Ihnnol.
0
1998,
Occanog,:,
43(.5), 1998, 885-895
by the American
Society
OF Limnology
and Oceanography,
Jnc.
Photochemically produced carboxylic acids as substrates for freshwater
bacterioplankton
Stefan Bertilsson and Lam J. Tranvik
Department of Water and Environmental
Studies, Linkoping
University,
S-58 183 Linkoping,
Sweden
Abstract
.
High-molecular-weight
dissolved organic matter is abundant in humic lakes and is a large potential source of
energy for heterotrophic organisms. These substances are hard to degrade enzymatically because of their high
aromaticity and complex structure. However, there is increasing evidence that photochemical processes render the
material more bioavailable. We demonstrate a substantial photochemical production of four carboxylic acids (oxalic,
malonic, formic, and acetic acid) in a humic lake. The combined production rate in the surface water of these four
acids was 19 pg C liter-l h-l with natural sunlight. Furthermore, based on radiotracer studies, we found that the
amount of carbon assimilated and oxidized to CO, from malonic, formic, and acetic acid exceeded bacterial carbon
production, sometimes by more than one order of magnitude. This implies that carboxylic acids were major bacterioplankton substrates. Nevertheless, under natural sunlight at the lake surface, microbial utilization of carboxylic
acids was substantially lower than the photochemical production of the acids. Hence, photochemically produced
carboxylic acids may accumulate in sunlight exposed environments and may also serve as bacterial substrates after
mixing into deeper layers, or during night.
-
Dissolved organic matter (DOM) from terrestrial sources
makes up a dominant fraction of the bioavailable organic
matter in the water column of many lakes (Wetzel 1984,
1995). Although algal primary production is traditionally
considered the major energy source for bacterioplankton, the
concentration of allochthonous DOM explains much of the
variation in bacterial density among lakes (Tranvik 1988).
Accordingly, additional energy from the watershed and from
the littoral zone is most likely needed to support the relatively high heterotrophic biomass of many oligotrophic lakes
(de1 Giorgio and Gasol 1995).
Generally, DOM of lakes is dominated by humic matter
(Thurman 1985). Therefore, it is likely that the high bacterial biomass in many humic lakes is dependent on the
utilization
of humic compounds. Accordingly,
bacterial
growth on isolated freshwater humic substances has been
demonstrated (e.g., Moran and Hodson 1990). The bacterial utilization of the energy and carbon stored in the
heterogeneous, recalcitrant humic compounds is poorly
understood. Because of their relatively high molecular
weight, the humic substances must be degraded into smaller units before they can be utilized by bacterial cells. Bacteria employ extracellular enzymes to cleave the macromolecules
into monomers
or oligomers
that are
subsequently easily transported across the cell membrane
(Chrost 1991). There are, however, alternative mechanisms that render the organic material less refractory and
thereby facilitate bacterial utilization of this potential carbon and energy source.
Photochemical processes have been pointed out as one
such catalytic mechanism, altering bioavailability
of natural organic matter (Geller 1986; Lindell et al. 1995). Kieber et al. (1989) observed a strong positive correlation
between photochemical
production
of pyruvate from
DOM in marine environments, and biological uptake of
this degradation intermediate. Furthermore, several other
carbonyl compounds (Kieber et al. 1989; Mopper et al.
1991), as well as low-molecular-weight
(LMW) carboxylic acids (Backlund 1992; Allard et al. 1994), have been
identified as important degradation intermediates. Wetzel
et al. (1995) demonstrated a substantial production of several LMW carboxylic acids when a leachate from senescent littoral plants was exposed to ultraviolet (UV) radiation from the sun or from artificial sources. Upon UV
irradiation, the organic matter was also more susceptible
to microbial degradation.
A recent study by Bertilsson and Allard (1996) supports
these observations, as abiotic production of several LMW
carboxylic acids was substantial when natural organic matter
in humic lake water was exposed to UV radiation of near
natural intensities and spectral distribution. Irradiation of humic water containing indigenous bacteria resulted in an increased bacterial biomass. These studies indicate a sequential
photochemical-microbial
degradation pathway. Although
there is substantial evidence for the photochemical production of LMW carboxyl and carbonyl compounds, including
indications of their importance as bacterial substrates (Kieber et al. 1989; Wetzel et al. 1995), we know little about
how much of total carbon and energy demand of aquatic
bacteria is supported by photochemically produced intermediates.
We studied the strictly photochemical production of LMW
carboxylic acid degradation intermediates in one humic and
one clearwater lake. In addition, we measured bacterial up-
Acknowledgments
This study was financed by grants from the Swedish Natural Research Council to Bert Allard and L.J.T. and from the Environment
and Climate program of the European Commission (MICOR project) to L.J.T. We thank Helen Wolrath for analytical assistance and
Bo Svensson and Julie Wilk for valuable comments. In addition,
the comments by two anonymous reviewers greatly improved the
manuscript.
885
886
Bert&on
take and respiration of carboxylic acids and compared these
rates with total bacterial production.
Materials and methods
Summary of experimental design-Several
experiments
were performed, each employing a separate original lake water sample. Subsamples were filter sterilized, analyzed for
initial chemical characteristics, and incubated either irradiated or in the dark. Subsequently, the strictly photochemical
production of LMW carboxylic acids was measured. In parallel, unfiltered water from the same initial water sample was
incubated under similar conditions (dark/irradiated). These
samples, containing the total microbial community of the
lake water, were used for kinetic studies of biological carboxylic acid utilization and bacterial production immediately
after the incubation.
Four of the experiments employed water from a humic
lake and an artificial source of UV radiation. Each of these
experiments was focused on detailed studies of the biological utilization of a separate carboxylic acid (oxalic, malonic,
formic, or acetic acid). Finally, two experiments were made
under natural sunlight with water from a humic and a clearwater lake, respectively. In these experiments, photochemical production and bacterial utilization of malonic, formic,
and acetic acid were performed simultaneously. Together
with estimates of bacterial production, the combined measurements of photochemical formation and bacterial utilization of carboxylic acids allowed us to gauge the relation
between photochemical production and microbial degradation of these substances, as well as the ratio between total
bacterial biomass carbon production and bacterial cycling of
carboxylic acid carbon.
Sampling and irradiation-Surface
water was collected
from the humic Lake Skarshultsjon and the clearwater Lake
Fiolen (Tranvik 1988) in July 1995. The lakes are oligotrophic and are situated in a boreal forest area in southern Sweden, close to the limnological field station at Aneboda
(57”07’N, 14”34’E). A 2-m Plexiglas tube was used to sample water from the upper 2-m layer of the lake. The water
was immediately transported to the laboratory in Mini-Q
prewashed glass bottles, sieved through a nylon net (mesh
size, 100 pm) to remove large organisms and particles, and
processed within 1 h of sampling.
Samples for studies of the abiotic, photochemical transformation of organic matter in filter-sterilized water (0.2 pm
Sarstedt Filtropur-L filters, prewashed with 100 ml Milli-Q)
were incubated in thoroughly rinsed and autoclaved 40-ml
quartz tubes that were sealed with silicone stoppers and horizontally positioned. Tubes were incubated for 8 h at in situ
temperature (I 8-20°C) at the water surface, either around
noon under natural sunlight in the lake or in loo-liter aquaria
at 13 cm from four UV-A emitting lamps (Philips TL4OW/
05). The irradiation path length was 4 cm, and dark controls
were covered with aluminum foil. The lamps generated a
mean irradiance at the water surface of 0.015 W rnp2 of
UV-B (10% of natural radiation from the sun during a clear
summer day in southern Sweden), 8.5 W rnp2 of UV-A (35%
of natural radiation), and 5.5 W m-2 of photosynthetically
and Tranvik
active radiation (PAR) (1.6% of natural radiation), measured
using a broad band radiometer (IL 1400, International Light).
The irradiance during incubations under natural sunlight was
recorded for each individual experiment using the same radiometer (PAR, UV-A, and UV-B). Subsamples for chemical
analyses (carboxylic acid concentrations, UV absorbance,
and fluorescence) and direct counts of bacteria were collected from the incubated triplicates. To account for a possible leakage of formic and acetic acid from the silicone
stoppers (Linde:il 1996), similar incubations were performed
with Milli-Q water. No significant leakage was observed under the studied conditions.
Aliquots of 4 liters of organism-containing water were
transferred to polypropylene plastic bags that were subsequently sealed with a thermal sealer. Preliminary experiments showed that none of the studied carboxylic acids (<5
pg liter- ‘) or any detectable amount of dissolved organic
carbon (DOC; <CO.1mg liter-l) was released to the enclosed
water upon exposure in the dark or in sunlight. One bag was
covered with black plastic shields to block UV radiation and
light, while the other was incubated without any shielding
(>90% transmittance at each wavelength between 250 and
600 nm), and the average radiation path length of the bags
was 4 cm. The bags were incubated simultaneously with the
filter-sterilized incubations (8 h at 18-20°C). Subsamples of
incubated, organism-containing dark and UV-irradiated water were retriet,ed for measurements of bacterial production
and carboxylic acid uptake. Quantitative carboxylic acid
analysis and bacterial direct counts were performed on samples from both dark and irradiated samples after the preincubation.
Chemical aE!aZyses-Concentrations of LMW carboxylic
acids were analyzed by capillary ion electrophoresis (Dahlen
et al. 1996). We focused on four acids (oxalic, malonic, formic, and acetic acid) that were found to be major products
formed during irradiation of humic water (Bertilsson and
Allard 1996 unpubl. data). Five milliliters of a sterile filtered
sample (0.2 pm Millex GV, Millipore) was spiked with 100
~1 of a concentrated internal standard solution (molybdate),
to a final concentration of 100 pg liter-l to compensate for
variations in irjection efficiency. One hundred microliters of
a concentrated octanesulfonate solution was added to a final
concentration of 70 PM to obtain isotachophoretic conditions during the electromigrative sampling (45 s at 5 kV).
Duplicate subsamples of 0.6 ml were added to polypropylene
vials for analysis. Analytes were separated on a Quanta 4000
capillary electrophoresis system (Millipore) equipped with
an 80-cm (72 cm to detector) fused silica capillary (Supelco)
with an inner diameter of 75 pm, using a buffer (pH 7.9)
containing 5 mM 1,2,4,-benzene tri-carboxylic acid with 0.5
mM of an electro-osmotic flow modifier (OFM-BT, Waters).
A separation voltage of 15 kV was applied with the anode
located at the capillary outlet on the detector side. Detection
of carboxylic acids was accomplished by universal indirect
UV detection at 254 nm, and a Millennium Chromatography
Manager System (Waters) was used for data acquisition and
processing. LIvIW carboxylic acids were identified by using
retention times and spiking selected samples with small volumes of concentrated carboxylic acid stock solutions. Stan-
UV production
of bacterial substrates
dard curves (5-1,000 pg liter-l) were prepared by dissolving
the four carboxylic acids, i.e., oxalic, malonic, formic, and
acetic acid, in the waters studied.
The concentration of DOC in initial water was measured
on a Shimadzu TOC-5000 analyzer. Samples were filtered
through prewashed 0.2-pm polyvinylidene
difluoride
(PVDF) membrane filters (Millex GV, Millipore), acidified
(HCl, pH 2), and purged with CO,-free air to remove inorganic carbon. Blanks and standard solutions were prepared
from Milli-Q water and hydrogen phthalate and were analyzed before and after each set of samples. For each analysis,
three to five injections were made on the carbon analyzer,
resulting in a coefficient of variation of 2% or less. The
absorbance of the filtered water was measured in a lo-mm
quartz cuvette on a Beckman DU 650 spectrophotometer
with Mini-Q water as a control. Fluorescence was measured
with a Shimadzu RF-1501 spectrofluorometer. Excitation
was at 355 nm and emission at 455 nm (band widths, 10
nm), and the fluorescence was calibrated against a quinine
sulfate solution. One quinine sulfate unit is the fluorescence
of a 0.01-mg liter-l solution of quinine sulfate in 0.1 M
H,SO,. Triplicate water samples from each treatment (i.e.,
dark and irradiated samples) were pooled before analysis of
absorbance and fluorescence.
Bacterial production and biomass-Total
heterotrophic
bacterial production was measured using the microcentrifuge
radiolabeled leucine incorporation technique (Smith and
Azam 1992). In humic lakes, saturated uptake rate of label
requires higher concentrations of leucine than normally used
(Tulonen 1993). In previous experiments in Skarshultsjon,
we found saturated uptake at 100 nM in the studied lake
(Lindell et al. 1996). Aliquots of 1.7 ml of preincubated
water were incubated with 100 nM L-(4,5-3H) leucine (15
nM Amersham 150 Ci mmol-‘, 1.0 mCi ml-l, and 85 nM
cold substrate). For each treatment (irradiated, dark), one
trichloracetic acid (TCA)-killed control (5% final concentration) and triplicate live samples were incubated for approximately 40 min, and the reaction was terminated by adding
TCA (5% final concentration). Samples were centrifuged at
14,000 X g for 10 min and aspirated. The remaining pellet
was subsequently washed once with 5% TCA and once with
80% ethanol. Thereafter, 0.5 ml of scintillation cocktail
(Quicksafe A, Zinsser Analytic) was added. Radioactivity of
incorporated leucine was measured by liquid scintillation
counting (LSC). Bacterial carbon production was calculated
using the conversion factors by Simon and Azam (1989).
Bacterial abundance was determined by epifluorescence
microscopy after staining with 4’6-diamidino-2-phenylindole (DAPI, Porter and Feig 1980). At a minimum, 250 cells
and 10 fields of view were counted from each slide.
Microbial carboxylic acid transformation kinetics-To
study the bacterial utilization of photochemical degradation
intermediates (oxalic, malonic, formic, and acetic acid), 14Clabeled intermediates (New England Nuclear; oxalic acid,
4.3 mCi/mmol; acetic acid, 55 mCi/mmol; formic acid, 55.8
mCi/mmol; and malonic acid, 56.7 mCi/mmol) were added
to light- or dark-incubated surface water that had previously
been sieved through a loo-pm net, at concentrations ranging
887
from 1 to 650 pg liter-l. Nonradioactive substrates were
added to achieve the highest concentrations (>50-100 pg
carboxylic acid liter- I, depending on substrate). Each of the
four acids was studied in a separate subsample. Triplicate 5ml aliquots were incubated in tubes with gas-tight silicone
stoppers. Formaldehyde-killed controls (2% final concentration) were incubated in parallel. After 1 h, the samples were
killed with formaldehyde (2% final concentration), and HCl
was injected into the sealed incubation tubes to lower pH to
approximately 3.4. The carbon dioxide was removed by gentle bubbling with air for 5 min and collected in a CO,-absorbing solution (Carbosorb, Packard; Tranvik 1993). Scintillation cocktail (Permafluor, Packard) was added, and
radioactivity of the released CO, was measured by LSC.
To measure the incorporation of 14C into microbial biomass, the remaining aqueous solution was filtered onto 0.2pm cellulose-acetate filters, which were subsequently rinsed
with an unlabeled solution of 1 mg liter-l of each of the
acids. To minimize adsorption, filters had previously been
soaked in this solution. The filters were dissolved in a scintillation cocktail (Filtercount, Packard), and incorporated 14C
was estimated by LSC.
Detailed kinetic experiments with Skarshultsjon water after artificial UV irradiation or dark incubation were conducted with each of the four carboxylic acids using eight
different carboxylic acid additions between 1 and 650 pg
liter-l. In addition, we performed two experiments under
natural sunlight in Skarshultsjon and in Fiolen, respectively,
where the transformations of malonic, formic, and acetic
acid were studied in parallel subsamples, while oxalic acid
was omitted because of methodological problems (see Results). Two concentrations of added carboxylic acid between
1 and 550 pg liter-l for the different substrates were used
in these experiments. We calculated the biological utilization
of carboxylic acids by the respiration-corrected kinetic approach (Hobbie and Crawford 1969). The maximal transformation rate of the studied substrate (V,,,,,) was derived from
the reciprocal of the slope of a linear regression, with added
concentration of substrate as the independent variable and
turnover time of added substrate (T,) as the dependent variable (modified Lineweaver-Burke plot; Wright and Hobbie
1966; Wright 1978). The intercept with the dependent axis
represents the turnover time at ambient concentration (T,,71b).
Uptake rates at ambient concentration (Van],,)were calculated
by dividing the ambient concentration measured by capillary
ion electrophoresis with Tamb.We present all parameters with
standard deviations calculated according to the procedures
described by Bevington (1969) for propagation of errors
when measured parameters are combined.
Abiotic, photochemical mineralization of oxalic, malonic,
formic, and acetic acid into CO, was studied by spiking separate, sterile filtered samples from Lake Skarshultsjon with
each of the 14C-carboxylic acids in autoclaved quartz tubes
to final concentrations of 100 pg carboxylic acid liter-l.
These tubes were then incubated at the surface of the lake
under natural sunlight and thus at in situ temperature for 4
h. Dark controls were incubated in parallel for comparison.
In the preceding experiments on bacterial carboxylic acid
transformations, there was substantial volatilization of oxalic
acid during the CO, removal. Hence, for the abiotic miner-
888
Bertilsson and Tranvik
Table 1. Initial chemical characteristics of surface waters for use in studies with an artifcial UV source (mean of four separate experiments
&SD) and under natural sunlight (individual bags incubated). Malonic acid was not detected.
Experimental
group
Artificial UV (Skarshult)
Sunlight (Skarshult)
Sunlight (Fiolen)
DOC
mg liter-’
12.9 + 1.46
12.4
6.8
Fluorescence
UV absorbance at
Carboxylic acid concentration
(pg carbon liter-I)
250 nm
365 nm
(QSU)
Oxalic acid
fiormic acid
Acetic acid
0.573 & 0.0013
0.553
0.120
0.122 ? 0.0007
0.115
0.017
18.3 2 0.12
17.98
3.77
0.98 +: 0.86
0.42
2.64
7.03 + 4.78
10.0
7.88
9.72 ? 1.64
16.8
18.5
alization studies, we modified the pH during CO, stripping
to 5.6 instead of the previously
used 3.4. With this method,
14Cactivities in controls were low (< 1% of initial 14Cactivity).
Results
Photochemical production of LMW carboxylic acids-The
accumulated irradiation during the incubations was 4.5 kJ
me2 of UV-B, 710 kJ m 2 of UV-A, and 9,800 kJ m-2 of
PAR for Skarshultsjon, while the corresponding values for
Fiolen were 1.8 kJ me2 of UV-B, 230 kJ rnF2 of UV-A, and
4,200 kJ m -2 of PAR due to cloudy weather. The total dose
in studies under artificial UV-A-emitting
lamps was 0.4 kJ
rnp2 of UV-B, 245 kJ m-2 of UV-A, and 158 kJ m-2 of PAR.
The initial characteristics of the five different water samples from Skarshultsjon that were used in the experiments
did not differ substantially in DOC, absorbance, or fluorescence (Table 1). In contrast, concentrations of DOC, as well
as the absorbance and fluorescence, were substantially lower
in the water obtained from Lake Fiolen compared to Skarshultsjon. Low amounts of oxalic, formic, and acetic acid
were found in all sampled waters, while malonic acid was
not detected.
A substantial photochemical production of carboxylic acids occurred in sterile filtered water from Skarshultsjon, both
when UV-A-emitting
lamps were used (Fig. 1A) and when
the water was exposed to natural sunlight (Fig. 1B). The
combined production of oxalic, malonic, formic, and acetic
acid under natural sunlight was 19 pg carboxylic acid carbon
liter- 1 h- l. Formic acid was the major degradation product,
accounting for about 50% of the produced carboxylic acid
carbon. Concomitant with the production of carboxylic acids, absorbance and fluorescence of the humic water decreased (Table 2). Only a minor production of carboxylic
acids could be demonstrated with water from the clearwater
Lake Fiolen (Fig. 1C). The background concentrations in the
lake water obscured the interpretation of results and made
quantification of the photochemical production of carboxylic
acids in this lake difficult.
Bacterial abundance at the end of incubations in the study
of carboxylic acid production in the absence of bacteria was
in most cases very low (<lo8 cells liter- I), but some samples
(both irradiated and dark controls) contained significant
numbers of very small bacterialike
particles ( lo*-10” liter-l,
compared to 3-7 lo’ cells liter-l of larger bacteria found in
live samples, Table 3). Because of their small biomass, these
apparently spherical cell-like particles with diameters of
CO.2 pm (corresponding to cell volumes of 0.03 pm”, to be
compared to volumes of 0.1-0.2 pm? typical of the indigenous bacteria of these lakes [Tranvik 19881) probably did
not seriously affect carboxylic acid concentrations. Their potential effect on the results would be conservative, as bacterial consumption of carboxylic acid during incubations
would decrease the apparent abiotic production of the acids.
Eight hours of artificial UV irradiation, with lamps emitting primarily in the UV-A region, did not significantly inhibit bacterial :?roduction in Lake Skarshultsjiin water (Table
3). In contrast, natural sunlight decreased bacterial production 62-80% in the two studied lakes.
Microbial transformation of photochemically produced
intermediates--The biological transformation of formic and
acetic acid in Skarshultsjiin water exposed to artificial UV
radiation and the corresponding controls followed MichaelisMenten kinetics within the studied concentration interval
(Fig. 2A). Formic acid was almost exclusively transformed
into CO,, and the fraction of totally transformed acids being
incorporated i-no microbial biomass never exceeded 2% in
either dark controls or irradiated samples. The average fraction of the metabolized labeled acetic acid that was respired
was 59%.
In contrast, the uptake of malonic acid did not follow
Michaelis-Menten kinetics at concentrations >35 pg malonic acid C liter-l (Fig. 2B). Below this concentration, the T,
was linearly related to added malonic acid concentration (r2
> 0.99, n = 7), but T, leveled off as transformation rates
increased linearly at higher concentrations. This may be due
to an alternatIve uptake mechanism, possibly by other organisms, such as diffusion-controlled
algal uptake (Wright
and Hobbie 1966). Since only concentrations below 35 pg
malonic acid C liter-’ were considered relevant for ambient
conditions (the highest ambient concentration detected in
lake water with organisms present was 26 pg malonic acid
C liter I ; Table 4), we used only these data and assumed
Michaelis-Menten kinetics when calculating Tamband V,m,,.
A substantial fraction of the total transformed malonic acid
was incorporated into microbial biomass (average, 78%),
whereas about 20% was given off as CO,.
High amounts of oxalic acid 14Cwere removed during the
bubbling procedure, including the formaldehyde-killed controls. This inclicates an abiotic transformation of oxalic acid
to CO, or, more likely, purging of the acid itself during the
bubbling intended for the removal of CO,. Furthermore, the
low specific activity of the oxalic acid prohibited the study
UV production
7 ,
7
c
L
Table 2. Photochemically induced decrease in fluorescence and
absorbance in studies with an artificial UV source (mean of four
separate expcrimcnts ? SD) and under natural sunlight (individual
bags incubated).
I
n Dark
0 Irradiated
6
889
of bacterial substrates
t
Photobleaching
(% of dark controls)
Fluorcscence
Experiment
Skarshultsjon, artificial UV
Skarshultsjon, natural sunlight
Fiolen, natural sunlight
I
-1
J
Oxalic
acid
Malonic
acid
Formic
acid
Acetic
acid
1 BDark
13Irradiated
T ‘1
3l
s
t
0
I
Oxalic
acid
Malonic
acid
Formic
acid
Acetic
acid
Total of
4 acids
LL
n Dark
q Irradiated
T
.c
=I6
3
p4
t
-.L
Oxalic
acid
Malonic
acid
Formic
acid
Acetic
acid
89 2 1.9
86
96
4 acids
“16
v4
k
0
95 +- 1.2 97 ” 0.8
87
91
96
100
(QSU>
Total of
22
‘2
250 nm
365 nm
Total of
4 acids
Fig. 1. Abiotic production rates (pg carboxylic acid C liter-l
h -I) of oxalic, malonic, formic, and acetic acid in irradiated samples
and dark controls of sterile filtered lake water. Experiments were
performed using artificial lamps and water from Skarshultsjon (A)
(n = 12; data were pooled from all experiments) and water from
Skarshultsjiin (B) or Fiolen (C) under natural solar radiation (n =
3). Error bars indicate standard deviation.
of biological utilization at environmentally relevant concentrations (< 11 pg oxalic acid C liter-l; Table 4).
In water from Skarshultsjon exposed to artificial UV, the
turnover time at ambient concentration (Ta,,,,,)of acetic acid
was 18.5 h L 8.9 (SE, n = 24), whereas it was 13.5 h 5
3.4 (n = 24) for the corresponding dark controls. Similarly,
Ta,,,,,of formic acid was 3.0 h + 1.2 (n = 24) in the light
and 1.7 h + 0.3 (n = 24) in dark controls. Tambof malonic
acid was 26.2 2 1.2 (n = 9) and 1.1 2 0.3 (n = 9) in
irradiated and dark samples, respectively. The estimated
Vamb,calculated from T:,“,,,and the ambient concentration of
the studied substrate (Table 4) in most cases approached the
V,,, (Table 5). Thus, in general, the bacteria were saturated
with respect to the uptake of the studied substrates.
For incubations under natural sunlight, the variance was
larger because of the use of fewer concentrations. This particularly applied to malonic acid, since the transformation of
this substrate did not follow Michaelis-Menten kinetics at
high concentrations. Nevertheless, in sunlight-exposed Skarshultsjon water, Vamhand V,,,,, of acetic and formic acid resembled the values found during the more detailed kinetic
studies (Table 5). The Van,,,of malonic acid was estimated
only in the irradiated samples, since the ambient concentration of malonic acid was below detection limit in the dark
samples. The transformation rates were substantially lower
in Fiolen (Table 5). There were no detectable concentrations
Table 3. Bacterial production (&SD of analytical triplicates) and
abundance in individual enclosures used in experiments focused on
bacterial utilization of separate carboxylic acids in Skarshultsjon
water under artificial UV radiation (*), and employing several carboxylic acids in parallel under natural sunlight (**) in Skarshultsjon
and Fiolen.
Carboxylic acid
experiment
Oxalic acid*
Malonic acid*
Formic acid*
Acetic acid*
SkWhultsjon**
Fiolen* *
Bacterial cells
(10” cells liter- I)
Bacterial production
(ng C liter-’ h-l)
Dark
35.0
36.9
32.4
48.7
38.2
41.3
-I- 3.6
2 1.5
-t- 0.4
2 0.8
2 1.4
2 2.4
Irradiated
34.2
32.6
26.4
45.4
14.4
8.2
”
Ir
2
2
+
f:
1.9
1.9
1.3
1.0
0.9
0.6
Dark
Irradiated
5.4
4.6
5.2
3.6
6.4
2.9
5.8
5.4
5.0
5.4
6.8
3.3
Bertilsson and Tranvik
890
60
n
A
s40
i
‘S
& 30
ti
5 20
I-
10
0
20
40
80
0
60
100
120
Added cone. (vg carboxylic acid-C litei’)
of malonic acid in any of the incubations with Fiolen water,
and therefore \‘am,,was not calculated. The Tam,,for formic
acid was not significantly different from 0 in dark incubations. Therefore, reliable estimates of Va,,,could not be made.
The total transformation of carboxylic acids was compared to the bacterial biomass production, as estimated by
leucine incorporation. The microbial transformation rates for
total carboxylic acid carbon ranged between 0.05 and 3.1 pg
liter-l h--l, whereas the bacterial carbon production amounted to 0.05-0.32 pg liter-l h-l. Among all the paired observations of rates of bacterial carboxylic acid transformation
and biomass production, the resulting ratio in units of carbon
was from 0.2 to 14. Hence, the transformation of carboxylic
acid carbon sometimes exceeded bacterial production by
more than an order of magnitude (Fig. 3). A major part of
this excessive metabolism of carboxylic acids could be directly attributed to the almost complete oxidation of formic
acid into CO,, whereas acetic and malonic acid were incorporated into bacterial biomass to a larger extent during experiments.
The relation between the abiotic production and the biotic
transformation (Valnb)of carboxylic acids was close to one in
incubations with artificial UV radiation. However, in sunlight
incubations, the abiotic production was substantially higher
than the biological transformation for each of the studied
acids (Fig. 4).
200
Carboxylic acid photooxidation-Four
hours of exposure
under natural sunlight resulted in the conversion of 27 pg
liter- * (25.5) of the added oxalic acid (100 pg liter-l) into
CO,, whereas no conversion could be observed in dark controls. No statistically significant (Student’s t-test, P > 0.05)
photooxidation of the other carboxylic acids was observed
during the 4-h incubation.
160
Discussion
80
40
0
20
0
40
60
80
Added cont. (vg carboxylic
100 120 140
acid-C liter-l)
Fig. 2. Examples of Wright-Hobbie plots of the relationship between added substrate concentration and turnover time of the substrate given for formic acid (A) and the corresponding plot for the
transformation kinetics observed for malonic acid (B) in water from
SkCirshultsjiin after artificial UV irradiation. Solid lines in (A) are
fitted from linear regression (r2 > 0.98, iz = 24) and in (B) from
third-order polynomial regression (r2 > 0.97, y1 = 24).
Photochemical production of carboxylic acids-Several
investigators have identified sunlight- or UV-induced production of short-chain carboxylic acids in natural, humicrich surface waters or in aqueous solutions containing humic
substances from various sources (Backlund 1992; Allard et
al. 1994; Wetzel et al. 1995). Furthermore, a substantial production of carbonyl compounds of low molecular weight has
been observed, mostly in marine environments (Kieber et al.
1989, 1990; Mopper et al. 1991). Thus, solar radiation degrades high-molecular-weight
organic compounds (humic
substances) into LMW molecules that are readily used as
carbon and energy sources by aquatic bacteria. Indeed, several studies have indicated such an indirect stimulation, observed as increased bacterial production (Lindell et al. 1995;
Wetzel et al. 1995; Bushaw et al. 1996).
Our kinetic data provide strong support for a major role
of LMW carboxylic acids in the photochemical-microbial
degradation pathway, demonstrating substantial bacterial utilization of photochemically produced carboxylic acids. In
the clearwater Lake Fiolen, formation of LMW carboxylic
acids was markedly lower than in the humic Sktirshultsjijn,
or even below detection limit (Fig. 3). This implies that the
photochemical formation of LMW carboxylic acids is linked
UV production
891
of bacterial substrates
Table 4. Ambient carboxylic acid concentration in organism-containing samples,after incubation in the dark or irradiated, for subsequent
use in bacterial carboxylic acid transformation studies (+SD, n = 2). nd, not detected.
Exneriment
Sktirshultsjiin, artificial UV
Sktishultsjiin, natural sunlight
Fiolen, natural sunlight
Initial concentration in biological incubations (pg C liter-‘)
Irradiated
Dark
Studied acid
Oxalic acid
Malonic acid
Formic acid
Acetic acid
Oxalic acid
Malonic acid
Formic acid
Acetic acid
Oxalic acid
Malonic acid
Formic acid
Acetic acid
to the presence of humic substances. Accordingly, Bertilsson
and Allard (1996) found that a substantial potential for photochemical production of LMW carboxylic acids in humic
water was lost when the >l kD fraction of the organic matter was removed by ultrafiltration.
Several previous reports of photochemical degradation of
humic substances into carboxylic acids were done with radiation sources emitting primarily in the UV-C region (i.e.,
at 250 nm; Backlund 1992; Allard et al. 1994; Corin et al.
1996). This study and a few others (Wetzel et al. 1995; Bertilsson and Allard 1996; Kulovaara 1996) employ natural
sunlight and UV lamps emitting at wavelengths and intensities comparable to natural sunlight. In addition, in several
of the previous studies, isolated humic substances (Allard et
al. 1994; Kulovaara 1996) or humic substances extracted
from senescent plants (Wetzel et al. 1995) were used. Here,
we used humic lake water that was subject only to filtration.
Extraction procedures, especially those that require pH manipulation, may have an impact on the structure of the organic matter. Our use of natural sunlight or lamps emitting
primarily in the UV-A in combination with unmanipulated
0.29 t 0.04
4.81
2.91
7.36
10.24
nd
3.18 -I 0.076
8.44 k 3.87
0.72 -I 0.11
2
5
!I
k
0.13
0.03
0.04
2.26
11.1 + 1.00
nd
21.1 5 1.96
25.8
47.9
46.7
2.62
52.6 +: 5.94
1.81 + 0.55
?
+
2
2
0.56
6.14
5.71
0.60
nd
14.4 + 3.69
18.6 + 0.65
nd
20.3 2 2.38
39.7 + 4.10
DOM most likely yields rates of photochemical carboxylic
acid formation resembling those occurring in the top layer
of the water column.
In sunlight-irradiated water from Skarshultsjon, the formation rate of carboxylic acids was about 19 pg carboxylic
acid C liter-l h-l. Assuming a first-order, concentration-dependent production of carboxylic acids over time, identical
sensitivity of all DOC components toward photochemical
conversion to carboxylic acids, and a complete biological
utilization of the formed photoproducts, the half-life of the
DOC pool was about 460 irradiation hours in the upper 4
cm of the lake. Extrapolation of results from short-term incubations to the entire DOC pool may yield an overestimate
of the degradation rate, since compounds particularly sensitive to photochemical conversion are probably degraded
initially. By contrast, it must be noted that this half-life is
calculated only with respect to four studied photoproducts,
whereas several other compounds, such as CO,, CO, pyruvate, aldehydes, etc., have been reported to be formed (e.g.,
Mopper et al. 1991; Wetzel et al. 1995; Graneli et al. 1996).
In the experiment with natural sunlight in Skarshultsjon, car-
Table 5. Maximum transformation rate (V,,,) and transformation rate at ambient concentration (Van,,,)of carboxylic acids in samples
preincubated in the dark or irradiated with artificial UV or natural sunlight. (*SD). Experiments where Valllbcould not be calculated due
to undetectable ambient concentrations or due to turnover times not significantly different from zero are denoted ne, not estimated.
Experiment/Studied acid
Sk&shultsj&r, artificial UV
Malonic acid
Formic acid
Acetic acid
Sktishultsjiin, natural sunlight
Malonic acid
Formic acid
Acetic acid
Fiolen, natural sunlight
Malonic acid
Formic acid
Acetic acid
Maximum rate (V,,,,)
(pg C liter-’ h-l)
Dark
Irradiated
0.245 IL 0.032
1.740 + 0.027
0.962 + 0.024
0.369 +: 0.029
2.01 2 0.031
0.596 2 0.033
0.231 : 0.062
1.16 2 0.087
0.191 k 0.146
0.996 + 0.237
0.733 2 0.335
0.373 f. 0.167
0.617 k 0.026
0.191 + 0.135
0.104 IL 3.138
0.092 2 0.150
0.022 k 0.124
Rate at ambient concentration ( Vamb)
(pg C liter-’ h- ‘)
Dark
Irradiated
1.83
0.624
“+”0.950
k 0.327
nc
1.20 : 0.345
0.111 ? 0.005
2.43 k 0.654
0.552 + 0.291
0.281 2 0.177
1.29 -e 0.465
0.121 +_ 0.027
ne
0.054 “+” 0.010
0.115 : 0.084
0.010 2 0.004
892
Bertilsson and Tranvik
Sk&shultsjBn
Artificial UV
Skarshultsjtin
\latural Sunlight
1
:iolen
H Dark Control: 5
4atural 0 Irradiated
sunlight
T
Tit
Studied Carboxylic acicl
Fig. 3. The ratio of individual carboxylic acid transformation at ambient concentration to total
bacterial production for malonic, formic, and acetic acid in irradiated incubations and dark controls
in natural sunlight and artificial UV radiation in Skarshultsjon and under natural sunlight in Fiolen.
“Not detected” refers to cases where Valnbcould not be calculated because of undetectable ambient
concentration. Experiments where the turnover time at ambient concentration (Tlmb) was not significantly different from 0, thus preventing Vamhto be calculated, are denoted “Tamb0.” Error bars
indicate standard deviation.
-
boxylic acid carbon photoproduction during 8 h corresponded to 1.2% of total DOC while there was a decrease in DOM
fluorescence of 14% (Table 2). If we use fluorescence as a
surrogate for DOC concentration in the humic lake, the photoproduction and further biological transformation of the
four studied carboxylic acids would account for 8.6% of the
photodegradation of DOC. Since photochemical bleaching
of fluorescence is typically more rapid than the corresponding decrease in DOC, we may have overestimated this decrease. Hence, the calculated fraction attributed to the measured carboxylic acids would be an underestimate.
The rates presented here derive from incubations within
the upper 4 cm of the water column. The rate of photochemical transformation decreases with depth because of the attenuation of radiation. Previously,
we found that the depth
attenuation of other photochemical transformations (fluorescence bleaching, Tranvik et al. in prep.; photolytic mineralization to CO,, Graneli et al. 1996) resembles the depth attenuation of UV-A. If we assume that this is also valid for
photochemical production of carboxylic acids, we can calculate the carboxylic acid production per unit of lake surface
area in the 3.5-m-deep, homothermic epilimnion of Skarshultsjon. We derived the depth-integrated carboxylic acid
production from it-radiance data collected with a UV-A sensor throughout the epilimnion (Lindell et al. 1996). If depth
attenuation
of carboxylic
acid photoproduction
is similar
to
the depth attenuation of UV-A, the sum of the production of
the four measured carboxylic acids amounted to 46 mg C
m-2 d-l during incubations. Because of the rapid attenuation
of short-waveler gth radiation, 76% of this production would
take place in the upper 10 cm of the water column. Previously, we found depth-integrated rates in this lake of microbial community respiration, algal primary production, and
photolytic CO, production of 460, 0.7-26, and 50.1 mg C
rn+ d-l, respectively (Tranvik 1989; GranCli et al. 1996).
These values are comparable to our results on carboxylic
acid production, with the conservative assumption that all of
the daily carboxylic acid production took place during the 8
h of incubation around noon. It is then clear that photolytic
carboxylic acid production is similar to the photochemical
production of CO, and of the same magnitude as algal primary production. Hence, even when integrated over depth
into the darker layers of the mixed water column, the photochemical production of the four measured carboxylic acids
appears to be a major source of labile DOC on sunny summer days in this lake. Considering that the carboxylic acids
most probably make up only a fraction of the total organic
and inorganic products of photodegradation of DOC, these
results further emphasize the importance of solar irradiation
in DOC dynamics.
Microbial utir’ization of carboxylic acids-At
ambient
concentrations in water from Skarshultsjon, formic, acetic,
UV production
4
893
of bacterial substrates
/
0 Artificial UV
H Natural Sunlight
M = Malonic
F = Formic
A = Acetic
2
4
6
8
10
12
Abiotic Production (pg C liter-’ h-l )
Fig. 4. Relationship between abiotic production and biological transformation of malonic, formic, and acetic acid under artificial UV radiation (open circles) and natural sunlight in Sktishultsjiin
and for formic and acetic acid in Lake Fiolen (solid squares). Error bars indicate standard deviation.
and malonic acids were transformed at rates close to V,,,,
both under artificial UV radiation and natural sunlight. Thus,
the uptake systems of the bacteria for these acids appear to
have been saturated. Formic acid was almost entirely mineralized without being converted into biomass, as about 98%
of the 14C-labeled substrate was mineralized into CO, during
l-h incubations. This may have been due to a direct degradation of formic acid into CO, by formatdehydrogenase or
similar enzymes (Gottschalk 1985), which may take place
on the outside of the cell membrane.
Compared to formic acid, a much smaller fraction (about
20%) of the metabolized malonic and acetic acid carbon was
mineralized into CO, during incubation with radiolabeled
substrates. However, the actual bacterial growth yield (i.e.,
biomass carbon produced per unit of organic carbon utilized)
during growth on these acids is probably substantially lower
than indicated by the results derived from l-h incubations
with radiotracers. During these short-term exposures to tracer substrates, isotopic equilibrium is not achieved. Hence,
the 14C: 12Cratio will be higher in incorporated than in respired carbon, resulting in an apparent growth yield substantially higher than the actual yield (Gtide 1984; Tranvik
and Hijfle 1987).
Because of the low specific activity of oxalic acid and its
volatilization during acidification and bubbling intended for
the removal of 14C02, we have no reliable data on the bacterial utilization of this substrate. However, after modifying
the 14C02-purging procedure, we could successfully study its
abiotic photooxidation. Oxalic acid was, in contrast to the
three other studied carboxylic acids, further photooxidized
into a form that was lost during more gentle acidification
and bubbling (most likely, CO,). Hence, the photochemical
production of oxalic acid was probably underestimated, as
the accumulation of oxalic acid was counteracted by further
photooxidation into CO,. Accordingly, Bertilsson and Allard
(1996) observed a steady-state concentration of oxalic acid
shortly after the onset of irradiation of humic water, while
other carboxylic acids continued to increase with increasing
UV dose. Oxalic acid is known to be easily oxidized further
into CO, (Baum 1987), which also supports these findings.
Possibly the other studied carboxylic acids were also subject
to photooxidation into CO, at low rates that were not detected by our methods and were insignificant in comparison
to bacterial utilization.
Relationship between bacterial biomass production and
utilization of carbo&ic acids-Although
there are several
reports of stimulated bacterial activity upon irradiation of
DOM, including studies where specific photochemically produced intermediates were identified (e.g., Wetzel et al. 1995),
we are aware of only one previous investigation of concomitant photochemical production and bacterial utilization of a
specific LMW organic compound, i.e., the study of pyruvate
transformations in marine waters by Kieber et al. (1989).
The uptake of pyruvate was estimated to account for 2-4%
of the total carbon flux to bacteria. In the present study, the
transformation of three detected photoproducts exceeded the
total bacterial production of C, sometimes by more than one
order of magnitude (Fig. 3). Thus, although much of the
carboxylic acid is mineralized into CO,, and even if a very
894
Bertilsson and Tranvik
low growth efficiency (< 10%) is assumed, all or most of
the bacterial carbon and energy demand can theoretically be
accounted for by the use of carboxylic acids.
The higher rates of photochemical production of carboxylic acids compared to bacterial uptake and mineralization
(Fig. 4) indicate accumulation of photochemically produced
intermediates at the surface during the light hours of the day.
Under natural sunlight, the photochemical production exceeded the bacterial transformation by one order of magnitude. These acids may then serve as bacterial substrates during the night or in deeper layers after mixing. We observed
sunlight inhibition by 62-80% of bacterial production in the
samples exposed to light in the lakes, as has also been previously observed (Herndl et al. 1993). This inhibition probably affects carboxylic acid metabolism as well, and occurs
also in the natural environment, although it may have been
exaggerated in the enclosed samples used in our experiments.
Although carboxylic acids, compared to sugars, for example, are poor in energy, the microbial oxidation of carboxylic acids appears to yield substantial amounts of energy
that was not used for synthesis of biomass. These exceedingly high rates of carboxylic acid carbon metabolism in
relation to total bacterial carbon production are surprising.
Bacterial production (measured by the leucine incorporation
method) depends on a number of assumptions and conversion factors, which may need caution (Kirchman 1993). Although we used a high concentration of leucine (100 nM)
sufficient to obtain saturation of leucine uptake in the studied
lakes (data not shown) and the most commonly used conversion factors (Simon and Azam 1989), it can not be excluded that we underestimated bacterial production and thus
overestimated the relative importance of carboxylic acids.
Still, as we sometimes found rates of carboxylic acid carbon
utilization that exceeded total bacterial carbon production by
more than an order of magnitude (Fig. 3), it is not likely
that even a drastic adjustment of the production values
would affect the major patterns of these results.
Several recent studies show the photochemical production
of carboxylic acids and other LMW compounds from DOM,
as well as the stimulation of bacteria due to photochemical
transformations of DOM. Here, we present evidence that carboxylic acids play an important role in this sequential photochemical-microbial
degradation of DOM, providing an
important carbon and energy source to freshwater bacterioplankton and simultaneously being a major intermediate in
the decomposition and turnover of DOM.
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Received: 30 January 1997
Accepted: 16 March I998