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Applied and Environmental Microbiology, January 2000, p. 133-139, Vol. 66, No. 1
Department of Civil and Environmental
Engineering1 and Department of Plant
Biology,2 Arizona State University, Tempe,
Arizona 85287
Received 5 April 1999/Accepted 28 October 1999
The feasibility of biologically removing nitrate from groundwater
was tested by using cyanobacterial cultures in batch mode under
laboratory conditions. Results demonstrated that nitrate-contaminated groundwater, when supplemented with phosphate and some trace elements, can be used as growth medium supporting vigorous growth of several strains of cyanobacteria. As cyanobacteria grew, nitrate was removed from the water. Of three species tested, Synechococcus sp.
strain PCC 7942 displayed the highest nitrate uptake rate, but all
species showed rapid removal of nitrate from groundwater. The nitrate uptake rate increased proportionally with increasing light intensity up
to 100 µmol of photons m Nitrate
(NO3 Nitrate removal from groundwater may be accomplished by bacterially
mediated denitrification, or chemically and physically based
technologies (26). These treatment processes, however, require input of external energy sources (e.g., electricity or organic
carbon) and/or chemical additives and generate concentrated waste
streams that must be disposed. Therefore, they are often problematic
and expensive (8, 20). Since nitrate may be taken up
effectively by photosynthetic microorganisms, such as cyanobacteria, which require mostly fixed nitrogen, inorganic carbon, and light for
growth, the use of photosynthetic organisms would minimize the need of
chemicals and fossil fuels for nitrate removal, thus leading to an
efficient resource recovery and recycling. However, limited information
on engineering photosynthetic system for treating nitrate-contaminated
drinking water supplies is available.
With cyanobacteria, nitrate is taken up by a common high-affinity
transport system involving the NrtABCD permease (an ABC-type transporter) and to a lesser extent enters the cells by diffusion (11, 32, 33). Once inside the cell, nitrate is reduced to nitrite by nitrate reductase, and nitrite is further reduced to ammonium by nitrite reductase (19). Ammonium is then
incorporated into carbon skeletons mainly through the operation of the
glutamine synthetase-glutamate synthase cycle (11). Fixed
nitrogen storage components, such as C-phycocyanin (5)
and/or cyanophycin (31), may be formed and accumulated in
the cells under certain physiological conditions. Regulation of nitrate
assimilation has been found to be controlled by several regulatory
products that affect expression of the nrtABCD,
narB (encoding nitrate reductase), and nirA
(encoding nitrite reductase) genes that together form the
nirA-nrtABCD-narB operon in several cyanobacteria (19,
41). Nitrate uptake can be influenced by availability of
phosphate ion (PO43 Microalgal treatment of wastewater has been investigated for over 4 decades as an environmentally sound alternative to remove nutrients and
heavy metals from wastewater sources. However, very few investigations
have considered applying this technology to the treatment of drinking
water (8, 20, 42). The chemical composition (e.g., the pH,
dissolved inorganic carbon, nutrient levels, and metals) of groundwater
differs from that of wastewater, and the feasibility of growing
microalgae (a term used for cyanobacteria and unicellular algae) in
groundwater has yet to be evaluated. The objectives of the present
study were, first, to determine whether groundwater can sustain
cyanobacterial development and, second, to determine the rate of
nitrate removal from the water. Third, the study was designed to
determine how nitrate uptake and cyanobacterial growth could be
affected by environmental and biotic factors. Several cyanobacterial
species were employed as a working model for the study because of their
rapid nitrate uptake and high growth rate (19) and their
ability to be genetically manipulated (44), which may lead
to performance improvements of cyanobacteria for drinking water remediation.
Organisms and culture conditions.
Three strains of
cyanobacteria were tested in this study: Synechocystis sp.
strain PCC 6803, Synechocystis minima CCAP 1480/4, and
Synechococcus sp. strain PCC 7942. Axenic stock cultures
were maintained in BG-11 growth medium (45). Unless stated
otherwise, all cultures were maintained at 32°C and 160 µmol of
photons m Experimental system.
The groundwater used in this study was
obtained from wells in the Phoenix, Ariz., metropolitan area. Typical
chemical composition of groundwater was as follows: 1.5 to 2.3 mM
NO3
0099-2240/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Removal of Nitrate from Groundwater by
Cyanobacteria: Quantitative Assessment of Factors Influencing
Nitrate Uptake
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
2 s1, which
parallels photosynthetic activity. The nitrate uptake rate was affected
by inoculum size (i.e., cell density), fixed-nitrogen level in the
cells in the inoculum, and aeration rate, with vigorously aerated,
nitrate-sufficient cells in mid-logarithmic phase having the highest
long-term nitrate uptake rate. Average nitrate uptake rates up to 0.05 mM NO3 h1 could be achieved at
a culture optical density at 730 nm of 0.5 to 1.0 over a 2-day culture
period. This result compares favorably with those reported for nitrate
removal by other cyanobacteria and algae, and therefore effective
nitrate removal from groundwater using this organism could be
anticipated on large-scale operations.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
) concentrations in groundwater have
increased globally (26). Wastewater, fertilizers, and
livestock farming are major sources of nitrate in groundwater supplies
(18). Groundwater in many locations is used as a supply for
drinking water, and high nitrate concentrations present a potential
risk to public health, particular to infants (13). In the
United States, the Environmental Protection Agency has set a maximum
contaminant level (MCL) for nitrate in drinking water of 0.71 mM (10 mg
of NO3-N liter1)
(35). Similar MCLs for nitrate have been established in
Canada and Europe. As a result, many public and private groundwater
supply wells have been shut off as drinking water sources because their nitrate levels exceed the MCL (26). As water demands in many urban and agricultural areas increase, technology for treating nitrate-contaminated groundwater is becoming increasingly urgent.
), which has an important
role in cellular energetics as part of ATP (adenosine triphosphate) and
which influences the activity of many enzymes required for cell
metabolism, including the nitrate reduction process (2, 19).
Also, nitrate uptake can be affected by spectral quality and light
intensity (3, 38), temperature (32, 42), and cell
density and nitrate level (28, 30), as well as physiological
acclimation of the culture (30).
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
2 s1.
, a negligible amount of dissolved
phosphorus, 1,500 mg of total dissolved solids liter1,
2.5 to 3.3 mM bicarbonate, and a pH of 7.4 ± 0.3. Groundwater was
passed through a microfilter (0.45-µm-pore-size nylon membrane; Cole-Parmer, Vernon Hills, Ill.) and stored in a Nalgene container at
4°C until it was used as a growth medium.
2 s1 were adjusted by covering the
surface of the glass columns by shade nets. The illumination intensity
was measured with a light meter (LiCor model LI-189).
1.
Before batch culture experiments were started, cells were harvested from this continuous-culture system by centrifugation and washed once
with distilled water. All experiments were run at least in triplicate.
Samples were collected three to four times a day, and cell density and
nutrient concentrations were monitored accordingly.
Growth analysis.
Cell density, monitored as the optical
density at 730 nm (OD730) of cyanobacterial suspensions,
was measured with a spectrophotometer (UV-160; Shimadzu, Kyoto, Japan).
Increments in optical density during certain time intervals were used
to calculate the specific growth rate (µ) by the equation µ = (ln X2 
ln
X1)/(t2 t1), where X1 and X2 are the
optical densities at times t1 and
t2, respectively.
Nutrient analysis.
Cyanobacterial suspensions were passed
through a filter (0.45-µm-pore-size nylon membrane; Cole-Parmer) to
remove cells and some other organic particles, and the filtrates were
stored at 20°C until elemental analysis was performed. Analyses
were done with a TRAACS 800 continuous-flow analyzer, by using
industrial method no. 824-87T for NO3 and
method no. J-004-88C for PO43 determinations
(BRAN+LUEBBE, Chicago, Ill.).
Nutrient and irradiance dependence of growth.
The specific
growth rate as a function of nutrient availability was fitted to the
kinetic growth model of Dugdale (10) by using a rectangular
hyperbola function described by the equation µ = µmax S/(KS + S), where
µmax is the maximum specific growth rate, S is
the nutrient concentration (e.g., PO43 or
NO3), and Ks is the
nutrient concentration at which half-maximal growth occurs. The
specific growth rate as a function of irradiance was also fitted to a
hyperbolic curve described by the equation µ = µmax I(Ik + I), where
Ik and I are the half saturation and incident irradiance, respectively (16).
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Cyanobacterial growth in groundwater. The viability of cyanobacteria in groundwater was determined. Three fairly well characterized species of cyanobacteria that do not fix nitrogen (Synechocystis sp. strain PCC 6803, Synechocystis minima CCAP 1480/4, and Synechococcus sp. strain PCC 7942) were precultured in BG-11 growth medium. Cells were harvested in the exponential growth phase and inoculated into glass columns containing groundwater. All three strains were found to grow quite well under these conditions, with doubling times during exponential growth between 10 and 20 h. During the time course of cultivation of Synechococcus sp. strain PCC 7942, the nitrate concentration in the groundwater decreased with a half-life of about 12 h. About 30 h after the start of the experiment, nitrate was virtually undetectable in the medium, indicative of an efficient nitrate uptake by the cells. Synechococcus sp. strain PCC 7942 was among the fastest growers in this experiment, and therefore it was used in all subsequent experiments reported in this paper.
Light intensity dependence of growth and nitrate removal.
Cell
growth and nitrate uptake rates as a function of light intensity were
investigated for Synechococcus sp. strain PCC 7942. Maximal
photoautotrophic growth rates of this strain increased with light
intensity, until reaching a saturation level (Fig. 1A). The light intensity
(Ik) at which the specific growth rate of the
cells is half the maximal value was estimated to be 50 µmol of
photons m2 s1. Prolonged exposure of the
culture with relatively low cell density (OD730, <0.1) to
180 µmol of photons m2 s1 was found to
cause photobleaching of the cells (data not shown). Nitrate uptake for
the same experiment is presented in Fig. 1B. The rate of nitrate uptake
closely paralleled the growth rate, with conditions of most vigorous
growth coinciding with conditions for the highest nitrate uptake rate.
On the basis of these results, an irradiance of 160 µmol of photons
m2 s1, causing nearly maximal growth and
nitrate uptake rates, was chosen for subsequent experiments.
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), 12 (
), 50 (
),
100 (
), and 180 (
) µmol of photons mLong-term survival of cyanobacteria in groundwater.
Growth of
Synechococcus sp. strain PCC 7942 eventually ceased after
being maintained in continuous culture in groundwater for about 7 days.
To identify the limiting nutrient, 0.32 mM sulfur (S) and magnesium
(Mg), 100 µM phosphate (P), and 1.0 ml of BG-11 trace element mix
liter1 or, as a control, BG-11 growth medium were added
to the Synechococcus sp. strain PCC 7942 culture, and growth
was monitored. Phosphate was the limiting nutrient: whereas addition of
0.32 mM MgSO4 or 1.0 ml of trace element mix
liter1 (both concentrations are equal to those present in
BG-11 growth medium) did not improve growth, supplementation with 150 µM PO43 alone caused the culture to resume
growth, albeit at a significantly lower specific growth rate than was
observed before nutrient limitation (data not shown). A similar growth
rate was also found in the culture that was transferred to BG-11 growth
medium, indicating that many cells lost much of their viability during
the period they were maintained in groundwater.
to
groundwater, Synechococcus sp. strain PCC 7942 cells were
able to grow and take up both nitrate and phosphate, and the culture
could go through at least four additional divisions in the
continuous-culture mode. Thereafter, growth as well as phosphate and
nitrate uptake ceased, although ca. 80 µM
PO43 and 1.2 mM NO3
remained in the medium (data not shown). At this time, addition of 0.3 ml of a trace element mix liter1 was sufficient to
recover cell growth. However, recovery did not occur upon addition of
any of the macronutrients (Mg, S, N, or P) alone. It was clear that,
after nitrate and phosphate, one or more trace elements became limiting
for long-term growth of Synechococcus sp. strain PCC 7942 in groundwater.
Phosphate requirement for cell growth and nitrate uptake.
To
determine how much phosphate is minimally needed for continued growth
at satisfactory rates, phosphate-deprived Synechococcus sp.
strain PCC 7942 cells were transferred to groundwater with ambient
nitrate (1.53 mM NO3) and to which 0.3 ml of
BG-11 trace element mix liter1 and various phosphate
concentrations (0, 6.5, 14.6, 53.6, or 105.2 µM
PO43) had been added. Figure
2 shows the growth of the culture as well
as nitrate and phosphate levels remaining in the medium as a function
of the added PO43 concentration. While little
growth was observed without external PO43
addition, addition of even modest amounts of phosphate (6.5 to 14.6 µM PO43) resulted in resumption of growth
at a significant rate. Little growth enhancement was observed upon a
further increase of the phosphate concentration above 53.6 µM (Fig.
2A). The phosphate concentration (Ks) leading to
half the maximal specific growth rate was 9 µM.
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15 µM), cellular nitrate removal rates from the medium continued to increase
significantly with increasing phosphate concentration, reaching a
maximum at the highest phosphate concentration assayed in this
experiment (105.2 µM) (Fig. 2B). Added phosphate was simultaneously
taken up rapidly by the cells (Fig. 2C). Even 105.2 µM
PO43, the highest level of phosphate tested,
was completely removed from the medium within the first 20 h of cultivation.
Not only was addition of phosphate required for uptake of nitrate and
simultaneous growth, but it also modified the pigment composition of
cells. A relatively high level of phycobilisome in the cells was
observed upon phosphate deprivation (no growth) and decreased upon
addition of low levels of phosphate. The dependency of dissolved
phosphate concentrations on phycobilisome levels reflects that these
pigment-protein complexes break down to facilitate some cell growth
under nutrient-limiting conditions and then increase significantly with
addition of higher phosphate levels. The amount of chlorophyll
a also declined upon addition of low phosphate concentrations and then increased again upon addition of more phosphate, but this change was somewhat smaller than that in the level
of phycobilisomes.
Growth and nitrate uptake as a function of nitrate
availability.
To determine nitrate uptake and cell growth of
Synechococcus sp. strain PCC 7942 at various initial nitrate
concentrations, tap water, which had an ionic composition similar to
that of groundwater except for a much lower
NO3 level, was used as growth medium after
supplementation with 100 µM PO43 and
various amounts of NaNO3. By using nitrate-deprived
Synechococcus sp. strain PCC 7942 cells as an inoculum,
growth was determined for different initial
NO3 concentrations. The simulated groundwater
(tap water plus NO3) sustained growth rates
of cyanobacteria identical to those in groundwater. The growth rate
clearly depended on the nitrate concentration in the medium (Fig.
3A). The maximal specific growth rate as
a function of nitrate concentration showed typical saturation kinetics (10), and the initial nitrate concentration
(Ks) at which the growth rate was half maximal
was 0.2 mM. Nitrate removal from the medium (taken up by the cells)
occurred at a significant rate upon addition of nitrate (Fig. 3B) and
then was almost independent of the initial nitrate concentration when
it was above 0.32 mM. However, higher maximal cell densities were
observed in cultures with higher initial NO3
concentrations. As shown in Fig. 1, that cells continued growing even
after nitrate had been deprived from the medium, indicating a rapid
accumulation of internal fixed-nitrogen reserves.
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), 1.61 mM (
). The light intensity was 160 µmol
mEffect of nitrate level of cells in inoculum. Rapid nitrate uptake has often been observed in nitrate-deprived cells over a time scale of minutes (19, 30). To determine whether nitrate-deprived or nitrate-sufficient cells were more appropriate for rapid, consistent, and complete removal of nitrate from groundwater, nitrate uptake and cell growth were monitored in parallel in both types of cells (Fig. 4). Nitrate-deprived cells were generated by culturing in nitrate-free BG-11 medium for 48 h, whereas nitrate-sufficient cells had been grown for 24 h in normal BG-11 medium. Before inoculation, both types of cells were harvested and washed twice with deionized water.
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1) was observed during the first 2 h of incubation
(Fig. 4A). However, after this short period of nitrate uptake, no
decline in nitrate concentration was detected in the medium for the
next 30 h, corresponding to a lag phase in cell growth of the same
length (Fig. 4B). At about 35 h after inoculation, rapid uptake of
nitrate and cell growth resumed. In contrast, the nitrogen-sufficient
culture exhibited a lower initial nitrate uptake rate (0.05 mM
h1). However, the latter showed a much shorter lag phase
in both nitrate uptake and growth than the former. After the lag phase had been overcome, the rate of nitrate uptake by cells was independent of whether cells were nitrate deprived or nitrate sufficient.
Nitrate uptake optimization.
Nitrate uptake and reduction
require energy and reducing power; therefore, aeration of the culture,
which provides CO2 and improves light availability of
individual cells for photosynthesis, was likely to be important for
most efficient nitrate uptake. Figure 5A
shows the average nitrate uptake during a cultivation period of 45 h as a function of aeration rate and initial cell density. Indeed, a
higher aeration rate yielded higher nitrate uptakes. The maximum rate
of nitrate uptake was close to 0.05 mM h1 in the culture
with an initial cell density of 0.5 to 1.0 OD730, and
declined when more concentrated cultures that were closer to stationary
phase were used. Similarly, the total amount of biomass produced
increased with aeration rate and with inoculum size, up to a maximal
level (Fig. 5B).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Nitrate removal by cyanobacteria.
Our results demonstrate the
potential of cyanobacteria to efficiently remove and utilize nitrate
from groundwater. The rate with which this uptake by cyanobacterial
cultures proceeds is such that within one or several days the nitrate
level in groundwater can be reduced from severalfold above MCL to
levels that are acceptable for drinking water standards. Moreover, no
nitrite or ammonia was detected in the cultures (data not shown),
indicating that the levels of these harmful intermediates are less than
0.1 mg liter1. The cyanobacterial strains tested in this
study are rather comparable in their ability to grow in groundwater and
in their nitrate uptake efficiency. However, we have observed that
cyanobacteria that can fix nitrogen from the air (for example,
Nostoc and Anabaena species) were very
inefficient in uptake of nitrate when nitrate was present in the medium
at relatively low concentrations. Moreover, the tested nitrogen-fixing
cyanobacteria excreted ammonium into the medium (data not shown), which
is known to be common for many nitrogen-fixing cyanobacteria
(4).
Nitrate removal from groundwater versus wastewater. Nitrate-contaminated groundwater differs from most wastewater in terms of the levels of other macronutrients, including phosphate. Whereas groundwater usually has little phosphate, wastewater generally contains a significant amount of this compound (9, 43). Indeed, the phosphate level apparently limits cyanobacterial growth in groundwater, influencing not only the overall growth rate but also the final cell density that the cyanobacterial culture reaches. The first few cell divisions after transfer to groundwater probably depend mostly on cell-internal phosphate reserves that have been carried over from the artificial growth medium (such as BG-11). Long-term growth and survival of the cells, however, require addition of phosphate.
The role of phosphate and trace elements.
Synechococcus
sp. strain PCC 7942 requires addition of about 9 µM phosphate to grow
at half-maximal rate. This appears to be higher by an order of
magnitude than the phosphate concentration required for some other
species (12, 36). Factors that may contribute to this
difference include the species used and the mode in which the cultures
are grown (batch versus continuous culture) as well as the cellular
phosphorus level at which the assay is conducted. The amount of nitrate
versus phosphate that is required to restore a half-maximal growth rate
differs in this strain by a factor of 22 [(Ks = 200 µM NO3)/(Ks = 9 µM PO43)], and this ratio is within the
range common for many algae and cyanobacteria, at which one nutrient
limitation changes over to the other (17, 38).
Interestingly, the phosphate concentration needed for half-maximal rate
of nitrate uptake was higher than the concentration sufficient for
maximal growth rates (15 and 9 µM, respectively). This may reflect
the requirement for cell vigor and health for optimal nitrate uptake.
Indeed, as shown in Fig. 4, under fully phosphate-sufficient
conditions, the amount of pigments was larger than at lower phosphate
levels. This difference presumably may lead to a difference in the
photosynthetic activity. Since nitrate uptake and assimilation require
ATP and reducing equivalents, photosynthetic activity is likely to be a
prerequisite for efficient nitrate uptake and reduction. It is
important to note that the Synechococcus sp. strain PCC 7942 culture was able to take up all phosphate from the medium within
20 h, even at the highest phosphate concentration available (105 µM). This time frame is similar to that needed for uptake of nitrate,
and the addition of small amounts of phosphate to the culture will not lead to secondary contamination of treated water by residual phosphate.
1 every few days was sufficient for
long-term maintenance of cyanobacterial cultures in groundwater, and
therefore trace elements are not a major limiting factor that would
impact larger-scale applications of cyanobacterial cultures.
Nitrate-starved cells versus nitrate-sufficient cells. It has been known that nitrogen-deprived cells generally show higher inorganic nitrogen uptake rates than nitrogen-sufficient cells in the short term (14, 30). However, after an initial nitrate uptake in nitrogen-deprived cells, these cells entered a lag phase in which no nitrate uptake occurred. Furthermore, the length of the lag phase increased with the time that cells were deprived of nitrate. In contrast, nitrogen-sufficient cells continued to take up nitrate. Therefore, results generated from short-term nitrate uptake experiments cannot be reliably used to determine conditions for optimal, sustained nitrate uptake rates. For long-term studies, the overall uptake rates of nitrate and perhaps also of other forms of fixed nitrogen are closely related to the growth rate of the cells, and a fixed nitrogen-sufficient inoculum, rather than nitrogen-deprived one, is preferred because of the lengthy lag phase observed after nitrogen deprivation. In addition, nitrogen deprivation may cause cell autolysis (7) and excretion of secondary metabolites (4, 6).
Light requirement.
The nitrate assimilation process is
ultimately driven by light energy, which provides, through
photosynthesis, ATP and reducing equivalents for nitrate uptake and
reduction. On the other hand, too much light can also be a source of
photooxidative damage. At low irradiance (approximately 6 µmol of
photons m2 s1), photosynthesis and
respiration occur at roughly the same rate (light compensation point),
and a lack of significant nitrate uptake is not unexpected. At about 50 µmol of photons m2 s1, the growth rate is
half maximal. The nitrate uptake rate as a function of light intensity
follows a similar pattern as the growth curve, consistent with the
requirement of photosynthesis for efficient nitrate uptake
(27). The light requirement for Synechococcus sp.
strain PCC 7942 growth appeared to be quantitatively similar to what
has been reported for many algal and cyanobacterial species
(15).
2
s1 and above, depending on the density of the culture),
photooxidative stress is introduced in cyanobacteria due to absorption
of excess light than cannot be used productively for photosynthesis.
Upon exposure to 180 µmol of photons m2
s1, the Synechococcus sp. strain PCC 7942 culture was bleached and killed within 48 h if the initial cell
OD730 were below 0.1. This is consistent with earlier data
that attributed the light-induced demise of a low-density culture
mainly to photooxidative damage (1, 23). Therefore, from a
practical standpoint, a scaled-up bioreactor powered by solar energy
will need to have a relatively long light path so that cells are
generally shielded by each other in order to prevent photo-bleaching.
In this context, aeration can mainly affect the light regime to which
individual cells in the culture is exposed (39), thereby
ensuring the maximal photosynthetic activity while at the same time
diminishing the inhibitory effect associated with high light
intensities (24).
Application potential.
Synechococcus sp. strain PCC 7942 and other non-nitrogen-fixing cyanobacteria appear to be very suitable
to remove nitrate from groundwater or other similar types of water
sources. When maintained at an optimal cell density (OD730,
0.5~1.0) and at an incident light intensity of 180 µmol of photons
m2 s1 at 32°C, the average nitrate uptake
rate was 0.05 mM NO3 h1 (Fig.
5A). For an initial nitrogen concentration of 1.5 mM
NO3, this uptake rate would reduce nitrate
levels below the MCL limit (0.71 mM NO3)
within 14 h and allow complete removal of this nutrient within 30 h. These values compare favorably with those reported in the literature (Table 1).
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ACKNOWLEDGMENTS |
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This work was funded by an Arizona State University Project Ingenhousz grant for collaborative research.
We are grateful to Tom Colella for his kind assistance in determining nitrate and phosphate concentrations.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Plant Biology, Arizona State University, Tempe, AZ 85287. Phone: (602) 965-3698. Fax: (602) 965-6899. E-mail: huqiang{at}imap4.asu.edu.
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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