FEMS Microbiology Ecology 25 (1998) 217^227
E¡ects of sunlight on occurrence and bacterial turnover of speci¢c
carbon and nitrogen compounds in lake water
Niels O.G. JÖrgensen a; *, Lars Tranvik 1;b , Heèlene Edling b , Wilhelm Graneèli b ,
Maîns Lindell b
a
Department of Ecology and Molecular Biology, Royal Veterinary and Agricultural University, Thorvaldsensvej 40,
DK-1871 Frederiksberg C, Denmark
b
Department of Ecology, Limnology, Ecology Building, Lund University, S-223 62 Lund, Sweden
Received 29 July 1997; revised 5 November 1997; accepted 11 November 1997
Abstract
The effects of solar radiation on concentrations and microbial utilization of various carbon and nitrogen compounds were
studied in July in a thermally stratified lake in southern Sweden. Exposure of bacteria-free water to natural sunlight in the
surface of the lake for 7 h around noon led to higher concentrations of inorganic carbon (39^80%), amino acids (0^23%) and
carbohydrates (0^15%), while lower concentrations of monosaccharides (0^38%), nitrate (0^23%) and urea (0^27%) were
measured. Ammonium was unchanged. Lake bacteria were inoculated into the irradiated water and into water that had not
been exposed to solar radiation (dark controls). The bacterial production was 35 to 80% higher during exponential growth (20
h after inoculation) in the irradiated samples than in the controls. The bacterial utilization of specific carbon and nitrogen
compounds in the irradiated samples differed from that in the controls, but the changes in the epilimnion and the hypolimnion
varied. Dominant nutrients to the bacteria were carbohydrates, amino acids, glucose and ammonium. In the controls a release
of combined amino acids (epilimnion) or carbohydrates (hypolimnion) occurred. An apparent non-biological removal of urea
in the irradiated hypolimnion samples was found, since the microbial urea degradation was only 1% of the reduction in
concentration. Our results suggest that biogeochemical cycling in natural waters is influenced by sunlight, due to changes of
microbially available components that were not reported previously, including amino acids, carbohydrates, nitrate and urea.
z 1998 Published by Elsevier Science B.V. All rights reserved.
1. Introduction
Dissolved organic matter (DOM) is the largest
reservoir of organic carbon in natural waters [1].
* Corresponding author. Tel.: +45 (35) 28 26 25;
Fax: +45 (35) 28 26 06; E-mail: [email protected]
1
Present address: Tema Vatten, Department of Water and
Environmental Studies, Linkoëping University,
S-581 83 Linkoëping, Sweden.
Only a minor fraction of the DOM is readily available for microbial utilization, the remainder being
recalcitrant (for a recent review, see SÖndergaard
and Middelboe [2]). In freshwater environments, a
signi¢cant portion of the recalcitrant material is humic substances, that include a variety of combined
organic molecules such as fatty acids, phenols, saccharides and amino acids [1,3].
Natural solar radiation has been found to induce
chemical transformation of DOM. Hence, the chro-
0168-6496 / 98 / $19.00 ß 1998 Published by Elsevier Science B.V. All rights reserved.
PII S 0 1 6 8 - 6 4 9 6 ( 9 7 ) 0 0 0 9 6 - 2
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mophoric fraction is degraded, resulting in a bleaching of DOM [4]. High-molecular-weight DOM compounds are photochemically degraded into smaller
molecules. All of the documented low-molecularweight organic photoproducts are carbonyl compounds (reviewed in Moran and Zepp [5]), such as
carboxylic acids [6] and aldehydes [7]. A light-mediated release of biologically available monosaccharides and amino acids from recalcitrant DOM may
also occur, but this has not yet been demonstrated.
Recently photochemical release of ammonium from
DOM has been suggested to be a signi¢cant process
regulating nitrogen availability in aquatic ecosystems
[8].
A radiation-induced modi¢cation of recalcitrant
DOM towards more bioavailable organic compounds a¡ects the cycling and mineralization of organic matter. Accordingly, the availability of DOM
as a substrate for heterotrophic bacteria is enhanced
by exposure of the DOM to solar radiation, resulting
in enhanced bacterial growth [9,10].
In the present study we examined the e¡ect of
solar radiation on the composition and bacterial utilization of lake water DOM. We demonstrate that
sunlight modi¢es the composition of natural DOM
and that this modi¢cation stimulates the bacterial
activity and the bacterial preference for speci¢c substrates. Our results suggest that photochemical reactions involving free and combined carbohydrates and
amino acids may a¡ect bacterial metabolism.
2. Material and methods
2.1. Sampling and experimental set-up
E¡ects of sunlight on microbial nutrient cycling
were studied in water from the thermally strati¢ed
clear-water Lake Skaërshult, southern Sweden [11], in
July 1994. Water was collected in the epilimnion (1
m, 26³C) and in the hypolimnion (7 m, 8³C) and
sequentially ¢ltered through Gelman type A/E glass
¢bre ¢lters and 0.2-Wm Gelman Supor ¢lters (Gelman Sciences, Ann Arbor, MI, USA). Finally, the
water was ¢lter-sterilized through 0.2-Wm Gelman
Vacucap ¢lters into autoclaved 190-ml quartz tubes.
The initial 100- to 500-ml ¢ltrates were discarded at
each ¢ltration to exclude a contamination with dis-
solved organics from the ¢lters. The tubes were
sealed with rubber plugs and exposed to natural sunlight at the surface of the lake for 7 h around noon.
The maximum light intensity was 0.14 W m32 (UVB, 280^320 nm), 26 W m32 (UV-A, 320^400 nm) and
315 W m32 (PAR, 400^750 nm), as measured with
an IL1400A radiometer with broadband sensors (International Light, Newburyport, MA, USA). After
exposure, 10% of the original bacterial densities
from the epilimnion and hypolimnion were reintroduced into the samples. The bacterial density was
estimated from volumes of the original lake water,
¢ltered through 6 0.7-Wm pore size GF/F ¢lters
(Whatman International, Maidstone, England). The
resulting bacterial cultures and an identical set of
control cultures with unirradiated water were transferred to a series of 100-ml £asks and incubated at in
situ temperature for 60 h. At 10- to 12-h intervals,
samples were taken from the £asks for analysis of
various microbiological and chemical parameters.
2.2. Microbiological analyses
Bacterial densities were determined by epi£uorescence microscopy after staining with DAPI [12]. For
measurement of the bacterial production, 1.7-ml triplicate samples and a killed control (contained 2%
formaldehyde) received [3 H]leucine and unlabeled
leucine to a ¢nal concentration of 100 nM [13,14].
After incubation periods of 30 to 120 min, the cells
were mixed with trichloroacetic acid (TCA), centrifuged and reextracted in TCA according to Kirchman [15] and Smith and Azam [16]. Finally, the radioactivity was counted by liquid scintillation.
Bacterial carbon and nitrogen production was derived from leucine incorporation and conversion factors presented by Simon and Azam [17] and assuming a bacterial C:N ratio of 5 [18].
Bacterial assimilation of dissolved free amino acids
(DFAA), glucose and fructose was determined from
net radiotracer incorporation and actual concentrations of the substances. Triplicate 5-ml water samples and a killed control (containing 2% formaldehyde) received 10 nCi of either [14 C]glucose,
[14 C]fructose or four 14 C-labelled amino acids at
equimolar concentrations (glutamic acid, serine, glycine and alanine). The added tracers did not exceed
1 nM in concentration, corresponding to a maximum
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of 1.5% of their natural concentrations. The incubations were terminated after 15 to 90 min by addition
of formaldehyde, followed by ¢ltration through 0.2Wm membrane ¢lters, rinsing with 0.2 Wm ¢ltered
lake water and counting of the radioactivity by
liquid scintillation.
Uptake (hydrolysis) of urea by the bacteria was
measured from production of 14 CO2 in triplicate
20-ml water samples to which 60 nCi [14 C]urea
(equals 54 nM l31 , or a maximum of 7% of the
natural concentrations) had been added. The samples were incubated in 100-ml serum bottles with
rubber membranes to which plastic cups were fastened with accordion-folded paper wicks under the
membrane. The uptake was stopped after incubation
periods of 75 to 210 min by injection of formaldehyde through the membranes. The samples were
acidi¢ed with 10% phosphoric acid to drive o¡
14
CO2 , after which the wicks were soaked with 500
ml of Carbosorb CO2 absorber (Packard Instruments, Groningen, The Netherlands). After 1 h on
a shaking table, the CO2 traps were transferred to
20-ml scintillation vials and radioassayed.
Bacterial L-glucosidase activity was estimated by
addition of the substrate analogue 4-methyl umbelliferyl-L-glucopyranoside (L-glucosidase-MUF) to a ¢nal concentration of 200 WM in 5-ml samples. After
an incubation period of 15 h (from 22 to 37 h after
the start of the experiment), the enzyme activity was
measured as the release of the £uorescent product
4-methylumbelliferone [19].
2.3. Chemical analysis
Concentrations of glucose, fructose and total dissolved carbohydrates (DCCHO, measured as eight
individual monosaccharides after hydrolysis) were
determined by high pressure liquid chromatography
(HPLC) and pulsed amperometric detection [20].
DFAA and dissolved combined amino acids
(DCAA, hydrolysed to individual DFAA according
to the procedure of JÖrgensen and Jensen [21]) were
measured as £uorescent o-phthaldialdehyde derivatives by HPLC [22,23]. Total dissolved nitrogen
(TDN) was measured on a Mitsubishi Total Nitrogen Analyzer TN-5 (Mitsubishi Kasei Corporation,
Tokyo, Japan) by high temperature oxidation and
chemoluminescense detection. Concentrations of dis-
219
solved organic nitrogen (DON) were calculated by
subtraction of dissolved inorganic nitrogen (DIN,
ammonium, nitrate and urea) from the TDN concentrations. Ammonium and nitrate was measured by
standard autoanalyser methods. Urea was determined by the diacetyl monoxime method of Price
and Harrison [24]. Dissolved organic carbon
(DOC) and dissolved inorganic carbon (DIC) were
measured by platinum-catalyzed high temperature
oxidation using a Shimadzu TOC 5000 total carbon
analyzer (Shimadzu Corporation, Kyoto, Japan)
[25].
3. Results and discussion
3.1. Light-induced e¡ects on natural pools of organic
and inorganic compounds
Exposure of lake water to solar radiation altered
the concentration of several organic and inorganic
compounds, relative to water incubated in the dark
(Fig. 1). No statistically signi¢cant changes of the
DOC concentrations were observed, but the dissolved inorganic carbon (DIC) pool increased by
80% (epilimnion) and 39% (hypolimnion). These increases indicate that a portion of the DOC was photooxidized to DIC. The observed production of DIC
is within the range previously measured in Swedish
lakes [25].
The sunlight increased the concentration of dissolved free and combined amino acids (DFAA and
DCAA), respectively, by 13^23% (DCAA only
changed in the epilimnion). Total dissolved combined carbohydrates (DCCHO) were raised by 15%
in the hypolimnion, but no changes were observed in
the epilimnion. The light did not change the concentration of free glucose, but fructose was reduced by
38% in the hypolimnion samples. Previously, it has
been demonstrated that solar radiation transforms
high molecular weight DOM into a variety of organic and inorganic molecules [5]. Our study indicates
that the list of DOM photoproducts should also include free and combined amino acids and carbohydrates, but apparently not free dissolved fructose and
glucose. The produced components may have been
associated with high-molecular-weight organic compounds, which in natural waters have been found to
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include both amino acids and saccharides [26,27].
Possibly, the changes in combined amino acids and
carbohydrates were due to an increased analytic
availability, following the photochemical conditioning of humic-bound DCAA and DCCHO. Fructose
has been shown to react with metals and form complexes in natural environments [28]. This may explain the observed reduction of the concentration
of free fructose in the hypolimnion samples.
The concentrations of total dissolved nitrogen and
dissolved organic nitrogen (TDN and DON) were
not in£uenced by the exposure to light (Fig. 1).
The DON pool was 2.5-fold higher in the epilimnion
than in the hypolimnion. The reduced DON pool in
the hypolimnion in relation to the epilimnion most
likely indicates a previous microbial degradation of
DON. The 5- and 11-fold higher amounts of nitrate
and ammonium, respectively, in the hypolimnion
than in the epilimnion, indicate that a mineralization
of organic nitrogen had occurred in the hypolimnion.
The concentration of ammonium in the water was
not in£uenced by the solar radiation, in contrast to
the recent ¢nding of Bushaw et al. [8]. Hence, their
suggestion of a signi¢cant photochemical release of
ammonium from natural DOM was not substantiated by our results. Nitrate in the hypolimnion light
samples declined 23% relative to the dark controls
while no changes were found in the epilimnion samples. Immediately after the photochemical incubation, the water was inoculated with bacteria. At
this stage, the bacterial production was extremely
low (not statistically di¡erent from zero; P 6 0.05,
t-test) (Fig. 2A), indicating that no signi¢cant bacterial activity occurred during the light exposure.
Hence, the chemical reduction of nitrate in the hypolimnion samples was most probably the result of a
non-biological reaction. Nitrate may have reacted
with nitrite to form NO2 [29], or it may have served
as an electron acceptor in the photo-oxidation of
organic or inorganic compounds. The concentration
of urea increased by 27% in the epilimnion samples,
but in the hypolimnion no signi¢cant changes occurred. Possibly the produced urea originated from
splitting of urea-aldehyde condensates. Urea can react with aldehydes to produce long-chain polymers
[30], and since aldehydes are known products of photolysis of DOM [8], such polymers may have been
present before photoreaction in the epilimnion, but
not in the hypolimnion.
Fig. 1. Concentrations of various dissolved organic and inorganic compounds in ¢lter-sterilized Lake Skaërshult water after 7 h incubation
in the dark (¢lled bars) or in sunlight (open bars) in quartz tubes (average concentrations from duplicate analysis of each of three tubes
shown). *Indicates a signi¢cant change (P 6 0.05; t-test).
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221
acids. Since the solar radiation only increased the
amino acid pools (DFAA and DCAA) by 0.44
WM, and only in the epilimnion samples, the higher
protein synthesis in the light samples could not alone
be supported by a photochemical amino acid production. Rather, photochemical production of lowmolecular-weight carbon compounds [6,7,29] provided carbon for anabolic pathways of the bacteria.
However, bioavailability of DCAA, e.g. associated
with humic matter, may have changed during the
light exposure, but this would not be detected by
the concentration measurements.
3.3. Light-induced e¡ects on bacterial utilization
of carbon compounds
Fig. 2. Cell-speci¢c protein synthesis (leucine incorporation) (A)
and bacterial abundance (B) in batch cultures with epilimnion
(Epi) or hypolimnion (Hypo) water incubated in the light and
in the dark. Means þ 1 S.D. shown (n = 3 replicates of each of
3 samples).
3.2. Light-induced stimulation of the bacterial
production
The cell-speci¢c protein synthesis (from incorporation of [3 H]leucine) peaked at 22 h, when the individual protein synthesis rate was 2-fold higher in the
light than in the dark exposed samples (Fig. 2A).
The bacterial production led to a 1.8- and 1.4-fold
higher cell abundance after 60 h in the epilimnion
and hypolimnion samples, respectively, in light
than in dark (Fig. 2B). Obviously substances in the
photolysed water stimulated the bacterial growth.
The higher leucine incorporation in the light samples
may indicate that the bacterial stimulation was due
to an enhanced uptake of amino acids. The bacterial
protein production during the entire 60-h incubation
period necessitated from 1.7 to 4.1 WM amino acids,
based on conversion of [3 H]leucine incorporation to
total protein synthesis [17]. The higher protein synthesis in the light-exposed samples required 1.3 (epilimnion) and 1.1 WM (hypolimnion) of these amino
Utilization of dissolved organic compounds by the
bacteria [31] was followed during the 60-h incubation
period in radiotracer experiments (DFAA, glucose,
fructose and urea), or from changes in the ambient
pools (DCAA and DCCHO), assuming that all variations in concentrations were due to bacterial activity. The bacterial uptake was converted to units of
carbon and nitrogen and related to the carbon and
nitrogen production of the bacteria. All presented
rates are net values (no respiration included), except
for DCAA and DCCHO, as discussed below. Uptake of nitrogen is presented in the following section.
The carbohydrate concentration in the epilimnion
samples decreased during the bacterial incubation
(Fig. 3) and the saccharide composition changed.
The most abundant carbohydrate saccharides in the
epilimnion were glucose, galactose, arabinose and
mannose, with relative proportions of 1.00, 0.67,
0.58 and 0.47, respectively. This composition had
remained unchanged during the exposure to sunlight
(P 6 0.05; t-test). The bacterial carbohydrate consumption of 1.14 WM during incubation of the light
samples was dominated by uptake of glucose and
mannose. The uptake resulted in an increase of galactose and arabinose to 1.01 and 0.78, respectively,
relative to glucose. In the dark controls, the carbohydrate pool was reduced by 1.6 WM during the incubation. A high uptake of mannose reduced its relative abundance from 0.47 to 0.20, while there was a
similar uptake of glucose, galactose and arabinose.
Carbohydrates were the single most common, identi¢ed carbon source to the bacteria, constituting
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Fig. 3. Bacterial production (biomass accumulation) and integrated consumption of nutrients (carbon and nitrogen) during the 60-h incubation period. Percentages indicate the integrated uptake of carbon or nitrogen relative to the biomass production. Numbers in parentheses indicate corresponding values corrected for release of DCAA, DCCHO or nitrate. Error bars indicate þ 1 S.D. (n = 3 replicates of
each of 3 samples).
from 43 to 67% of the measured bacterial carbon
incorporation in the epilimnion (Fig. 3).
The radioisotope uptake in the epilimnion samples
showed that free glucose and fructose together made
up 7% (dark) to 17% (light) of the total carbon uptake, but glucose was 7-fold more important than
fructose. Incorporation of DFAA constituted about
25% of the bacterial carbon uptake, and they were a
more important bacterial substrate than DCAA.
Dominant DFAA were aspartate, serine and glycine
at the start of the experiment in both the light and
the dark samples, but due to the uptake, variations
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in composition occurred during the incubation. In
the dark samples, an increased concentration of
DCAA indicated a release of combined amino acids.
This release led to a change in the DCAA composition. Initially, common DCAA in the light and dark
samples were aspartate, glutamate, serine, glycine
and alanine, but the proportion of glutamate and
lysine increased signi¢cantly after the DCAA release.
The high activity of the exoenzyme L-glucosidase in
the dark controls (see below) may explain the observed DCAA production in these samples, assuming
that the DCAA were amino acids of the L-glucosidase enzyme.
In the hypolimnion, the bacterial uptake of amino
acids and saccharides in the light samples resembled
that in the epilimnion light (Fig. 3) but in the dark
controls, the carbohydrate concentration increased
from 35 to 60 h. This increase equaled 40% of the
bacterial carbon uptake. Relative to glucose, the initial proportions of galactose, arabinose and mannose
were 0.54, 0.49 and 0.51, respectively. As in the epilimnion samples, no e¡ects of sunlight on the saccharide composition was found (P 6 0.05; t-test). In
the light samples, bacterial uptake reduced the carbohydrate concentration by 1.03 WM. A large uptake
of mannose reduced its relative proportion from 0.51
to 0.23, while the uptake of the three other saccharides was similar. The carbohydrate release of 0.67
WM in the dark controls was caused by a production
of mainly arabinose and galactose, increasing their
proportions relative to glucose from 0.54 and 0.49,
to 0.76 and 0.65, respectively.
Galactose and glucose are among the major components of bacterial exopolysaccharides [32]. Therefore, the release of galactose may indicate a production of exopolysaccharides by bacteria in the
hypolimnion dark samples. A carbohydrate production quantitatively similar to the present release, and
also including galactose, previously has been observed in cultures of natural bacterioplankton [20].
The bacterial utilization of amino acids in the hypolimnion was 1.8-fold higher in the dark than in the
light. The uptake of DFAA in the two cultures was
similar, but the DCAA uptake was signi¢cantly reduced in the light (Fig. 3). Most of the DCAA uptake occurred during the last third of the 60-h incubation period. Initially the DFAA pool was
dominated by aspartate, glycine and arginine but at
223
60 h, glycine and lysine were most abundant. Major
DCAA during the entire 60-h incubation were aspartate, glutamate, glycine and arginine.
Free and combined saccharides and amino acids
met a larger portion of the bacterial carbon demand
in the epilimnion than in the hypolimnion. After
correction for release of DCAA and DCCHO, saccharides and amino acids were found to sustain 55%
(light) and 72% (dark controls) of the bacterial carbon demand in the epilimnion. The corresponding
values for the hypolimnion samples were 31 and
20%. The bacterial carbon production was determined from the incorporation of [3 H]leucine.
The calculated contributions of DCAA and
DCCHO to the bacterial carbon demands probably
are overestimates, as the uptake was based on concentration changes. When using changes in concentration it cannot be determined whether the substances taken up were incorporated into cell components
or respired. Bacterial production and isotope uptake
of DFAA, glucose and fructose are presented as net
rates, not including respiration. If respiration of
DCAA and DCCHO was similar to that of
DFAA, glucose and fructose taken up by the bacteria (these respiration percentages ranged from 15 to
27%; JÖrgensen, unpublished data), carbon incorporation from DCAA and DCCHO may be similarly
overestimated. The bacterial growth e¤ciency (the
fraction of the utilized organic carbon transformed
to biomass) in similar cultures from the same lake in
previous experiments was found to be 7 to 30%, i.e.
the respiration was 70 to 93% [11,33]. If the bacteria
had a similar, low growth e¤ciency in this study, 15
to 27% respiration of amino acids and saccharides
implies that a large fraction of other components
taken up by the bacteria was respired. Generally,
the respiration by natural bacterial assemblages consumes at least roughly as much carbon as does biomass accumulation [34].
Use of a substrate analogue for measuring extracellular L-glucosidase activity (indicator of cellulose
degradation to low molecular-weight saccharides)
showed a similar enzyme activity in the epilimnion
light and the hypolimnion light and dark samples,
but in the epilimnion dark there was a 4- to 5-fold
higher activity (Table 1). The enhanced enzyme activity is in accordance with the high uptake of
DCCHO in these samples (Fig. 3). Thus, cellulose
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Fig. 4. Inorganic nitrogen dynamics during the incubation. A:
Uptake of urea, determined from hydrolysis of 14 C-labeled urea.
B: Changes in ammonium. C: Concentration changes of urea,
nitrate and ammonium, and bacterial uptake of urea in the four
cultures. In the hypolimnion samples, the urea uptake is magni¢ed 10U. All error bars indicate þ 1 S.D. (n = 3 replicates of
each of 3 samples).
or more likely cellulose-derived compounds probably
were taken up by the bacteria in the epilimnion dark
samples during the experiment.
3.4. Light-induced e¡ects on bacterial utilization of
nitrogen compounds
Uptake of nitrogen by bacteria was variable. In
the epilimnion, DFAA-N and DCAA-N equaled
48% of the bacterial nitrogen demand in the light
samples, while inorganic nitrogen corresponded to
21% (Fig. 3). In the dark samples, nitrogen equivalent to 30% of the bacterial production was released
as DCAA. This loss of amino nitrogen apparently
was compensated for by a relatively high uptake of
free amino acids and inorganic nitrogen, corresponding to 113% of the bacterial nitrogen production.
DFAA made up 48% of the uptake, while inorganic
nitrogen (ammonium, nitrate and urea) accounted
for the remaining uptake. The DFAA uptake was
higher than that in the other epilimnion and hypolimnion cultures, in which 15 to 28% of the nitrogen
uptake originated from DFAA, and the uptake of
DIN was 3-fold higher than in the epilimnion light
samples. The maximum urea uptake (hydrolysis) was
2-fold higher in the dark than in the light samples,
but it peaked at 47 h in both cultures (Fig. 4A).
In the hypolimnion, ammonium was the dominant
nitrogen source, making up from 51% (dark) to 61%
(light) of total nitrogen assimilation (Figs. 3 and 4B).
The uptake of urea was insigni¢cant (Fig. 4A),
whereas there was a production of nitrate, probably
due to a bacterial oxidation of ammonium to nitrate
(nitri¢cation, see below). Total amino nitrogen met
18% (light) and 40% (dark) of the bacterial nitrogen
production.
A large fraction of the bacterial nitrogen production was sustained by uptake of the studied nitrogen
compounds. When considering the release of DCAA
and nitrate, from 54% (hypolimnion, light) to 92%
(hypolimnion, dark) of the bacterial nitrogen production was accounted for. Accordingly, at most
about half of the nitrogen utilized by the bacteria
resided in complex bulk DON that could not be
characterized as free or combined amino acids.
Bacterial uptake of urea equaled 4.5% (light) and
16% (dark) of the bacterial nitrogen production in
the epilimnion, but only 0.4% in the hypolimnion
samples. The urea concentrations decreased up to
0.8 WM during the incubation (data not shown),
Table 1
Cell-speci¢c L-glucosidase activity (10318 mol L-glucosidase-MUF cell31 h31 )
Epilimnion light
Epilimnion dark
Hypolimnion light
Hypolimnion dark
0.40 þ 0.35
2.12 þ 0.63
0.60 þ 0.26
0.67 þ 0.60
L-glucosidase-MUF (4-methyl umbelliferyl-L-glucopyranoside) was added to 5-ml water samples to a ¢nal concentration of 200 WM. The
samples were incubated for 15 h (from 22 to 37 h after start of the experiment).
Means þ 1 S.D. shown (n = 4).
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but the measured decreases did not match the
amounts of urea taken up by the bacteria, measured
with the urea radioisotope. The bacterial urea hydrolysis (production of 14 CO2 from [14 C]urea)
peaked at 36 to 47 h, with rates of 3.5 to 9 nM
h31 (epilimnion) and 0.2 to 0.4 nM h31 (hypolimnion) (Fig. 4A). Since we did not measure an actual
incorporation of urea nitrogen into the bacteria, but
only the urease activity, it may be argued that the
bacteria did not take up ammonium produced by the
urea hydrolysis. However, the maximum urease rates
coincided with non-detectable concentrations of ammonium in the epilimnion (Fig. 4B). If ammonium
produced by the hydrolysis of urea was not taken up
by the bacteria but released, the ammonium concentration in the epilimnion samples was expected to
increase by 0.2 to 0.4 WM due to urea hydrolysis
after 47 h of incubation. Since we did not detect
ammonium at 47 h, we believe that the present approach did measure the actual incorporation of urea
nitrogen into the bacteria. The coincidence of a maximum urea hydrolysis rate and absence of ammonium in the epilimnion samples suggests that the
low ammonium concentrations stimulated the urea
hydrolysis. Despite a nitrate level of 0.6 to 1.0 WM
in the epilimnion, the uptake of nitrate was lower
than that of urea.
The isotope-based uptake of urea equaled 19%
(light) and 45% (dark) of the urea decline in the
epilimnion and about 1% in the hypolimnion (Fig.
4C). This discrepancy suggests that bacterial incorporation was not the only process responsible for
removal of urea. It is probable that the reduction
of urea was due to a nonbiological polymerization.
Urea reacts with aldehydes to form polymers [30].
Since aldehydes are common products of the photolysis of DOM [8], this reaction may have been involved in reducing the free urea pool in our samples.
We speculate that urea may also have produced nitrogen for the observed nitri¢cation in the hypolimnion samples (Fig. 4C). Hydrolysis of urea to ammonium (and bicarbonate) can provide a nitrogen
source to nitrifying bacteria [35]. The present coincidence between reduction of ammonium and nitri¢cation suggests, however, that nitrate in the hypolimnion cultures was produced by oxidation of
ammonium (Fig. 4C). Alternatively, the decrease of
urea that according to the radiotracer experiments
225
was not accompanied by a microbiological hydrolysis, could have involved transformation of nitrogen
by nitrifying bacteria. If so, the carbon moiety of
urea was metabolized to other compounds than
CO2 , as indicated by an insigni¢cant production of
14
CO2 from the added urea radioisotope.
3.5. Relations between bacterial production and
uptake of speci¢c C and N compounds
The stimulated bacterial production in the lightexposed water was generally not re£ected in proportional increases in uptake of amino acids, saccharides
and inorganic nitrogen. The 1.4- to 1.8-fold higher
bacterial production in the light samples increased
the net contribution of amino acid and saccharide
carbon to the bacterial carbon production from 20
to 31% in the hypolimnion samples but in the epilimnion, the amino acid and saccharide contribution
was reduced from 72 to 55% (Fig. 3). The net uptake
of nitrogen from amino acids, ammonium, nitrate
and urea, relative to the bacterial nitrogen production, was 14% (epilimnion) to 38% (hypolimnion)
lower in the light cultures than in the dark cultures
(Fig. 3). Also, the higher concentrations of DFAA,
DCAA and urea, and the reduction of fructose after
the initial light exposure (Fig. 1) apparently do not
in£uence the uptake rates of the compounds, when
compared to the dark controls. These ¢ndings all
suggest that other substances, e.g. photochemically
produced low-molecular substances that were not
detectable by our methods, were essential nutrients
for the bacteria in the light samples.
The observed bacterial preference for DFAA over
DCAA agrees with other studies [36,37], but a higher
preference for DFAA over ammonium [38] was not
con¢rmed by our observations. The large uptake of
ammonium in the hypolimnion samples may be related to utilization of carbohydrates, together creating a favourable C/N ratio for bacterial cell production [39,40]. However, other organic compounds
formed by photooxidation may have stimulated ammonium uptake. Uptake of urea equaled 25% of
ammonium uptake in the epilimnion, but in the hypolimnion the high ammonium pool probably reduced the utilization of urea nitrogen.
Our observations demonstrate that cycling of carbon and nitrogen by natural bacterioplankton is
FEMSEC 881 4-3-98
226
N.O.G. JÖrgensen et al. / FEMS Microbiology Ecology 25 (1998) 217^227
stimulated by solar radiation. The quantitative e¡ect
of sunlight on the biogeochemical £ux in a natural
ecosystem will depend on the light penetration depth
and the residence time of the water receiving reactive
light. In addition to the stimulatory e¡ect of sunlight
on bacteria, caused by photochemical conditioning
of DOM, solar light may also inhibit bacteria, e.g.
due to DNA damage [41,42]. Hence, in a range of
lakes with greatly di¡erent light attenuation, there
was a 1^24% depth-integrated inhibition of bacterioplankton production due to sunlight [43]. However,
inhibition occurs during exposure to light, while the
bacterial substrates that are photochemically produced from recalcitrant DOM can be utilized by
bacteria after mixing to deeper layers protected
from short wavelength light, or during night. Thus,
solar inhibition of bacteria may alter the temporal
and spatial patterns of bacterial utilization of photoproduced substrates, but does probably not a¡ect the
total extent of the sequential photochemical-microbial degradation of DOM.
This paper demonstrates that sunlight can modify
the composition of dissolved carbon and nitrogen
compounds utilized by bacterioplankton (amino
acids, carbohydrates, nitrate and urea) in lake water,
and that some of the modi¢ed substances may lead
to an altered bacterial metabolism. Our study was
restricted to one ecosystem during a summer strati¢cation and thus seasonal e¡ects were not examined.
However, we hope that the present results will make
other aquatic ecologists aware of the potential e¡ects
of sunlight on bacterial cycling of dissolved compounds in natural waters.
Acknowledgments
We wish to thank R.E. Jensen for skillful technical
assistance and N. Kroer for providing analysis of
TDN. This study was supported by The Danish Strategic Environmental Research Program to N.O.G.J.,
the Swedish Natural Science Research Council to
W.G. and L.T., and the Environment and Climate
Program of the European Commission (MICOR
project) to L.T.
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