the importance of anammox and codenitrification in agricultural...
TRANSCRIPT
THE IMPORTANCE OF ANAMMOX AND CODENITRIFICATION IN AGRICULTURAL SOIL
Andrew M. Long
A Thesis Submitted to the University of North Carolina Wilmington in Partial Fulfillment
of the Requirements for the Degree of Master of Science
Department of Biology and Marine Biology
University of North Carolina Wilmington
2011
Approved by
Advisory Committee
Stuart Borrett Lawrence Cahoon
Bongkeun Song Chair
Accepted by
Dean, Graduate School
iii
TABLE OF CONTENTS
LIST OF TABLES...........................................................................................................................v
LIST OF FIGURES.......................................................................................................................vii
CHAPTER 1....................................................................................................................................1
ABSTRACT.....................................................................................................................................2
SOIL NITROGEN CYCLE.............................................................................................................3
Traditionally Studied N Pathways in Soil............................................................................4
Newly Described N Pathways in Soil..................................................................................6
Anammox Bacteria Characteristics..........................................................................7
Anammox Detection Methods.................................................................................9
Codenitrifier Characteristics..................................................................................11
Codenitrification Detection Methods.....................................................................12
Study Objectives................................................................................................................13
REFERENCES..............................................................................................................................14
CHAPTER 2..................................................................................................................................20
ABSTRACT...................................................................................................................................21
INTRODUCTION.........................................................................................................................23
MATERIALS AND METHODS...................................................................................................27
Sample Collection..............................................................................................................27
DNA extraction, PCR amplification, Cloning, and Sequencing........................................28
Quantitative PCR of hzo and nosZ genes and Fusarium oxysporum.................................29
15N-Tracer Incubation Experiments...................................................................................30
Statistical Analysis.............................................................................................................31
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RESULTS......................................................................................................................................33
Physical and Chemical Properties of Soils Collected from Agricultural Fields................33
Detection and Phylogeny of Anammox Bacteria in Agricultural Soils.............................33
Abundance of anammox and denitrifying bacteria and fungal codenitrifiers....................37
29N2 and 30N2 production from 15N tracer incubation experiments....................................38
DISCUSSION................................................................................................................................43
Diversity of Anammox Bacteria in Agricultural Soils......................................................43
Quantification of N2 producing microorganisms..............................................................44
Detection and N2 production from 15N-labeled incubation experiments...........................45
REFERENCES..............................................................................................................................48
CHAPTER 3..................................................................................................................................53
ABSTRACT...................................................................................................................................54
INTRODUCTION.........................................................................................................................56
MATERIALS AND METHODS...................................................................................................59
Sample Collection..............................................................................................................59
DNA extraction and Quantitative PCR of hzo and nosZ genes and F.oxysporum............60
15N-Tracer Incubation Experiments...................................................................................61
Statistical Analysis.............................................................................................................62
RESULTS......................................................................................................................................64
Physical and Chemical Properties of Soils Collected from Agricultural Fields................64
Abundance of anammox and denitrifying bacteria and fungal codenitrifers.....................64
29N2 and 30N2 production from 15N tracer incubation experiments....................................67
DISCUSSION................................................................................................................................74
v
REFERENCES..............................................................................................................................76
vi
LIST OF TABLES
Table Page
1. Physical and Chemical Characteristics of Samples Soils Chapter 2............................................................................................................................37 2. Molecular detection of anammox bacteria and N2 production rates from soil slurry incubations....................................................................39
3. Abundance of Denitrifiers, Anammox Bacteria, and F.oxysporum based in Agricultural Soils...........................................................................42 4. Physical and Chemical Characteristics of Samples Soils Chapter 3............................................................................................................................68 5. Abundance of Denitrifiers, Anammox bacteria, and F.oxysporum in Beaufort, NC..................................................................................................................69 6. Abundance of Denitrifiers, Anammox bacteria, and F.oxysporum in Currituck, NC.................................................................................................................70 7. N2 Production in Beaufort, NC
Chapter 3............................................................................................................................72 8. N2 Production in Currituck, NC
Chapter 3............................................................................................................................73
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LIST OF FIGURES Figure Page
1. Chemical and microbial processes in the soil nitrogen cycle......................................................................................................8
2. Phylogenetic tree of planctomycete 16S rRNA genes detected in North Carolina soil samples............................................................................38
3. Phylogenetic tree of translated HZO sequences detected from agricultural soils........................................................................40 4. PCoA Plot of HZO sequences found in agricultural soils.................................................................................................................41
5. 29N2 and 30N2 production from 15NO3
- incubation experiments......................................................................................................43
6. PCA Plot Comparing 29N2 and 30N2 production rates, microbial abundance and soil characteristics.....................................................................45
7. Sampling Locations in Beaufort and Currituck Transects in North Carolina...............................................................................................64
9. Abundance of Anammox and Denitrifying bacteria, and the Codenitrifying fungus, F.oxysporum, across the Beaufort and Currituck transects...............................................71
11. N2 Production Rates from 15NO3
- Incubations across the Beaufort and Currituck transects.......................................................................................74 12. PCA plot comparing the N2 production, the abundance of N-removing organisms, and the physical and chemical parameters of the agricultural field in Beaufort, NC................................................................................76
13. PCA plot comparing the N2 production, the abundance of N-removing organisms, and the physical and chemical parameters of the agricultural field in Currituck, NC...............................................................................77
CHAPTER 1
The Changing of the Guard: Traditionally Studied vs. Newly Described Pathways in the Soil
Microbial Nitrogen Cycle
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ABSTRACT
Nitrogen is an essential macronutrient for life. However, high loads of nitrogen fertilizers in
agricultural fields cause serious environmental problems and alter various ecosystems. Since
microorganisms play a vital role in nitrogen transformation, it becomes increasingly important to
understand the processes involved in soil nitrogen cycle. Previous studies have revealed the
importance of nitrification, denitrification, nitrogen fixation and dissimilatory nitrate reduction to
ammonia (DNRA) in agricultural N-cycling. Nitrification has been described in both bacteria
and archaea. Studies have revealed that while archaeal nitrifiers are more abundant in
agricultural soils, bacterial nitrifiers may be more active in the nitrification pathway. DNRA was
shown to be higher than denitrification in some soils. Nitrogen fixation is carried out by
cyanobacteria in biological crusts in arid soils, free-living azotobacter and rhizobia associated
with root nodules in agricultural soils. Denitrification has been revealed as a widespread N-
removing pathway in soils. Two nitrogen removal pathways have been recently described in
soils. Anaerobic ammonium oxidation (anammox) and codenitrification convert fixed N to N2
gas. Anammox bacteria belong to the phylum Planctomycetes and have a number of unique
cellular features such as the membrane bound organelle-like structure called the anammoxosome
and ladderane lipids. Anammox bacteria may be detected through the use of molecular markers
and 15N stable isotope labeled substrate incubations. Although bacteria and fungi mediate
codenitrification, fungal codenitrification has been shown to be the dominant N removal pathway
in some soils. Codenitrification produces N2O and N2 from the reduction of NO2- by a co-
substrate, which can be NH4+, azide, hydroxylamine, or salicylhydroxamic acid. New methods to
detect and quantify fungal codenitrification should be developed for a better understanding of the
soil nitrogen cycle.
3
SOIL NITROGEN CYCLE
Nitrogen, a group 15 element, has the ability to exist in 5 oxidation states. Although some
states are more stable than others, all states can exist in nature. This occurs due to the property of
nitrogen that allows its oxidation state to be correlated to kinetics rather than thermodynamic
equilibria (Jetten et al., 2009). Different nitrogen species are used by organisms to generate
cellular energy by serving as an electron donor or acceptor in metabolic pathways. Nitrogen is
assimilated as a macronutrient, which is evident in the reduced chemical formula of an organism,
CH2O0.5N0.15.
Soil nitrogen cycle
The soil nitrogen cycle is a complex, multi-processed system that while heavily studied, is
not completely understood (Figure 1). The effects of anthropogenic influences on the nitrogen
cycle are paramount to gaining an understanding of its importance. These effects are wide
reaching in the biosphere but most pervasive in agricultural systems. As described by Vitousek et
al. (1997), the application of inorganic nitrogen fertilizers has engendered widespread changes in
the global nitrogen cycle. Fertilizer inputs have doubled the rate of nitrogen uptake in terrestrial
environments and increased the activity of denitrifiers, which in turn increases the amount of the
greenhouse gas N2O in the atmosphere. Inorganic nitrogen fertilizers have also contributed to the
acidification of soils, streams, rivers, and some lakes, as well as increased the rate of nitrogen
transfer from rivers to estuaries and oceans. The effects of inorganic nitrogen fertilizers have
worked in concert with myriad other factors in decreasing biodiversity (Vitousek et al., 1997).
4
Figure 1. Chemical and microbial processes in the soil nitrogen cycle
The excess nitrogen in agricultural fields due to over-fertilization has a number of fates,
which include but are not limited to: plant uptake, leaching, and being fed into the microbial
nitrogen cycle. The microbial nitrogen cycle can be attributed to myriad well-known processes
including nitrification, DNRA, ammonium uptake, assimilatory nitrate reduction, nitrogen
fixation, and denitrification, as well as newly described pathways such as codenitrification, and
anammox (Figure 1).
Traditionally studied N pathways in soils
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Nitrification is the stepwise aerobic oxidation of ammonium (NH4+) to nitrite (NO2
-) and nitrate
(NO3-). The presence of an ammonium monooxygenase (Amo), which catalyzes the oxidation of
NH4+, has been described in both bacteria (McTavish et al., 1993) and archaea (Venter et al.,
2004). Bacterial nitrification is carried out by two distinct chemolithoautotrophic groups of
bacteria. The first group of bacteria (AOB) oxidizes NH4+, while the second group of bacteria
(NOB) oxidizes NO2-. According to 16S rRNA gene phylogenies, AOB belong to three genera,
Nitrosomonas, Nitrosospira, and Nitrococcus (Head et al., 1993). Four genera of NOB have been
described, Nitrobacter, Nitrospina, Nitrococcus and Nitrospira (Teske et al., 1994). Ammonia
oxidizing archaea (AOA) are less diverse than ammonia oxidizing bacteria (AOB), and have
only been described in Crenarchaeota (Konneke et al., 2005). Using quantitative PCR, AOA
have been shown to be more abundant than AOB in soil (Leininger et al., 2006). However, using
DNA stable isotope probing (DNA-SIP), there is evidence that while AOA are numerically
dominant, AOB are functionally dominant in agricultural soil (Jia and Conrad, 2009).
Both DNRA and assimilatory nitrate reduction generate NH4+ as an end product. DNRA
differs from assimilatory nitrate reduction in that it requires reduced conditions with an electron
donor and does not have an organic nitrogen intermediate (Gardner et al., 2006). Ammonium
uptake by plants and other organisms ties up available NH4+ in biomass, which may then be
recycled through decomposition. DNRA can occur in bacteria (Chang and Morris, 1962),
protozoa (Hadas et al., 1992), and fungus (Zhou et al., 2002). In some soils, such as upland
tropical forest soils, the rate of DNRA can exceed the rates of nitrification and denitrification,
which causes an increase of NH4+, which can then be fed into other processes in the nitrogen
cycle, such as nitrification (Silver et al., 2001).
Nitrogen fixation produces bio-available nitrogen from N2 gas. Nitrogen fixation in soils is
6
mediated through free-living bacteria and plant endosymbionts. These discoveries, pioneered by
Martinus Beijerinck and Sergei Winogradsky near the turn of the twentieth century, led to further
works in nitrogen fixation. Free-living nitrogen fixers in soils include cyanobacteria, which can
grow in biological crusts in arid soils (Harper and Marble, 1988) and azotobacter (Beijerinck,
1901). However, some azotobacter can form symbiotic relationships with plants (Kass et al.,
1971). Rhizobia form root nodules after a signaling cascade with legumes (Verma et al., 1992).
Nitrogen fixation in agricultural soils is a heavily studied field that has been primarily focused on
symbiotic nitrogen fixation in legumes. Legume yield can be used to estimate nitrogen fixation at
the field level. Using this approach, Yang et al. (2010), calculated that up to 300 kg N ha-1 per
year might be attributed to biological N-fixation associated with legumes in agricultural soils, but
much less on average.
As more inorganic nitrogen is added to agricultural fields, the processes by which nitrogen
is removed become a clear focus. Three of these processes, denitrification, anammox, and
codenitrification, are involved in the removal of nitrogen from the ecosystem by producing
dinitrogen gas (N2). Denitrification is the anaerobic process in which nitrate (NO3-) is converted
to N2O and N2. It is a widespread process that occurs in various terrestrial and aquatic
ecosystems where nitrate and carbon are available and oxygen is scarce (Davidson and
Seitzinger, 2006).
Newly described N pathways in soils
Anammox, a more recently described process in the nitrogen cycle, produces N2 through
anaerobic oxidization of NH4+ by NO2
- reduction (Mulder et al., 1995; Van de Graaf et al.,
1995). Codenitrification, which is mediated by a common soil fungus (Fusarium oxysporum) and
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some bacteria (e.g. Streptomyces antibioticus), produces N2 and N2O through the reduction of
NO2- coupled to unknown metabolic conversions of some other nitrogen compound (azide,
ammonium, salicylhydroxamic acid, or hydroxylamine (Tanimoto et al., 1992; Kumon et al.,
2002; Spott and Strange, 2011). Codenitrification may even dominate the removal of nitrogen in
some soils, such as grassland soils (Laughlin and Stevens, 2002).
Anammox bacteria characteristics
Bacteria that belong to the family Brocadiaceae in the phylum Planctomycetes mediate the
anammox reaction. Five candidate genera have been described from enrichment cultures:
Candidatus “Brocadia” (Strous et al., 1999), Candidatus “Kuenenia” (Schmid et al., 2000),
Candidatus “Scalindua” (Schmid et al., 2003), Candidatus “Anammoxoglobus” (Kartal et al.,
2007), and Candidatus “Jettenia” (Quan et al., 2008). Anammox bacteria have several unique
physiological features including a unique cell plan, cell division proteins and lipids. Using an
electron microscope, it was observed that anammox bacteria have a unique intracellular structure
coined the “anammoxosome.” This compartment takes up most of the cell by volume. However,
this cell plan is not exclusive to anammox bacteria and is similar to other members of the phylum
Planctomycetes, to which anammox bacteria belong (Kartal et al., 2008). Members of this
phylum contain intracellular membranes and complex compartmentalization in comparison to a
typical bacterium (Fuerst, 2005).
Typically, planctomycetes have two to three cellular compartments; the outer most is
called the “paryphoplasm,” which is analogous to the periplasm in Gram-negative bacteria. They
differ in that the periplasm is outside the cytoplasmic membrane and the paryphoplasm is inside
the cytoplasmic membrane (Lindsay et al., 2001). In most planctomycetes, the innermost cellular
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compartment is the “riboplasm.” This compartment contains the nucleoid and the ribosomes and
is therefore the center of DNA replication, translation, and transcription (Strous et al. 1999;
Lindsay et al. 2001). In the case of anammox bacteria, the third compartment is the
anammoxosome, which is the proposed locus of the anammox reaction (van Niftrik et al. 2008a;
2008b).
Anammox bacteria contain lipids in their anammoxosome membrane that are unique in
nature. These “ladderane” lipids consist of hydrocarbon chains with linearly concatenated
cyclobutane rings with the ladderanes in a cyclohexane ring. These ladderane lipids contain both
ester-linkages and ether-linkages, which breaks from the common wisdom that ether-linkages are
exclusive to archaea while ester-linkages are exclusive to bacteria and eukaryotes (Sinninghe
Damsté et al., 2002). Based on molecular modeling, Sinninghe Damsté et al. (2002) described
tightly-pack ladderane lipids in the anammoxosome. This unusually high density prevents this
membrane from being permeable to apolar compounds. Since the metabolism of anammox
bacteria involves gaseous molecules and the toxic intermediate hydrazine, this tightly packed
membrane may allow the anammoxosome to retain these substrates (Jetten et al., 2009).
Anammox bacteria have a very slow cell cycle, with a division time of once every 11-20
days as compared to that of Escherichia coli, which divides on the order of minutes (Strous et al.,
1999). The organisms are obligate anaerobes that cannot tolerate oxygen conditions above 2 µM
(Strous et al., 1999). These two characteristics make it quite difficult to culture the organisms,
but it is only possible to obtain an enrichment culture of the microbes with the sequencing batch
reactor (SBR) method (Strous et al., 1999). Typically, anammox bacteria derive their energy
from the 1:1 conversion of ammonia and nitrite to dinitrogen gas. This is a thermodynamically
favorable equation and is more favorable than aerobic ammonium oxidation (Strous et al., 1999).
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Anammox bacteria have a very high affinity for their substrates, and can grow at concentrations
of less than 5µM (Strous et al., 1999). The metabolic activity is quite low in comparison and may
be the reason for the slow growth rate (Jetten et al., 2009).
The unique metabolism of anammox bacteria has an overall reaction of ammonium being
oxidized by nitrite to form water and dinitrogen gas (Strous et al. 1999). The free energy of this
reaction is -357 kJ/mol (Strous et al., 1999). Genome analysis in Ca. “Kuenenia stuttgartiensis”
showed several genes encoding for proteins might be involved in the metabolic processes in
anammox bacteria (Jetten et al., 2009). These genes include: a cd1 type nitric oxide: nitrite
oxidoreductase (nirS), and nine paralogues of hydroxylamine/hydrazine oxidoreductase
(hao/hzo) (Hooper et al., 1997). One reaction has yet to be associated with a particular gene, the
step is the combination of ammonium and nitric oxide to form hydrazine. A “hydrazine
hydrolase” is proposed to be the enzyme that combines ammonium with nitric oxide to form
hydrazine (Jetten et al., 2009). The proposed mechanism for the anammox reaction starts with
nitrite being reduced nitric oxide through NirS. This nitric oxide molecule is then combined with
ammonium by hydrazine hydrolase to form hydrazine. The next step is catalyzed by HZO and
converts hydrazine to dinitrogen gas, which also produces four electrons and four protons and
creates the proton motive force across the anammoxosome membrane (Jetten et al., 2009).
Anammox detection methods
Anammox has been detected using anammox 16S rRNA genes and 15N stable isotope
incubation techniques in a number of environments including marine sediments (Dalsgaard and
Thamdrup, 2002; Hietanen and Kuparinen, 2008; Rich et al., 2008), oxygen-minimum zones
(Dalsgaard et al., 2003; Kuypers et al., 2003; 2005; Stevens and Ulloa, 2008), freshwater
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marshes (Penton et al., 2006; Humbert et al., 2010), rivers (Zhang et al., 2007), meromictic lakes
(Schubert et al., 2006), river estuaries (Risgaard-Peterson et al., 2004; Trimmer et al., 2003; Dale
et al., 2009), permafrost (Penton et al., 2006). Anammox bacteria have also been found in
various soil types, which include permafrost soils (Penton et al, 2006), reductisol, agricultural
soils (Humbert et al, 2010), peat soils (Hu et al, 2011), and rice paddy soils (Zhu et al, in press).
However, estimating the importance of anammox in soil N cycling is somewhat complicated due
to codenitrification, which can use the same substrates as anammox.
The use of 16S rRNA genes to detect and identify anammox bacteria in environmental
samples is not without inherent flaws. Low diversity of anammox bacteria in marine ecosystems
has been suggested to be an artifact of biases within the coverage and specificity of the existing
anammox 16S rRNA gene primer sets (Amano et al., 2007; Dale et al., 2009; Li et al., 2010).
New 16S rRNA gene primers and PCR protocols have been used to detect higher diversity of
anammox in coastal marine sediment (Amano et al., 2007), estuaries (Dale et al., 2009) and
various soil types (Humbert et al., 2010). However, the existing anammox 16S rRNA gene
primer sets have been shown to amplify 16S rRNA genes belonging to unidentified
planctomycetes in soil (Humbert et al., 2010).
While the initial detection of anammox bacteria primarily based on the 16S rRNA genes,
the functional genes encoding dissimilatory nitrite reductase (nirS) and hydrazine oxidize (hzo)
were recently used as alternative genetic markers for the detection of anammox bacteria in the
environment (Lam et al., 2009; Schmid et al., 2008). Hydrazine oxidize (HZO) is an octahaem
cytochrome c protein that was proposed for the oxidation of hydrazine (N2H4) to N2 (Schalk et
al., 1998; Strous et al., 2006; Shimamura et al., 2007). Multiple homologous sequences of hzo
genes were found in the genome of “Ca. K. stuttgartiensis” (Strous et al., 2006). Based on
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phylogenetic analysis, the hzo gene sequences were branched into three distinct clusters (Schmid
et al., 2008). The genes assigned to cluster 1 were found in all of the known candidate genera
and two that have been verified to be active (Schmid et al., 2008; Shimamura et al., 2007). The
hzo genes in cluster 1 have been used as genetic marker to detect anammox bacteria in several
ecosystems. Li et al., (2010) found that primers for hzo genes may provide better coverage of
anammox bacteria genera than primers for anammox 16S rRNA genes. The existing hzo primers
have been shown to produce phylogenies comparable to those of phylogenies based on anammox
16S rRNA genes in high-temperature petroleum reservoirs (Li et al., 2010), river sediments
(Hirsch et al., 2011), marine sediments (Dang et al., 2010; Li et al., 2010; Hirsch et al., 2011),
oxygen minimum zones (Schmid et al., 2008), and bioreactors (Schmid et al., 2008). However,
soil anammox communities have not been examined using this genetic approach although hzo
gene can be more specific and selective genetic marker for anammox bacteria (Hirsch et al.,
2011). In addition, there has yet to be confirmation of the presence and importance of anammox
in agricultural soil N cycling.
The contribution of anammox to the total N2 production (%anammox) can be calculated
by measuring the rates of both anammox and denitrification using 15N tracer incubation
techniques (Thamdrup and Dalsgaard, 2002). The %anammox has been shown to vary across
aquatic environments, from being functionally absent to the dominant pathway with up to 79%
of the N removed by anammox (Engstrom et al, 2005). However, estimating %anammox in soils
may become difficult because anammox and codenitrification can simultaneously generate 29N2
from the addition of 14NH4+ and 15NO3
-/15NO2- in 15N tracer incubation experiments.
Codenitrifier characteristics
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While codenitrification has been shown to occur in bacteria, the process has been studied
more thoroughly in fungi. Codenitrification has been described in three members of the order
Hypocreales: F. oxysporum (Tanimoto et al., 1992), Fusarium solani (Shoun et al., 1992), and
Cylindrocarpon tonkinense (Shoun et al., 1992). F. oxysporum is a filamentous, spore-forming
fungus that has shown the ability to exist as a saprophyte in aerobic soils and can degrade lignin
and other complex carbohydrates (Rodriguez et al., 1996). F. oxysporum is ubiquitous in soils,
having been found from grassland soils and tropical forests to desert soils and tundra (Stoner,
1981).
The fungal codenitrification pathway has yet to be fully characterized. A nitric oxide reductase,
cytochrome p450nor, has been shown to produce N2O from NO with a co-substrate (azide or
NH4+) (Su et al., 2004). This enzyme has also been shown to catalyze the formation of N2O in
the fungal denitrification pathway. In the fungal denitrification pathway, a copper-containing
nitrite reductase (nirK) associated with the mitochondrion may be involved (Kim et al., 2010).
The likely pathway of codenitrification therefore proceeds through the reduction of NO2- to NO,
catalyzed by a fungal nirK, followed by the conversion of NO and a co-substrate to N2O. N2O
may be the end product or a nitrous oxide reductase may convert the N2O to N2.
Codenitrification detection methods
Studies have reported potential rates of codenitrification assessed by using 15N tracer
incubation experiments with various combinations of labeled codenitrification substrates
(Tanimoto et al., 1992; Laughlin and Stevens, 2002; Spott and Strange, 2011). Under
denitrifying conditions with 15N labeled substrates, codenitrification will produce 45N2O and
29N2. Using these methods, codenitrification has been shown to occur in pure cultures of
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F.oxysporum (Tanimoto et al., 1992) and S. antibioticus (Kumon et al., 2002) as well as in
grassland soils (Laughlin and Stevens, 2002) and agricultural soils (Spott and Strange, 2011).
Laughlin and Stevens further resolved the fungal contribution towards the nitrogen gas flux with
the use of fungicides (2002). To detect and quantify fungal codenitrifiers in agricultural soils, a
quantitative PCR approach may be taken. While no functional gene markers for codenitrification
exist, F. oxysporum, a fungal codenitrifier, has been detected in soils using PCR of the ITS
region in rRNA genes (Mishra et al., 2003). Using these same primers targeting the highly
conserved ITS region, quantitative PCR may be used to quantify F. oxysporum in soils (Jimenez-
Fernandez et al., 2010).
Study Objectives
Anammox bacteria and codenitrifiers have been detected in soils (Penton et al., 2006;
Humbert et al., 2010; Laughlin and Stevens, 2002; Spott and Strange, 2011) and in the case of
codenitrification, significant activity has been ascribed to the process (Laughlin and Stevens,
2002). Agricultural soils are rich in inorganic nitrogen species, which are utilized by anammox
and codenitrification. This study had three objectives. The first was to assess which of the three
N-removal pathways was dominant in agricultural soils. The second was to assess the
contribution of anammox and codenitrification towards the production of N2 in agricultural soils,
and the third objective was to assess what geochemical and microbial features influence the
removal of N from agricultural soils.
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Silver WL, Herman DJ, Firestone MK (2001). Dissimilatory nitrate reduction to ammonium in upland tropical forest soils. Ecol 82(9):2410-2416 Sinninghe Damsté JS, Strous M, Rijpstra WIC, Hopmans EC, Geenevasen JAJ, van Duin ACT, van Niftrik LA and Jetten MSM (2002). Linearly concatenated cyclobutane lipids form a dense bacterial membrane. Nature 419:708–712. Spott O, Strange CF (2011). Formation of hybrid N2O in a suspended soil due to co-denitrification of NH2OH. J Plant Nutr Soil Sci 000: 1-14. Stevens H, Ulloa O (2008). Bacterial diversity in the oxygen minimum zone of the eastern tropical South Pacific. Environ Microbiol 10: 1244-1259. Stoner MF (1981). Ecology of Fusarium in noncultivated soils. Pages 276-286 in: Fusarium: Diseases, Biology, and Taxonomy. P.E. Nelson, T.A. Toussoun and R.J. Cook, eds. The Pennsylvania State University Press, University Park. Strous M, Fuerst JA, Kramer EHM, Logemann S, Muyzer G, van de Pas-Schoonen KT et al (1999). Missing lithotroph identified as new planctomycete. Nature 400: 446-449. Strous M, Pelletier E, Mangenot S, Rattei T, Lehner A, Taylor MW et al (2006). Deciphering the evolution and metabolism of an anammox bacterium from a community genome. Nature 440: 790-794. Tanimoto T, Hatano K, Kim DH, Uchiyama H, Shoun H (1992). Co-denitrification by the denitrifying system of the fungus Fusarium-oxysporm. FEMS Microbiol Lett 93: 177-180. Teske A, Alm E, Regan JM, Toze S, Rittmann BE, Stahl DA (1994). Evolutionary relationships among ammonia- and nitrite-oxiziding bacteria. J Bacteriol 176: 6623-6630. Thamdrup B, Dalsgaard T (2002). Production of N-2 through anaerobic ammonium oxidation coupled to nitrate reduction in marine sediments. Appl Environ Microbiol 68: 1312-1318. Trimmer M, Nicholls JC, Deflandre B (2003). Anaerobic ammonium oxidation measured in sediments along the Thames estuary, United Kingdom. Appl Environ Microbiol 69: 6447-6454. Vandegraaf AA, Mulder A, Debruijn P, Jetten MSM, Robertson LA, Kuenen JG (1995). Anaerobic oxidation of ammonium is a biologically mediated process. Appl Environ Microb 61: 1246-1251. Van Niftrik L, Geerts WJC, van Donselaar EG, Humbel BM, Webb RI, Fuerst JA, Verkleij AJ, Jetten MSM and Strous M (2008a). Linking ultrastructure and function in four genera of anaerobic ammonium-oxidizing bacteria: Cell plan, glycogen storage, and localization of cytochrome c proteins. J Bacteriol 190:708–717.
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Van Niftrik L, Geerts WJC, van Donselaar EG, Humbel BM, Yakushevska A, Verkleij AJ, Jetten MSM and Strous M (2008b). Combined structural and chemical analysis of the anammoxosome: A membrane-bounded intracytoplasmic compartment in anammox bacteria. J Struct Biol 161:401–410. Venter JC, Remington K, Heidelberg et al. (2004). Environmental genome shotgun sequencing of the sargasso sea. Science 304:66-74. Verma DPS, Hu CA, Zhang M (1992). Root Nodule Development - origin, function and regulation of nodulin genes. Physiologia Plantarum 85(2):253-265. Vitousek PM, Aber JD, Howarth RW, Likens GE, Matson PA, Schindler DW et al (1997). Human alteration of the global nitrogen cycle: Sources and consequences. Ecol Appl 7: 737-750. Yang JY, Drury CF, Yang XM, et al. (2010). Estimating biological N-2 fixation in Canadian agricultural land using legume yields. Agriculture Ecosys Environ 137(1-2):192-201. Zhang Y, Ruan XH, den Camp H, Smits TJM, Jetten MSM, Schmid MC (2007). Diversity and abundance of aerobic and anaerobic ammonium-oxidizing bacteria in freshwater sediments of the Xinyi River (China). Environ Microbiol 9: 2375-238. Zhou ZM, Takaya N, Nakamura A, Yamaguchi M, Takeo K, Shoun H (2002). Ammonia fermentation, a novel anoxic metabolism of nitrate by fungi J Biol Chem 277(3):1892-1986 Pages: 1892-1896 Zhu G, Wang S, Wang Y, Wang C, Risgaard-Petersen N, Jetten MSM, and Yin C (in press). Anaerobic ammonia oxidation in a fertilized paddy soil. ISME J (19 May 2011) doi:10.1038/ismej.2011.63
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CHAPTER 2
Co-occurring Anammox, Denitrification and Fungal Codenitrification in Agricultural Soils
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ABSTRACT
Codenitrification and anammox are two processes that have yet to be fully explored in
agricultural soils. The contribution of these two processes to the flux of N2 gas may prove to be
significant in global nitrogen cycle. In order to assess the contribution of codenitrification and
anammox to N2 production, molecular and stable isotope analyses were conducted with soil
samples collected from eight different agricultural fields in the US. The abundance of
denitrifying and anammox bacteria was calculated based on quantitative PCR of nitrous oxide
reductase (nosZ) and hydrazine oxidase (hzo) genes, respectively, while the internal transcribed
spacer (ITS) of Fusarium oxysporum, codenitrifying fungus, was quantified to estimate
codenitrifier abundance. 15N tracer incubation experiments (15NO3- or 15NH4
+) were used to
estimate the rates of 29N2 production from anammox and codenitrification and 30N2 from
denitrification. The abundance of nosZ and hzo genes in soils ranged from 3.21 x 106 to 7.88 x
106 copies g-1, and 4.99 x 103 to 1.57 x 104 copies g-1, respectively. The ITS of F. oxysporum was
found to range from 0 to 3.12 x 105 copies g-1. The potential denitrification rates ranged from
8.38 to 84.24 nmoles N2g-1d-1. The combined rates of anammox and codenitrification ranged
from 5.592 to 295.4 nmoles N2g-1d-1, while the potential anammox rates ranged from 0.013 to
0.484 nmoles N2g-1d-1. The percent of N2 produced as 29N2 in the stable isotope incubations
ranged from 32-77.9%. This suggests that anammox and codenitrification may be key processes
in agricultural N cycling. In addition, a coupling between codenitrification and denitrification
was proposed an alternative linked pathway of N2O production and consumption in agricultural
soils based on correlation analysis of Q-PCR and 15N tracer incubation experiments. Further
study with new method development is necessary to estimate differential contribution of
anammox, codenitrification and denitrification in total N2 and N2O production in agricultural
22
soils.
23
INTRODUCTION
The soil nitrogen cycle is a complex, multi-processed system that while heavily studied,
is not completely understood. Anthropogenic influences on the nitrogen cycle are increasingly
importance in gaining a more complete view of its processes. As described by Vitousek et al.,
(1997) the application of inorganic nitrogen fertilizers has engendered widespread changes in the
global nitrogen cycle. Fertilizer inputs have doubled the rate of nitrogen uptake in terrestrial
environments and increased the activity of denitrifiers, which in turn has increased the amount of
nitrous oxide (N2O), a greenhouse gas, in the atmosphere. Inorganic nitrogen fertilizers have
also contributed to the acidification of soils, as well as increased the rate of nitrogen transfer
from streams and rivers to estuaries and oceans (Vitousek et al., 1997). These anthropogenic
effects are wide reaching in the biosphere but perhaps most pervasive in agricultural systems.
The excess nitrogen in agricultural fields has a number of fates, which include but are not
limited to: plant uptake, leaching, and processing by the microbial nitrogen cycle. The microbial
nitrogen cycle can be attributed to a myriad of processes, which include ammonification,
nitrification, dissimilatory nitrate reduction to ammonium (DNRA), ammonium uptake,
assimilatory nitrate reduction, nitrogen fixation, denitrification, codenitrification, and anaerobic
ammonium oxidation (anammox). As more inorganic nitrogen is added to agricultural fields, the
clear quantification of the processes that remove it becomes increasingly important.
Three processes, denitrification, codenitrification, and anammox, are involved in the
removal of nitrogen from soils through the production of N2O or dinitrogen gas (N2).
Denitrification is an anaerobic process in which nitrate (NO3-) is converted to N2O and N2. It is a
24
widespread process that occurs in various terrestrial and aquatic ecosystems where nitrate and
labile carbon are available and oxygen is scarce (Davidson and Seitzinger, 2006).
Codenitrification, a process that is yet to be fully characterized, produces N2 and N2O
through the reduction of nitrite (NO2-) coupled to other nitrogen compounds including azide,
ammonium (NH4+), salicylhydroxamic acid, and hydroxylamine (Tanimoto et al., 1992; Spott
and Strange, 2011). Codenitrification can occur in both fungi (e.g. Fusarium oxysporum) and
bacteria (e.g. Streptomyces antibioticus) (Tanimoto et al., 1992; Kumon et al., 2002). 15N tracer
incubation methods have been used to measure codenitrification in grassland and agricultural
soils. (Laughlin and Stevens, 2002; Spott and Strange, 2011). Codenitrification was estimated to
contribute up to 92% of the N2 produced in the grassland soils (Laughlin and Stevens, 2002).
Anammox, a more recently described process in the nitrogen cycle, produces N2 through
anaerobic oxidization of NH4+ by NO2
- reduction (Mulder et al., 1995; Van de Graaf et al.,
1995). Anammox has been detected in a number of aquatic ecosystems, which include marine
sediments (Dalsgaard and Thamdrup, 2002; Hietanen and Kuparinen, 2008; Rich et al., 2008),
oxygen-minimum zones (Dalsgaard et al., 2003; Kuypers et al., 2003; 2005; Stevens and Ulloa,
2008), freshwater marshes (Penton et al., 2006; Humbert et al., 2010), rivers (Zhang et al., 2007),
meromictic lakes (Schubert et al., 2006), and river estuaries (Risgaard-Peterson, et al. 2004;
Trimmer et al. 2003; Dale et al., 2009). Anammox bacteria have also been found in various soil
types, which include permafrost soils (Penton et al., 2006), reductisol, agricultural soils
(Humbert et al., 2010), peat soils (Hu et al., 2011), and rice paddy soils (Zhu et al., in press).
However, estimating the importance of anammox in soil N cycling is somewhat complicated due
to codenitrification, which can use the same substrates as anammox.
25
The contribution of anammox to the total N2 production (%anammox) can be calculated
by measuring the rates of both anammox and denitrification using 15N tracer incubation
techniques (Thamdrup and Dalsgaard, 2002). The %anammox has been shown to vary across
aquatic environments, being the dominant pathway up to 79% (Engstrom et al., 2005). However,
estimating %anammox in soils may become difficult because anammox and codenitrification can
simultaneously generate 29N2 from the addition of 14NH4+ and 15NO3
-/15NO2- in 15N tracer
incubation experiments. The contribution of anammox and codenitrification in N2 production can
be presented as a percent rate of 29N2 (% 29N2) in the total N2 production by anammox,
codenitrification and denitrification. Alternatively, molecular methods quantifying anammox
bacteria, denitrifiers, and codenitrifiers can be used to estimate the genetic potential of three
different N2 producing microbes in soil ecosystems.
Molecular detection and quantification of anammox bacteria was primarily based on 16S
rRNA genes. The functional genes encoding dissimilatory nitrite reductase (nirS) and hydrazine
oxidase (hzo) have recently been used as alternative genetic markers for anammox bacterial
detection and quantification in the environment (Lam et al., 2009; Schmid et al., 2008; Dang et
al., 2011; Hirsh et al., 2011; Li et al., 2011). The gene encoding nitrous oxide reductase (nosZ)
has been used to quantify N2 producing denitrifying bacteria in soils and sediments (Henry et al.,
2006; Dandie et al., 2008; Chon et al., 2011). A functional genetic marker for codenitrification
has not been developed yet. Alternatively, ribosomal RNA genes for codenitrifying organisms
can be targeted for molecular quantification in environmental samples. F.oxysporum is the most
studied microorganism that carries out codenitrification (Tanimoto et al., 1992; Su et al., 2004).
Detection and quantification of F. oxysporum was demonstrated in inoculated soil samples
26
(Jimenez-Fernandez et al., 2010) by targeting the internal transcribed spacer (ITS) region of F.
oxysporum rRNA genes.
By combining molecular quantification and 15N tracer incubation techniques, the
significance of the three different N2 producing pathways can be assessed in soils. We
hypothesize that the microbes responsible for anammox, codenitrification, and denitrification
will be present in agricultural soil, that denitrifiers and codenitrifying fungi will be more
abundant in agricultural soils sue to their ability to compete aerobically, and that although
denitrification may be the dominant N-removing pathway, anammox and codenitrification will
contribute to the removal of N in agricultural soils. In order to test the proposed hypotheses, we
examined anammox, denitrification and codenitrification in agricultural soils collected from
North Carolina, Iowa, Indiana and Kansas. The presence of anammox bacteria was accessed
based on hzo gene detection. Anammox and denitrifying bacteria were detected and quantified
using quantitative PCR (qPCR) of hzo and nosZ genes, respectively, while the detection and
abundance of codenitrifying organisms was estimated based on the presence and number of F.
oxysporum species in soils. The rates of anammox, denitrification and codenitrification co-
occurring in soils were measured using 15N tracer incubation experiments and their contribution
to total N2 production in agricultural soils were estimated based on relative abundance of
microbes responsible for three N2 production pathways.
MATERIALS AND METHODS
Sample Collection
The soil samples were collected in triplicate with a core sampler (10.16 cm diameter) in eight
different agricultural fields in the United States: Pasquotank County, NC (N36° 07’ 30.249”,
W76° 10’ 10.776”), Beaufort County, NC (N35° 27.681’, W076° 55.0926’), Currituck County,
27
NC (N36° 23’ 09.77”, W76° 07’ 18.82”), Rockingham County, NC (36° 23’ 47.199” N, 79° 39’
28.827” W), Tippecanoe County, IN (N40° 29’ 20.027”, W87° 00’ 07.256”), Jewell County, KS
(N39° 56.1008’, W098° 2.1027), Story County, IA (N42° 1.401’, W093° 37.5373’), and Boone
County, IA (N41° 55.186’,W 93° 44.891’). All fields had a history (> 5 years) of maize,
soybean, and/or wheat production following typical regional management practices. The sites
selected had no organic fertilizer additions in at least the five years preceding this study.
Sampling depths at sites varied between a total depth of 60 and 150 cm depending on the depth
of the water table. The soil horizon breaks at each site, either surface, mid, and deep, or surface
and deep cores were sampled, and specific depths are listed in Table 1. The layers were
homogenized separately. Two grams of each layer were stored in 2mL micro centrifuge tubes in
a -80ºC freezer for DNA analysis while the rest was stored in a 4ºC cold room in sealed mason
jars for rate measurements. Additional bulk samples from the same depths and locations were
used for soil characterization. The soil texture and nutrient profiles are reported in Table 1. The
soil texture was measured using a hydrometer (Day, 1965). A 1:1 soil to water slurry was used to
measure the pH (McLean, 1982). Organic matter was measured from the loss on ignition at
360ºC (Schulte and Hopkins, 1996). The inorganic N (NH4+ and NO3
-) was measured using a 1N
KCl cadmium reduction (Dahnke, 1990). The phosphorus was measured through the use of the
Mehlich III soil test (Mehlich, 1984). The physical and chemical properties of the sampled soils
were analyzed by Joshua Heitman and colleagues at North Carolina State University (Raleigh,
NC).
DNA extraction, PCR amplification, Cloning, and Sequencing
The soil DNA extraction proceeded with a ZR Soil Microbe DNA kit (Zymo Research
28
Corporation. Orange, CA) using the manufacturer’s instructions. The soil DNA concentration
was measured with a Quant-It™ PicoGreen® dsDNA Assay Kit, according to the manufacturer’s
protocol (Invitrogen, Carlsbad, CA). PCR of the anammox bacteria16S rRNA gene was
conducted by following the method of Dale et al. (2009). PCR of hzo gene was conducted with
the primer combination of hzocl1F1 and hzocl1R2 (Schmid et al., 2008) with some
modifications. The PCR mixture was a 25µl volume reaction containing 12.5µl GoTaq® Green
Master Mix (Promega, Madison, WI), 1µl of each primer (10 µM), and 1µl of DNA as the
template (10-100 ng). The PCR cycle began with a 5 min, 95°C denaturation step, followed by
40 cycles of denaturation at 94°C for 45 s, a primer annealing step for 1 min at 50°C, concluding
with a 1 min extension step at 72°C. Gel electrophoresis on 1.0% agarose gel was used to
examine the PCR products, which were subsequently purified using the Wizard® SV Gel and
PCR Clean-Up System (Promega, Madison, WI) using the manufacturer’s instructions. The
purified amplicons were cloned using the Perfect PCR Cloning Kit (5Prime, Gaithersburg, MD).
Clone libraries for the following sites were constructed: Beaufort, NC, Currituck, NC,
Pasquotank, NC, Jewell, KS, Tippecanoe, IN, and Boone, IA. Separate clone libraries for the
depth profiles of Beaufort and Pasquotank, NC were created. The clones were sequenced using
BigDye terminator (Applied Biosystems, Foster City, CA) and an ABI 3130xl automated genetic
analyzer (Applied Biosystems, Foster City, CA). NCBI BLAST (http:/www.ncbi.nih.gov) was
used to find closely related sequences. The sequences, along with closely related reference
sequences, were aligned using clustalW (http:/www.ebi.ac.uk/clustalw/). MEGA version 4.0
(Tamura et al., 2007) was utilized to create neighbor-joining trees with boot-strapping of 16S
rRNA gene sequences. The hzo sequences were translated into protein sequences and MEGA
29
was utilized to create a Dayhoff model tree with bootstrapping. Similarities were calculated
using EBI EMBOSS (http:/www.ebi.ac.uk).
Quantitative PCR of hzo and nosZ genes and Fusarium oxysporium
DNA samples extracted from the top layer of the eight different sites were utilized for
qPCR analysis. Abundance of anammox and denitrifying bacteria were quantified using primers
targeting the hzo gene (HZOQPCR1F (5’-AAGACNTGYCAYTGGGGWAAA-3’) and
HZOQPCR1R (5’-GACATACCCATACTKGTRTANACNGT-3’)) and nosZ gene (NOSZ2F
and NOSZ2R; Henry et al., 2006), respectively. Abundance of the codenitrifying fungus F.
oxysporum, was measured by targeting the ITS region using the primers FOF1 and FOR1
designed by Mishra et al.,(2003). The qPCR standards were generated by serial dilution of the
plasmids carrying the respective gene targets. All qPCR utilized GoTaq qPCR Master Mix Green
(Promega, Madison, WI) and a 7500 Real Time PCR System (Applied Biosystems, Foster City,
CA). The PCR cycling for hzo genes included an initial denaturation step for 10 minutes at 95ºC
followed by 50 cycles of 95ºC for 45 sec, 53ºC for 45 sec, 72ºC for 35 sec, and a measurement
step for 35 sec at 75ºC. The PCR cycling for nosZ genes started with an initial denaturation step
for 10 minutes at 95ºC followed by 50 cycles of 95ºC for 45 sec, 55ºC for 45 sec, 72ºC for 35
sec, and a measurement step for 35 sec at 80ºC. The PCR cycling for F. oxysporum ITS region
began at an initial denaturation step for 2 minutes at 95ºC followed by 40 cycles of 95ºC for 1
minute, 65ºC for 30 sec, 72ºC for 30 sec, and a measurement step for 10 sec at 79ºC. PCR
specificity and primer-dimer formation were monitored with analysis of disassociation curves.
All qPCR reactions were performed in triplicate.
15N-Tracer Incubation Experiments
30
The rates of 29N2 and 30N2 production were measured and calculated using a modified
method of Thamdrup and Dalsgaard (2002). Approximately 2 g of soil was transferred to 12 mL
Exetainer tubes® (Labco, High Wycombe, UK) and mixed with 2 mL of milli-Q water to
generate saturated soil slurries. The tubes were sealed with gas-tight septa and flushed with He
gas. The tubes with soil slurries were left over night in attempt to reduce the background NOx.
The remaining background nitrate and nitrite levels were measured using reduction by Vanadium
(III) and chemiluminescent detection developed by Framan and Hendrix (1989) with an Antec
model 7020 nitric oxide analyzer (Antek Instruments, Houston, TX). After sitting over night, the
tubes were vacuumed and flushed with He gas three times. The tubes had He-flushed stock
solutions of Na15NO3 (99.5 atm%; Cambridge Isotope Laboratory, Andover, MA) added to give
a final concentration of 1 mM 15NO3-. Time-course incubations were carried out in duplicate
(time points: 0, 1, 2, 3, and 5 hr). The incubation was killed with saturated ZnCl at each time
point. The samples were run on a continuous flow isotope ratio mass spectrometer (Thermo
Finnigan Delta V; Thermo Scientific, Waltham, MA) that was in line with an automated gas
bench interface (Finnigan Gas Bench II). All samples from a single site were run continuously.
29N2 and 30N2 production rates were calculated from the samples amended with 15NO3-. The
background nitrate levels were taken into account for the rates of 29N2 and 30N2 production along
with tracer dilution as described by Thamdrup and Dalsgaard (2002). Anammox and
codenitrification are considered to mediate 29N2 production while 30N2 is generated from
denitrification.
In order to measure potential anammox rates in soils, 29N2 production was measured
using serum bottles with 15NH4+ addition. Approximately 5 g of soil was transferred to 30 mL
Wheaton serum bottles (Sigma-Aldrich, St. Louis, MO) and mixed with 5 mL of milli-Q water to
31
make saturated soil slurry. The bottles were sealed with gas-tight butyl rubber stoppers and
flushed with He gas. After an overnight incubation, the headspace of each serum bottle was
vacuumed and flushed with He gas. The serum bottles were injected with He-flushed stock
solutions of (15NH4)2SO4 (99.2 atm%; Cambridge Isotope Laboratory, Andover, MA) to give a
final concentration of 1 mM 15NH4+. The headspace gas in the serum bottles (5 mL) was sampled
at the beginning (0 hr) and end (24 hr) of incubation and transferred to a He-filled 12 mL
Exetainer tube (Labco, High Wycombe, UK) using a gas-tight syringe (Hamilton Company,
Reno, NV). The samples were run on a continuous flow isotope ratio mass spectrometer (Thermo
Finnigan Delta V; Thermo Scientific, Waltham, MA). The rate of 29N2 production was calculated
after accounting for tracer dilution based on the background ammonium concentration measured
according to Dahnke (1990) (Table 1).
Statistical analysis
UniFrac (http://bmf2.colorado.edu/unifrac/), a computer program, was utilized to assess
the relationship of soil anammox bacteria to known anammox bacterial genera (Lozupone and
Knight, 2005). The rates calculated from the 15N soil slurry incubation experiments, the
abundance of N-removing organisms calculated based on qPCR data, and the soil characteristics
were used for Principle Component Analysis (PCA) in the Canoco program (version 4.5,
Microcomputer Power, Ithica, NY). Pearson correlation values were calculated from the data
collected from the incubation experiments, the qPCR abundance levels and the soil characters
using Microsoft Excel (Redmond, WA). R-squared and P-values were calculated from linear
regression analyses using Microsoft Excel.
32
RESULTS Physical and Chemical Properties of Soils Collected from Agricultural Fields
Soil samples were collected from 8 agricultural fields in NC, IA, IN and KS. Physical and
chemical properties of the soil samples were examined to determine the environmental factors
influencing anammox, codenitrification and denitrification in agricultural fields. The
measurements of organic matter, NH4+, NO3
-, and other chemical parameters are reported in
Table 1. High concentrations of NH4+ and NO3
- were characteristic of all soil samples, ranging
from 1.1 to 10.2 mg/kg for NH4+ and from 1.3 to 5.9 mg/kg for NO3
-. The overall trend found
was higher levels of NH4+ and NO3
- in the surface soils than deeper soils. Soil pH varied
considerably from the more acidic soils in North Carolina (ranging from 5.2-6.3) to the more
neutral to basic soils in midwestern states (ranging from 5.9-8.2). The physical characteristics of
the sample sites were variable. Soil textures range from sandy loam in Beaufort, NC to silt loam
in Currituck, NC to clay loam in Pasquotank, NC.
Detection and Phylogeny of Anammox Bacteria in Agricultural Soils
Anammox bacterial 16S rRNA genes were initially targeted for detection in the soil
samples following the methods of Dale et al. (2009). Planctomycetes 16S rRNA genes were
33
detected but the sequences obtained were not closely related to known anammox bacteria (Figure
1).
Figure 1. Phylogenetic Tree of Planctomycete 16S rRNA genes detected in North Carolina soil samples.
Alternatively, hzo genes were targeted to detect anammox bacteria in agricultural soils
using the method described by Schmid et al. (2008). The PCR with hzocl1F1 and hzocl1R2
primers generated amplicons with a length of 470 bp. Cloning and sequencing of the amplicons
confirmed the detection of only hzo genes associated with hzo genes in cluster 1 (Schmid et al.,
2008). Based on the hzo gene detection, anammox bacteria were found to be widespread, but not
ubiquitous across the eight sample sites at four states (Table 2). Anammox bacteria were
detected in the top 30 cm of Beaufort, NC, Pasquotank, NC, Currituck, NC, Boone, IA,
Tippecanoe IN, and Jewel, KS as well as in 25-60 cm in Pasquotank, NC, from 30-86 cm in
Beaufort and 60-100 cm in Pasquotank, NC. Overall, anammox bacteria were detected most
often in the top 45 cm of soils.
34
Phylogenetic analysis of the translated HZO sequences showed that soil anammox
bacteria were closely related to “Ca. Jettenia,” sharing 94.1-100% sequence similarity (Figure 2).
None of the sequences were associated with the HZO sequences found in “Ca. Scalindua spp,”
“Ca. Anammoxoglobus spp,” “Ca. Kuenenia spp,” or “Ca. Brocadia spp.” The HZO sequences
were grouped in six clades based on phylogenetic affiliation. One clade (Jettenia clade) clusters
with “Ca. Jettenia,” while the other five clades (I-V) cluster independently. Clade I, II and V
were associated with HZO sequences found in the Upper Cape Fear River Estuary (Hirsch et al.,
2011) while, the HZO sequences in Clade III were closely related to those found in North
Carolina groundwater (Hirsch et al., 2011). Clade IV was associated with HZO sequences
detected in an anammox reactor (Quan et al., 2009).
35
Figure 2. Phylogenetic tree of translated HZO sequences detected from agricultural soils.
36
Weighted UniFrac clustering analysis using the HZO sequences obtained from all the
sample sites supported the results of phylogenetic analysis (Figure 3). The first principle
component (PC1) explained 44.1% of the sequence variability while the second principle
component (PC2) explained 14.9% of the variability. All the HZO sequences detected in soils
clustered together with the sequences found in “Ca. Jettenia” and Planctomycete KSU-1.
Figure 3. PCoA plot of HZO sequences found in agricultural soils
Abundance of anammox and denitrifying bacteria and fungal codenitrifers
Quantitative PCR was performed on DNA samples from the top 30 cm of all the sample
sites using primers specific for hzo and nosZ genes for anammox and denitrifying bacteria,
respectively. The ITS region specific to F. oxysporum was targeted to quantify a group of
codenitrifiers. The hzo genes were detected in all 8 different soil samples by qPCR although the
PCR with hzocl1F and 1R primers did not detect the hzo genes in the soils collected in
37
Rockingham, NC and Story, IA (Table 2). Overall abundance of the hzo gene ranged from 4.99 x
103 to 1.57 x 104 copies per gram of soil (Table 3). The highest hzo abundance was recorded
from Boone, IA while the lowest hzo abundance was recorded from Tippecanoe, IN. Abundance
of the nosZ gene ranged from 3.21 x 106 to 7.88 x 106 copies per gram of soil (Table 3). The
highest nosZ abundance was recorded from Beaufort, NC while the lowest nosZ abundance was
recorded from Boone, IA. Abundance of F. oxysporum ranged from below the detection limit to
3.12 x 105 copies per gram of soil (Table 3). The highest F. oxysporum abundance was recorded
from Currituck, NC while the lowest abundance was recorded from Story, IA. The presence of F.
oxysporum was not detected in soil samples collected from Boone, IA, and Jewell, KS.
29N2 and 30N2 production from 15N tracer incubation experiments
Potential anammox, codenitrification, and denitrification rates in soil samples were
measured using two different 15N substrates (15NH4+ or 15NO3
-) (Table 2). The 29N2 production
from 15NH4+ tracer incubations indicates the presence of anammox in soils samples while the
29N2 and 30N2 productions from 15NO3- tracer incubations were used to calculate the combined
potential rates of anammox and codenitrification and the potential rate of denitrification,
respectively (Figure 4).
38
Figure 4. 29N2 and 30N2 production from 15NO3- incubation experiments. A: Beafort, B:
Currituck, C: Pasquotank, D: Boone, E: Jewell, F: Tippecanoe
The 29N2 production rates from anammox and codenitrification varied from 5.592 to
295.4 nmoles N2g-1d-1, while the denitrification rates ranged from 8.38 to 84.24 nmoles N2g-1d-1.
The %29N2 ranged from 32.1 to 77.9%. Both the lowest potential 29N2 and denitrification rates
were from the surface profile of Boone, IA while both the highest potential 29N2 and
denitrification rates were from the surface profile of Beaufort, NC. The highest %29N2 was found
39
in Beaufort, NC while the lowest was found in Pasquotank, NC. The overall trend was higher
potential 29N2 and denitrification rates in North Carolina as compared to the other states in the
sample set.
Both anammox and codenitrification can utilize NH4+ anaerobically in the presence of
NO2-. However, anammox produces N2 as an end product without generating N2O as an
intermediate, while codenitrification produces N2O as either an intermediate or as the preferred
end product. Therefore, the 29N2 production during the 15NH4+ incubations with 14NO3
- rich soils
may be derived from anammox rather than codenitrification. Based on the hzo gene detection
experiment, the surface soils from each sample site were selected for the 15NH4+ incubations. The
potential anammox rate ranged from 0.013 to 0.484 nmoles N2g-1d-1 based on 29N2 production
from this incubation conditions. Using both the 29N2 produced from the 15NH4+ and 15NO3
-
incubation experiments, the 29N2 produced from anammox was estimated to represent from 0.004
to 5.636% of the total 29N2 production. The lowest potential rate of 29N2 production was in the
surface profile of Beaufort, NC and the highest potential rate was in the surface profile of
Tippecanoe, IN. Overall, the potential 29N2 rates from North Carolina were lower than the 29N2
production rates from the other states.
Weighted and normalized Principle Component Analysis (PCA) was conducted to
determine correlations among the soil characteristics, the potential denitrification rates, the
potential 29N2 rates calculated from the 15NO3- incubation experiments, and the potential 29N2
rates calculated from the 15NH4+ incubation experiments measured in agricultural soils as well as
the abundance of N-removing microorganisms (Figure 5). The potential denitrification rates were
negatively correlated with the levels of Na+ (σ = -0.719, R2 = 0.517, P-value = 0.021). The
potential 29N2 production rates calculated from the 15NO3- incubation experiments were
40
correlated with OM (σ = 0.252, R2 = 0.064, P-value = 0.859) and NH4+ (σ = -0.535, R2 = 0.286,
P-value = 0.166). The potential 29N2 production rates calculated from the 15NH4+ incubation
experiments exhibited a negative correlation with the concentration of NO3- (σ = -0.927, R2 =
0.860, P-value = 0.004). The abundance of nosZ genes in the sampled agricultural fields was
strongly correlated to the 29N2 production rates calculated from the 15NO3- incubation
experiments (σ = 0.911, R2 = 0.828, P-value = 0.042). The abundance of hzo genes showed no
strong correlations to any N2 production rates although weak levels of correlation to 29N2
production from 15NH4+ (σ = -0.103, R2 = 0.011, P-value = 0.465) and 15NO3
- (σ = 0.236, R2 =
0.056, P-value = 0.952) were exhibited. There was a strong negative correlation between the
abundance of F. oxysporum rRNA ITS and the 29N2 production rates calculated from the 15NH4+
incubation experiments (σ = -0.801, R2 = 0.642, P-value = 0.019).
41
Figure 5. PCA Plot Comparing 29N2 and 30N2 production rates, microbial abundance and soil characteristics.
42
DISCUSSION
Diversity of Anammox Bacteria in Agricultural Soils
The diversity of anammox bacteria in terrestrial ecosystems was reported as higher than
that of anammox bacteria in marine ecosystems based on 16S rRNA genes (Humbert et al.,
2010). Low diversity of anammox bacteria in marine ecosystems has been suggested to be an
artifact of biases within the coverage and specificity of the existing anammox 16S rRNA gene
primer sets (Amano et al., 2007; Dale et al., 2009; Li et al., 2010). New 16S rRNA gene primers
and PCR protocols have been used to detect higher diversity of anammox in coastal marine
sediment (Amano et al., 2007), estuaries (Dale et al., 2009) and various soil types (Humbert et
al., 2010). Li et al. (2010), through primer evaluation of both anammox 16S rRNA genes and hzo
genes, found that primers for hzo genes may provide better coverage of anammox bacteria
genera than primers for anammox 16S rRNA genes. The existing hzo primers have been shown
to produce phylogenies comparable to those of phylogenies based on anammox 16S rRNA genes
(Schmid et al., 2008; Li et al., 2010; Hirsh et al., 2011). In addition to these factors, the existing
anammox 16S rRNA gene primer sets have been shown to amplify 16S rRNA genes belonging
to unidentified planctomycetes in soil (Humbert et al., 2010). Our results based on 16S rRNA
genes are consistent with this and provided no anammox 16S rRNA gene sequences. Therefore
the diversity of anammox bacteria in agricultural soil was largely explored with primers targeting
hzo genes. Using these methods, the diversity of anammox translated HZO sequences was found
in the agricultural soils sampled to be low, with only “Ca. Jettenia” present. This was in contrast
to the findings of Humbert et al. (2010), which show high diversity of anammox bacteria across
terrestrial ecosystems with “Ca. Jettenia,” “Ca. Brocadia,” and “Ca. Kuenenia.”
43
A number of selective pressures may have contributed to the lack of diversity in
anammox bacteria in the agricultural soils. Paramount in these factors is tillage, which introduces
air into the soil and disrupts the structural integrity and horizons of the soil profile. Tillage is
known to have a detrimental effect on the abundance of denitrifiers in the soil, with up to 60%
less denitrifiers in tilled versus untilled soils (Doran, 1980). Another selective pressure in
agricultural fields that may have an effect on soil anammox diversity is the long-term application
of inorganic nitrogen fertilizers. The application of NH4+-based fertilizers and NO3
--based
fertilizers alter denitrifier community structures (Enwall et al., 2005). The selective pressures in
agricultural soils may have allowed for “Ca. Jettenia,” a mesophilic anammox bacterium
enriched from anoxic sludge in high NO3- and NH4
+ concentrations, to become the dominant
anammox bacteria in the soils tested (Quan et al., 2008).
Quantification of N2 producing microorganisms
The qPCR of hzo genes detected anammox bacteria in each of the sampled agricultural
fields. The abundance of anammox bacteria was near that found in Jiaozhou Bay, China (2.0 x
104 - 8.7 x 105 copies hzo g–1 sediments) (Dang et al., 2010). Denitrifiers were also detected in
every sampled agricultural field through the use of qPCR amplification of nosZ genes. The
abundance of denitrifiers detected in the agricultural fields sampled in this study fall into the
ranges of denitrifier abundance described in other agricultural soils (105-107 gene copies g-1)
(Henry et al., 2006). From qPCR, F. oxysporum was not detected in every agricultural soil
sampled. The abundance of F. oxysporum in the sampled soils was greater than that (102-104
gene copies g-1) found in inoculated soil samples (Jimenez-Fernandez et al., 2010). This is the
first attempt to quantify F. oxysporum using qPCR in non-inoculated agricultural soils. Sequence
44
analysis of the gene amplicons confirmed the identity to be ITS region sequences with 100%
sequence similarity to F. oxysporum strains (Unpublished data).
Detection and N2 production from 15N-labeled incubation experiments
Due to the majority of the molecular detection occurring in the top 30 cm of the soils, the
surface soils were selected for the 15NO3- tracer incubation experiments. The combined potential
anammox and codenitrification rates from the surface soils fall into the range of potential
anammox rates (19.2 to 384 nmole N2 g-1d-1) described in Chesapeake Bay sediments (Rich et
al., 2007). The potential denitrification rates based on the production of 30N2 are quite low. While
N2 is the end product of denitrification, N2O is also emitted. In agricultural soils, N2O can
contribute as much as 78% of the gases produced from denitrification (Stevens and Laughlin,
1998). Therefore the total N removal capacity of denitrifiers was not completely explored in this
study. The range of the potential denitrification rates in the selected soils falls in line with the
range of potential denitrification rates reported in the sediments of the Cape Fear River Estuary
(Dale et al., 2009). The %29N2 ranged from 32.1-77.9% of the total N2 produced. Although this is
consistent with the upper range of the percentage of N2 produced from anammox found in
coastal marine sediments (Engstrom et al., 2005), it is more complicated to estimate %anammox
in soils because this high percentage of 29N2 from the total N2 production can be attributed to a
number of processes. One explanation, which operates under the assumption that all 29N2 was
produced from the anammox reaction, would be that anammox was an important contributor and
may have even dominated the production of N2 in the tested agricultural soils. However, based
on the results of the qPCR abundance of hzo genes, this explanation seems unlikely. If the 29N2
production from the 15NO3- experiments was solely from anammox, the rate per anammox cell
ranged from 1.424-95.291 pmoles N per anammox cell per day. Zhu et al. (in press), reported
45
ranges from 2.9-21 fmoles N per anammox cell per day in rice paddy soils. Therefore, it seems
more likely that the 29N2 produced from the 15NO3- incubation experiments was from more than
one source. Codenitrification may also produce 29N2. The presence and abundance of Fusarium
oxysporum, a known codenitrifier, in the soil samples suggests that codenitrification may
contribute to the production of 29N2. While other codenitrifiers proceed to the production of N2,
F. oxysporum has shown a tendency to produce N2O under incubation conditions with NH4+ and
NO2- amendments (Tanimoto et al., 1992). This, coupled with the strong positive correlation
exhibited between the abundance of the nosZ gene and the production of 29N2 in the 15NO3-
incubation experiments, suggests that there might be a coupling of codenitrification and
denitrification, in which codenitrification can produce 45N2O under incubation conditions with
15NO2- and 14NH4
+ amendments, (Tanimoto et al., 1992; Laughlin and Stevens, 2002; Spott and
Strange, 2011). The 45N2O produced through codenitrification may have thereafter been utilized
by denitrifiers to produce 29N2 in the 15NO3- incubation experiments. Alternatively,
codenitrification utilizes different nitrogen substrates rather than NH4+ in soils and mediates the
29N2 production in 15NO3- incubation experiments. Negative correlation between NH4
+ and 29N2
production rates might provide evidence for this speculation.
The 15NH4+ tracer incubation experiments detected active anammox bacteria and
codenitrifiers in all of the sample sites in the study. The N2 production rates using these
incubation conditions were significantly lower than the N2 production rates calculated from the
15NO3- incubation experiments. The 29N2 production rates from the 15NH4
+ incubation experiment
may have been more representative of anammox than of codenitrification. This may be the case
due to codenitrification producing N2O as an end product rather than N2 with NH4+ as a substrate
(Tanimoto et al., 1992). Denitrification will preferentially use the lighter 44N2O rather than the
46
45N2O produced by codenitrification to produce more 28N2 than 29N2. The negative correlation
between the 29N2 production from the 15NH4+ incubation experiments and the abundance of F.
oxysporum ITS further suggested that the 29N2 produced under these incubation conditions is
reflective of the N2 produced by anammox.
In this study, anammox bacteria were found to be widespread based on molecular
detection and ubiquitous based on 15NH4 tracer incubation experiments. The anammox bacteria
present in these agricultural soils exhibited low diversity. Anammox bacteria and codenitrifiers
in agricultural soils were found to be active and may have contributed up to 77.9% of the N2
produced depending on the environmental variables. Anammox bacteria seem to be a minor
component of N2 production in agricultural soils as estimated by qPCR. Future studies must
address the contribution of codenitrification to the production of N2 in agricultural soils as well
as develop a method to separate this contribution from the production of N2 from anammox. The
rate of N2O production from codenitrification can be calculated through the use of stable isotope
incubations (Spott and Strange, 2011). Constraining the production of N2O via denitrification
could be further resolved by additional isotopic characterization of N2O produced during tracer
incubations (Trimmer et al., 2006). Codenitrification produces 45N2O when incubated with a 15N-
labeled substrate while denitrification produces 46N2O. This characteristic allows the processes to
be resolved from one another. Characterization and development of molecular probes targeting
genes in the codenitrification pathway such as the fungal nirK and cytochrome p450nor may
prove useful in gaining further understanding the abundance and activity of codentrifiers in soils.
All of these approaches should be incorporated into future studies.
47
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Mehlich, A (1984). Mehlich-3 soil test extractant: A modification of Mehlich-2 extractant. Commun. Soil Sci. Plant Anal. 15:1409-1416. McLean, EO (1982). Soil pH and lime requirement. p. 199-223. In AL Page et al. (ed.) Methods of soil analysis, part 2. Agronomy Monogr. 9, 2nd ed. ASA and SSSA, Madison, WI. Mishra PK, Fox RTV, Culham A (2003). Development of a PCR-based assay for rapid and reliable identification of pathogenic Fusaria. FEMS Microbiol Lett 218: 329-332. Mulder A, Vandegraaf AA, Robertson LA, Kuenen JG (1995). Anaerobic Ammonium Oxidation Discovered in a Denitrifying Fluidedized-Bed Reactor. FEMS Microbiol Ecol 16: 177-183. Penton CR, Devol AH, Tiedje JM (2006). Molecular evidence for the broad distribution of anaerobic ammonium-oxidizing bacteria in freshwater and marine sediments. Appl Environ Microb 72: 6829-6832. Quan ZX, Rhee SK, Zuo JE, Yang Y, Bae JW, Park JR (2008). Diversity of ammonium-oxidizing bacteria in a granular sludge anaerobic ammonium-oxidizing (anammox) reactor. Environ Microbiol 10: 3130-3139. Rich JJ, Dale OR, Song B, Ward BB (2008). Anaerobic ammonium oxidation (Anammox) in Chesapeake Bay sediments. Microb Ecol 55: 311-320. Risgaard-Petersen N, Meyer RL, Schmid M, Jetten MSM, Enrich-Prast A, Rysgaard S (2004). Anaerobic ammonium oxidation in an estuarine sediment. Aquat Microb Ecol 36: 293-304. Schalk J, Oustad H, Kuenen JG, Jetten MSM (1998). The anaerobic oxidation of hydrazine: a novel reaction in microbial nitrogen metabolism. FEMS Microbiol Lett 158: 61-67. Schloss PD and Handelsman J (2005). Introducing DOTUR, a computer program for defining operational taxonomic units and estimating species richness. Appl Environ Microbial 71: 1501-1506. Schmid M, Twachtmann U, Klein M, Strous M, Juretschko S, Jetten M (2000). Molecular evidence for genus level diversity of bacteria capable of catalyzing anaerobic ammonium oxidation. Syst Appl Microbiol 23: 93-106. Schmid M, Walsh K, Webb R, Rijpstra WIC, van de Pas-Schoonen K, Verbruggen MJ (2003). Candidatus "Scalindua brodae", sp nov., Candidatus "Scalindua wagneri", sp nov., two new species of anaerobic ammonium oxidizing bacteria. Syst Appl Microbiol 26: 529-538. Schmid MC, Hooper AB, Klotz MG, Woebken D, Lam P, Kuypers MMM (2008). Environmental detection of octahaem cytochrome c hydroxylamine/hydrazine oxidoreductase genes of aerobic and anaerobic ammonium-oxidizing bacteria. Environ Microbiol 10: 3140-3149.
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Schubert CJ, Durisch-Kaiser E, Wehrli B, Thamdrup B, Lam P, Kuypers MMM (2006). Anaerobic ammonium oxidation in a tropical freshwater system (Lake Tanganyika). Environ Microbiol 8: 1857-1863. Schulte, EE, and BG Hopkins (1996). Estimation of soil organic matter by weight Loss-On-Ignition. p. 21-32. In FR Magdoff, MA Tabatabai, and E.A. Hanlon, Jr. (ed.) Soil organic matter: Analysis and interpretation. Special publication No. 46. Soil Sci. Soc. Am. Madison, WI. Shimamura M, Nishiyama T, Shigetomo H, Toyomoto T, Kawahara Y, Furukawa K (2007). Isolation of a multiheme protein with features of a hydrazine-oxidizing enzyme from an anaerobic ammonium-oxidizing enrichment culture. Appl Environ Microb 73: 1065-1072. Stevens H, Ulloa O (2008). Bacterial diversity in the oxygen minimum zone of the eastern tropical South Pacific. Environ Microbiol 10: 1244-1259. Stevens RJ, Laughlin RJ (1998). Measurement of nitrous oxide and di-nitrogen emissions from agricultural soils. Nutr Cycl Agroecosys 52: 131-139. Strous M, Fuerst JA, Kramer EHM, Logemann S, Muyzer G, van de Pas-Schoonen KT (1999). Missing lithotroph identified as new planctomycete. Nature 400: 446-449. Strous M, Pelletier E, Mangenot S, Rattei T, Lehner A, Taylor MW (2006). Deciphering the evolution and metabolism of an anammox bacterium from a community genome. Nature 440: 790-794. Tamura K, Dudley J, Nei M, Kumar S (2007). MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24: 1596-1599. Tanimoto T, Hatano K, Kim DH, Uchiyama H, Shoun H (1992). Co-Denitrification by the Denitrifying System of the Fungus Fusarium-oxysporm. FEMS Microbiol Lett 93: 177-180. Thamdrup B, Dalsgaard T (2002). Production of N-2 through anaerobic ammonium oxidation coupled to nitrate reduction in marine sediments. Appl Environ Microbiol 68: 1312-1318. Trimmer M, Nicholls JC, Deflandre B (2003). Anaerobic ammonium oxidation measured in sediments along the Thames estuary, United Kingdom. Appl Environ Microbiol 69: 6447-6454. Trimmer M, Risgaard-Petersen N, Nicholls JC, Engstrom P (2006). Direct measurement of anaerobic ammonium oxidation (anammox) and denitrification in intact sediment cores. Mar Ecol Prog Ser 326: 37-47. Vandegraaf AA, Mulder A, Debruijn P, Jetten MSM, Robertson LA, Kuenen JG (1995). Anaerobic Oxidation of Ammonium is a Biologically Mediated Process. Appl Environ Microb 61: 1246-1251. Vitousek PM, Aber JD, Howarth RW, Likens GE, Matson PA, Schindler DW et al. (1997). Human alteration of the global nitrogen cycle: Sources and consequences. Ecol Appl 7: 737-750.
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Zhang Y, Ruan XH, den Camp H, Smits TJM, Jetten MSM, Schmid MC (2007). Diversity and abundance of aerobic and anaerobic ammonium-oxidizing bacteria in freshwater sediments of the Xinyi River (China). Environ Microbiol 9: 2375-2382. Zhu G, Wang S, Wang Y, Wang C, Risgaard-Petersen N, Jetten MSM, and Yin C (in press). Anaerobic ammonia oxidation in a fertilized paddy soil. ISME J (19 May 2011) doi:10.1038/ismej.2011.63.
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CHAPTER 3
Spatial Variation of Anammox, Codenitrification, and Denitrification Across Two Agricultural
Fields in North Carolina
53
ABSTRACT
Three microbial pathways responsible for the production of dinitrogen gas (N2) in soil are
anammox, codenitrification, and denitrification. Denitrification is the most heavily studied and is
known to be a major producer of nitrous oxide (N2O) and N2 in soils. Codenitrification and
anammox were recently discovered and their importance in soil nitrogen cycle has yet to be fully
explored. In order to determine the geochemical and microbial features that influence
denitrification, anammox, and codenitrification, two agricultural fields in North Carolina were
studied using a transect approach. Quantitative PCR (qPCR) of nitrous oxide reductase (nosZ)
and hydrazine oxidase (hzo) genes were conducted to quantify the abundance of denitrifying and
anammox bacteria in soils, respectively. The number of potential codenitrifying microorganisms
was estimated based on the qPCR of the ITS region of rRNA specific to Fusarium oxysporum,
which was reported as the first codenitrifier. Soil slurry incubation experiments with 15NH4+
addition were used to assess potential anammox activity while 15NO3- addition experiments were
used to calculate the potential rates of anammox, codenitrification, and denitrification in the soil
samples. Abundance of denitrifiers in the transect from an agricultural field in Beaufort County
ranged from 1.36 x 104 to 1.78 x 108 nosZ copies g-1, while anammox bacteria ranged from 6.8 x
102 to 6.3 x 104 hzo copies g-1, and. F. oxysporum ranged from 1.58 x 105 to 6.92 x 106 copies g-
1. The transect from Currituck County had a range of 1.57 x 107 to 2.80 x 108 g-1 for denitrifiers,
3.76 x 102 to 1.63 x 104 g-1 for anammox bacteria, and 1.08 x 104 to 3.76 x 106 g-1 for F.
oxysporum per g of soil. The potential denitrification rates in the Beaufort transect ranged from
4.44 to 732 nmoles N2 g-1d-1 while the rates of anammox and codenitrification were 1.19 to 62.78
nmoles N2 g-1d-1. The Currituck transect had a range of 13.19 to 884 nmoles N2 g-1d-1 for
denitrification and 1.001 to 229.8 nmoles N2 g-1d-1 for anammox and codenitrification. Overall,
54
the qPCR and 15N soil slurry incubation experiments show denitrification as the dominant N2
production pathway across the two transects. However, the contribution of anammox and
codenitrification varies in significance both vertically and horizontally across the two transects
with at least one hot spot where their contribution to the N2 production exceeds denitrification.
This suggests that anammox and codenitrification may represent key processes in the soil N
cycle.
55
INTRODUCTION
Three microbial processes including denitrification, codenitrification and anammox are
responsible for the removal nitrogen as dinitrogen gas (N2) from terrestrial ecosystems.
Denitrification is the dissimilatory reduction nitrate (NO3-) to nitrous oxide (N2O) and N2.
Codenitrification produces N2 and N2O through the reduction of nitrite (NO2-) coupled to
unknown metabolic conversions of a nitrogen co-substrate, which have been shown to include
azide, ammonium (NH4+), salicylhydroxamic acid, and hydroxylamine (Tanimoto et al., 1992;
Spott and Strange, 2011). Anammox produces N2 through the anaerobic oxidation of NH4+
coupled to NO2- reduction. Anammox (for example, Humbert et al., 2010; Chapter 2),
codenitrification (for example, Laughlin and Stevens, 2002; Spott and Strange, 2011; Chapter 2),
and denitrification (for example, Henry et al., 2006; Philippot et al., 2009; Chapter 2) have been
found to occur in agricultural soils based on molecular and stable isotope analyses.
Of the three processes, codenitrification is the least studied and its pathway has not been
full characterized. Codenitrification may be mediated either by fungus (e.g. Fusarium
oxysporum) or bacteria (Streptomyces antibioticus) (Tanimoto et al., 1993; Kumon et al., 2002).
A nitric oxide reductase, cytochrome P450nor, has been shown to produce N2O from NO with a
co-substrate (azide or NH4+) (Su et al., 2004). This enzyme has also been shown to catalyze the
formation of N2O in the fungal denitrification pathway. Codenitrification can be differentiated
from denitrification through the use of 15N tracer incubation experiments. Under anaerobic
conditions, codenitrification will produce 45N2O and 29N2 when presented with 15NO3- and
14NH4+ while denitrification will produce 46N2O and 30N2. Anammox and codenitrification will
produce 29N2 when confronted with one 15N-labeled substrate and a 14N-labeled substrate. This
makes the differentiation between anammox and codenitrification impossible in soil samples. In
56
order to better characterize the N-removing pathways in a soil sample, stable isotope data must
be complimented with molecule data of the organisms involved in the processes.
MATERIALS AND METHODS
Sample Collection
The soil samples were collected with a core sampler (10.16 cm diameter) across two
transects in different agricultural fields in the United States: Beaufort County, NC (N35°
27.681’, W076° 55.0926’), Currituck County, NC (N36° 23’ 09.77”, W76° 07’ 18.82). Both
fields had a history (> 5 years) of maize, soybean, and/or wheat production following typical
regional management practices. The sites selected had no organic fertilizer additions in at least
the six years preceding this study. Cores were taken every 20m inter-row from ditch to ditch in
the Beaufort transect while in the Currituck transect, uniform ditches allowed for half the field to
be sampled. Cores were again taken every 20m in the Currituck transect except CUP1, which
was taken in-row midfield, while CUP2 was taken inter-row. CUP3 was 20m from CUP2 (Figure
1). Sampling depths at sites went to 30 cm. Each core was separated into 3 layers, ranging from
0-10 cm to 10-20 cm to 20-30 cm. The layers were homogenized separately. Two grams of each
layer were stored in 2mL micro centrifuge tubes in a -80ºC freezer for DNA analysis while the
rest was stored in a 4ºC cold room in sealed mason jars for rate measurements. Additional bulk
samples from the same depths and locations were used for soil characterization. The soil texture
and nutrient profiles are reported in Table 1. The soil texture was measured using a hydrometer
(Day, 1965). A 1:1 soil to water slurry was used to measure the pH (McLean, 1982). Organic
matter was measured from the loss on ignition at 360ºC (Schulte and Hopkins, 1996). The
inorganic N (NH4+ and NO3
-) was measured using a 1N KCl cadmium reduction (Dahnke, 1990).
The phosphorus was measured through the use of the Mehlich III soil test (Mehlich, 1984).
57
Extraction of DNA and Quantitative PCR of hzo and nosZ genes and Fusarium oxysporium
The soil DNA extraction proceeded with a ZR Soil Microbe DNA kit (Zymo Research
Corporation. Orange, CA) using the manufacturer’s instructions. The soil DNA concentration
was measured with a Quant-It™ PicoGreen® dsDNA Assay Kit, according to the manufacturer’s
protocol (Invitrogen, Carlsbad, CA).
DNA samples extracted from the top layer of the eight different sites were utilized for
quantitative PCR (qPCR) analysis. Abundance of anammox and denitrifying bacteria were
quantified using primers targeting the hzo gene (HZOQPCR1F and HZOQPCR1R; Chapter 2)
and nosZ gene (NOSZ2F and NOSZ2R; Henry et al, 2006), respectively. Abundance of the
codenitrifying fungus F. oxysporum, was measured by targeting the ITS region using the primers
FOF1 and FOR1 designed by Mishra et al., (2003). Q-PCR standards were generated by serial
dilution of the plasmids carrying the respective gene targets. All qPCR utilized GoTaq qPCR
Master Mix Green (Promega, Madison, WI) and a 7500 Real Time PCR System (Applied
Biosystems, Foster City, CA). The PCR cycling for hzo genes included an initial denaturation
step for 10 minutes at 95ºC followed by 50 cycles of 95ºC for 45 sec, 53ºC for 45 sec, 72ºC for
Figure1.SamplingLocationsinBeaufortandCurrituckTransectsinNorthCarolina
58
35 sec, and a measurement step for 35 sec at 75ºC. The PCR cycling for nosZ genes included an
initial denaturation step for 10 minutes at 95ºC followed by 50 cycles of 95ºC for 45 sec, 55ºC
for 45 sec, 72ºC for 35 sec, and a measurement step for 35 sec at 80ºC. The PCR cycling for the
F. oxysporum ITS rRNA region included an initial denaturation step for 2 minutes at 95ºC
followed by 40 cycles of 95ºC for 1 minute, 65ºC for 30 sec, 72ºC for 30 sec, and a measurement
step for 10 sec at 79ºC. Primer specificity and primer-dimer formation was monitored with
analysis of disassociation curves. All qPCR reactions were performed in duplicate.
15N-Tracer Incubation Experiments
The rates of 29N2 and 30N2 production were measured and calculated using a modified
method of Thamdrup and Dalsgaard (2002). Approximately 2 g of soil was transferred to 12 mL
Exetainer tubes® (Labco, High Wycombe, UK) and mixed with 2 mL of milli-Q water to
generate saturated soil slurries. The tubes were sealed with gas-tight septa and flushed with He
gas. The tubes with soil slurries were left over night in attempt to reduce the background NOx.
The remaining background nitrate and nitrite levels were measured using reduction by Vanadium
(III) and chemiluminescent detection developed by Framan and Hendrix (1989) with an Antec
model 7020 nitric oxide analyzer (Antek Instruments, Houston, TX). After sitting over night, the
tubes were vacuumed and flushed with He gas three times. The tubes had He-flushed stock
solutions of Na15NO3 (99.5 atm%; Cambridge Isotope Laboratory, Andover, MA) added to give
a final concentration of 1 mM 15NO3-. Time-course incubations were carried out in duplicate
(time points: 0, 3, and 5 hr). The incubation was killed with saturated ZnCl at each time point.
The samples were run on a continuous flow isotope ratio mass spectrometer (Thermo Finnigan
Delta V; Thermo Scientific, Waltham, MA) that was in line with an automated gas bench
interface (Finnigan Gas Bench II). All samples from a single site were run continuously. 29N2 and
59
30N2 production rates were calculated from the samples amended with 15NO3-. The background
nitrate levels were taken into account for the rates of 29N2 and 30N2 production along with tracer
dilution as described by Thamdrup and Dalsgaard (2002). Anammox and codenitrification are
considered to mediate 29N2 production while 30N2 is generated from denitrification.
In order to measure potential anammox rates in soils, 29N2 production was measured
using serum bottles with 15NH4+ addition. Approximately 5 g of soil was transferred to 30 mL
Wheaton serum bottles (Sigma-Aldrich, St. Louis, MO) and mixed with 5 mL of milliQ water to
create saturated slurry. The bottles were sealed with gas-tight butyl rubber stoppers and flushed
with He gas. After an overnight incubation, the headspace of each serum bottle was vacuumed
and flushed with He gas. The serum bottles were injected with He-flushed stock solutions of
(15NH4)2SO4 (99.2 atm%; Cambridge Isotope Laboratory, Andover, MA) to give a final
concentration of 1 mM 15NH4+. The headspace gas in the serum bottles (5 mL) were sampled at
the beginning (0 hr) and end (24 hr) of incubation and transferred to a He-filled 12 mL Exetainer
tube (Labco, High Wycombe, UK) using a gas-tight syringe (Hamilton Company, Reno, NV).
The samples were run on a continuous flow isotope ratio mass spectrometer (Thermo Finnigan
Delta V; Thermo Scientific, Waltham, MA). The rate of 29N2 production was calculated after
accounting for tracer dilution based on the background ammonium concentration measured
according to Dahnke (1990) (Table 1).
Statistical analysis
The rates calculated from the 15N soil slurry incubation experiments, the abundance of N-
removing organisms calculated using qPCR, and the soil characteristics were used to create a
Principle Component Analysis (PCA) plot utilizing the Canoco program (version 4.5,
Microcomputer Power, Ithica, NY). Pearson correlation values were calculated from the data
60
collected from the incubation experiments, the qPCR abundance levels and the soil characters
using Microsoft Excel (Redmond, WA). R-squared and P-values were calculated from linear
regression analyses using Microsoft Excel.
61
RESULTS
Physical and Chemical Properties of Soils Collected from Agricultural Fields
Soil samples were collected from 2 agricultural fields in NC. Physical and chemical
properties of the soil samples were examined to determine the environmental factors influencing
anammox, codenitrification and denitrification in agricultural fields. The measurements of
organic matter, pH, NH4+, NO3
-, Mg, P, Fe, and K are reported in Table 1. High concentrations
of NH4+ and NO3
- were characteristic of all soil samples, ranging from 12.2 to 30.9 mg/kg for
NH4+ and from 6.8 to 118.1 mg/kg for NO3
-. Soil pH was acidic across both transects, ranging
from 4.7 to 5.8. Soil textures were sandy loam in Beaufort, NC and silt loam in Currituck, NC.
Abundance of anammox and denitrifying bacteria and fungal codenitrifiers
Q-PCR was performed on DNA samples from the top 30 cm of all the samples using
primers specific for hzo and nosZ genes for anammox and denitrifying bacteria, respectively. The
ITS region specific to F. oxysporum was targeted to quantify potential codenitrifiers. The DNA
extracted from BEP6A, BEP6B, BEP6C, BEP7B and BEP7C were not amplifiable. Overall
abundance of the hzo gene in the Beaufort transect ranged from 1.35 x 103 to 1.26 x 105 copies
per gram of soil (Figure 2, Table 2). The hzo genes in BEP1A, BEP3C, and BEP4C were not
detected. The highest hzo abundance was found in BEP1B while the lowest hzo abundance was
recorded from BEP5C. Abundance of the nosZ gene in the Beaufort transect ranged from 1.36 x
62
104 to 1.78 x 108 copies per gram of soil (Figure 2). The highest nosZ abundance was in BEP3C
while the lowest nosZ abundance was in BEP7A. Abundance of F. oxysporum in the Beaufort
ranged from 4.18 x 104 to 6.92 x 106 copies per gram of soil (Figure 2). F. oxysporum was absent
from BEP1B, BEP1C, BEP2C, BEP4B, BEP4C, and BEP5C based on q-PCR. The highest F.
oxysporum abundance was recorded from BEP2A while the lowest abundance was found in
BEP7A.
Overall abundance of the hzo gene in the Currituck transect ranged from 6.02 x 102 to
1.72 x 104 copies per gram of soil (Figure 2, Table 3). CUP2A, CUP4C, and CUP5B did not
have detectable number of anammox bacteria. The highest hzo abundance was recorded from
CUP6A while the lowest hzo abundance was found in CUP1C. Abundance of the nosZ gene in
the Currituck transect ranged from 1.86 x 104 to 2.80 x 108 copies per gram of soil (Figure 2).
63
The highest nosZ abundance was recorded from CUP6A while the lowest nosZ abundance was
recorded from CUP1C. Abundance of F. oxysporum in the Currituck transect ranged from 1.08 x
104 to 3.76 x 106 copies per gram of soil (Figure 2). F.oxysporum was absent from CUP1C,
CUP4C, CUP6A, CUP6B, and CUP6C. The highest abundance of F. oxysporum was recorded
from CUP1A while the lowest abundance was found in CUP3C.
64
Figure 2. Abundance of Anammox bacteria, Denitrifying bacteria and the Codenitrifying fungus, Fusarium oxysporum across the Beaufort and Currituck transects 29N2 and 30N2 Production from 15N tracer incubation experiments
Potential anammox, codenitrification, and denitrification rates in soil samples were
measured using two different treatments of 15N substrates (15NH4+ or 15NO3
-) (Figure 3, Table 3).
The 29N2 production rates from anammox and codenitrification using 15NO3- in the Beaufort
transect varied from 1.05 to 62.78 nmoles N2g-1d-1, while the denitrification rates ranged from
65
1.41 to 731.2 nmoles N2g-1d -1. The %29N2 ranged from 1.8 to 45.6%. The lowest potential 29N2
rate was from BEP1C while the lowest denitrification rate was from BEP7C. Both the highest
potential 29N2 rate and denitrification rates were from BEP5A. The highest %29N2 was found in
BEP7C while the lowest was found in BEP4A. The vertical trend in the Beaufort transect was
higher denitrification rates in the 0-10cm depth with the lowest rates at the 20-30cm depth. The
29N2 production followed this trend. Both denitrification and 29N2 production were highest near
the middle of the field in Beaufort.
The 29N2 production rates from anammox and codenitrification using 15NO3- in the
Currituck transect varied from 1.00 to 229.8 nmoles N2g-1d-1, while the denitrification rates
ranged from 5.59 to 845.5 nmoles N2g-1d -1 (Figure 3, Table 4). The %29N2 ranged from 0.26 to
64.4%. The lowest potential 29N2 rate was from CUP5A while the lowest denitrification rate was
66
from CUP4C. The highest potential 29N2 rate was from CUP5C while the highest denitrification
rate was from CUP3B. The highest %29N2 was found in CUP5C while the lowest was found in
CUP2B. The vertical trend in the Currituck transect was higher denitrification rates in the 10-
20cm depth with the lowest rates at the 20-30cm depth, while the 29N2 production was highest in
the 20-30cm depth and lowest at the 0-10 cam depth. Higher denitrification rates were located in
between the edge of the field and the mid field point while 29N2 production was relatively
constant across the horizontal transect in Currituck.
The potential anammox rates in the Beaufort transect ranged from 0.0009 to 0.229
nmoles N2g-1d-1 based on 29N2 production using 15NH4+ while the potential anammox rates in the
Currituck transect ranged from 0.0006 to 0.152 nmoles N2g-1d-1. Using both the 29N2 produced
from the 15NH4+ and 15NO3
- incubation experiments, the 29N2 produced from anammox was
estimated to account from 0.016 to 5.002% of the total 29N2 production in the Beaufort transect
and 0.003 to 7.589% of the total 29N2 production in the Currituck transect. The lowest potential
67
29N2 production rate in the Beaufort transect was from BEP1B while the lowest potential 29N2
production rate in the Currituck transect was from CUP3A. The highest potential 29N2 production
rate in the Beaufort transect was from BEP3B while the highest potential 29N2 production rate in
the Currituck transect was from CUP2B.
Figure 3. N2 production rates from 15NO3- incubations across the Beaufort and Currituck
transects
68
Weighted and normalized Principle Component Analysis (PCA) was conducted to
determine the soil characteristics influencing abundance and activities of anammox, denitrifying
and codenitrifying organisms in soils (Figures 4 and 5). PCA was also conducted to determine
correlations between the measured potential N2 production rates and the measured abundance of
N2 producing microorganisms (Figures 4 and 5). The principle component 1 explained 99.9% of
the variation in the Beaufort transect, while the principle component 2 explained 0.1% of the
variation. The 29N2 production from the 15NO3- incubation experiments (σ = -0.664, R2 = 0.441,
P-value = 0.029), the 30N2 production from the 15NO3- incubation experiments (σ = -0.730, R2 =
0.625, P-value = 0.011), the 29N2 production from the 15NH4+ incubation experiments (σ = -
0.669, R2 = 0.447, P-value = 0.031) all exhibited a negative correlation with the concentration of
total potassium (K). The 30N2 production from the 15NO3- incubation experiments (σ = -0.534, R2
= 0.295, P-value = 0.042), and the 29N2 production from the 15NH4+ incubation experiments (σ =
-0.525, R2 = 0.276, P-value = 0.043) also exhibited a negative correlation with the concentration
of NO3-. There were no strong or significant correlations exhibited between the N2 production
rates and the abundance of N-removing microbes in the Beaufort transect.
69
Figure 4. PCA plot comparing the N2 production, the abundance of N-removing organisms, and the physical and chemical parameters of the agricultural field in Beaufort, NC `
The principle component 1 explained 95.2% of the variation in the Currituck transect
while the principle component 2 explained 4.8% of the variation. The 29N2 production from the
15NO3- incubation experiments and the 30N2 production from the 15NO3
- incubation experiments
exhibited no significant correlations. The 29N2 production from the 15NO3- incubation
experiments in the Currituck transects showed a weak correlation with the abundance of
F.oxysporum (σ = 0. 497, R2 = 0.247, P-value = 0.311), while the 30N2 production from the
15NO3- incubation experiments showed a weak correlation with the abundance of nosZ (σ =
0.148, R2 = 0.022, P-value = 0.093). The 29N2 production from the 15NH4+ incubation
experiments in the Currituck transects showed a correlation with the abundance of hzo (σ =
0.507, R2 = 0.257, P-value = 0.049). The 29N2 production from the 15NH4+ incubation
experiments exhibited no other significant correlations.
70
Figure 5. PCA plot comparing the N2 production, the abundance of N-removing organisms, and the physical and chemical parameters of the agricultural field in Currituck, NC
71
DISCUSSION
The abundance and activity of anammox, denitrification, and codenitrification were
found to vary both vertically and horizontally in the two transects. Overall, the trend in the soil
profile was higher activities and gene abundance in the top 10 cm and lower activities and
abundance in the deeper soils. For denitrifiers and codenitrifiers, two groups of organisms that
compete aerobically as well as anaerobically, the oxygen availability in the field may contribute
to their increased abundance in the top 10 cm on agricultural soils (Tiedje, 1988).
The abundance of anammox bacteria based on the qPCR of hzo genes was near that
found in Jiaozhou Bay, China (2.0 x 104 - 8.7 x 105 copies hzo g–1 sediments) and within the
range reported in Chapter 2 (Dang et al., 2010). The abundance of denitrifiers based on qPCR of
nosZ in the two transects fell into the ranges of denitrifier abundance described in other
agricultural soils (105-107 nosZ gene copies g-1) (Henry et al., 2006; Chapter 2). The abundance
of F. oxysporum in the sampled soils was greater than that (102-104 gene copies g-1) found in
inoculated soil samples (Jimenez-Fernandez et al., 2010) but within the range reported in
Chapter 2. The combined potential anammox and codenitrification rates from both transect fell
into the range of potential anammox rates described in Chesapeake Bay sediments and in
Chapter 2 (Rich et al., 2007). The potential denitrification rates based on the production of 30N2
in both transects exceed the range reported in Chapter 2 and were in the range of denitrification
reported from other agricultural soils (Enwall et al., 2005).
In the Beaufort transect, K is negatively correlated with the production of 30N2 and 29N2
from both incubation conditions. The production of 30N2 and the production of 29N2 from the
15NH4+ incubation experiments exhibited a negative correlation with the concentration of NO3
-.
This may be due to the amount of fertilizer in the agricultural field in Beaufort exceeding the
72
capacity of denitrifiers, codenitrifiers, and anammox bacteria to remove it. The rate of these N-
removal processes may be uncoupled to the concentration of NO3- due to the magnitude of NO3
-
in the system.
In the Currituck transect, the production of 29N2 from the 15NH4+ incubation experiments
was correlated with the abundance of hzo genes. This correlation may suggest a coupling of the
microbial abundance and ecosystem processing. This is consistent to the findings of Philippot et
al. (2009), who described a relation between the magnitude of N2O produced in soil and the
abundance of N2O-producing denitrifiers. This correlation may also suggest that the 29N2 from
15NH4+ incubations may be more associated with anammox than codenitrification.
Codenitrification may use other substrates than NH4+ and may preferentially produce N2O
(Tanimoto et al., 1992). This correlation is consistent with the findings of chapter 2.
In summary, the dominant N-removing pathway in both transects was denitrification. The
contribution of anammox and codenitrification varied spatially and within the soil profile of both
transects. In at least one area, a “hot-spot”, of codenitrification and anammox existed and the
production of 29N2 exceeded the production of N2 due to denitrification. The controlling nutrient
in the Beaufort transect appeared to be K for all the processes. In the Currituck transect, the
abundance of hzo correlated to the 29N2 production from the NH4+ incubations. The results of this
study underpin the necessity of resolving codenitrification from anammox in agricultural soils as
well as the importance comparing the abundance of microorganisms with the rate of their
respective ecosystem functions.
73
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