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Applied and Environmental Microbiology, July 2001, p. 2916-2921, Vol. 67, No. 7
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.7.2916-2921.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
The Leucine Incorporation Method Estimates
Bacterial Growth Equally Well in Both Oxic and Anoxic Lake
Waters
David
Bastviken1,* and
Lars
Tranvik2
Department of Water and Environmental
Studies, Linköping University, SE 581 83 Linköping,1 and Department of
Limnology, Uppsala University, SE 752 36 Uppsala,2 Sweden
Received 23 January 2001/Accepted 12 April 2001
 |
ABSTRACT |
Bacterial biomass production is often estimated from incorporation
of radioactively labeled leucine into protein, in both oxic and anoxic
waters and sediments. However, the validity of the method in anoxic
environments has so far not been tested. We compared the leucine
incorporation of bacterial assemblages growing in oxic and anoxic
waters from three lakes differing in nutrient and humic contents. The
method was modified to avoid O2 contamination by performing
the incubation in syringes. Isotope saturation levels in oxic and
anoxic waters were determined, and leucine incorporation rates were
compared to microscopically observed bacterial growth. Finally, we
evaluated the effects of O2 contamination during incubation
with leucine, as well as the potential effects of a headspace in the
incubation vessel. Isotope saturation occurred at a leucine
concentration of above about 50 nM in both oxic and anoxic waters from
all three lakes. Leucine incorporation rates were linearly correlated
to observed growth, and there was no significant difference between
oxic and anoxic conditions. O2 contamination of anoxic
water during 1-h incubations with leucine had no detectable impact on
the incorporation rate, while a headspace in the incubation vessel
caused leucine incorporation to increase in both anoxic and
O2-contaminated samples. The results indicate that the
leucine incorporation method relates equally to bacterial growth rates
under oxic and anoxic conditions and that incubation should be
performed without a headspace.
| |
INTRODUCTION |
Bacterial production in aquatic
systems is commonly estimated from the rate of incorporation of
radioactively labeled leucine into bacterial protein (10,
13). This method has been extensively tested in oxic waters
(12, 14, 30, 31). It is fast and technically simple and
has gradually become very popular compared to methods such as those
based on the frequency of dividing cells (9) or
incorporation of radioactively labeled thymidine into bacterial DNA
(7, 20).
Bacterial production methods cannot be applied to anoxic environments
without caution. It has been shown that several important metabolic
groups of bacteria in anoxic environments do not incorporate thymidine
(8, 11, 24-26, 34). In field studies, the thymidine method frequently yields lower estimates of bacterial production than
the leucine method in anoxic environments (18, 33). A proposed reason, apart from differential uptake, is that thymidine to a
large extent is incorporated into proteins instead of DNA under anoxic
conditions (18, 33). The leucine method has been used in
anoxic water columns (2, 18) and has recently also been
applied to sediment bacteria (5, 6, 17, 33), but no
systematic comparison of how it performs under different oxygen conditions has been presented.
In addition to potential metabolic constraints on leucine incorporation
into anaerobic bacteria, there is also the issue of sensitivity to
O2 contamination. It seems to be very difficult to create anoxic conditions during the leucine incubation when following the protocols developed for oxic conditions (15,
31), and any headspace in the incubation vessels containing
O2 may corrupt the samples.
The purpose of this study was to assess the applicability of the
leucine method to both anoxic and oxic conditions in freshwater. We
developed a modified protocol to avoid O2
contamination, assessed isotope saturation levels of bacterial
assemblages developing in oxic and anoxic waters, and simultaneously
measured bacterial growth by microscopy and by the
[3H]leucine incorporation method in similar
lake water with or without the presence of O2. In
addition, we evaluated the effect of short-term O2 contamination during
[3H]leucine incubations of anoxic samples.
| |
MATERIALS AND METHODS |
Bacterial regrowth cultures.
Bacterial production was
measured in cultures containing natural bacterial assemblages growing
in filtered water, which was retrieved from three different lakes
during summer stratification in 1999. The lakes were chosen to be
different in terms of nutrient and dissolved organic carbon
concentrations. In situ average water column total phosphorous
concentrations were 15, 52, and 38 mg liter
1,
and dissolved organic carbon concentrations were 19.8, 17.9, and 9.4 mg
liter1, in Lillsjön, Mårn, and
Illersjön, respectively. All lakes had anoxic hypolimnetic water.
Water column composite samples were collected in 10-liter polyethylene
carboys using a submersible pump (Amazon 10; Awimex International). The
water was then transported to the laboratory and stored at 2°C until
used in the experiments. In the isotope dilution experiments the water
was transferred directly into infusion bottles (see below) without
nutrient addition and filtering, and isotope dilution experiments were
performed after 1 week at 15°C. The water for other experiments was
spiked with phosphate
(Na2HPO4) and ammonium
(NH4Cl) to make the organic carbon the limiting
resource (additions gave minimal final concentrations of 350 µg of P
liter1 and 1.5 mg of N
liter1, respectively) and filtered through a
glass fiber filter (Gelman A/E) to reduce bacterial numbers and remove
bacterivores. As a first step to remove O2, the
water was then purged with N2 (N52; Air Liquide)
(<1 ppm of O2) for 1 h. Thereafter, the
water was transferred to 330-ml infusion bottles (Laboratory Service
Provider B.V.; 250 ml of water per bottle) with a dispenser pump while continuously flushing the bottles with N2.
Directly after transfer the bottles were capped with 17-mm-thick butyl
rubber stoppers, which were tested to be gas tight after being pierced
more than 100 times with syringe needles (Microlance, 0.6 by 25 mm;
Becton-Dickinson). The stoppers were secured with aluminum caps.
In a second oxygen removal step, the capped bottles for the anoxic
treatments were attached via syringe needles to a gas exchange device
with which the bottles were evacuated until vigorous bubble formation
occurred upon tapping of the bottles with a plastic rod. Evacuation was
followed by N2 addition to about 2 atm of pressure. The cycle of evacuation and N2 addition
was repeated at least nine times. The purging and the gas exchange
cycles removed oxygen to below the levels of detection by both Winkler
titration and an oxygen electrode (Orion model 835; detection limit of
0.005 mg liter1) with little disturbance of the
organic matter and bacteria compared to alternative oxygen removal
methods such as boiling at 100°C or addition of reducing agents such
as sulfide. After the second removal step, excess
N2 was released from the anoxic bottles through a
syringe needle until ambient air pressure was reached. To ensure that
bottles for oxic treatments were oxygenated, they were subjected to gas
exchange cycles with air instead of N2.
During the experiments, samples for measuring leucine incorporation,
bacterial biomass, and biovolume or for measuring oxygen
content were
withdrawn from the bottles with plastic syringes
(see below for
details). To maintain atmospheric pressure within
the bottles, air or
N
2 corresponding to the sample volume was
added
upon sampling in the oxic and anoxic bottles, respectively.
Syringes
used for sampling anoxic bottles were flushed at least
three times with
N
2 prior to
sampling.
Leucine incorporation method.
The leucine incorporation
protocols of Smith and Azam (31) and Kirchman
(15) were used with some minor modifications. Instead of
using Eppendorf tubes, we incubated the samples in 2-ml plastic
syringes (Plastipak syringes having a rubber seal of the piston and
Discardit II syringes made only of plastic, both from
Becton-Dickinson). Syringes were kept in plastic bags with
N2 for at least 24 h prior to use to reduce
the amount of O2 dissolved in the plastic.
Samples of 1.7 ml were drawn directly from the experiment bottles into
the 2-ml syringes, and 50 µl of leucine solution (purged with
N2 to remove O2) was
injected into each syringe through its tip using a Hamilton syringe.
For control samples, 90 µl of 100% trichloroacetic acid (TCA) was injected prior to the leucine injection. Immediately after injections, syringe needles (Microlance, 0.5 by 16 mm; Becton-Dickinson) were connected to the incubation syringes and the air in the needles was
replaced with sample water by gently pushing the syringe piston until
the first droplet of the sample could be seen at the end of the needle.
The syringe was then capped by insertion halfway into a 10-mm-thick
rubber stopper. Syringes were thoroughly shaken and rotated to mix the
sample and then incubated in the dark at 15°C. Incubations were
stopped after about 60 min by transferring the samples from the
syringes into 2-ml Eppendorf vials holding 90 µl of 100% TCA.
Control samples were transferred to similar vials without TCA. The
subsequent processing of the samples, including washing in 5% TCA and
80% ethanol, was performed as described by Smith and Azam
(31). Radioactivity was measured after addition of
scintillation cocktail in a Beckman LS 1801 scintillation counter, correcting quench by the H number method.
Bacterial production was estimated as described by Kirchman
(
15), according to the equation BP = LI · 131.2 · (% Leu)
1 · (C/protein) · ID, where BP is bacterial production, LI is the
leucine incorporation rate (moles liter
1
h
1), 131.2 is the formula weight of leucine, % Leu is the fraction
of leucine in protein (0.073) (
15),
C/protein is the ratio of
cellular carbon to protein (0.86)
(
15), and ID is the isotope
dilution (a value of 1 is used
here when comparing bacterial estimates
from leucine incorporation and
microscopy). [
3H]leucine (37 MBq/mol; Amersham
Pharmacia Biotech product no.
TRK 510) was diluted to various extents
with cold leucine prior
to experiments due to economic reasons. The hot
leucine comprised
10 to 100% of the total leucine, depending on the
final leucine
concentration chosen. The final leucine concentration
varied in
the isotope saturation experiment (see below) but was 100 nM
in
the other
experiments.
Isotope saturation.
Unlabeled leucine, naturally occurring
in the water or from intracellular leucine synthesis, causes dilution
of the added radioactively labeled leucine. Such isotope dilution
significantly decreases the uptake of labeled leucine unless leucine is
added at a concentration high enough to make the isotope dilution
negligible. To find such saturating concentrations, we compared leucine
incorporation at final added concentrations ranging from 5 to 257 nM in
both oxic and anoxic bacterial regrowth cultures originating from the three different lakes studied. For each bottle and leucine addition, leucine incorporation was measured in triplicate samples and one killed
control using Plastipak syringes as described above.
Leucine incorporation versus observed change in biomass.
To
compare how leucine incorporation relates to bacterial growth in oxic
and anoxic waters, leucine incorporation and the change in bacterial
biomass were measured simultaneously in five oxic and five anoxic
bacterial regrowth cultures from each lake. Twice daily, 5-ml samples
for determination of bacterial abundance and biovolume were withdrawn
from all cultures and preserved by adding borax-buffered formaldehyde
to a 5% final concentration. Bacterial abundance was determined daily
by counts of DAPI (4',6'-diamidino-2-phenylindole)-stained cells using
an epifluorescence microscope (27) in two replicates per
lake and oxygen regimen to monitor bacterial growth. When microscopic
counting indicated that the cultures had reached the exponential growth
phase, leucine incorporation was measured in triplicate samples and one
killed control per bottle using Plastipak syringes as described above.
The experiment was terminated after about 10 days. Bacterial abundances
in all samples were determined using flow cytometry
(Becton-Dickinson
FACSCalibur and CellQuest 3.1 software) after
staining with Syto 13 (Molecular Probes) as described by del Giorgio
et al. (
3).
Volumes (
V) of 100 to 400 bacterial cells per replicate
bottle during the exponential growth phase were determined using
image-analyzed fluorescence microscopy as described by Bertilsson
et
al. (
1) and converted to bacterial dry weight
(
mb) using
the equation
mb = 435 ×
V0.86 (
16). To estimate
bacterial carbon biomass, we assumed that
carbon comprised 50% of
bacterial dry
weight.
For each culture the natural logarithm of bacterial abundance was
plotted against time. The slope of this curve, representing
the
intrinsic growth rate (
k) (
19), was calculated
using at
least three of the data points closest in time to the leucine
incorporation measurements. The change in bacterial biomass during
the
leucine incorporation experiments was calculated according
to the
equation
BB = BA
0 ·
k ·
t ·
Ccell, where
BB is the change
in
bacterial carbon biomass during the leucine incorporation experiment,
BA
0 is the bacterial abundance at the start of
the leucine incorporation
experiment,
k is the intrinsic
growth rate,
t is the time of the
leucine incorporation
experiment, and
Ccell is the average
carbon
content in each bacterial
cell.
Effects of O2 contamination, headspace, and different
incubation vessels.
The effect of short-term exposure to
O2 during incubation was investigated using an
anoxic regrowth culture (water from Lillsjön). Samples were
incubated in syringes (six replicates and one killed control) that were
deliberately contaminated with different amounts of
O2. Contamination with O2
corresponding to 5 to 76% atm saturation was achieved by introducing a
0.3-ml headspace of the appropriate mixture of N2
and air, right after the leucine addition but before mixing of the
sample. Each level of O2 contamination was
introduced into three additional syringes (not amended with radioactive
leucine) for control of the attained O2
concentration. We also tested different incubation vessels (Plastipak
syringes or Discardit II syringes from Becton-Dickinson or screw cap
Eppendorf vials from Sarstedt).
The oxygen concentration in the syringes was measured
spectrophotometrically at 430 nm (
28). Appropriate amounts
of Winkler
reagents were introduced through syringe tips using 50-µl
Hamilton
syringes. Spectrophotometric O
2
measurements were calibrated against
oxygen electrode measurements
(Orion model 835; detection limit,
0.005 mg
liter
1) in N
2-purged
anoxic water and against Winkler titration in water
with an
O
2 content of 25 to 100% atm saturation. The
resulting
calibration curve was linear over the whole interval (0 to
100%
atm saturation; O
2 concentration [% atm
saturation] = 80.83 ·
A430 
1.14;
r2 = 0.995).
It took almost 4 h to start all of the leucine incubations for the
experiment on effects of O
2 contamination, and
the bacterial
production rate in the sampled culture increased slowly
during
this period. Hence, replicates of each O
2
contamination level
were started individually in a semirandomized
design to make sure
that there was no bias among the different
contamination levels
regarding initial bacterial production. To account
for the drift
in the bacterial production during the experiment, a
linear regression
analysis of all bacterial production
measurements versus incubation
start times was done. Individual
correction factors for each starting
time were derived from the
regression slope (i.e., the residuals)
to normalize bacterial
production to the average production based
on all
measurements.
| |
RESULTS AND DISCUSSION |
Experimental setup and O2 concentrations.
The
procedure for deoxygenating and sealing the infusion bottles allows
water to be kept anoxic for very long times, and in previous tests of
diffusion through the stoppers, no transport of
O2 into bottles could be detected during the test
period (2 months). At various stages during the experiments, the
O2 level was checked using three methods: (i)
with an O2 electrode during preparation of
cultures, (ii) by Winkler titration of water in infusion bottles prior
to and after experiments, and (iii) by spectrophotometric measurements
of water in syringes used for measuring leucine incorporation.
O2 concentrations in the anoxic bottles or
syringes were always below the detection limit regardless of the method
used. In addition, a calculation based on the biomass yield in the
anoxic batch cultures, oxic respiration of glucose, and a bacterial
growth efficiency of 40% (a conservative value since the median for
freshwaters is reported to 26% [4]) shows that any
O2 contamination sufficient to support
significant bacterial growth would have been detected during
O2 measurements. This confirms that anoxic
conditions were reached and maintained during the experiments.
Isotope saturation.
The changes in leucine incorporation rate
with increasing leucine concentration were similar in waters from all
lakes (Fig. 1). The leucine incorporation
reached a saturation level at a leucine concentration of about 50 nM
under both oxygen conditions. Hence, a leucine concentration of 100 nM
was chosen for both oxic and anoxic treatments to ensure saturation.
Jørgensen (12) also found isotope saturation at above 50 nM in waters from two eutrophic lakes, and the saturating concentration
of leucine in humic water is typically high (30 nM or more)
(32) compared to saturating leucine concentrations
commonly found in marine waters (15, 30).
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FIG. 1.
Leucine uptake at different concentrations by bacteria
in waters from three lakes, Illersjön (A), Mårn (B), and
Lillsjön (C), under both oxic and anoxic conditions (circles and
diamonds, respectively). Bars show ±1 standard deviation
(n = 3).
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In waters from two of the lakes, the oxic leucine incorporation
appeared to be higher at a leucine concentration of about
250 nM than
at 50 to 100 nM (Fig.
1A and B), indicating that there
may be a
secondary increase in the leucine incorporation rate
at high leucine
concentrations. Bi- or multiphasic modes of leucine
incorporation in
both pelagic and sediment environments have been
reported (reference
6 and references therein). This may be
due to diffusion
gradients of enzymes and substrates or to physiological
heterogeneity
of the microbial community, including the presence
of eucaryotes taking
up leucine at higher concentrations. A secondary
increase in leucine
incorporation was not seen in the anoxic cultures,
indicating a
difference in leucine biochemistry between oxic and
anoxic microbial
communities.
Leucine incorporation versus observed change in biomass.
Exponential growth started within 48 h in all cultures. The
leucine incorporation was measured at least once per bottle during the
exponential growth phase (Fig. 2).
Leucine incorporation rates were proportional to rates of increase in
bacterial abundance during the period of
[3H]leucine incorporation (Fig.
3A). When analyzed separately, the rates
of increase in cell numbers and leucine incorporation were as closely
related in oxic as in anoxic cultures (by linear regression, r2=0.86 under both oxygen conditions).
In addition, the slopes of the regressions were not significantly
different (by two-way analysis of variance [ANOVA] with the method
[leucine incorporation or bacterial counting] and the
O2 regimen as independent factors, P = 0.58), suggesting similar ratios of leucine
incorporation to increase in cell number under both
O2 conditions. Similar results are obtained when
comparing bacterial carbon production derived from leucine measurements
and from the increase in bacterial carbon biomass calculated from cell
abundance and size, but the use of bacterial volume estimates
introduced some minor additional variation (oxic,
r2 = 0.84; anoxic,
r2 = 0.82) (Fig. 3B). These results
imply that leucine incorporation corresponded well to bacterial biomass
production under both oxic and anoxic conditions in waters from three
very different lakes.
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FIG. 2.
Bacterial growth curves during the batch culture
experiments. The arrows indicate the time of leucine incorporation
measurements. Bars show ±1 standard deviation (n = 5). Curve labels refer to lake names (see the text) and to anoxic
(anox) and oxic (ox) treatments.
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FIG. 3.
(A) Leucine incorporation versus change in bacterial
abundance (y = 27.79 · x + 0.77 and r2 = 0.86 for the anoxic
samples; y = 26.96 · x + 1.17 and r2 = 0.86 for the oxic samples
[linear regression]). (B) Bacterial production from the leucine
incorporation (BPleu) versus observed biomass change
(BPobserved) (y = 0.90 · x + 0.15 and r2 = 0.82 for the anoxic samples; y = 1.05 · x + 0.05 and r2 = 0.84 for the oxic samples [linear regression]). Grey and black symbols
denote oxic and anoxic conditions, respectively. Triangles, circles,
and squares represent experiments with water from Illersjön,
Lillsjön, and Mårn, respectively (see text for information about
lakes). The line corresponds to the 1:1 relationship.
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Effects of O2 contamination, headspace, and different
incubation vessels.
Throughout the 3.5-h experimental period the
bacterial production increased by about 70% (Fig.
4). When this drift was corrected for
(see Materials and Methods), the results showed that deliberate O2 contamination did not affect leucine
incorporation (by ANOVA, P = 0.57) (Table
1).
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FIG. 4.
Drift of bacterial production (determined by leucine
incorporation) with time in the experiment testing impacts of
O2 contamination, type of incubation vessel, and headspace
in the incubation vessels.
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TABLE 1.
Drift-corrected bacterial production in the experiment
testing impacts of O2 contamination, type of incubation
vessel, and presence of a headspace during incubation
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Theoretically, O
2 contamination could affect
bacterial production in several ways. The sudden introduction of
O
2 into the
samples may inhibit certain anaerobic
bacteria that are sensitive
to changes in the composition of dissolved
gases. Alternatively,
O
2 may increase substrate
availability by allowing the use of
oxygenases, a group of powerful
extracellular enzymes used in
the primary attack of substrates
(
29). The sudden presence of
O
2
possibly also induces a metabolic shift in the microbial community
favoring oxygen respiration at the expense of anoxic respiration
(nitrate, iron, manganese, and sulfate respiration), methanogenesis,
and fermentation processes, which all yield less energy per unit
of
substrate used than oxygen respiration (
35). Thus, an
O
2 intrusion causing such a metabolic shift
should result in altered
bacterial production. However, we found that
the O
2 saturation
level did not significantly
affect the leucine incorporation by
bacteria from anoxic water during
the 60 min of incubation with
leucine. One possible explanation of
these results is that effects
of O
2 contamination
develop too slowly to be registered during
short incubation times.
Alternatively, it is possible that most
of the bacteria active
in the anoxic water were facultative anaerobes.
Interestingly,
microbial processes regarded to be obligately anaerobic,
such as
methanogenesis and denitrification, seem to be quite tolerant
to
temporary O
2 intrusion, although process rates
are often reduced
under oxic conditions (
21-23).
The presence of a headspace during incubation yielded a small increase
(9%) in leucine incorporation (Table
1) compared to
incubation with no
headspace (by ANOVA,
P = 0.031). Possibly this
was due
to more vigorous mixing of the samples when bubbles were
present,
causing increased substrate availability. This headspace
effect can
easily be avoided by the use of syringes instead of
Eppendorf tubes
when measuring leucine incorporation under both
oxic and anoxic
conditions.
Leucine incorporation was on average 11% lower in Plastipak syringes
than in Discardit II syringes and Eppendorf vials (by
ANOVA,
P = 0.002) (Table
1). The rubber sealing the piston in
the Plastipak syringes may have caused a decrease in bacterial
growth
rates. Inhibition of bacterial production by rubber stoppers
has been
previously reported (
24). We thus recommend the use
of
Discardit II or other syringes without rubber
sealing.
Anoxic leucine incorporation rates were consistently lower than oxic
rates in the saturation level experiments (Fig.
1), while
oxic and
anoxic rates were equal in all other experiments. This
difference is
probably due to the different pretreatments of the
water. In the
isotope dilution experiment, original lake water
was transferred
directly into the experimental bottles, and possibly
bacterial growth
was limited to different extents in oxic and
anoxic waters by
remineralization processes. Presumably, bacterivores
were more active
in the oxic cultures, yielding a greater remineralization
and therefore
higher bacterial growth rates in the presence of
O
2. The other experiments included addition of
inorganic nutrients
and filtration, resulting in low initial numbers of
bacteria,
and a period of exponential growth without limitation by
nutrients
or organic substrate in any of the oxygen
regimens.
Comparative studies of bacterial production in oxic and anoxic
environments, e.g., at different depths of stratified water
bodies,
rely on methods that deliver comparable results for all
samples.
Despite the widespread use of the leucine method for
anoxic waters, the
validity of the method in these environments
has not been evaluated
previously. We describe a convenient procedure
for headspace-free
incubation in plastic syringes, and we conclude
that the leucine
incorporation method used in this way works equally
well in both oxic
and anoxic
waters.
| |
ACKNOWLEDGMENTS |
We thank Stefan Bertilsson, Sofia Kallner, and Ramunas
Stepanauskas for assistance during various parts of the work. Håkan Olsson generously provided information which helped us select lakes to sample.
This study was funded by the Swedish Natural Science Research Council.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Water and Environmental Studies, Linköping University, SE 581 83 Linköping, Sweden. Phone: 46 13 282960. Fax: 46 13 133630. E-mail: david.bastviken{at}tema.liu.se.
| |
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Applied and Environmental Microbiology, July 2001, p. 2916-2921, Vol. 67, No. 7
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.7.2916-2921.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
