ASSESSING THE ROLE OF ANAMMOX IN A NITROGEN CONTAMINATED AQUIFER
James B. Taylor
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
2012
Approved by
Advisory Committee
Lawrence Cahoon Michael Mallin
Bongkeun Song
Chair
Accepted by
Dean, Graduate School
iii
TABLE OF CONTENTS
CHAPTER 1
LIST OF TABLES ...........................................................................................................................v
LIST OF FIGURES ....................................................................................................................... vi vi
INTRODUCTION ...........................................................................................................................2
The Nitrogen Cycle ..............................................................................................................2
History..................................................................................................................................7
Morphology and Functionality ..........................................................................................10
Ecology ..............................................................................................................................14
CHAPTER 2
ABSTRACT ...................................................................................................................................22
INTRODUCTION .........................................................................................................................23
METHODS AND MATERIALS ...................................................................................................28
Sample Collection in August of 2011 ................................................................................28
DNA Extraction ................................................................................................................28
PCR amplification, Cloning, and Sanger Sequencing .......................................................31
Quantitative PCR of functional genes in anammox, denitrifying and nitrifying bacteri. ..30
15N-Tracer Incubation Experiments ..................................................................................32
Statistical analyses .............................................................................................................33
RESULTS .....................................................................................................................................34
Vertical Geochemical Distribution ....................................................................................34
Abundance of anammox, denitrifying, and nitrifying bacteria in the aquifer. ................. 36
Potential rates of anammox and denitrification Based on 15
N Isotope Pairing..................39
Statistical analysis of Rates, bacterial abundance and geochemical parameters ...............44
Phylogeny of Anammox Bacterial communities ...............................................................50
DISCUSSION ...................................................................................................... .........................54
iv
Anammox and denitrifier Quantification ...........................................................................54
N2 Potential Rates via Anammox and Denitrification .......................................................57
Diversity and phylogeny of Anammox bacteria ................................................................59
CONCLUSIONS............................................................................................................................61
Future considerations .........................................................................................................61
LITERATURE CITED ..................................................................................................................63
v
LIST OF TABLES
Table Page
1. Geochemical vertical distribution of F575 sampling site ........................................................35
2. Copy numbers per ng DNA of functional genes and 16S RNA ..............................................37
3. Percent abundance of bacteria that contain specific functional gene ......................................38
4. Potential rates compared to percent anammox ........................................................................43
5. Pearson product-moment correlation coefficient analysis between the log of AMX/DNF
bacterial abundance and geochemical variables ......................................................................46
6. Pearson product-moment correlation coefficient analysis between anammox and
denitrification rates, % anammox and geochemical variables .................................................48
7. Pearson product-moment correlation coefficient analysis between anammox and
denitrification rates and relevant bacterial abundances ...........................................................49
8. Estimated gene abundance in 1L of groundwater samples ......................................................55
vi
LIST OF FIGURES
Figure Page
1. Nitrogen cycling in a marine ecosystem ....................................................................................3
2. The 5 genera of anammox bacteria within the planctomycetes phylum ....................................9
3. Vertical cross-section of the anammox cell and anammoxosome ...........................................13
4. Aerial depiction of the contaminant plume ..............................................................................26
5. Copy number per ng DNA, N2 production rates and geochemical parameters arranged
vertically along the MLS in the aquifer ...................................................................................41
6. Ratio of rates to gene abundance, N2 production rates and geochemical parameters arranged
vertically along the MLS in the aquifer ...................................................................................43
7. CCA plot showing possible correlations between bacterial abundance and geochemical
variables ...................................................................................................................................45
8. CCA plot showing possible correlations between bacterial abundance and geochemical
variables ...................................................................................................................................47
9. Phylogenetic tree of hzsA gene sequences detected from two different depths .......................52
10. Phylogenetic tree of anammox bacterial 16S rRNA sequences detected from two different
depths .......................................................................................................................................53
vii
ACKNOWLEDGEMENTS
I would like to thank Dr. BK Song for his steadfast support and guidance, and the
members of Song lab for all the help throughout the years. I would like acknowledge my
committee members, Dr. Larry Cahoon and Dr. Mike Mallin for their expertise and guidance
through the many intricacies of the project.
I would like to thank my family and friends for working around my long hours and their
never ending love and support.
This research is supported by the NSF Geobiology & Low Temperature Geochemistry
Program. I would like to acknowledge the field scientists at USGS, and Denis LeBlanc for his
on-site expertise.
I would like to dedicate my thesis to my mother, Lynn Taylor, for without her none of
this would have been possible.
Chapter 1
The Anammox tale: An in-depth review of the ecology, morphology and ecological impact of
anaerobic ammonium oxidation
2
INTRODUCTION
The Nitrogen Cycle
Nitrogen is an essential element that exists in up to 9 different oxidation states, utilizes
several different environmental transport/storage pathways and contains many ways to change
species (See review: Jetten 2009). In order to achieve every oxidation state, the nitrogen must
pair with an atom of oxygen, hydrogen, or other nitrogen. This flexibility of the nitrogen atom
allows for many combinations of unique molecules that represent every oxidation state. Different
nitrogen species are used by organisms generating cellular energy by serving as an electron
donor or acceptor in metabolic pathways. Nitrogen is then assimilated as a macronutrient, which
is evident in the reduced, basic chemical formula of an organism, CH2O0.5N0.15 (Review: Jetten
2009).
The global nitrogen cycle operates by keeping a smaller, fixed/biologically available pool
of nitrogen in constant flux with the monstrous amount of atmospheric nitrogen (in the N2 form).
The flux and balance of this system controls the amount of fixed nitrogen in marine and
terrestrial ecosystems. This delicate balance is important for many reasons, including the role of
nitrogen as a limiting nutrient in primary production (Vitousek and Howarth, 1991).
The nitrogen cycle is mainly composed up of five catabolic processes (nitritification,
nitratification, denitrification, dissimilatory nitrate reduction to ammonia (DNRA) and anaerobic
ammonium oxidation (anammox), three anabolic processes (ammonium uptake, assimilatory
nitrate reduction and nitrogen fixation), and ammonification (Figure 1).
3
Figure 1. Nitrogen cycling in a marine ecosystem. (Francis et al., 2007).
4
Although nitrogen makes up 78% of our atmosphere, most is unavailable to organisms
due to the strong triple bond that holds the two N atoms together. In order to be able to utilize
this N, it must be fixed. N fixation can either occur abiotically by lightning or biologically by
certain microorganisms such as some bacteria, actinomycetes, cyanobacteria and blue-green
algae. One of the biomarkers for this process is the nifH gene in microorganisms which codes for
the reductase component of nitrogenase, the enzyme necessary to fix N (Zehr et al., 2007). The
nitrogenase enzyme complex is inhibited by oxygen, so many N fixing bacteria utilize a
symbiotic relationship with plant roots that contain the oxygen-molecule stealing
leghaemoglobin to create a low-oxygen environment (Herridge et al., 1995). Once the N is
biologically fixed, the new organic N can be mineralized to ammonium by the process of
ammonification. The nitrogen is now in an inorganic state. Plants may assimilate the ammonium
to turn back into organic nitrogen for cellular processes (Herridge et al., 1995).
Nitrification is an important piece of nitrogen speciation, transport, and removal in soils
and watersheds (Hill & Shackleton, 1989; Fisk & Fahey, 1990). During the process of
nitrification, ammonium is converted to nitrate. This is a two-step process that involves two
different types of bacteria, ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria
(NOB). The AOB convert ammonium to nitrite in an aerobic (though not always) environment.
The two dominant bacteria genera that carry out the first step in the nitrification process are
Nitrosospira and Nitrosomonas, both betaproteobacteria. Certain Gammaproteobacteria like the
Nitrosococcus can also oxidize ammonium, but are usually found in marine systems (Teske et
al., 1994). Recently it was discovered that Archaea can also oxidize ammonium (AOA). Two of
the main enzymes in this process are ammonia monoxygenase (AMO) which catalyzes the
5
oxidation of ammonium to hydroxylamine, and hydroxylamine oxidoreductase (HAO) which
oxidizes hydroxylamine to nitrite.
The second half of the nitrification process is mediated by NOB, which oxidize the
intermediate nitrite to nitrate. Nitrobacter spp. are the main genus thought to carry out the nitrite
oxidation process. Nitrite oxidoreductase (NOR) is the enzyme responsible for the oxidation of
nitrite (Meincke et al., 2004).
When oxygen is not present, nitrate becomes a very attractive electron acceptor for nitrate
respiring microorganisms. The process of the dissimilatory reduction of nitrate is called
denitrification, a very important process in the global geochemical nitrogen cycle. The process is
utilized by bacteria, Archaea, and even fungi (Kobayashi et al., 1996). The nitrate respiration
begins with the reduction of nitrate to nitrite, using the enzyme nitrate reductase (nar). The next
stage is the reduction of nitrite to nitric oxide, using the enzyme nitrite reductase (nor). The next
step is the reduction of nitric oxide to nitrous oxide. This reduction is interesting because the
enzyme that catalyzes this reaction, nitric oxide reductase (nor), is the enzyme that creates
formation of the dinitrogen bond between the two nitrogen atoms of nitrous oxide (Bothe et al.,
2000). Nitrous oxide is not always reduced to dinitrogen gas by all denitrifiers because not all
denitrifiers possess the ability for that last reduction step (Bothe et al., 2000). However, if the
denitrifier does possess the nitrous oxide reductase gene (nos), the final step of denitrification is
complete and the dinitrogen gas is released to the atmosphere as it is not biologically available.
The denitrification process is normally carried out by heterotrophic bacteria such as Paracoccus
denitrificans and various pseudomonads (Carlson & Ingraham 1983).
6
Dissimilatory reduction of nitrate to ammonium (DNRA) process occurs in low oxygen
settings similar to denitrification, and both processes can even compete for available nitrate. This
process is mediated by prokaryotes carrying the nrfA gene. (See review: Simon 2002). One of the
main concerns about DNRA is that NO3- is transformed into another mineral N form which is
less mobile and may conserve N in the ecosystem, unlike denitrification (Buresh and Patrick,
1978; Tiedje, 1988). King and Nedell (1985) determined that DNRA may occur in areas with
elevated concentrations of organic electron donors and low nitrate. The ratio between the
available electron donors and electron acceptors is an important factor to determine if DNRA or
denitrification is a dominant (Tiedje et al., 1982; Smith and Zimmerman, 1981) as DNRA,
anammox, and denitrification are competing for the same oxidants (Payne 1973; Burgin and
Hamilton, 2007). When simply comparing the potential free energy of total denitrification (-2669
kJ mol−1
glucose) and DNRA (–1796 kJ mol−1
glucose), denitrification should be favored over
DNRA (Gottschalk, 1986). However, in certain ecosystems such as anoxic sediments with
sulfidic conditions (Christensen et al., 2000), DNRA has the advantage over denitrification since
more electrons can be transferred per mole of nitrate (Tiedje et al., 1982). There are several
genera of soil bacteria capable of DNRA, which are either obligate anaerobes (Clostridium),
facultative anaerobes (Citrobacter, Enterobacter, Erwinia, Escherichia, Klebsiella) or aerobes
(Bacillus, Pseudomonas) (Tiedje et al.,1988).
The last process of the N cycle is another form of dinitrogen gas production, where
dinitrogen is created through the anaerobic oxidation of ammonium (anammox) (Van de Graaf et
al., 1995; Jetten et al., 2001; Dalsgaard et al., 2003; Kuypers et al., 2003). The anammox bacteria
are members of Planctomycetes phylum in which 5 genera have been described. Anammox
bacteria utilize a unique organelle like structure lined with ladderane lipids called the
7
anammoxisome to carry out the anammox process using the volatile hydrazine as an
intermediate. The anammox process is important because it does not create nitrous oxide as a
byproduct, unlike denitrification (Jetten 2008).
ANAMMOX UNRAVELED
History
Several decades before the discovery of anammox bacteria, Austrian theoretical
chemist Engelbert Broda (1977) put forth the idea that organisms could be capable of oxidizing
ammonium by using nitrite or nitrate as an electron acceptor. Richards (1965) even demonstrated
the mysterious and unexplainable loss of ammonium in anoxic fjords a decade before Broda’s
prediction. In 1995, Mulder et al. discovered the same phenomena in a bioreactor in the
Netherlands. It was not until 1999 that Strous et al., (1999) purified anammox cells from an
enrichment culture and the anammox bacteria examination could begin in earnest. The first
discovered anammox bacterium belongs to the order of Planctomycetales (Strous et al., 1999)
and was named Candidatus Brocadia anammoxidans. At the time of this review, five anammox
genera have been described, with 16S rRNA gene sequence identities of the species ranging
between 87 and 99% (Jetten et al., 2009).
Even with as few as 87% identity to one another in some anammox bacteria, the
organisms still exist in the same monophyletic cluster (Brocadiales) and the same order
(Planctomycetales). The anammox branch lies inside the phylum Planctomycetes (Strous et al.,
8
1999; Schmid et al., 2005; 2007) (figure 2). Four “Candidatus” anammox genera have been
enriched from bioreactors: “Kuenenia” (Schmid et al., 2000; Strous et al., 2006), “Brocadia”
(Strous et al., 1999; Kuenen and Jetten., 2001; Kartal et al., 2008), “Anammoxoglobus” (Kartal
et al., 2007b) and “Jettenia” (Quan et al., 2008). The last anammox genus, “Candidatus
Scalindua” (Kuypers et al., 2003; Schmid et al., 2003; van de Vossenberg et al., 2008), has often
been detected in marine sediments and oxygen minimum zones (Dalsgaard et al., 2005; Schmid
et al., 2007). Within the genus “Scalindua”, two marine molecular isolates have been identified.
9
Figure 2. The 5 genera of anammox bacteria within the planctomycetes phylum (Song,
Unpublished)
10
Morphology and functionality
Since their discovery anammox bacteria have been known as slow-growers, with a
division time of once every 11-20 days (Strous et al., 1999). Escherichia coli, for example,
divide once every 20 or so minutes. The anammox bacteria are obligate anaerobes and cannot
tolerate oxygen conditions above 2 μM (Strous et al., 1999). With such a slow growth and low
oxygen tolerance, it is quite difficult to culture the organisms, but it is possible to grow an
enrichment culture of the microbes with the sequencing batch reactor (SBR) method (Strous et
al., 1999). This type of reactor can keep a stable batch for more than a year.
Anammox bacteria usually gain their energy from the 1:1 chemolithotropic conversion of
ammonia and nitrite to dinitrogen gas. The overall reaction includes 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). This is a thermodynamically favorable reaction and is more
favorable than the aerobic oxidation of ammonium (Strous et al., 1999). However, the metabolic
rate is relatively low, which could account for the slow growth rate (Jetten et al., 2009).
Anammox bacteria are known for having a high affinity for their substrates, and can grow at
concentrations of less than 5μM (Strous et al., 1999).
Strous et al., (2006) was able to piece together the genome of Candidatus “Kuenenia
stuttgartiensis” in order to further understand the anammox metabolic processes and genes.
These genes include a cd1 type nitric oxide: nitrite oxidoreductase (nirS), and nine paralogues of
hydroxylamine/hydrazine oxidoreductase (hao/hzo) (Hooper et al., 1997). The stepwise
combination of ammonium and nitric oxide to form hydrazine gene has yet to be found, but a
“hydrazine hydrolase” later to be “hydrazine synthase” enzyme is proposed to combine
11
ammonium with nitric oxide to form hydrazine (Jetten et al., 2009; Kartal et al., 2011). The
current mechanism for the anammox reaction begins with nitrite being reduced to nitric oxide
through nirS. The second step would be the reduction of NO and its concurrent condensation
with ammonium to produce N2H4 (Hydrazine). The next step is the oxidation of hydrazine to
dinitrogen gas, which produces four electrons and four protons and thus creates the proton
motive force across the anammoxosome membrane (Jetten et al., 2009; Kartal et al., 2011).
Recently hydrazine synthase (hzs), has been used as a biological marker for anammox discovery
and is an integral aspect of the anammox process. hzs catalyzes the synthesis of hydrazine from
nitric oxide and ammonium.
Anammox bacteria have many extraordinary and unique features that separate this
organism from other bacteria, even other Planctomycetes. Anammox have a unique cell plan and
unique lipids that have been recently studied in the past decade. Anammox bacteria have a
unique intracellular structure called the “anammoxosome” (figure 3). This compartment occupies
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 (Kartal et al., 2008). Members of the
Planctomycetes have intracellular membranes and complex compartmentalization in comparison
to a typical bacterium (Fuerst, 2005). The cytoplasm in anammox bacteria is thus divided into
three cytoplasmic compartments separated by single bilayer membranes; the outer most is called
the “paryphoplasm”, similar to the periplasm in Gram-negative bacteria. They are different 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
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;
12
Lindsay et al., 2001). When dealing with anammox bacteria however, the third compartment is
the anammoxosome, which is the proposed location of the anammox reaction (van Niftrik et al.,
2008a; 2008b).
Anammox bacteria contain lipids in the previously aforementioned anammoxosome
membrane that are unique to anammox and not found elsewhere in nature. These “ladderane”
lipids consist of hydrocarbon chains with linearly concatenated cyclobutane rings with the
ladderanes in a cyclohexane ring. The concatenated cyclobutane ring systems are unique in
nature. These ladderane lipids have ester-linkages and ether-linkages, which are interesting
because ether-linkages are were thought to be exclusive to Archaea while ester-linkages are
exclusive to bacteria and eukaryotes (Sinninghe Damsté et al., 2002). Based on molecular
modeling, the ladderane lipids were described as tightly-packed in the anammoxosome
(Sinninghe Damsté et al., 2002). This unusually high density prevents this membrane from being
permeable to apolar compounds. Because the metabolism of anammox bacteria involves gaseous
molecules and the toxic intermediate hydrazine, this tightly packed membrane may allow the
anammoxosome to minimize the loss of the substrates (Jetten et al., 2009).
13
Figure 3: Vertical cross-section of the anammox cell and anammoxosome (Kuypers et al., 2003).
14
Ecology
Molecular detection methods Many methods are available for the detection of anammox
bacteria and their activity in the natural environment and man-made ecosystems (Risgaard-
Petersen et al., 2003; Schmid et al., 2005). Anammox have been detected in many environments,
including: marine sediments (Dalsgaard and Thamdrup, 2002; Rich et al., 2008), oxygen-
minimum zones (Dalsgaard et al., 2003; Kuypers et al., 2003; 2005), freshwater marshes (Penton
et al., 2006), permafrost (Penton et al., 2006), rivers (Zhang et al., 2007), river estuaries
(Risgaard-Peterson et al., 2004; Dale et al., 2009). PCR amplification with non-specific16S
rRNA gene-targeted primers followed by phylogenetic analysis is a common technique to
discover the identity of unknown organisms in environmental samples. However, anammox
bacteria may not be fairly represented in general 16S rRNA gene clone libraries since the
“universal” primer set for 16S rRNA gene amplification can result in low identification. Using a
more specific primer, i.e. amx386F (an anammox specific primer) along with a general
eubacterial reverse primer or a specific anammox reverse primer (i.e. amx820R) can increase
relative amounts of anammox 16S rRNA gene sequences (Schmid et al., 2000; 2007; Penton et
al., 2006).
More recently however, more functional PCR approaches using primers amplifying
anammox genes encoding hydroxylamine/hydrazine oxidoreductase (HAO/HZO), hydrazine
synthase (HZS), and nitrous oxide reductase (NIR) proteins showed that these genes are suitable
targets for molecular ecological studies on both aerobic and anaerobic ammonium-oxidizing
bacteria (Haranghi et al., 2012; Li et al., 2010; Quan et al., 2008; Schmid et al., 2008). Li et al.,
(2010) and Haranghi et al., (2012) found that primers for hzo and hzs genes may provide better
coverage of anammox bacteria genera than primers for anammox 16S rRNA genes, respectively.
15
Another approach to quantifying and qualifying anammox bacteria in the environment is
fluorescent in situ hybridization (FISH). FISH is useful for visual identification of anammox
cells and has been used successfully in several studies (Helmer-Madhok et al., 2002; Schmid et
al., 2000, 2003). Schmid et al., (2003) was able to create a probe that was very effective at
hybridizing to 16S rRNA of anammox bacteria. As more valid anammox sequences become
available, probe designs will continue to improve (Jetten et al., 2009). Two advanced FISH
techniques are; the Raman-FISH, which combines stable-isotope Raman spectroscopy and FISH
for the single cell analysis of identity and function (Pätzold et al., 2008), and the CARD-FISH,
(Catalyzed reporter deposition) has also been used to detect anammox because it is more
sensitive than the traditional FISH technique (Hannig et al., 2007).
15N Tracer incubation methods Anammox contribution to the total N2 production (%anammox)
can be calculated by measuring the rates of both anammox and denitrification using 15
N tracer
incubation techniques (Thamdrup and Dalsgaard, 2002). The 15
N tracer incubation has been the
technique of choice for the detection of anammox activity. When environmental samples
including soil, sediment and water, are incubated under an anoxic environment with 15
N-
substrates, one of several reactions can happen. For example, the addition of 15
NH4+ paired with
14NO2
-, or
15NO3
-/NO2
- with
14NH4
+ will generate
29N2. This specific molecular weight of
dinitrogen gas is indicative of the anammox reaction and has been used successfully in several
studies since anammox bacteria utilize one molecule each of NH4+ and NO3
-/NO2
- (Dalsgaard et
al., 2003; Kuypers et al., 2003; Thamdrup and Dalsgaard 2002). The addition of 15
NO3-/NO2
-
with 14
NH4+
could also generate 30
N2 as an end production of denitrification, which uses two
molecules of NO3-/NO2
-. In this method, rates of N2 production via anammox or denitrification
may be measured simultaneously in the same sample via continuous flow isotope ratio mass
16
spectrometer (Thermo Finnigan Delta V; Thermo Scientific, Waltham, MA) in line with a gas
bench interface. 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). The 15
N2 (14
N15
N) isotope technique can also be modified to
include the addition of inhibitors. Differential effects of acetylene and methanol on anammox
and denitrification helped solidify a more solid understanding of the main pathways of N2
production in marine sediments (Jensen et al., 2007).
Ladderane Lipid analysis The ladderane lipids are so unique and otherwise unknown in nature,
they are an ideal biomarker for anammox bacteria (Lanekoff & Karlsson, 2010; Rattray et al.,
2008). The intact ladderane glycerolphospholipids are thermally unstable (Li & Gu, 2011;
Sinninghe Damsté et al., 2005), however, they degrade into predictable breakdown products
(Rush et al., 2011). The 13
C content of anammox lipids is around 45% depleted when compared
to their carbon source, while lipids from other autotrophic organisms are around 20 to 30%
depleted (Rattray et al., 2010; Rush et al., 2011). Recently, lipid analysis has become a viable
method for detecting anammox bacteria in natural environments (Brandsma et al., 2011; Byrne et
al., 2009; Hu et al., 2011; Jaeschke et al., 2007; Jaeschke et al., 2009; Lanekoff & Karlsson,
2010).
Importance of anammox in marine ecosystems Nitrogen cycling in marine ecosystems is very
important due to N being the limiting agent of primary productivity. The ammonium is converted
to nitrate via nitrite through the process of nitrification (as described above), and the nitrite and
nitrate are converted to nitrogen gas anaerobically through the process of anammox or
denitrification (Kuypers et al., 2003; 2005). The first discovery of anammox in anoxic waters
was by Kuypers et al. (2003) in the largest anoxic basin on the planet, the Black Sea. To confirm
17
the presence of anammox, Kuypers et al (2003) utilized several techniques listed above including
nutrient analysis, FISH, 15
N potential rates, and 16S rRNA sequence analysis. After this
development, similar ecosystems such as the Peruvian and Benguela upwelling systems also
yielded the existence of anammox (Kuypers et al., 2005; Thamdrup et al., 2006). In the Benguela
system there was no significant denitrification activity reported, which would leave anammox as
the sole sink of fixed nitrogen in the entire system.
The water chemistry of oxygen minimum zones (OMZs) would seem to support
anammox over denitrification as seen in the OMZs of the Peruvian and Chilean upwelling (Galán
et al., 2009; Hamersley et al., 2007; Lam et al., 2009; Thamdrup et al., 2006; Ward et al., 2009).
In these OMZs, anammox rates were highest in zones of depleted nitrate and accumulating
nitrite, even with little to no ammonium (Hamersley et al., 2007; Schmid et al., 2005; Thamdrup
et al., 2006). Anammox rates were at their highest closer to the top of the OMZ (Lam &
Kuypers, 2011), and positively correlated with cell densities (Hamersley et al., 2007; Kuypers et
al., 2005).
The discovery of anammox in anoxic waters allowed researchers to explore other anoxic
marine ecosystems for anammox presence. Soon after, anammox was found in marine/estuary
sediment (Risgaard-Petersen et al., 2004; Risgaard et al., 2004; Meyer et al., 2005; Tal et al.,
2005; Amano et al., 2007; Hietanen, 2007; Schmid et al., 2007; Rich et al., 2008). Within these
systems the anammox reaction was estimated to contribute more than 50% of all marine nitrogen
losses, which would indicate the anammox was far more important to the global nitrogen cycle
than previously thought.
18
Importance of anammox in freshwater and terrestrial ecosystems The impact of anammox
bacteria have also been explored in freshwater and terrestrial ecosystems such as freshwater
marshes (Penton et al., 2006; Humbert et al., 2010), rivers (Zhang et al., 2007), lakes (Schubert
et al., 2006), and river estuaries (Risgaard-Peterson, et al., 2004; Trimmer et al. 2003; Dale et al.,
2009), various soil types, which include permafrost soils (Penton et al., 2006), reductised,
agricultural soils (Humbert et al., 2010), peat soils (Hu et al., 2011) and groundwater (Clark et
al., 2008). Humbert et al., (2010) showed that terrestrial anammox diversity is much greater than
marine diversity using molecular methods.
The first anammox found in lake ecosystems was in Lake Tanganyika, the second largest
lake in the world, by Schubert et al. (2006). Incubations with stable 15
NO3- indicated that
anammox was discovered in the suboxic water. FISH revealed up to 13,000 anammox bacteria
ml-1
and the presence of ladderane lipids. Phylogenetic analyses of 16S rRNA genes indicated
the presence of sequences most closely related to the known anammox bacterium “Candidatus
Scalindua brodae”. Anammox rates were similar to those reported for the marine water column
and up to 13% of the produced N2 could be attributed to the anammox bacteria.
Estuarine sediments are an important ecosystem that serves as a “catch” for N runoff
from terrestrial ecosystems (Costanza et al., 1998). Up to 26% of N2 production from estuarine
sediments is estimated to originate from anammox (Meyer et al., 2005; Risgaard-Petersen et al.,
2004; Trimmer et al., 2006). This would make anammox a significant aspect of the estuarine
nitrogen cycle.
In regards to agricultural soil, the application of inorganic nitrogen fertilizers has created
widespread changes in the global nitrogen cycle. Fertilizer inputs have doubled the rate of
19
nitrogen uptake in terrestrial environments and increased the activity of denitrifiers, which in
turn has increased the production of nitrous oxide (N2O), a greenhouse gas. Inorganic nitrogen
fertilizers have also contributed to the increased acidification of soils, and increased the rate of
nitrogen transfer from streams and rivers to estuaries and oceans (Vitousek et al., 1997).
Importance of anammox in groundwater While studies have shown that anammox have been
found in ammonium contaminated groundwater (Humbert et al., 2010; Smits et al., 2009), there
are very few studies showing the impact of anammox on groundwater, how anammox bacteria
interact within an aquifer community, and the amount of N2 anammox bacteria are responsible
for moving. Only one other study, Moore et al. (2011), has addressed the rates, community
structure and abundance of anammox bacteria in a contaminated aquifer. Groundwater
ecosystems, very much unlike river or marine systems, have very long exposure times to active
microbial communities, depending on the aquifer characteristics. Combined with the surplus of
ammonium and nitrite/nitrate and low concentrations of organic carbon the slow-growing
anammox of this aquifer may be one of the best sites to study anammox bacteria. Groundwater
systems are vastly important and the United States uses 79.6 billion gallons per day of fresh
groundwater for public supply, private supply, irrigation, livestock, manufacturing, mining,
thermoelectric power, and other purposes (USGS, 2009). The removal of nitrogen from
contaminated aquifers is understudied, and the possibility of remediation through microbial
means must be studied further. This experiment is one of the first studies to couple the rates,
abundance, and geochemical factors of anammox in a groundwater ecosystem.
In order to narrow the scope of the experiment, three focused questions were created.
Each question was accompanied by a hypothesis based on previously available knowledge and
data. To test each hypothesis, three tasks were created. The first research question asked was
20
which nitrogen removal pathway in a contaminated aquifer, anammox or denitrification, would
be the dominant pathway. The question was aimed to explore exactly how important anammox
bacteria are with regards to nitrogen removal. Based on preliminary geochemical data we
hypothesized that anammox would be the dominant removal process in the aquifer due to the
relatively high concentrations of nitrite and ammonium, necessary substrates for the anammox
reaction to proceed. In order to test this, the rates of anammox and denitrification will be
measured using 15
N isotope pairing techniques. The second research question was to determine
which environmental factors would favor anammox in nitrogen removal. We hypothesized that
higher concentrations of ammonium and nitrite will enhance anammox contributions to nitrogen
removal. This hypothesis is based on the conventional thought that more substrate availability
would directly impact the rates of nitrogen removal. To address this, we will compare anammox
rates and gene abundance to relevant geochemical features measured by the United States
Geological Survey along the same vertical gradient. The final research question is a more
ecological focused question, asking which anammox genera are more prevalent within the
groundwater ecosystem. Based on research in a similar contaminated aquifer, we hypothesized
that bacteria assigned to the genus Brocadia would be dominant anammox population in
groundwater ecosystem. We tested this by examining the anammox community structures using
the 16S rRNA and an anammox specific functional gene as genetic markers. By combining these
three results and cross-referencing the data, the mystery of anammox bacterial activity in
contaminated aquifers will be revealed. Thus, I have conducted the examination of the
importance of anammox in groundwater N cycle for my masters thesis research.
21
CHAPTER 2
Molecular and stable isotope characterization of anammox and denitrification in a nitrogen-
contaminated aquifer, Cape Cod, Massachusetts
22
ABSTRACT
Groundwater nitrogen contamination is a widely known problem and a serious concern
for public health and ecosystem management. Denitrification and anaerobic ammonium
oxidation (anammox) are microbial pathways capable of removing nitrate and nitrite from
groundwater ecosystems. Anammox may have a significant role in removing N since organic
carbon in groundwater might be limited to support denitrification. In order to evaluate the
importance of anammox in N removal and to identify anammox hot spots in aquifers,
groundwater microbial communities were examined at a nitrogen contaminated sand and gravel
aquifer on Cape Cod, MA. The U.S. Geol. Survey has constructed multilevel sampling systems
and a database of water quality parameters at this aquifer. Groundwater samples were collected
from multiple depths of a selected sampling well (F575) spanning from 6.2 m above to 6.7 m
below sea level. Molecular methods were used to examine anammox community structures and
to quantify the abundance of anammox and denitrifying bacteria along the depth profile. 15
N
tracer incubation was conducted to measure the rates of anammox and denitrification.
Quantitative PCR (qPCR) assays of hydrazine synthase genes (hzsA) estimated that anammox
bacterial gene abundance ranged from 2.5x103 to 2.5x10
5 copies ng
-1 DNA while abundance of
denitrifying bacteria genes were 1.9x103 to 1.1x10
4 copies ng
-1 DNA based on qPCR of nitrous
oxide reductase genes (nosZ). Anammox rates were found to range from negligible to 6.4 ±
0.048 nmoles of N2 L-1
D-1
while denitrification ranged from 0.38 ± 0.005 to 34.9 ± 0.05 nmoles
of N2 L-1
D-1
. Anammox was estimated to contribute up to 64% of the total N2 production in the
examined aquifer site. Thus, this study clearly demonstrates that the anammox plays a significant
role in microbial nitrogen removal in the aquifer.
23
INTRODUCTION
Groundwater nitrogen contamination is a widely known problem and a serious concern
throughout the United States and other areas of the world. Nitrogen contamination usually occurs
in places of heavy agricultural activities through the use of synthetic fertilizers, or of wastewater
spills from septic tanks and an old sewer system (Savard et al., 2009). In some parts of the world
groundwater is the only source of drinking water, and nitrate contaminated water can cause
methemoglobinemia in infants (also known as Blue baby syndrome), due to the presence of
methemoglobin in the blood (Umezawa et al., 2008). Nitrates and nitrites in groundwater
designated as drinking water have also been linked to several types of cancers in the digestive
tract, a result of the carcinogenic nature and formation of nitrosamines (Harte et al., 1991; Nash,
1993; Khaniki et al., 2008). The United States has issued a Federal maximum containment level
of 10 mg NO3-N L-1
(US Environmental Protection Agency, 1986). In lieu of expensive physical
removal techniques that could harm the ecosystem, there are only two different microbial
pathways that will remove nitrogen from a system biologically via denitrification or anaerobic
ammonium oxidation (anammox). It was long thought that denitrification was the main source of
removal, but now anammox may be more important significant in nitrogen removal than
previously thought (Kuypers et al., 2005).
Denitrification proceeds with nitrate reduction to nitrite, via nitrate reductase. Nitrite
reductase will reduce nitrite to nitric oxide and nitric oxide reductase catalyzes the
transformation to nitrous oxide (Rinaldo et al., 2007). Denitrification is finally completed when
nitrous oxide is reduced to dinitrogen gas via nitrous oxide reductase. A concern with the
denitrification is the production of N2O (300 times more potent than CO2 as a greenhouse gas) as
a result of incomplete denitrification (IPCC, 2001). N2O is a known greenhouse gas, which is
24
detrimental to the environment. Denitrifying bacteria can be detected by targeting the specific
functional genes that facilitate the reduction of nitrate to dinitrogen gas. Because denitrifiers are
taxonomically very diverse, 16S rRNA-based phylogenetic analysis is inappropriate to explore
their diversity. Functional marker genes such as the nitrite reductase genes nirK and nirS, which
code for copper and cytochrome cd1-containing nitrite reductases, respectively, the key enzymes
in the denitrification process (Zumft, 1997), had been targeted to study denitrifier communities
in several different habitats. These include groundwater (Yan et al., 2003), soils (Priemé et al.,
2002; Rösch et al., 2002), river sediments (Taroncher-Oldenburg et al., 2003), marine sediments
(Braker et al., 2000; Braker et al., 2001; Liu et al., 2003) and seawater (Jayakumar et al., 2004).
The gene encoding nitrous oxide reductase (nosZ) has also been used to examine and quantify
N2O respiring denitrifying bacteria in sediments and soils (Henry et al., 2006; Dandie et al.,
2008; Chon et al., 2011).
Anammox bacteria were discovered in a waste water reactor in the 1990’s and are mainly
recognized for converting ammonium and nitrite into dinitrogen gas (Jetten et al., 1998).
Anammox bacteria offer a separate concurrent pathway from denitrifying bacteria and skip the
step of producing N2O gas. They produce N2 through anaerobic oxidization of NH4+ by NO2
-
reduction (Mulder et al., 1995; Van de Graaf et al., 1995). Nitrogen contaminated aquifers are
ripe with ammonium and nitrite central to anammox, and can provide ideal microbial habitats for
anammox bacteria (Miller et al., 2009). The anammox proceeds in bacteria that are members of
the family Brocadiaceae in the phylum Planctomycytes. There are five candidate genera that
have been described from enrichment cultures: Candidatus “Brocadia”, Candidatus “Jettenia”,
Candidatus “Kuenenia”, Candidatus “Anammoxoglobus”, and Candidatus “Scalindua” (Jetten et
al, 2009).
25
16S rRNA genes have been mainly used for detecting and identifying anammox bacteria
in environmental samples (Jetten et al., 2009). However, 16S rRNA is highly conserved, which
can limit their use in examining the full range of anammox diversity in an environment (Hirsh et
al., 2011). Most of the published PCR methods for anammox detection amplified the 16S rRNA
genes of non-anammox bacteria belonging to Planctomycetes as well as anammox bacteria,
although some methods of detection work better than others (Dale et al., 2009; Penton et al.,
2006). Targeting the functional genes of anammox bacteria provides a more reliable method of
detection, especially in environments with low anammox abundance (Kartal et al., 2011). There
are three main catalytic proteins that have been explored for biomarkers for anammox functional
detection; nitrite reductase (nirS: Strous et al., 2006), hydrazine synthase (hzsA gene: Harhangi et
al., 2012) and hydrazine oxidoreductase (hzo gene: Hirsch et al., 2011; Li et al., 2010).
At the MMR study site in Cape Cod, MA a plume of groundwater contamination was
created by discharge of treated wastewater from 1936 to 1995. The study aquifer is a shallow
sand and gravel aquifer that contains a large contamination plume. This site was chosen out of all
the other MMR sites due to its differing biogeochemical nature along the plume gradient. The
plume is at least a kilometer wide, 7 kilometers long and up to 25 meters deep (Miller et al.,
2009). The USGS began sampling groundwater and recording site data in 1990. The resulting
plume is estimated to be around 6 kilometers and 1 kilometer wide. The main sampling site,
F575, is located in the ‘Gravel pit’ approximately 100 yards down-gradient from the initial point
of contamination site, S469 (figure 4). There are several sampling wells spaced in between the
two main sites. This study site has a rich history, with years of geochemical data collection that
has been previously collected by the USGS on the site and the unique physiological
characteristics that a contaminated aquifer provides.
26
Figure 4. Aerial depiction of the contaminant plume (Miller et al., 2008).
27
The first objective of the study was to evaluate the importance of anammox in N removal
and identify anammox hotspots in a N contaminated aquifer by quantifying bacterial abundance
and N production rates. The hotspots should hypothetically be located in depths where the
bacterial abundance and rates are located. The second objective was to determine the
geochemical and microbial factors influencing anammox activities in groundwater by cross-
referencing the abundance/rates with geochemical data. The anammox hotspots of elevated
abundance and rates should be located in the depths where the precursors of the anammox
reaction (ammonium and nitrite/nitrate) are located. The third objective was to compare
anammox community structures under different geochemical conditions by conducting
phylogenetic analyses of 16S rRNA and hzsA genes in different depths. Based on studies from
similar contaminated aquifers, the bacteria in the aquifer should be closely related to bacteria
from the genus Brocadia (Moore et al., 2011).
28
METHODS AND MATERIALS
Sample Collection in August of 2011
All samples were collected from site F575 located approximately 100 meters down-
gradient from the initial treated wastewater seepage infiltration beds. The samples were collected
on duplicate 0.20 micron Sterivex cartridge filters (EMD Millipore, Billerica, MA) from two
adjacent multi-level samplers located within 2 horizontal meters of one another. The access
screens ranged vertically approximately 47ft from an altitude of 24.4 to -21.9ft MSL. The MLS
consisted of 14 screened ports evenly spaced inside the anoxic zone of the aquifer. The ports
cover a wide range of geochemical features, including a nitrate/nitrite zone, an ammonium zone,
and an N-deficient zone with no available inorganic or organic N available (Figure 5). The
groundwater is subsequently pumped via peristaltic pump (Geo pump, Geotech Environmental
Equipment, Denver, CO). The amount of groundwater pumped through each filter is 4 Liters or
until clogged, whichever occurs first. The filters were flushed with an empty syringe to discharge
any extra water inside the filter and immediately placed onto dry ice until transferred to a -80ºC
freezer for further DNA extraction. Triplicate filter samples were also collected for incubation
analyses. Four liters of groundwater were pumped through each filter and submerged inside an
airtight glass container filled completely with site water. The containers were kept on ice until
stored in a 4ºC cold room. The physical and chemical properties of the water samples were
analyzed by Dr. Richard Smith at United States Geological Survey. (USGS, DOI, Reston, VA)
DNA extraction.
The filters were removed from -80ºC storage and the plastic outer cartridge cut open with
a commercial glass cutter (Home Depot). The filter paper was removed and divided into halves.
Small incisions were made along the paper to facilitate shredding. The filter paper, along with
29
800μl of lysis solution (5Prime, Gaithersburg, MD) was added to 2mL microcentrifuge tubes and
incubated at 70ºC for 30 minutes. The contents were decanted into 2mL bead beating tubes (MP
Biomedical, Santa Ana, CA) and shaken at max speed (6.5) for 30 seconds in Model FP120 high
speed bench top cell disrupter (Thermo Fisher Scientific, Waltham, MA). The tubes were
centrifuged at 10,000 x g and 600μl were extracted and placed into new 1.5mL microcentrifuge
tubes. RNAse solution (3U: 5Prime, Gaithersburg, MD) was added to each tube and the mixtures
were incubated at 37ºC for 15 minutes after being inverted several times. Protein precipitation
solution (600μl; 5Prime, Gaithersburg, MD) was added and the tubes were spun at 10,000 xg
until the protein pellet was compact. The supernatant was poured into new tubes and 600μl of
100% cold isopropanol were added. The tubes were incubated at 4ºC overnight. The tubes were
centrifuged at 10,000xg for 15 minutes and then aspirated twice. The DNA pellets were hydrated
with 50μl of DNA hydration solution (5’). The extracted DNA concentration was measured
using a Quant-It™ PicoGreen® dsDNA Assay Kit (Invitrogen, Carlsbad, CA).
PCR amplification, Cloning, and Sanger Sequencing
PCR was performed to amplify anammox bacterial 16S rRNA genes and hzsA genes in
two distinct depths (7.44m MSL and 2.48m MSL). These depths were chosen for the large
quantity of NO2/3(7.44m) and NH4 (2.48m), respectively (Table 1). The 16S rRNA genes of
anammox bacteria were amplified using the primers amx385F and amx820R (Schmid et al.,
2000; 2003). The PCR began with an initial denaturation step of 95°C for 5 min, 30 cycles of
denaturation (94°C for 30 sec), primer annealing (45°C for 30 sec), extension (72°C for 1 min)
and a final extension step for 5 min at 72°C. Detection of hzsA genes was conducted using the
primers hzsA526F and the hzsA1857R (Harhangi et al., 2012). The hzsA PCR mixture was a
30
25μl reaction containing 12.5μl GoTaq Green Master Mix (Promega, Madison, WI), 1μl of DNA
as the template, and 1μl of each primer (10 μM). The thermal profile started with a denaturation
step of 5 min at 96°C, followed by 30 cycles of denaturation (1 min at 96°C), primer annealing
(1 min), and extension (1.5 min at 72°C), and finally a last extension step of 5 min at 72°C
(Harhangi et al., 2012). To differentiate the PCR products, gel electrophoresis on agarose gel
(1.0%) was used, which were then purified using the Wizard® SV Gel PCR clean-up System
(Promega, Madison, WI) using the manual instructions included. The purified products were
cloned using the pGEM®
-T Easy Vector Systems PCR Cloning Kit (Promega, Madison, WI).
Clone libraries for the M02-10P port (10.5m depth) and M01-01PT port (15.6m depth) 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 Search (http:/www.ncbi.nih.gov) was used to determine which sequences were
closely related. The sequences, along with reference sequences that are closely related, were
aligned using clustalW (http:/www.ebi.ac.uk/clustalw/). MEGA version 5.0
(http://www.megasoftware.net/) was used to create neighbor-joining trees with 1000x bootstrap
of anammox 16S rRNA and hzsA gene sequences.
Quantitative PCR of functional genes in anammox, denitrifying and nitrifying bacteria
A total of 13 DNA samples extracted from descending sequential depths of groundwater
were used to quantify the abundance of anammox, denitrifying and nitrifying bacteria (Table 2).
All of the qPCR was conducted with the GoTaq qPCR Master Mix Green (Promega) and a 7500
Real Time PCR System (Applied Biosystems, Foster City, CA). The anammox bacteria
abundance was quantified by the qPCR of the hzsA using the primers hzsA1597F (5’-
WTYGGKTATCARTATGTAG-3’) and hzsA1829R (5’-TCATACCACCARTTGTA-3’) as
described by Harhangi et al., (2012). The hzsA qPCR started with an initialization step of 3 min
31
at 96°C, followed by 50 cycles of denaturation (30 sec at 96°C), primer annealing (45 sec at
53°C), and extension (35 sec at 72°C), and finally a signal detection step at 75°C for 35 sec. For
the dissociation curve, the temperature ramped from 55°C to 95°C (at the instrument default
rate). Denitrifying bacterial abundance was measured by targeting nosZ for N2O respiring
bacteria while nirS and nirK genes were quantified for nitrite respiring denitrifiers. The nosZ
qPCR was conducted with the primers nosZ2F (5’ - CGCRACGGCAASAAGGTSMSSGT - 3’)
and nosZ2R (5’ - CAKRTGCAKSGCRTGGCAGAA - 3’) (Henry et al., 2006). The nosZ qPCR
began 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 fluorescent signal detection step for 35 sec at
80ºC. For the dissociation curve, the temperature ramped from 55°C to 95°C (at the instrument
default rate). Q-PCR of nirK gens was performed with the primers nirK-q-F (5’-
TCATGGTGCTGCCGCGYGA) (Santoro et al., 2006) and nirK1040 (Henry et al., 2004), while
nirS qPCR was conducted with the primers nirS1F (Braker et al., 1998) and nirS-q-R (5’-
TCCMAGCCRCCRTCRTGCAG) (Mosier et al., 2010). The nirS qPCR began with an initial
denaturation step for 15 minutes at 95ºC followed by 38 cycles of 95ºC for 15 sec, 62.5ºC for 30
sec, 72ºC for 30 sec, and a fluorescent signal detection step for 35 sec at 84ºC. For the
dissociation curve, the temperature ramped from °C to 95°C (at the instrument default rate). The
nirK qPCR began with an initial denaturation step for 15 minutes at 95ºC followed by 9
touchdown cycles of 95ºC for 15 sec, 68ºC for 30 sec (including an auto-increment of -1.0),
81.5ºC for 30 sec, followed by 35 cycles of 95ºC for 15 sec, 60ºC for 60 sec, 81.5ºC for 30 sec,
and a fluorescent signal detection step for 10 sec at 86ºC. For the dissociation curve, the
temperature ramped from 55°C to 95°C (at the instrument default rate). The Eubacterial 16S
qPCR for total bacterial abundance used the primers EU 341F and 685R (Muyzer et al., 1993)
32
The Eubacterial 16S qPCR began with an initial denaturation step for 10 minutes at 95ºC
followed by 40 cycles of 55°C for 30 sec and a fluorescent signal detection step for 35 sec at
72ºC. For the dissociation curve, the temperature ramped from 55°C to 95°C (at the instrument
default rate). Abundance of aerobic ammonia oxidizing bacteria was also quantified by targeting
ammonium monooxygenase genes (amoA) using the primers AMO1F (5’ - GGGG
TTTCTACTGGTGGT - 3’) and AMO2R (5’ -CCCCTCKGSAAAGCCTTCTTC - 3’)
(Rotthauwe et al., 1997). The initial denaturation began at 95ºC for 10 min followed by 50 cycles
at 95ºC for 15 sec, primer annealing at 53ºC for 45 sec, and extension at 72ºC for 30 sec and a
fluorescent signal detection step for 35 sec at 86ºC. For the dissociation curve, the temperature
ramped from 55°C to 95°C (at the instrument default rate). All of the PCR products were run on
1% agarose gels along with a 1kb DNA ladder to confirm the correct size of PCR products. All
of the qPCR reactions were completed in triplicates. PCR specificity and primer-dimer
formation were monitored with analysis of disassociation curves. QPCR standards of each gene
were generated via serial dilution of the PCR product carrying the targeted gene.
15N-Tracer Incubation Experiments
Anammox and denitrification rates in groundwater communities were measured using a
modified method of isotope pairing method described by Thamdrup and Dalsgaard (2002). The
filter cartridges with concentrated biomass were split open using a commercial glass cutter, and
the whole membrane filters were aseptically removed and placed inside 12 mL Exetainer tubes
and completely submerged beneath 2mL of the corresponding site water. The Exetainer tubes
were capped, sealed air-tight with new rubber septa, and flushed for 10 minutes with Helium gas.
The Exetainer tubes were incubated at room temperature overnight to eliminate any residual
33
NO3-/NO2
-. After overnight incubation, the presence of residual NO3
-/NO2
- were examined using
Vanadium (III) reduction and chemiluminescent detection developed by Braman and Hendrix
(1989) with an Antec 7020 nitric oxide analyzer (Antek Instruments, Houston, TX). After the
overnight incubation, the tubes were vacuumed for 5 minutes and flushed completely with
Helium gas for 5 minutes. The tubes had Helium-flushed stock solutions of Na15
NO3 (99.5
atm%; Cambridge Isotope Laboratory, Andover, MA) and 14
NH4SO4 (98 atm%; Sigma-Aldrich,
St. Louis, MO) added to give a final concentration of 0.1 mM of 15
NO3 + 14
NH4. The incubations
proceeded forward in duplicate and were collected in three time points: 0hr, 6hr, and 12hr. At
each collection, the incubations were stopped completely with the addition of 0.5 mL of 4M
ZnCl2 solution. The samples were stored upside down in water until run simultaneously on a
continuous flow isotope ratio mass spectrometer (Thermo Finnigan Delta V; Thermo Scientific,
Waltham, MA) connected to a gas bench interface. The production rates of 29
N2 and 30
N2 were
measured simultaneously in each sample tube. The 29
N2 production is indicative of anammox
activity, while the 30
N2 production shows only denitrification activity based on the gas
measurements from 15
NO3- and
14NH4
+ incubation experiments. The rates of
29N2 and
30N2
production were calculated using a modified method of Thamdrup and Dalsgaard (2002).
Statistical analyses
To compare the correlation between rates, abundances, and geochemical parameters, two
Canonical Correspondence Analysis (CCA) plots were created to demonstrate possible
statistically significantly correlated variables. To further test this, Pearson product-moment
correlation coefficient analysis was utilized via Sigma plot 11.0 (Systat Software inc., Germany).
The Pearson analysis will measure the strength of the linear dependence between two variables.
34
RESULTS
Vertical Geochemical distribution
The USGS catalogued the geochemistry along the MLS ports sampled for the project.
The geochemical data is listed according to decreasing depth and lower altitude MLS ports in
Table 1. The dissolved oxygen concentration was below detection throughout the studied MLS
ports. The shallowest MLS ports (altitudes 7.40 – 6.20m MSL) contain the largest concentrations
of NO3- and NO2
-. The highest concentration of NO3
- is 132 µM at altitude 7.44m MSL and the
lowest is negligible. The NO3- decreases gradually to a N-deficient zone where very little organic
or inorganic N exists. After N-deficient zone, there was an NH4+ spike (ranging from 7 to 38
µM) from altitudes 0.95 to -3.63m MSL that persisted through four MLS ports. NO3- increased
steadily in altitudes below -3.63m MSL (30 µM+).
35
Sam
ple
Po
rtA
ltit
ud
eC
hlo
rid
eN
itri
teN
itra
teS
ulf
ate
Am
mo
niu
mM
agn
esiu
mT
DN
Ino
rgan
ic N
Org
anic
ND
OC
(met
ers
MS
L)
(uM
)(u
M)
(uM
)(u
M)
(uM
)(u
M)
(uM
)(u
M)
(uM
)(u
M)
FS
W 5
75-M
02-0
9Y7.
4418
78
132
120
073
158
140
1812
7
FS
W 5
75-M
02-1
0P6.
8120
68
9114
70
7410
999
1012
4
FS
W 5
75-M
02-1
1Gn
6.2
246
1264
135
061
8576
911
0
FS
W 5
75-M
02-1
2R5.
5651
60
113
90
485
14
84
FS
W 5
75-M
02-1
3Bu
4.93
498
00
129
045
40
488
FS
W 5
75-M
02-1
4Bk
4.29
475
00
128
041
50
593
FS
W 5
75-M
02-1
5W3.
6643
40
014
00
386
06
97
FS
W 5
75-M
01-0
1PT
2.48
312
00
159
036
70
791
FS
W 5
75-M
01-0
2Gn
T0.
9532
60
015
67
3915
87
84
FS
W 5
75-M
01-0
3RT
-0.5
728
60
116
227
3732
293
89
FS
W 5
75-M
01-0
4Bu
T-2
.11
265
00
140
3837
4038
285
FS
W 5
75-M
01-0
5Bk
T-3
.63
396
00
106
3044
3230
263
FS
W 5
75-M
01-0
6WT
-5.1
650
20
067
1943
2519
656
FS
W 5
75-M
01-0
7O-6
.69
608
030
610
4835
305
46
Table 1. Geochemical vertical distribution of F575 sampling site
36
Abundance of anammox, denitrifying, and nitrifying bacteria in the aquifer
Abundance of anammox and denitrifying bacteria was measured based on qPCR of
functional genes. The qPCR assays of hzsA genes estimated that anammox bacterial gene
abundance ranged from 2.5x103 to 2.5x10
5 copies ng
-1 DNA (Table 2). The highest copy number
of hzs genes was found in the ammonium-rich 2.5 MSL altitude and the lowest in the
geochemical N-deficient zone altitude between 6.8 and 4.3 MSL (Figure 5). The abundance of
nosZ genes quantified in the aquifer ranged from 1.9x103 to 1.1x10
4 copies ng
-1 DNA. The
largest quantity of N2O respiring denitrifying bacteria containing the nosZ gene appears at
altitude 4.3m MSL, while the lowest abundance appears in the N-deficient zone altitudes (Figure
5). The qPCR results of nirK (9.23 x 100 to 2.64 x10
3 copies ng
-1 DNA) genes and nirS (3.0 x10
2
to 2.17 x105 copies ng
-1 DNA) genes were combined to estimate the abundance of NO2
- respiring
denitrifiers (Table 2). The highest number of nir genes was found at the 2.5m MSL altitude and
the lowest in the N-deficient zone (Table 2). The qPCR of amoA genes quantified AOB
abundance ranging from 1.6 x10-2
to 5.4x101 copies ng
-1 DNA (Table 2). The highest abundance
of AOBs were discovered in the ammonium zone (2.5 MSL) and no AOBs were found in several
ports, including (7.4 MSL to 6.2 MSL and -2.71 MSL to -3.63 MSL) (Table 2). Eubacterial 16S
rRNA gene abundance ranged from 2.6 x106 to 1.8 x10
7 copies ng
-1 DNA. The abundance of
anammox bacteria ranged from 1.1 to 40.5% of all bacteria detected by eubacterial 16S rRNA
qPCR. Bacteria containing the nirS/nirK genes ranged from 0.25 to 25.6% of all bacteria
detected, and bacteria containing nosZ genes only ranged from 0.03 to 1.56% of all bacteria
(Table 3).
37
Alti
tude
am
oAam
oA s
tdev
eub.
16S
eub.
16S
std
evni
rKni
rK s
tdev
nirS
nirS
std
evhz
shz
s st
dev
nosZ
nosZ
std
ev
copy
no.
per
ng
DN
Aco
py n
o. p
er n
g D
NA
copy
no.
per
ng
DN
Aco
py n
o. p
er n
g D
NA
copy
no.
per
ng
DN
Aco
py n
o. p
er n
g D
NA
7.44
2.10
E-0
21.
39E
-03
1.06
E+
076.
30E
+05
5.21
E+0
25.
45E
+01
1.40
E+0
43.
74E
+02
7.09
E+0
46.
79E
+03
4.84
E+0
32.
20E
+03
6.81
1.60
E-0
21.
31E
-03
9.10
E+0
71.
54E
+06
9.81
E+0
03.
28E
+00
2.98
E+0
28.
38E
+01
2.46
E+0
34.
54E
+02
1.86
E+0
32.
90E
+02
6.2
1.99
E-0
21.
75E
-04
9.90
E+0
61.
91E
+05
6.39
E+0
17.
47E
+01
7.30
E+0
41.
63E
+04
1.45
E+0
51.
12E
+04
1.89
E+0
32.
70E
+02
5.56
1.34
E+0
02.
28E
+00
1.87
E+0
83.
81E
+06
4.16
E+0
21.
56E
+02
1.19
E+0
56.
79E
+03
6.54
E+0
45.
35E
+03
3.48
E+0
34.
61E
+02
4.93
2.58
E+0
04.
43E
+00
7.53
E+0
65.
00E
+05
2.35
E+0
24.
41E
+01
8.21
E+0
42.
77E
+03
5.11
E+0
47.
03E
+03
4.94
E+0
38.
62E
+02
4.29
1.10
E-0
11.
54E
-01
3.44
E+0
71.
16E
+05
2.42
E+0
24.
03E
+02
2.12
E+0
44.
24E
+03
1.08
E+0
41.
45E
+03
7.38
E+0
36.
28E
+02
3.66
3.88
E-0
11.
32E
-01
1.44
E+0
85.
70E
+05
2.64
E+0
35.
39E
+02
1.43
E+0
54.
83E
+04
1.02
E+0
52.
25E
+04
1.05
E+0
42.
58E
+03
2.48
1.05
E+0
11.
65E
+01
1.80
E+0
81.
42E
+05
6.44
E+0
28.
47E
+01
2.17
E+0
55.
87E
+03
2.44
E+0
54.
61E
+04
5.26
E+0
32.
39E
+02
0.95
3.59
E-0
23.
17E
-02
8.61
E+0
79.
45E
+05
2.11
E+0
21.
76E
+02
1.31
E+0
53.
11E
+03
1.44
E+0
54.
15E
+03
5.18
E+0
31.
46E
+02
-0.5
74.
40E
+01
7.60
E+0
11.
66E
+08
2.30
E+0
61.
76E
+02
2.92
E+0
21.
41E
+05
1.75
E+0
48.
10E
+04
1.76
E+0
42.
01E
+03
1.59
E+0
2
-2.1
11.
99E
-02
8.15
E-0
41.
00E
+07
1.58
E+0
53.
45E
+02
1.94
E+0
27.
44E
+04
2.80
E+0
34.
83E
+04
6.79
E+0
32.
16E
+03
1.74
E+0
2
-3.6
36.
15E
-02
7.00
E-0
22.
56E
+06
3.57
E+0
42.
21E
+01
1.61
E+0
11.
30E
+04
8.98
E+0
29.
20E
+04
8.95
E+0
31.
94E
+03
1.88
E+0
2
-5.1
6N
/AN
/AN
/AN
/AN
/AN
/AN
/AN
/AN
/AN
/AN
/AN
/A
-6.6
95.
38E
+01
2.02
E+0
11.
91E
+07
1.47
E+0
59.
23E
+00
6.10
E-0
12.
07E
+04
8.09
E+0
21.
35E
+04
4.74
E+0
35.
17E
+03
1.03
E+0
2
Table 2. Copy numbers per ng DNA of functional genes and 16S RNA
38
Table 3. Percent abundance of bacteria that contain specific functional gene
Altitude nirK nirS hzsA nosZ
meters (MSL) % % % %
7.44 0.15 4.1 17.4 1.31
6.81 0.01 0.2 1.2 1.56
6.2 0.02 15.5 28.1 0.40
5.56 0.09 25.6 11.7 0.75
4.93 0.04 14.6 7.4 0.88
4.29 0.11 3.3 1.1 1.14
3.66 0.17 9.1 5.7 0.67
2.48 0.07 22.5 24.6 0.55
0.95 0.04 17.6 17.6 0.70
-0.57 0.01 2.4 1.2 0.03
-2.11 0.03 6.1 3.2 0.18
-3.63 0.01 6.6 40.5 0.98
-6.69 0.00 2.3 1.6 0.53
39
Potential rates of anammox and denitrification Based on 15
N Isotope Pairing
In order to determine the effectiveness of microbial activity on N removal in the aquifer,
the potential rates of anammox and denitrification were measured using 15
NO3- and
14NH4
+. Any
29N2 production was considered from anammox activity, while
30N2 production was considered
from denitrification (Figure 5). The 29
N2 production rates varied from as high as 6.39 nmol L-1
D-
1 to as low as below detection. The lowest rate of
29N2 production occurred at altitude 6.2m MSL,
a nitrate rich, zero-ammonium depth. The location of the highest rate of 29
N2 production is
altitude -5.2m MSL, where the nitrate plume begins to appear. The second highest rate of 29
N2
production is altitude 2.5m MSL, the transition zone between the N-deficient zone and NH4+
zone and also the location of the highest abundance of the anammox bacteria via the hzsA gene.
The average rate of 29
N2 production inside the ammonium zone is 3.73 ±0.038 nmol L-1
D-1
,
while the average rate of production outside of the ammonium zone is 0.40 ±0.007 nmol L-1
D-1
.
The 30
N2 production rates varied from as high as 34.9 nmol L-1
D-1
to as low as 0.38 nmol L-1
D-1
.
The lowest rate of 30
N2 production occurred in the N-deficient zone, while the highest 30
N2
production rate appears within the ammonium cloud (Figure 5). By comparing the production of
29N2 to the total N2 produced, an estimate of % anammox is calculated. The altitude with the
largest impact of anammox is in the N-deficient zone at altitude 4.9m MSL, where the anammox
constitutes 64% of total N2 production. The altitude with the least impact is in the ammonium
zone at altitude 0.95 MSL, where anammox is responsible for only 3% of total N2 production.
Overall, the rates of 30
N2 production are much higher in the ammonium and nitrate zones than in
the N-deficient zone. Both the rates and gene abundance show decline in the N-deficient zone,
and both show marked increases within the zones of either nitrate or ammonium. Based on the
gene abundance and rate measurements, the ratios of rates per gene copy can be calculated at
40
each depth by dividing the rate by the copy number per ng of DNA extracted. Ratio of anammox
rate over hzo gene abundance ranged from 0 to 289 fmol N2 per gene copy number. Ratio
denitrification rate over nosZ gene abundance ranged from 7.6 x101 to 1.4 x 10
4 fmol N2 per gene
copy number (Figure 6).
41
Figure 5. Copy number per ng DNA, N2 production rates and geochemical parameters arranged
vertically along the MLS in the aquifer. Bacterial abundance was not available from the -5.2m
MSL altitude.
42
Figure 6. Ratio of rates to gene abundance, N2 production rates and geochemical parameters
arranged vertically along the MLS in the aquifer. Bacterial abundance was not available from the
-5.2m MSL altitude.
43
Table 4. Potential rates compared to percent anammox
Altitude
Mean N2
production via
AMX
Stdev N2
production via
AMX
Mean N2
production via
DNF
Stdev N2
production via
DNF Total N2 production Percent Anammox
meters (MSL) nmol N2 L-1 d-1 nmol N2 L-1 d-1 nmol N2 L-1 d-1 %
6.2 0 0 20.0 4.72 20.0 0
5.56 0.28 0.1 1.8 1.70 2.1 13.3
4.93 0.67 0.2 0.4 0.12 1.0 64.0
4.29 0.46 0.3 0.7 0.38 1.2 38.6
3.66 0.60 0.3 1.8 1.90 2.4 24.8
2.48 5.58 1.1 12.8 2.06 18.4 30.3
0.95 1.04 0.1 26.6 8.67 27.6 3.8
-0.57 2.27 0.7 15.0 3.36 17.3 13.2
-2.11 3.71 1.5 31.1 1.25 34.8 10.6
-3.63 3.26 1.4 15.9 3.92 19.1 17.0
-5.16 6.39 2.8 34.9 4.23 41.3 15.5
-6.69 3.91 0.7 17.1 3.58 21.0 18.6
44
Statistical analysis of Rates, bacterial abundance and geochemical parameters
The statistical analyses using Pearson product-moment correlation coefficients between
anammox and denitrification rates against geochemical data and abundance data against
geochemical data are tabulated in tables 4 and 5, respectively. In order to determine which
variables would be good candidates for statistical inference two Canoco Correspondent Analysis
(CCA) plots were created, figures 7 and 8, respectively. Rates and bacterial abundances are
tabulated in Table 7.
The CCA plot showing possible correlations between bacterial abundance and
geochemical variables displays a positive correlation between abundance of nirS and hzsA genes,
and inorganic N compounds such as ammonium and nitrite (Figure 7). However, Pearsons
correlation analysis shows no significance between the gene abundances and ammonium and
nitrite. Alternatively, there are significant negative relationships between nirK and nosZ gene
abundances to TDN and inorganic N, which maintaining a positive correlation with organic
carbon.
CCA also demonstrates possible correlations among anammox and denitrification rates,
and geochemical variables (Figure 8). Anammox rates are significantly negatively correlated
with organic carbon. Percent anammox showed is significant negative correlation with TDN and
inorganic N. Denitrification rates are significantly correlated with the concentration of
ammonium (Table 6).
The correlation between potential rates of anammox and denitrification the respective
genes abundances responsible for the N2 production (ie; hzs vs anammox rate, and denitrification
rate vs nosZ, nirK, and nirS) were tested using Pearsons product-moment to determine whether
any significant trends occurred (Table 7).
45
Figure 7. CCA plot showing possible correlations between bacterial abundance and geochemical
variables.
46
Table 5. Pearson product-moment correlation coefficient analysis between the log of AMX/DNF
bacterial abundance and geochemical variables.
Nitrite Nitrate Ammonium TDN Inorganic N Organic N DOC
hzsA r 0.03 -0.24 -0.24 -0.30 -0.35 0.39 0.42
p 0.931 0.478 0.477 0.363 0.296 0.232 0.202
nosZ r -0.34 -0.43 -0.57 -0.72 -0.76 0.29 0.18
p 0.304 0.186 0.070 0.012 0.006 0.390 0.603
nirS r -0.16 -0.40 -0.39 -0.55 -0.60 0.37 0.55
p 0.645 0.227 0.239 0.077 0.052 0.265 0.083
nirK r -0.27 -0.54 -0.31 -0.65 -0.68 0.21 0.61
p 0.422 0.089 0.362 0.030 0.021 0.533 0.045
47
Figure 8. CCA plot showing possible correlations between bacterial abundance and geochemical
variables..
48
Table 6. Pearson product-moment correlation coefficient analysis between anammox and
denitrification rates, % anammox and geochemical variables.
Nitrite Nitrate Ammonium TDN Inorganic N Organic N DOC
AMX Rate r -0.34 -0.23 0.42 0.00 0.02 -0.16 -0.63
p 0.283 0.482 0.179 0.991 0.953 0.623 0.029
DNF Rate r 0.14 0.15 0.59 0.51 0.52 0.04 -0.36
p 0.675 0.642 0.044 0.088 0.085 0.905 0.248
AMX per cell rate r -0.16 0.28 0.00 0.20 0.22 -0.16 -0.74
p 0.650 0.407 0.992 0.561 0.524 0.631 0.009
DNF per cell rate r 0.21 0.21 0.54 0.51 0.54 -0.22 0.08
p 0.542 0.536 0.090 0.111 0.089 0.511 0.825
% AMX r -0.38 -0.38 -0.34 -0.60 -0.60 -0.15 0.04
p 0.224 0.220 0.276 0.039 0.041 0.636 0.899
49
Table 7. Pearson product-moment correlation coefficient analysis between anammox and
denitrification rates and relevant bacterial abundances.
hzs nosZ nirS nirK Eub 16S
AMX Rate r 0.295 - - - -0.04
p 0.378 - - - 0.906
DNF Rate r - -0.548 -0.0361 -0.378 -0.28
p - 0.0811 0.916 0.252 0.397
50
Phylogeny of Anammox Bacterial communities
In order to elucidate anammox community structure for the aquifer, two depths were
selected for anammox community analysis based on 16S rRNA and hzsA genes. Each depth was
selected specifically for their geochemical properties and the effect on the community. The first
altitude, 7.44 MSL, contains a surplus of TDN including quantities of NO3- and NO2
-, while the
second altitude, 2.48 MSL, has very low TDN and is in the transition zone between the N-
deficient zone and ammonium-rich plume. Primarily, anammox bacterial 16S rRNA genes were
targeted to determine which genera were dominant. Based on the anammox 16S rRNA detection,
the sequences obtained are closely related to the terrestrial Candidatus Kuenenia spp., and
Candidatus Brocadia spp. and clustered further away from Candidatus Anammoxoglobus and
Candidatus Jettenia spp.. The “Deep” (2.48m MSL) community and “Shallow” (7.44m MSL)
community are 5 meters apart, yet contain 2 slightly distinct bacterial communities (96.0-99.8%
similarity). The Deep anammox 16S rRNA community and Shallow community clustered away
from established anammox species. The communities are most similar to the Candidatus
Kuenenia spp. (Shallow: 94.5-95.0%; Deep: 94.4-94.7%) and Candidatus Brocadia spp.
(Shallow: 95.2- 95.8%; Deep: 95.0-95.6%) while less similar from Candidatus Jettenia spp.
(Shallow: 93.3-93.7%; Deep: 93.1-93.5%) and other anammox species (Figure 10). The hzsA
genes were independently used to compare the anammox community structures in both MLS’s.
Anammox bacteria were similar to Candidatus Brocadia spp., while maintaining a large distance
from the Candidatus Kuenenia spp.. The “Deep” (2.48m MSL) hzsA community and “Shallow”
(7.44m MSL) hzsA community are 5 meters apart, yet contain 2 distinct bacterial communities
(87.6-98.1% similarity). The Deep hzsA community and the Shallow hzsA community both
cluster away from other known anammox genera. The Cape Cod sequences related relatively
51
closest to Candidatus Brocadia spp. (Shallow: 83.4-83.6%; Deep: 82%) and Candidatus Jettenia
spp. (82.0-82.4%) than the Candidatus Kuenenia spp. (Shallow: 79.2-79.3%; Deep: 79.3-79.8%)
(Figure 9). Both phylogenies clustered away from the out group Candidatus Scalindua spp., the
marine sediment genera, which was not expected to be in a freshwater aquifer. The phylogenetic
tree of hzsA genes used 15 sequences from the 7.44m depth and 16 sequences from the 2.48m
depth. The 16S rRNA tree used 11 sequences from the 7.44m depth and 5 sequences from the
2.48m depth. The discrepancy between the numbers of sequences between the two trees lays in
the failure of the anammox specific 16S rRNA gene primers to only create true anammox
sequences. In the anammox 16S rRNA tree, both depths align close to the groundwater
communities in the UK (Smits et al., 2009), and are similar, albeit much less so, to sediment
samples taken from Cape Fear River estuary sediment (Dale et al., 2009). Comparative analysis
of anammox communities using the hzsA gene is difficult due to lack of hzsA sequence databases
because the primers have not been available for long.
52
Figure 9. Phylogenetic tree of hzsA gene sequences detected from two different depths.
F575 HZS 09Y 28
F575 HZS 09Y 30
F575 HZS 09Y 05
F575 HZS 09Y 12
F575 HZS 09Y 26
F575 HZS 09Y 06
F575 HZS 09Y 10
F575 HZS 09Y 07
F575 HZS 09Y 29
F575 HZS 09Y 02
F575 HZS 09Y 03
F575 HZS 09Y 27
F575 HZS 01PT 38
F575 HZS 01PT 04
F575 HZS 01PT 05
F575 HZS 01PT 08
F575 HZS 01PT 39
F575 HZS 01PT 33
F575 HZS 09Y 11
F575 HZS 09Y 31
F575 HZS 01PT 10
F575 HZS 01PT 37
F575 HZS 01PT 03
F575 HZS 01PT 01
F575 HZS 01PT 40
F575 HZS 01PT 02
F575 HZS 01PT 36
F575 HZS 01PT 34
F575 HZS 01PT 16
F575 HZS 01PT 35
F575 HZS 09Y 15
Candidatus Brocadia anammoxidans
Candidatus Brocadia fulgida
JN703690.1| Anammox bacterium enrichment culture clone BS23
Candidatus Jettenia asiatica
AB365070.1| Planctomycete KSU-1
Candidatus Anammoxoglobus propionicus
Candidatus Kuenenia stuttgartiensis
Candidatus Scalindua
99
52615399
87
82
60
67
73
53
0.01
53
Figure 10. Phylogenetic tree of anammox bacterial 16S rRNA sequences detected from two
different depths.
F575 09Y 21
F575 09Y 23
F575 09Y 06
F575 09Y 19
F575 09Y 17
F575 09Y 08
F575 09Y 04
F575 09Y 01
F575 09Y 14
AJ871747.1| UK Groundwater
F575 09Y 22
F575 MO1 01PT 04
F575 09Y 24
F575 MO1 01PT 48
F575 MO1 01PT 35
F575 MO1 01PT 07
F575 MO1 01PT 27
FJ490112.1| Cape Fear River estuary
Candidatus Kuenenia stuttgartiensis
GQ359859.1| CANON Reactor
Candidatus Brocadia frugida
Candidatus Brocadia anammoxidans
Candidatus Anammoxoglobus propionicus
Candidatus Jettenia asiatica
Candidatus Scalindua wagneri
Candidatus Scalindua sorokinii
Candidatus Scalindua brodae
Planctomyces maris
100
99
95
70
95
99
93
93
64
83
76
97
66
67
57
0.005
54
DISCUSSION
Anammox and denitrifier Quantification
Anammox bacterial quantification was mainly conducted with fluorescent in situ
hybridization of 16S rRNA (FISH) and qPCR of 16S rRNA genes (Schmid et al., 2005).
However, non-specific detection of 16S rRNA genes may lead to overestimation of anammox
bacterial abundance in various environmental samples and similar flaws (Amano et al., 2007;
Dale et al., 2009; Li et al., 2010). The functional genes including nirS, hzo and hzs have been
recently used as alternative genetic markers to detection and quantify anammox bacteria (Strous
et al., 2006; Harhangi et al., 2012; Hirsch et al., 2011; Li et al., 2010). The use of the hzsA gene
as a functional marker for anammox bacterial detection is more advantageous than using
hydrazine oxidase (hzo), as a single anammox bacterial cell carries a single copy of hzsA genes
unlike hzo (Harhangi et al., 2012). The hzsA genes are unique for anammox bacteria while
homologues genes of the hzo or nirS genes can be found in in non-anammox bacteria (Li et al.,
2010; Schmid et al., 2008). Because the abundance of the hzsA gene maintains a 1:1 ratio with
the number of anammox cells, it is possible to compare the abundances of hzsA genes alongside
the total cell counts provided from the FISH enumerations or hzsB quantification (Table 8).
55
Table 8. Estimated gene abundance in 1L of groundwater samples.
56
Overall, the abundance of anammox bacteria calculated from hzsA quantification
(6.5x105 to 7.6x10
7 copies L
-1) proved to be lower than in the freshwater Pearl river (via hzsB
quantification:1.3 x108 to 2 x10
9 copies g
-1)(Wang et al., 2012), the Golfo Dulce (via FISH:1.4
x1010
- 8.4 x1010
anammox cells L-1) (Schmid et al., 2007), but an order of magnitude less than
the numbers found via FISH in the Black Sea (Kuypers et al., 2003) and the Benguela OMZ
(Kuypers et al., 2005).
Abundance of denitrifiers in the aquifer was measured using qPCR of nirS, nirK and
nosZ genes. The nirS gene copies were one to two-fold lower than those measured in the
Arabian Sea Oxygen Minimum zone (1.9 to 5.6 × 108 L
−1 seawater) (Jayakumar et al., 2009).
However, nirK abundance was comparable to that of the Arabian Sea OMZ (1 x 104 to 4 x 10
5
L−1
seawater) (Jayakumar et al., Unpublished). The nosZ quantification was surprisingly low,
given the relatively (relative to anammox) high denitrification rates in the aquifer. The nosZ
relative abundance compared to total 16S rRNA copy numbers was around 0.5%, similar to
denitrifier abundance described in several different environments (Kandeler et al., 2006;
Henderson et al., 2010; Henry et al., 2006). Eubacterial 16S rRNA genes ranged from 2.44 x107
to 3.11 x108, an order of magnitude higher than the abundance found in a similar aquifer (Moore
et al., 2011). AmoA copy numbers were much lower when compared to Eastern Tropical South
Pacific (ETSP) and the Arabian Sea OMZs (Bouskill et al., 2012). This could be due to the very
low concentration of dissolved oxygen in this segment of the aquifer, which would inhibit
ammonium oxidation. When correlation analysis was conducted, several obvious patterns were
sought after, though for little gain. Dissolved organic carbon, important for the denitrification
showed no clear trend when correlated to the nosZ abundance, but was significantly correlated
with nirK abundance. Oddly enough, both nirK and nosZ abundance were negatively correlated
57
with DIN and TDN, which is a surprising finding. Nitrate is a necessary component of the
denitrification process and it is counterintuitive that the correlations would be negative.
Anammox gene abundance (hzs) showed no clear trend when compared to dissolved organic
nitrogen and other environmental parameters.
When functional gene abundances are compared to total eubacterial 16S rRNA gene
abundances, the hzsA gene was found in up to 40% of all bacterial cells detected. However, in
the depths where the hzsA gene relative abundance was highest, the 29
N2 production rates were
not always elevated as a result. This would further support that idea that potential rates do not
increase with abundance, and possibly that the hzsA qPCR could detect anammox bacteria that
are simply inactive, or less efficient in N2 production.
N2 Potential Rates via Anammox and Denitrification
The potential rates of N2 production varied greatly among the ports, based on the
geochemical availability of DON and DIN. Ports located in the geochemical N-deficient zone
clearly demonstrated much lower rates of N2 production than ports located in either the
nitrate/nitrite cloud or the ammonium plume (Figure 5). The anammox rates in the N-deficient
zone ranged from 0 to 0.67 ±0.01 nmol L-1
D-1
. Outside of the N-deficient zone, the rates ranged
from 1.04 ±0.01 nmol L-1
D-1
to 5.58 ±0.05 nmol L-1
D-1
. Anammox potential rates in similar
environments are much higher, including a study with 751 nmol L-1
d-1
from a similar
contaminated aquifer (Moore et al., 2011), 72 - 438 nmol L-1 d-1 from anoxic bottom water
(Dalsgaard et al., 2003), and other anoxic marine systems such as the Benguela upwelling system
(96 – 192 nmol L-1
d-1
)(Kuypers et al., 2005) . The most similar rate findings are in the anoxic
waters off northern Chile where anammox rates ranged from 4 nmol L-1
d-1
up to 16.8 nmol L-1
58
d-1
. Potential denitrification rates in the aquifer are much higher compared to anammox potential
rates. The highest rates are found in the ammonium zone, with the lowest rates found in the N-
deficient zone, a common theme found throughout the experiment. Denitrification is by far the
greater producer of total N2 in the aquifer. At the nitrate-rich depth, denitrification actually
contributes 100% of all N2. In the same aquifer, Smith et al. (2004) used a one dimensional
transport model to elucidate a denitrification rate and determined that in a provided nitrate-rich
depth, the rate of N2 production was 6.5-10 nmol N2 L-1
d-1
, which is comparable, albeit lower
than the measured potential rates in this experiment. DeSimone and Howes (1996) used several
different methods for determining in situ denitrification rates in the same aquifer (though from a
different area) and determined rates to be from 100-350 nmol N2 L-1
d-1
, an order of magnitude
higher than rates established in this study. In a wastewater-contaminated karst aquifer in Florida,
Griggs et al. (2003) found an average rate of 2 x103 nmol N2 L
-1 d
-1, several orders of magnitude
greater than rates in the Cape Cod aquifer. A groundwater 15
N addition study by Tobias et al.
(2001) found rates ranging from 180 – 235 nmol N2 L-1
d-1
.
Anammox does control the majority of N2 in one site, with 64% of all N2 being created
by anammox. At 8 of the 11 ports, anammox contributes between 10 – 30% of all N2 production.
This contribution is within the low range of N2 production by anammox in anoxic bottom water
(19-35%) (Dalsgaard et al., 2003) and comparable to the two contaminated aquifers sampled by
Moore et al. (2011) ranging between 18.0 ±6.5% and 35.7 ±13.6%. The data shows that depths
lacking DIN have the lowest rates of denitrification and highest percentages of anammox, which
could indicate that anammox has a better tolerance for substrate loss then denitrifying bacteria.
When Pearsons product-moment correlation coefficient was calculated, neither the
abundance of the genes encoding for denitrification or anammox correlated to their respective
59
rate counterparts. It should seem intuitive that larger abundances of bacteria would yield higher
rates of N2 production, but it appears that is not the case. This finding might indicate that
individual bacteria may not be consistent in single cell N2 production, or that individual bacteria
are not as efficient as others. When geochemical parameters such as total dissolved (TDN) and
inorganic dissolved (DIN) nitrogen concentrations were correlated with the rates, per cell rates,
and % anammox the conventional thinking would be that the rates would all increase as the
TDN/DIN increased. No significant correlations were found within the rates, however percent
anammox shows a negative correlation with both TDN and DIN. This may be indicative of
strong competition with denitrifying bacteria over the available nutrients. The denitrification rate
does not correlate with DIN or TDN, but with the ammonium concentration. Denitrifiers to not
require ammonium, but if ammonium oxidation is present perhaps the denitrifiers will quickly
consume any available nitrate created by the ammonium oxidizers. This would leave behind the
believable scenario of large amounts of inactive denitrifying organisms waiting in depths of low
nitrate and high ammonium. Anammox overall rates and anammox per cell rates significantly
decreased as DOC increased, which aligns with the idea that when DOC is present in the aquifer
denitrifying bacteria are dominant. These results suggest that merely looking at geochemical data
may provide a starting point for analysis but is not a direct indication of rates/gene abundance.
Diversity and phylogeny of Anammox bacteria
Two depths were sampled for phylogenetic analysis based upon their geochemical
properties, one depth containing a large amount of nitrate/nitrite whereas the other contained
small amounts of ammonium, yet no nitrate/nitrite. Cloning and sequencing revealed an
interesting split in the diversity of the anammox community. Harhangi et al. (2012) reported that
60
when using the hzsA_526F/hzsA_1857R primers for the hzsA gene the sequences reportedly
yielded very similar findings to 16S rRNA sequences, both agreeing on a “Candidatus Brocadia
spp.”. However, upon phylogenies of two different sites using anammox 16S rRNA and hzsA
sequences it is clear that the results are not congruent. The hzsA phylogenetic tree (Figure 9)
shows a clear separation between the Cape Cod sequences and known anammox genera, with the
deeper, ammonium-rich community showing similarity to the Candidatus Brocadia spp. clade,
while clustering farthest from the Candidatus Kuenenia spp. clade (Figure 9). The 16S rRNA
phylogeny shows the both the shallow and deep communities clustering away from previously
established genera, but relatively similar to the Candidatus Kuenenia spp. and Candidatus
Brocadia spp. (Figure 9). The Phylogenies are inconsistent with regards to the Candidatus
Kuenenia spp. placement, differing from the results reported by Harhangi et al. (2012). It was
first hypothesized that the anammox of the Cape Cod aquifer would be mostly dominated by
Candidatus Brocadia spp., due to Brocadia being the dominant genera in similar sites (Moore et
al., 2011), however there is a discrepancy between the phylogenies of 16S rRNA and hzsA genes.
Schmid et al. (2007) illustrated that when it comes to displaying diversity, the 16S rRNA gene
has the distinct disadvantage of lacking any functional analysis. However, due to detection of the
hzsA gene being relatively new and 16S rRNA having been universally used to classify
anammox bacteria we can confirm that the dominant anammox genera in the aquifer are similar
related to Candidatus Brocadia spp. based on 16S rRNA genes.
However while that supports the third hypothesis, it does not tell the entire story. The
<95% similarities between the Cape Cod communities and previously established genera and the
placement of the separate phylotypes on the phylogenetic tree would indicate the possibility of a
new genera or species of anammox bacteria in the aquifer.
61
CONCLUSION
The specific MLS ports used were chosen for several reasons; limited availability of
dissolved oxygen which would inhibit microbial nitrate reduction and a robust range of organic
and inorganic N concentrations that would provide substrate for anammox or denitrification
hotspots. The vertical geochemistry was cross-referenced with potential rate data, functional
gene quantification distribution, and anammox population analyses to elucidate a complete
survey of the sample aquifer. Contrary to conventional thinking, rates and bacterial abundance
show no correlation. It has been shown before that rates and abundance do not necessarily match
the in situ activity (Penton et al., 2006). It is clear that potential rates or bacterial abundance do
not tell the entire story of the aquifer by themselves, but combining rates with abundance and
geochemical features show a more complete picture. The anammox potential rates, while low, do
not complement the high bacterial abundance yet in low DIN altitudes contribute up to 64% of
all N2 production which makes anammox a very important feature in the Cape Cod aquifer.
Future considerations
Another way to compare the rates and abundances would be as a ratio. The ratio of
denitrification rates and anammox rates is a normalization process that better tells the story of the
individual anammox or denitrification cell. While merely glancing at the rates alone, it would
appear that several depths in the ammonium available zones show decreased rates, where rates
should be higher. When normalized to the copy numbers available and compared the rates per
copy numbers in the previously low rates depths are higher, illustrating that the rates are lowered
from a lack of gene abundance. The flaw in DNA extraction is the efficiency of the process,
where not all DNA may be recovered which may repress the abundance numbers. By
62
normalizing the process to rates per copy numbers, the ‘true’ rates can be analyzed and the
depths show a much more cohesive and likely trend when compared to the geochemical vertical
nitrogen distribution. It is possible that each anammox cell or denitrification cell possibly utilize
available nitrate/nitrite/ammonium more or less efficiently, depending on the geochemical
concentration (Figure 6).
63
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