a relatively narrow intermediate layer. The profiles as measured by the chemical species distribution were similar, though Mn showed a narrower and S a deeper oxidized layer, probably relating to the redox conditions needed to reduce the oxidized compounds present in the soil; sulfate, for example, requires lower Eh values to be reduced than do ferric oxides.
Manganese, Iron, and Sulfur did not diffuse appreciably in these experiments, but Nitrogen compounds did. These authors propose the following process is occurring in these flooded soils:
These authors measured the thickness of the oxidized layer in flooded soils. For Eh measurements, a platinum electrode was pushed down through the soil at a rate of 2mm/hour, sufficiently slowly for the electrode tip to reach near-equilibrium conditions as it descended. Concentrations of reduced and oxidized forms of Manganese, Iron, Sulfur, and Nitrogen were also measured.
Within a few days of submergence, soils showed a clear redox-potential profile as measured by the platinum electrode, with the oxidized layer above the reduced layer, and a transition from above 200 mV to below 200 mV across a relatively narrow intermediate layer. The profiles as measured by the chemical species distribution were similar, though Mn showed a narrower and S a deeper oxidized layer, probably relating to the redox conditions needed to reduce the oxidized compounds present in the soil; sulfate, for example, requires lower Eh values to be reduced than do ferric oxides.
Manganese, Iron, and Sulfur did not diffuse appreciably in these experiments, but Nitrogen compounds did. These authors propose the following process is occurring in these flooded soils:
“…ammonium diffusion from the reduced layer to the oxidized layer -> ammonium oxidation to nitrate (nitrification) -> nitrate diffusion from the oxidized layer to the reduced layer -> denitrification…”To explain the observation that nitrate was absent from the reduced layer, and never very abundant in the oxidized layer, and that ammonium was rapidly depleted in the oxidized layer. In this system, nitrification and denitrification are occurring simultaneously at different positions and redox potentials.
Wednesday, January 27, 2010
Yates et al. 2007
Yates TT, Si BC, Farrell RE, Pennock DJ. 2007. Time, location, and scale dependence of soil nitrous oxide emissions, soil water, and temperature using wavelets, cross-wavelets, and wavelet coherency analysis. Journal of Geophysical Research 112, D09104.
These authors analyzed a dataset of soil parameters and N2O emission using three subtly-different wavelet-based statistical techniques. There were two main purposes to this study; first, to examine the predictive relationships (if any) between soil parameters such as water filled pore space (WFPS) or temperature and N2O emissions; second, to evaluate the utility of these 3 wavelet techniques in analyzing this type of data.
N2O emission data is characterized by high variance in space and time, and frequent extreme values. These characteristics make many sophisticated geospatial statistical techniques not suitable, and the high spatial and temporal autocorrelation of many soil parameters eliminates many other techniques. These authors describe these limitations and some of the techniques that have been employed, and settle on 3 varieties of wavelet analysis.
Wavelet techniques are related to Fourier-transforms, and they appear to be highly complex and sophisticated methods to transform data for analysis, rather than being analytical methods per se. A large fraction of this paper is concerned with detailed description of the parameters of the transformation, and the interpretation of the results. One of the key advantages of these techniques is they usually allow examination of data across a broad range of spatial scales, thus permitting identification of the spatial scale at which important soil processes occur. Beyond that, I did not understand much of this paper.
Besides the interpretation of the differences between the 3 wavelet techniques, which was quite frankly beyond my understanding, the main result of this study was that the soil parameters that can predict N2O emissions in this landscape vary through the season. Early, around snowmelt and soil thawing, soil temperature is predictive of emissions. Later in the season, temperature loses its usefulness, and individual landscape features may present WFPS as predictive, but not in a global sense. By mid-summer, the soil parameters measured in this study no longer bore any relationship to N2O emissions. This loss of predictive value shows how complex this system is, and shows how some modeling efforts need to change in order to improve estimates of landscape-scale N2O processes.
Besides demonstrating my ignorance of advanced geospatial statistical techniques, this paper is primarily useful to me for its clear introduction describing the basic controls on and processes of N2O production in soils. My previous understanding centred on the role of water in restricting O2 availability in soils leading to changes at both the community and cell-physiology levels and consequently N2O production patterns in space and time appears to be essentially correct, and is reinforced by the early introduction section of this paper and the references therein.
These authors analyzed a dataset of soil parameters and N2O emission using three subtly-different wavelet-based statistical techniques. There were two main purposes to this study; first, to examine the predictive relationships (if any) between soil parameters such as water filled pore space (WFPS) or temperature and N2O emissions; second, to evaluate the utility of these 3 wavelet techniques in analyzing this type of data.
N2O emission data is characterized by high variance in space and time, and frequent extreme values. These characteristics make many sophisticated geospatial statistical techniques not suitable, and the high spatial and temporal autocorrelation of many soil parameters eliminates many other techniques. These authors describe these limitations and some of the techniques that have been employed, and settle on 3 varieties of wavelet analysis.
Wavelet techniques are related to Fourier-transforms, and they appear to be highly complex and sophisticated methods to transform data for analysis, rather than being analytical methods per se. A large fraction of this paper is concerned with detailed description of the parameters of the transformation, and the interpretation of the results. One of the key advantages of these techniques is they usually allow examination of data across a broad range of spatial scales, thus permitting identification of the spatial scale at which important soil processes occur. Beyond that, I did not understand much of this paper.
Besides the interpretation of the differences between the 3 wavelet techniques, which was quite frankly beyond my understanding, the main result of this study was that the soil parameters that can predict N2O emissions in this landscape vary through the season. Early, around snowmelt and soil thawing, soil temperature is predictive of emissions. Later in the season, temperature loses its usefulness, and individual landscape features may present WFPS as predictive, but not in a global sense. By mid-summer, the soil parameters measured in this study no longer bore any relationship to N2O emissions. This loss of predictive value shows how complex this system is, and shows how some modeling efforts need to change in order to improve estimates of landscape-scale N2O processes.
Besides demonstrating my ignorance of advanced geospatial statistical techniques, this paper is primarily useful to me for its clear introduction describing the basic controls on and processes of N2O production in soils. My previous understanding centred on the role of water in restricting O2 availability in soils leading to changes at both the community and cell-physiology levels and consequently N2O production patterns in space and time appears to be essentially correct, and is reinforced by the early introduction section of this paper and the references therein.
Tuesday, January 26, 2010
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.
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.
Pennock 2004
Pennock DJ. 2004. Designing field studies in soil science. Canadian Journal of Soil Science 84: 1-10.
This author reviews the major issues surrounding field-based (as opposed to strictly laboratory-based) research, focusing on issues specific or of greatest importance to soil science. Soil science’s history could perhaps be described as a fusion of physical geography and geology with agronomy, and many published studies in the soil science journals show these roots. Following the lead of previous authors, who have included ecologists, statisticians, and philosophers and historians of science, this author divides field research into 2 major categories, broadly manipulative studies and mensurative studies. Manipulative studies are, under some definitions including one tentatively employed in this paper, the only type of study that qualify for the name “experiment”, and involve complete control over experimental conditions by the researcher. Treatments in an experiment are directly related to replication, and can be applied with great precision. Mensurative studies are those that at least partly use features of the environment beyond the control of the researcher to test hypotheses or discover new information. The key feature of a mensurative study is that the features of interest are clearly defined but not controlled (i.e. not randomized) by the person conducting the study.
Replication, and avoiding pseudoreplication, is of great importance in all types of studies. However, the replication built into a manipulative experiment in the form of repeated application of treatments is distinct from the replication of a mensurative study using repeated features of the environment. That these are different types of replication is stated in this paper, but I found no more detail or explanation than that. Pseudoreplication in this paper is discussed little in the context of independence of samples; rather the discussed risk is of attempting to draw inferences beyond the inference space of the study. This is a problem in both major types of study, and can be avoided by carefully determining and describing the inference space, and expanding that space by greater replication; too-small sample sizes are quite simply labeled as unpublishable in this paper, a sentiment I can agree with.
Determining the required sample size is a major issue for all types of studies. In this author’s presentation, this is an early step in the design of the study, after the biological and statistical questions have been established but before data collection begins. There is some discussion here as well of statistical power (the chance of avoiding a Type II error, that is of failing to reject a false null hypothesis) and recommendations of flexibility regarding especially alpha values (the chance of making a Type I error, that is of rejecting a null hypothesis that is not false). For a number of reasons, some of which are practical and logistical, alpha values larger than the ubiquitous 0.05 are encouraged, because in many cases the consequences of the 2 types of error are not even, and one may wish to concentrate on reducing the probability of a Type II error.
This paper describes 10 commonly-encountered study designs in soil science and related disciplines, and then discusses study-design concerns common to all such as replication and the need to clearly define study units, samples, populations, and other important aspects. Finally, this author presents the conclusions from all of these examples and considerations in the form of a short list of key recommendations. Quoting directly:
This author reviews the major issues surrounding field-based (as opposed to strictly laboratory-based) research, focusing on issues specific or of greatest importance to soil science. Soil science’s history could perhaps be described as a fusion of physical geography and geology with agronomy, and many published studies in the soil science journals show these roots. Following the lead of previous authors, who have included ecologists, statisticians, and philosophers and historians of science, this author divides field research into 2 major categories, broadly manipulative studies and mensurative studies. Manipulative studies are, under some definitions including one tentatively employed in this paper, the only type of study that qualify for the name “experiment”, and involve complete control over experimental conditions by the researcher. Treatments in an experiment are directly related to replication, and can be applied with great precision. Mensurative studies are those that at least partly use features of the environment beyond the control of the researcher to test hypotheses or discover new information. The key feature of a mensurative study is that the features of interest are clearly defined but not controlled (i.e. not randomized) by the person conducting the study.
Replication, and avoiding pseudoreplication, is of great importance in all types of studies. However, the replication built into a manipulative experiment in the form of repeated application of treatments is distinct from the replication of a mensurative study using repeated features of the environment. That these are different types of replication is stated in this paper, but I found no more detail or explanation than that. Pseudoreplication in this paper is discussed little in the context of independence of samples; rather the discussed risk is of attempting to draw inferences beyond the inference space of the study. This is a problem in both major types of study, and can be avoided by carefully determining and describing the inference space, and expanding that space by greater replication; too-small sample sizes are quite simply labeled as unpublishable in this paper, a sentiment I can agree with.
Determining the required sample size is a major issue for all types of studies. In this author’s presentation, this is an early step in the design of the study, after the biological and statistical questions have been established but before data collection begins. There is some discussion here as well of statistical power (the chance of avoiding a Type II error, that is of failing to reject a false null hypothesis) and recommendations of flexibility regarding especially alpha values (the chance of making a Type I error, that is of rejecting a null hypothesis that is not false). For a number of reasons, some of which are practical and logistical, alpha values larger than the ubiquitous 0.05 are encouraged, because in many cases the consequences of the 2 types of error are not even, and one may wish to concentrate on reducing the probability of a Type II error.
This paper describes 10 commonly-encountered study designs in soil science and related disciplines, and then discusses study-design concerns common to all such as replication and the need to clearly define study units, samples, populations, and other important aspects. Finally, this author presents the conclusions from all of these examples and considerations in the form of a short list of key recommendations. Quoting directly:
1. A clear definition of the research question is the initial (and most critical) step. This definition dictates the type of research design that is appropriate and the specific design issues associated with different research types.
2. The appropriateness of a given research design can be judged only after a thorough review of what is known about the research question. Exploratory pattern studies can be very informative at an early stage of research, but yield little new information for well-established research topics. Equally, the imposition of a set of treatments if little is known of the processes controlling responses is unlikely to produce comprehensive interpretations.
3. There is never a good reason for haphazard sampling – the rationale for selecting sampling points in pedological, soil geomorphic, or inventory studies should be clearly stated.
4. A clear definition of the population and the elements that comprise the population under study is very important.
5. The definition of the population dictates the extent of the study and the physical or temporal space that the results pertain to, which is critical to avoid pseudoreplication.
6. The sample support, spacing, and extent of the study must be consistent with what is known of the processes controlling the phenomena being studied.
7. The construction of hypotheses for formal testing should be based on sound physical or biological reasoning, and sufficient samples should be taken to allow reliable testing of the alternative hypotheses.
8. The exclusion of phenomena because they cannot be replicated is inherently limiting to the expansion of our knowledge of soils. Innovative approaches must continue to be developed and applied so that we can expand the scale at which field studies can be undertaken.
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