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.
Wednesday, February 24, 2010
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.
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.
Monday, February 22, 2010
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:
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.
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.
Friday, February 19, 2010
Liptzin 2006
Liptzin D. 2006. A banded vegetation pattern in a High Arctic community on Axel Heiberg Island, Nunavut, Canada. Arctic, Antarctic, and Alpine Research 38: 216-223.
This author attempted to explain the observation of banded vegetation on a slope that lacked the usual factors that generate such patterns. In temperate and tropical locations, banded vegetation, also known as “tiger stripes”, forms on shallow slopes in dry areas with a consistent direction of water flow. Plants at a position on the slope increase water retention and facilitate further colonization by plants. Similarly, some locations experience consistent wind direction carrying sea spray that kills trees at some positions. In cold environments, patterned ground from cryoturbation on shallow slopes can also lead to banded vegetation. However, the study site in this paper lacks all of these features, including cryoturbation despite the presence of permafrost within 50cm at most locations.
Some aspect of soil properties is the obvious explanatory hypothesis, which this author explores after describing the transects measuring plant diversity and the soil pits used to examine soil properties. In general, features that would normally be expected to influence plant diversity and abundance such as soil moisture or exchangeable cation levels, had no significant impact in the various statistical tests employed in this study. However, soil type did have some effect, as a few species of plants were found only on sandy soil, and nitrogen levels were negatively correlated with species richness.
The discussion section of this paper is an excellent example of a chain of logical reasoning working through a series of potential explanations. While this paper is interesting, it’s only relevant to my own studies in a narrow area around potential starting points in looking for explanations for whatever patterns I may find in my biogeography studies in 2010. However, this paper seems remarkably suitable as an introduction to the basics of modern soil science research, and may be relevant to my not-quite-mothballed interest in an undergraduate course about the current state of the scientific literature.
This author attempted to explain the observation of banded vegetation on a slope that lacked the usual factors that generate such patterns. In temperate and tropical locations, banded vegetation, also known as “tiger stripes”, forms on shallow slopes in dry areas with a consistent direction of water flow. Plants at a position on the slope increase water retention and facilitate further colonization by plants. Similarly, some locations experience consistent wind direction carrying sea spray that kills trees at some positions. In cold environments, patterned ground from cryoturbation on shallow slopes can also lead to banded vegetation. However, the study site in this paper lacks all of these features, including cryoturbation despite the presence of permafrost within 50cm at most locations.
Some aspect of soil properties is the obvious explanatory hypothesis, which this author explores after describing the transects measuring plant diversity and the soil pits used to examine soil properties. In general, features that would normally be expected to influence plant diversity and abundance such as soil moisture or exchangeable cation levels, had no significant impact in the various statistical tests employed in this study. However, soil type did have some effect, as a few species of plants were found only on sandy soil, and nitrogen levels were negatively correlated with species richness.
The discussion section of this paper is an excellent example of a chain of logical reasoning working through a series of potential explanations. While this paper is interesting, it’s only relevant to my own studies in a narrow area around potential starting points in looking for explanations for whatever patterns I may find in my biogeography studies in 2010. However, this paper seems remarkably suitable as an introduction to the basics of modern soil science research, and may be relevant to my not-quite-mothballed interest in an undergraduate course about the current state of the scientific literature.
Thursday, February 18, 2010
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.
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.
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.
Wednesday, February 17, 2010
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.
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.
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.
Friday, February 12, 2010
Kellman and Kavanaugh 2008
Kellman L, Kavanaugh K. 2008. Nitrous oxide dynamics in managed northern forest soil profiles: is production offset by consumption? Biogeochemistry 90: 115-128.
These authors measured surface flux and subsurface profiles of N2O at a number of paired sites in the managed forest of Nova Scotia. Half of the sites were clear-cut harvested three years before the study, the other half more than 50 years previously. Climate factors such as air temperatures and solar radiation were consistent across the study area. Fluxes and profiles were measured periodically through a 9-month snow-free period in 2005, from early March to late November.
Surface fluxes were measured by pulling samples into evacuated containers from chambers mounted on permanent collars. Similarly, profiles were measured by sampling from permanent probes buried in the walls of soil pits. Actual measurement of gas concentrations were in the laboratory using a gas chromatograph system.
The soil probes consist of 50cm PVC tubes, covered with a “water resistant porous membrane” (could they be using Gore-tex?) and buried in the walls of pits at depths of 0, 5, 20, and 35cm, with 0 at the mineral soil-organic layer interface. This provides a 50cm-long sampling space at four depths, replicated across 40 sites.
The relationship between profile N2O concentrations and surface flux was almost always non-significant. These authors attribute this lack of correlation to consumption of N2O in the soil profile. In contrast, the studies that have linked CO2 profiles to surface flux have relied on the (probably true) assumption that CO2 is not consumed in the soil, and moves through diffusion in a manner that can be predicted from soil physics. N2O profiles that include regions of consumption are complicated by the biological and chemical factors that control production and consumption, as well as movement. All of this leads to a disconnection between soil N2O cycling and surface-atmosphere exchange.
This paper should almost certainly be included in the introduction, methods, and/or discussion section(s) of my pits & probes manuscript. This is one of the few studies I have found that examined N2O in soil profiles; most others appear to focus on CO2 or in some cases the biogeochemistry of CH4.
These authors measured surface flux and subsurface profiles of N2O at a number of paired sites in the managed forest of Nova Scotia. Half of the sites were clear-cut harvested three years before the study, the other half more than 50 years previously. Climate factors such as air temperatures and solar radiation were consistent across the study area. Fluxes and profiles were measured periodically through a 9-month snow-free period in 2005, from early March to late November.
Surface fluxes were measured by pulling samples into evacuated containers from chambers mounted on permanent collars. Similarly, profiles were measured by sampling from permanent probes buried in the walls of soil pits. Actual measurement of gas concentrations were in the laboratory using a gas chromatograph system.
The soil probes consist of 50cm PVC tubes, covered with a “water resistant porous membrane” (could they be using Gore-tex?) and buried in the walls of pits at depths of 0, 5, 20, and 35cm, with 0 at the mineral soil-organic layer interface. This provides a 50cm-long sampling space at four depths, replicated across 40 sites.
The relationship between profile N2O concentrations and surface flux was almost always non-significant. These authors attribute this lack of correlation to consumption of N2O in the soil profile. In contrast, the studies that have linked CO2 profiles to surface flux have relied on the (probably true) assumption that CO2 is not consumed in the soil, and moves through diffusion in a manner that can be predicted from soil physics. N2O profiles that include regions of consumption are complicated by the biological and chemical factors that control production and consumption, as well as movement. All of this leads to a disconnection between soil N2O cycling and surface-atmosphere exchange.
This paper should almost certainly be included in the introduction, methods, and/or discussion section(s) of my pits & probes manuscript. This is one of the few studies I have found that examined N2O in soil profiles; most others appear to focus on CO2 or in some cases the biogeochemistry of CH4.
Thursday, February 11, 2010
Lamb et al. 2006
Lamb EG, Cahill JF, Dale MRT. 2006. A nonlinear regression approach to test for size-dependence of competitive ability. Ecology 87: 1452-1457.
These authors describe a statistical analytical technique, “nonlinear regression”, that potentially provides more information than linear regression techniques including ANCOVA. The basic linear regression formula includes the intercept and the slope as parameters; nonlinear regression adds an exponent parameter. In this paper, these parameters are referred to as k1 (intercept), k2 (slope), and k3 (exponent), in the formula
These authors tested their technique on three example datasets from previous studies. The first example is most thoroughly examined, and involves a plant competition experiment. One key feature of all of the example analysis is the dataset must be paired, such that each data point on the x axis corresponds to a partner data point on the y axis. In the plant competition example, individual plants that did not experience competition are paired with individuals that did, because each pair of plants was grown in a communal pot that was treated at the pot level with manipulations such as fertilizer application. The continuous dataset is plant size, measured as the absolute gain in mass over the course of the growing season.
The exponent parameter k3 can take on any value between negative and positive infinity, to describe curves that may be accelerating, saturating, or straight. At values of 1 or -1, k3 is not informative as a parameter, and should be discarded from the model. This analysis is based on a model-building and model-testing technique, where models with various values for the three parameters are tested against null models and each other in an iterative fashion to find the model that best fits the data.
This approach is likely to be useful in the analysis of some of the data collected at Alexandra Fjord in 2009.
These authors describe a statistical analytical technique, “nonlinear regression”, that potentially provides more information than linear regression techniques including ANCOVA. The basic linear regression formula includes the intercept and the slope as parameters; nonlinear regression adds an exponent parameter. In this paper, these parameters are referred to as k1 (intercept), k2 (slope), and k3 (exponent), in the formula
y = k1 + k2x^k3
where y is the response variable and x is the explanatory variable.These authors tested their technique on three example datasets from previous studies. The first example is most thoroughly examined, and involves a plant competition experiment. One key feature of all of the example analysis is the dataset must be paired, such that each data point on the x axis corresponds to a partner data point on the y axis. In the plant competition example, individual plants that did not experience competition are paired with individuals that did, because each pair of plants was grown in a communal pot that was treated at the pot level with manipulations such as fertilizer application. The continuous dataset is plant size, measured as the absolute gain in mass over the course of the growing season.
The exponent parameter k3 can take on any value between negative and positive infinity, to describe curves that may be accelerating, saturating, or straight. At values of 1 or -1, k3 is not informative as a parameter, and should be discarded from the model. This analysis is based on a model-building and model-testing technique, where models with various values for the three parameters are tested against null models and each other in an iterative fashion to find the model that best fits the data.
This approach is likely to be useful in the analysis of some of the data collected at Alexandra Fjord in 2009.
Thursday, February 4, 2010
Michelsen et al. 1999
Michelsen, A., Graglia, E., Schmidt, I.K., Jonasson, S., Sleep, D., and Quarmby, C. 1999. Differential responses of grass and a dwarf shrub to long-term changes in soil microbial biomass C, N and P following factorial addition of NPK fertilizer, fungicide and labile carbon to a heath. New Phytologist 143(3): 523-538.
These authors measured the responses of two tundra plants, one graminoid and one shrub, to additions of fertilizer, labile carbon, and a fungicide over four years in a heath in subarctic Sweden. The underlying hypothesis is that differences in growth strategy between these two groups of plants would be reflected in their responses to changes in microbial biomass, also measured by these authors.
Molecules inaccessible to plants by virtue of being incorporated into microbial cells in soils dominate their respective pools. For example, in this study, microbial N concentrations were up to 50 times higher than free, inorganic N such as ammonium. Similar ratios were found for C and P. Rapid incorporation of these nutrients into microbes therefore constitutes competition between microbes and plants. Consistent with the variety of ecological and growth strategies that plants have evolved interacting with each other and with the abiotic features, plant strategies impact their interactions with microbes and how they react to such competition.
As predicted, the graminoid Festuca ovina responded rapidly to changes in nutrient levels. Added N from fertilizers led to increased ground cover by this grass, while added labile C, in the form of sugar, led to decreased ground cover, presumably due to a rapid increase in microbial biomass and associated increased microbial uptake of soil N and P. In contrast, the shrub Vaccinium uliginosum did not appreciably react to changes in nutrient levels, consistent with a long-view growth strategy and successful escape from severe competition with soil microbes. This may have been mediated by the mycorrhizal associations the shrub has but the graminoid lacks.
This paper, interestingly, does not cite any of the work by Wardle or Nilsson, the authors who severely criticized the earlier work by this research group (Michelsen et al. 1995).
These authors measured the responses of two tundra plants, one graminoid and one shrub, to additions of fertilizer, labile carbon, and a fungicide over four years in a heath in subarctic Sweden. The underlying hypothesis is that differences in growth strategy between these two groups of plants would be reflected in their responses to changes in microbial biomass, also measured by these authors.
Molecules inaccessible to plants by virtue of being incorporated into microbial cells in soils dominate their respective pools. For example, in this study, microbial N concentrations were up to 50 times higher than free, inorganic N such as ammonium. Similar ratios were found for C and P. Rapid incorporation of these nutrients into microbes therefore constitutes competition between microbes and plants. Consistent with the variety of ecological and growth strategies that plants have evolved interacting with each other and with the abiotic features, plant strategies impact their interactions with microbes and how they react to such competition.
As predicted, the graminoid Festuca ovina responded rapidly to changes in nutrient levels. Added N from fertilizers led to increased ground cover by this grass, while added labile C, in the form of sugar, led to decreased ground cover, presumably due to a rapid increase in microbial biomass and associated increased microbial uptake of soil N and P. In contrast, the shrub Vaccinium uliginosum did not appreciably react to changes in nutrient levels, consistent with a long-view growth strategy and successful escape from severe competition with soil microbes. This may have been mediated by the mycorrhizal associations the shrub has but the graminoid lacks.
This paper, interestingly, does not cite any of the work by Wardle or Nilsson, the authors who severely criticized the earlier work by this research group (Michelsen et al. 1995).
Wednesday, February 3, 2010
Wardle and Nilsson 1997
Wardle DA, Nilsson M-C. 1997. Microbe-plant competition, allelopathy and arctic plants. Oecologia 109: 291-293.
These authors critique Michelsen et al. (1995), a study that came to several important conclusions regarding the interactions between Arctic plants and the soil microbial communities. This is a very negative review of that paper, in which these authors question almost all of the conclusions of Michelsen et al. (1995).
These authors make two main criticisms. First, they question the measures of soil microbial activity used by the earlier paper. Second, they question the conclusions regarding the allelopathy of Empetrum hermaphroditum. Soil microbial activity was measured by Michelsen et al. (1995) in two ways: soil respiration, and soil ergosterol content. Neither approach is necessarily informative about one of Michelsen et al.’s (1995) main claims, that soil microbial biomass was increased by the addition of plant leaf extracts. There are a number of studies, many of them with Nilsson as a co-author, in which a lack of association between microbial biomass and soil respiration was demonstrated. Furthermore, ergosterol is presented by Michelsen et al. (1995) as an indicator of fungal biomass, but previous work by Newell and colleagues (e.g. Newell and Fallon, 1991; Newell 1992) showed that ergosterol is not a reliable indicator of biomass nor is it useful as a proxy measure of soil fungal activity; the ratio of ergosterol to fungal biomass is highly variable.
From the reference list in this short paper, it appears that Wardle and Nilsson had, by early 1997, completed a considerable body of work regarding the allelopathic and other ecological interactions of E. hermaphroditum in sub-arctic environments. The conclusion by Michelsen et al. (1995) that the chemicals released by this plant have a greater impact on microbial communities than potential surface-plant competitors is not supported by this work by Wardle, Nilsson, and their colleagues.
The conclusion of Michelsen et al. (1995) that currently has the most direct bearing on my own work is that key plant traits often possessed by prostrate shrubs in tundra ecosystems such as a high root: shoot ratio and storage of nutrients such as nitrogen in the roots allow those plants to escape from or outcompete soil microorganisms. This conclusion was not addressed by these authors, but given the devastation inflicted upon the other conclusions, my confidence in the utility of Michelsen et al. (1995) in addressing issues of interactions between Cassiope tetragonal and soil microbial communities has been shaken.
These authors critique Michelsen et al. (1995), a study that came to several important conclusions regarding the interactions between Arctic plants and the soil microbial communities. This is a very negative review of that paper, in which these authors question almost all of the conclusions of Michelsen et al. (1995).
These authors make two main criticisms. First, they question the measures of soil microbial activity used by the earlier paper. Second, they question the conclusions regarding the allelopathy of Empetrum hermaphroditum. Soil microbial activity was measured by Michelsen et al. (1995) in two ways: soil respiration, and soil ergosterol content. Neither approach is necessarily informative about one of Michelsen et al.’s (1995) main claims, that soil microbial biomass was increased by the addition of plant leaf extracts. There are a number of studies, many of them with Nilsson as a co-author, in which a lack of association between microbial biomass and soil respiration was demonstrated. Furthermore, ergosterol is presented by Michelsen et al. (1995) as an indicator of fungal biomass, but previous work by Newell and colleagues (e.g. Newell and Fallon, 1991; Newell 1992) showed that ergosterol is not a reliable indicator of biomass nor is it useful as a proxy measure of soil fungal activity; the ratio of ergosterol to fungal biomass is highly variable.
From the reference list in this short paper, it appears that Wardle and Nilsson had, by early 1997, completed a considerable body of work regarding the allelopathic and other ecological interactions of E. hermaphroditum in sub-arctic environments. The conclusion by Michelsen et al. (1995) that the chemicals released by this plant have a greater impact on microbial communities than potential surface-plant competitors is not supported by this work by Wardle, Nilsson, and their colleagues.
The conclusion of Michelsen et al. (1995) that currently has the most direct bearing on my own work is that key plant traits often possessed by prostrate shrubs in tundra ecosystems such as a high root: shoot ratio and storage of nutrients such as nitrogen in the roots allow those plants to escape from or outcompete soil microorganisms. This conclusion was not addressed by these authors, but given the devastation inflicted upon the other conclusions, my confidence in the utility of Michelsen et al. (1995) in addressing issues of interactions between Cassiope tetragonal and soil microbial communities has been shaken.
Michelsen et al. 1995
Michelsen A, Schmidt IK, Jonasson S, Dighton J, Jones HE, Callaghan TV. Inhibition of growth, and effects on nutrient uptake of arctic graminoids by leaf extracts – allelopathy or resource competition between plants and microbes? Oecologia 103: 407-418.
These authors conducted an experiment to examine the potential allelopathic effects of plant leaf extracts among a few species of arctic tundra plants. Three arctic/subarctic plants suspected of releasing phytotoxic compounds upon their competitors including Cassiope tetragonal were harvested and their leaves and branches ground up to make leaf extracts. Three species of arctic graminoids (Carex bigelowii, Festuca vivipara, Luzula arcuata) were then treated with these extracts while growing in either sterilized or non-sterilized soil in greenhouses in southern Sweden. All of the graminoids, the soil they grew in, and two of the leaf extract-providing plants came from a montaine subarctic hillside in northern Sweden; the third leaf extract came from Betula pubescens ssp. tortuosa, a birch, were collected from individuals growing near the tree line at 450m altitude near Abisko Scientific Research Station, above the Arctic Circle in a subarctic ecosystem.
The experimental design was factorial, with two soil types (sterilized vs. non-sterilized) and four leaf-extract treatments including a control of distilled water. In addition to growth of the graminoids, measurements were made of the chemicals in the leaf extracts and soils, nutrient uptake by excised roots, soil ergosterol content, and soil respiration. Excised roots take up nutrients in a manner directly correlated to the nutrient-limitation status of the plant; more phosphorus-starved plants, for example, have roots that more rapidly take up phosphorus when offered. Soil ergosterol content is a measure of fungal biomass, while soil respiration was taken as a measure of total microbial activity.
Sterilized soil had higher extractable nutrients, probably as a result of the breakdown of microbial cells during autoclaving. Nitrate levels were negligible, both in soils and in leaf extracts; nitrate was a component of the dilute nutrient water used to maintain the plants while growing. Some mycorrhizae were found, but they covered less than 1% of the roots in non-sterilized soil, and had no impact on other measured parameters.
The highest growth of all three test graminoids was recorded in sterilized soil with no extract added (i.e. distilled water added instead of leaf extract solution). Plants growing under these conditions experienced no inhibition from the materials of other plants, and did not compete with soil microbes for nutrients, at least during the early stages of the experiment before microbes recolonized the sterilized soils. Recolonization was much faster by prokaryotes than by fungi, as measured by the contrast in soil respiration rates and soil ergosterol contents. Recolonization also varied between leaf extract treatments, with a negative correlation between microbial activity and plant growth; strongly growing plants were able to outcompete colonizing microbes, while poorly growing plants were further inhibited by colonizing and rapidly growing microbes.
All three leaf extracts significantly reduced growth of all three graminoid species. However, it is not clear that allelopathy alone was responsible for this effect. The results of this study indicate that competition between plants and microbes also played a major role. In particular, the components of the leaf extracts, especially labile carbon and (in the case of the Betula extract) phosphorus, appeared to stimulate the microbial community, increasing competitive pressure on the plants. Added nitrogen, for example, appears not to have much benefited the plants, as their roots were N-limited when grown in non-sterilized soil even though the leaf extracts included high concentrations of inorganic nitrogen.
The susceptibility of plants to this combined effect of allelopathy and microbial competition probably varies by species. Plant traits of particular importance are probably the root: shoot ratio, in which plants with more robust roots are less harmed, and the storage of nutrients such as nitrogen in plant roots, in which plants with a growth strategy favouring nutrient storage rather than immediate use are less harmed. Such traits appear to be widespread among the dominant plants of the Arctic tundra, including Cassiope tetragonal and probably other prostrate shrubs. Many of these plants may form associations with mycorrhizal fungi, which provide some protection against microbial competition.
This paper is directly relevant to the discussion section of my current-high-priority Pits & Probes manuscript. The patterns of soil respiration and microbial GHG activity under some of the lowland communities are consistent with successful competition against soil microbes by Cassiope tetragonal and possibly Salix arctica plant roots.
This paper was critiqued quite harshly by Wardle and Nilsson (1997).
These authors conducted an experiment to examine the potential allelopathic effects of plant leaf extracts among a few species of arctic tundra plants. Three arctic/subarctic plants suspected of releasing phytotoxic compounds upon their competitors including Cassiope tetragonal were harvested and their leaves and branches ground up to make leaf extracts. Three species of arctic graminoids (Carex bigelowii, Festuca vivipara, Luzula arcuata) were then treated with these extracts while growing in either sterilized or non-sterilized soil in greenhouses in southern Sweden. All of the graminoids, the soil they grew in, and two of the leaf extract-providing plants came from a montaine subarctic hillside in northern Sweden; the third leaf extract came from Betula pubescens ssp. tortuosa, a birch, were collected from individuals growing near the tree line at 450m altitude near Abisko Scientific Research Station, above the Arctic Circle in a subarctic ecosystem.
The experimental design was factorial, with two soil types (sterilized vs. non-sterilized) and four leaf-extract treatments including a control of distilled water. In addition to growth of the graminoids, measurements were made of the chemicals in the leaf extracts and soils, nutrient uptake by excised roots, soil ergosterol content, and soil respiration. Excised roots take up nutrients in a manner directly correlated to the nutrient-limitation status of the plant; more phosphorus-starved plants, for example, have roots that more rapidly take up phosphorus when offered. Soil ergosterol content is a measure of fungal biomass, while soil respiration was taken as a measure of total microbial activity.
Sterilized soil had higher extractable nutrients, probably as a result of the breakdown of microbial cells during autoclaving. Nitrate levels were negligible, both in soils and in leaf extracts; nitrate was a component of the dilute nutrient water used to maintain the plants while growing. Some mycorrhizae were found, but they covered less than 1% of the roots in non-sterilized soil, and had no impact on other measured parameters.
The highest growth of all three test graminoids was recorded in sterilized soil with no extract added (i.e. distilled water added instead of leaf extract solution). Plants growing under these conditions experienced no inhibition from the materials of other plants, and did not compete with soil microbes for nutrients, at least during the early stages of the experiment before microbes recolonized the sterilized soils. Recolonization was much faster by prokaryotes than by fungi, as measured by the contrast in soil respiration rates and soil ergosterol contents. Recolonization also varied between leaf extract treatments, with a negative correlation between microbial activity and plant growth; strongly growing plants were able to outcompete colonizing microbes, while poorly growing plants were further inhibited by colonizing and rapidly growing microbes.
All three leaf extracts significantly reduced growth of all three graminoid species. However, it is not clear that allelopathy alone was responsible for this effect. The results of this study indicate that competition between plants and microbes also played a major role. In particular, the components of the leaf extracts, especially labile carbon and (in the case of the Betula extract) phosphorus, appeared to stimulate the microbial community, increasing competitive pressure on the plants. Added nitrogen, for example, appears not to have much benefited the plants, as their roots were N-limited when grown in non-sterilized soil even though the leaf extracts included high concentrations of inorganic nitrogen.
The susceptibility of plants to this combined effect of allelopathy and microbial competition probably varies by species. Plant traits of particular importance are probably the root: shoot ratio, in which plants with more robust roots are less harmed, and the storage of nutrients such as nitrogen in plant roots, in which plants with a growth strategy favouring nutrient storage rather than immediate use are less harmed. Such traits appear to be widespread among the dominant plants of the Arctic tundra, including Cassiope tetragonal and probably other prostrate shrubs. Many of these plants may form associations with mycorrhizal fungi, which provide some protection against microbial competition.
This paper is directly relevant to the discussion section of my current-high-priority Pits & Probes manuscript. The patterns of soil respiration and microbial GHG activity under some of the lowland communities are consistent with successful competition against soil microbes by Cassiope tetragonal and possibly Salix arctica plant roots.
This paper was critiqued quite harshly by Wardle and Nilsson (1997).
Moffat et al. 1990
Moffat AJ, Johnston M, Wright JS. 1990. An improved probe for sampling soil atmospheres. Plant and Soil 121: 145-147.
These authors describe a new design of soil gas probe they produced to examine soil O2 levels, especially at landfills and other potentially polluted sites. The design is fairly complex, with moving parts. Essentially, the probe is driven into the soil to the desired depth, and then lifted slightly to create a void below the tip. The top of the probe is then rotated and tapped to open the tip, and a gas sample can be drawn from a tube on the side. These authors tested this probe by measuring O2 concentrations in a column of sand that had been flushed with pure N2; anoxic conditions were recorded in the deeper parts of this column.
These authors describe a new design of soil gas probe they produced to examine soil O2 levels, especially at landfills and other potentially polluted sites. The design is fairly complex, with moving parts. Essentially, the probe is driven into the soil to the desired depth, and then lifted slightly to create a void below the tip. The top of the probe is then rotated and tapped to open the tip, and a gas sample can be drawn from a tube on the side. These authors tested this probe by measuring O2 concentrations in a column of sand that had been flushed with pure N2; anoxic conditions were recorded in the deeper parts of this column.
Farías et al. 2009
Farías L, Fernández C, Faúndez J, Cornejo M, Alcaman ME. 2009. Chemolithoautotrophic production mediating the cycling of the greenhouse gases N2O and CH4 in an upwelling ecoystem. Biogeosciences 6: 3053-3069.
Nitrous oxide accumulates in these waters, indicative of greater production than consumption. These authors state that N2O reduction can only occur through a single identified pathway, that of total denitrification that takes NO3- or NO2- all the way to N2, and only under extremely limited O2 conditions; they cite Elkins et al. (1978) and Farías et al. (2009). Variation in space and time in dissolved N2O patterns do suggest some consumption is occurring, but in general production outweighs consumption.
“Chemolithoautotrophy is the non-photosynthetic biological conversion of C1 molecules (usually CO2 or CH4) into organic matter."These authors studied the chemolithoautrophic community in the south-eastern Pacific ocean, at a long-term research position on the small continental shelf of Chile. At this position, the water is about 90m deep, and is part of one of the largest and most productive upwelling regions of the world, where productivity is extremely high. For about 70% of the year, surface winds drive upwelling that brings nutrients such as NO3- up to the photic zone, allowing massive planktonic productivity. Some of these plankton are non-photosynthetic prokaryotic autotrophs, using inorganic molecules such as NH4+, NO2-, and HS- as electron donors to drive the energy-intensive process of fixing inorganic carbon, chiefly CO2, as organic matter. These organisms require oxygen as an electron acceptor, thus they are all aerobic organisms and do not thrive in anaerobic environments. Assimilation of CO2 in the dark is the diagnostic signal of the presence of these organisms. Some chemolithoautrophs use CH4 in addition to or instead of CO2, but always under aerobic conditions; anaerobic methanotrophs are outside the scope of this study, and have not been identified in this system.
Nitrous oxide accumulates in these waters, indicative of greater production than consumption. These authors state that N2O reduction can only occur through a single identified pathway, that of total denitrification that takes NO3- or NO2- all the way to N2, and only under extremely limited O2 conditions; they cite Elkins et al. (1978) and Farías et al. (2009). Variation in space and time in dissolved N2O patterns do suggest some consumption is occurring, but in general production outweighs consumption.
Kostina et al. 1994
Kostina NV, Stepanov AL, Umarov MM. 1994. Study of the complex of nitrous oxide-reducing microorganisms in the soil. Eurasian Soil Science 26: 81-87.
These authors extracted microorganisms from a range of soils apparently collected from various places in Russia and produced cultures of organisms capable of reducing nitrous oxide. The only nitrogen source in culture vials was N2O, and conditions were rendered anaerobic by flushing with argon gas.
Most cultures gradually lost their ability to reduce N2O, especially mixed-species cultures. A few pure strains were isolated that did not show this loss, and maintained high levels of activity in storage.
Pseudomonas spp. and Bacillus spp. contributed the vast majority of N2O-reducing activity in all soils, with other groups including Aeromonas spp., Micrococcus spp., Flavobacterium spp., Erwinia spp., and an organism identified as “similar to Corynebacterium” also showing some activity. Neither actinomycetes nor eukaryotes were found in any of the cultures capable of N2O-reduction, indicating this is a physiological process not possessed by these organisms.
This is an English translation of a paper that was probably originally in Russian: Pochvovedeniye 1993 25: 72-76.
These authors extracted microorganisms from a range of soils apparently collected from various places in Russia and produced cultures of organisms capable of reducing nitrous oxide. The only nitrogen source in culture vials was N2O, and conditions were rendered anaerobic by flushing with argon gas.
Most cultures gradually lost their ability to reduce N2O, especially mixed-species cultures. A few pure strains were isolated that did not show this loss, and maintained high levels of activity in storage.
Pseudomonas spp. and Bacillus spp. contributed the vast majority of N2O-reducing activity in all soils, with other groups including Aeromonas spp., Micrococcus spp., Flavobacterium spp., Erwinia spp., and an organism identified as “similar to Corynebacterium” also showing some activity. Neither actinomycetes nor eukaryotes were found in any of the cultures capable of N2O-reduction, indicating this is a physiological process not possessed by these organisms.
This is an English translation of a paper that was probably originally in Russian: Pochvovedeniye 1993 25: 72-76.
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