ELSEVIER
FEMS Microbiology
Ecology
18 (1995) 45-50
Microbial utilization of lipids in lake water
Ryszard J. Chr6st
*,
Adam J. Gajewski
Department of Microbial Ecology, Institute of Microbiology of Warsaw University, Karowa 18, PL-00-927 Warsaw 64, Poland
Received
18 January
1995; revised 12 June 1995; accepted
12 June 1995
Abstract
This report
measurements
evaluates
the importance
of lipids as a source of substrates for metabolism of heterotrophic bacteria based on
of uptake of [ 3H]pa1mitic acid and lipase activity in lakes of differing degrees of eutrophication. The highest
rates of utilization of lipids by bacteria (i.e. specific and actual rates of uptake of [3H]pa1mitic acid and specific lipase
activity) were observed in pelagic water of mesotrophic lake where the highest concentration and the shortest turnover time
of natural pool of palmitic acid was found. Lipids in highly eutrophicated lakes were less important for metabolism of
aquatic bacteria1 communities. Lipid content in lake water depended on species composition of plankton in the studied lakes.
Keywords: Aquatic
bacteria;
Palmitic acid uptake; Lipolysis;
Lake
1. Introduction
Many constituents of the dissolved organic matter
(DoM) in aquatic environments
serve as an energy
and nutrient source for metabolism of heterotrophic
microorganisms
[1,2]. DOM consists of an innumerable amount of a variety of organic compounds and
among them proteins,
peptides,
polysaccharides,
lipids, nucleic and other organic acids, phosphoric
esters and humic substances
predominate
[2-41.
Lipids contribute from 3% to 55% to the DOM pool
in aquatic environments
[5,6]. Identification
of
sources of organic matter, their significance and fate,
is important to the understanding
of functioning
of
aquatic ecosystems. Such objectives can be achieved
by the studying of microbial metabolism of natural
lipid compounds and their derivatives.
l
Corresponding
author. Fax: (04822) 268 982.
0168.6496/95/%09.50
0 1995 Federation
SSDI 0168-6496(95)00039-9
of European
Microbiological
Enzymatic hydrolysis of lipids must precede their
utilization
by bacteria
because
only the final
monomeric products of hydrolysis (e.g. palmitate)
can be transported and metabolized in the cytoplasm
[7]. Until now there is no information available on
simultaneous
hydrolysis of lipids and subsequent
uptake of the products of lipolysis by microorganisms in natural waters. Therefore the importance of
lipids and long-chain
fatty acids in bacterial
metabolism in natural waters is almost unknown.
The present study reports on: (1) the maximal and
actual rates of uptake of palmitic acid, final product
of lipolysis by bacteria and (2) its uptake constants
(K,), (3) turnover time and (4) magnitude of natural
pool of palmitic acid in lake water. We compared the
rates of uptake of palmitic acid with bacterial abundance and lipase activity, and physico-chemical
properties of water in lakes of differing degrees of
eutrophication.
It was also intriguing for us to know
how natural assemblages of aquatic bacteria assimiSocieties. All rights reserved
46
R.J. Chr&,
late water insoluble,
emulsified
short-time incubation experiments.
A.J. Gajewski/FEMS
substrate
during
2. Materials and methods
2.1. Sample collection
Water samples were taken from ten sampling sites
located in the pelagial zone along the length of three
lakes: Kisajno (area 19.8 km*, max. depth 24 m),
Beldany (area 12.4 km*, max. depth 46 m) and
Jagodne (area 9.1 km*, max. depth 34 m) during late
summer stratification period. Using a 4.5 liter sampler, water samples were collected from lake surface
(depth 0.5 m). S amples from each sampling site were
mixed together (vol/vol)
and the obtained integrated
sample was used as a representative sample for the
whole lake. Large phytoplankton
and zooplankton
were removed immediately
after sample collection
by filtration of water through a 100 pm plankton
net.
2.2. Uptake kinetics of [3H]palmitic
acid
Ethanol solution
of [9,10(n)-3H]palmitic
acid
(spec. activity 53 Ci mmol ‘, Amersham) was used
to determine the kinetics of its uptake. The stock
solution of [3H]palmitic acid was diluted with ethanol
(95% vol/vol)
in a series of increasing concentrations of substrate before the use in experiments.
Ten-ml triplicate water samples were supplemented
with an increasing amount of radiolabelled palmitic
acid yielding final concentrations:
0.94, 1.89, 4.72,
18.87 and 47.17 nmol 1-l in assay. Samples were
incubated in situ (18°C) for 6 h and fixed with 4%
Table 1
Physico-chemical
and biological
period (September)
properties
Microbiology
Ecology 18 (1995) 45-50
(final cont.) buffered neutral formaldehyde.
After
incubation,
samples were filtered through 0.2 ,um
membrane filters (Schleicher-Schuell,
Germany). Filters retaining radioactive particles were rinsed three
times with 5 ml of 95% ethanol. Radioactivity was
assayed by liquid scintillation
counting in a Beckman LS 3500. The kinetic parameters of uptake of
[“Hlpalmitic
acid, i.e. maximal velocity of uptake
(V,,,) and half-saturation constant (K,) were calculated from the direct plot of reaction velocity versus
substrate concentration using the IBM PC computer
program ‘Enzfitter’ (Elsevier-Biosoft,
UK) to determine the best fit of the rectangular hyperbola [8].
Turnover time (T,) of uptake of palmitic acid was
calculated according to the formula: Tt = KJV,,,.
2.3. Isotope
dilution
[3H]palmitic acid
analysis
of
of lake water sampled from pelagial zone of the studied lakes during late summer stratification
Lake Kisajno
Lake Betdany
Lake Jagodne
Water transparency
Temperaturk CC) .
Oxygen (mg 0, 1 DOC(mgCl-‘)
Chlorophyll a ( pg
Number of bacteria
2.0
14.5
8.0
6.8
15.3
1.16
1.2
14.5
9.7
8.7
30.5
2.45
0.7
14.5
7.4
15.5
34.9
14.94
’)
1-
1)
(X lo6 cells ml-‘)
of
Assay of isotope dilution [9] was applied to estimate natural pool [9,10] of palmitic acid which
controlled
actual
rates of uptake
[11,12]
of
[3H]palmitic acid by natural assemblages of aquatic
bacteria. Ten-ml triplicate water samples were supplemented with a constant amount (final cont. 7.55
nmol 1-l ) of [3H]palmitic acid in a series of six
known increasing concentrations
(final cont.: 0, 5,
10, 25, 50, 100 nmol 1-l) of unlabelled palmitic
acid. Samples were incubated and processed as described above. Actual rates of uptake of [3H]palmitic
acid and pool of natural lipids were calculated from
an isotope dilution plot [ll] according to Chrost and
Overbeck [12]. The slope of an isotope dilution plot
provides a measure of uptake velocity of the substrate. The negative interception
with the X-axis
provides an estimate of the effective pool of natural
Parameter
(m)
uptake
R.J. Chr&c, AJ. Gajewski/
palmitic acid that competes with [ 3H]palmitic
bacterial uptake system.
FEMS Microbiology
Ecology 18 (1995) 45-50
47
1,600
acid in
2.4. Other analyses
Water transparency
was determined
by Secchi
disc visibility.
Temperature
and dissolved oxygen
concentration
in lake water were measured in situ
with oxygen meter (model 54, Yellow Spring Instr.).
Dissolved organic carbon (DOC) was analyzed with
the use of a Beckman Total Organic Carbon Computational System (Tocomaster mod. 915-B). Chlorophyll a, extracted with 90% methanol, was measured
spectrophotometrically
[ 131. Bacterial cell numbers
were determined in epifluorescence
microscope after
DAF’I staining [14]. Lipase activity was assayed
spectrophotometrically
according to the procedure of
Boon [15] modified by Gajewski et al. [7].
800
400
0
0
4
‘d
Lake Kisajno
/
I
10
20
30
40
,
1,600
E
V ,,=943 (k29) pmol 1-l h-’
K,=7 (f 1) run01 I-’
H
I&
1,200
a
800
ZI
400
Lake Jagodne
‘t
3. Results and discussion
Basic physical, chemical and biological properties
of water of the studied lakes varied markedly depending on the trophic status of lake (Table 1). On
the basis of limnological analyses, Lake Kisajno was
recognized as mesotrophic system and Lake Beldany
and Lake Jagodne were eutrophic and highly eutrophic reservoirs respectively.
The uptake rates of [3H]palmitic acid by natural
bacterial assemblages followed Michaelis-Menten
kinetics (Fig. 1). The highest V,,, of uptake rate of
L3H]palmitic acid was determined in eutrophic Lake
Beldany (Fig. lC>. V,,, values of uptake in Lake
Kisajno and Lake Jagodne were 22% and 57% lower,
respectively, than found in Lake Beldany.
The K, values determined on samples from Lake
Kisajno and Lake Beldany were similar and they
Table 2
Actual rates of uptake of [3H]palmitic
dissolved organic carbon (PAL,/DOC),
Lake
Kisajno
Beldany
Jagodne
v,
(pm01 I-’
1160
100
607
h-l)
J
0
10
r3H1Palmitic
20
30
acid (m-no1 1-l)
Fig. 1. Kinetics of uptake of [3H]palmitic acid by microplankton
in surface water of pelagial of the studied lakes during late
summer stratification.
Vmax: maximal rates of uptake; K,: uptake
constant; f : standard deviation.
acid (V,), concentration
of palmitic acid in lake water (PAL,) and its contribution
and lipase activity in the studied lakes. f : standard deviation
Specific V,
(amol cell-’
1.00
0.04
0.04
h-r)
40
PAL”
(nmoll~‘)
PAL,/DOC
(So)
Lipase
(nmoII_t
155( + 28)
7(*0.1)
81 ( f 0.5)
0.43
0.01
0.10
65 (+7)
105 (* 12)
176 (+9)
to the pool of
h-r)
48
R.J. Chrdst, A.J. Gajewski/FEMS
were approximately 4.5 times lower than that calculated for samples from Lake Jagodne (Fig. 1). Therefore, turnover time of uptake of palmitic acid (q)
was considerably higher in Lake Kisajno (18 hours)
and Lake Beldany (13 h) than in Lake Jagodne (7 h).
Turnover time of uptake of palmitic acid was inversely related to the degree of eutrophication of the
studied lakes.
The actual rates of uptake of palmitic acid (V,)
were the highest and almost reached V,,, value in
mesotrophic Lake Kisajno, whereas V, values calculated for eutrophic water samples were significantly
lower (Table 2, Fig. 1). This suggested that the
concentration of fatty acids in water of Lake Kisajno
almost saturated bacterial transport systems. Estimated size of the pool of palmitic acid naturally
present in lake water confirmed the above assumption (Table 2). Both highest content of palmitic acid
and its contribution to the total pool of DOC in lake
water were determined in samples from Lake Kisajno
(Table 2). Moreover, in these samples we found also
the lowest number of bacteria which meant that
bacteria in less eutrophicated lake had notably higher
specific uptake rates (i.e. uptake calculated per bacterium) of palmitic acid than bacteria in more eutrophicated lakes (Jagodne and BeIdany; Table 2).
High concentration
of palmitic acid in water of
unpolluted,
mesotrophic
Lake Kisajno and consequently its important role in bacterial metabolism
may be explained by widespread distribution of lipids
which are products of photosynthesis of many species
of diatoms [16,17] and other microalgae [17-191.
This observation agrees well with the recent studies
on origin and composition of lipids in marine system
[4]. Diatoms contributed
22% to the total algal
biomass in the water column of Lake Kisajno. In
eutrophic lakes Beldany and Jagodne we observed
predomination
of cyanobacteria ( Anabaena sp., Oscillatoria sp.). Diatoms constituted only 8.4% and
1.1% of the total phytoplankton
biomass in Lake
Jagodne and in Lake Beldany, respectively.
Since
cyanobacteria
contain markedly less lipids than diatoms [18], different lipid concentrations
in water of
the studied lakes may therefore reflect differences in
species composition of phytoplankton communities.
Freshwater invertebrates are also another important source of fatty acids and lipids in lake water
[20]. Neutral and polar lipids are common con-
Microbiology
Ecology 18 (1995) 45-50
stituents of planktonic animals [21] and their content
in zooplankton is strictly dependent on concentration
and composition of edible phytoplankton
in lake. In
lakes the content of lipids in zooplankton increases
in late summer and autumn when edible algae (diatoms, green algae and chrysophytes) predominate in
phytoplankton
community
[22]. These algae were
present only in Lake Kisajno and not in other lakes
studied. Data on the concentration of dissolved fatty
acids in freshwaters are extremely rare. Analyses of
fatty acids in seawater by means of gas-liquid chromatographic
technique
showed concentrations
of
palmitic acid in the range of 105-458 nmol 1~ 1 [23].
Our estimates of content of palmitic acid determined
by an isotope dilution assay revealed a similar range
of its concentration in lake water.
Lipolytic activity of natural bacterial assemblages
varied from 65 nmol 1-l h-’ (Lake Kisajno) to 176
nmol 1-l h-’ (Lake Jagodne; Table 2). Activity of
lipase was positively proportional to degree of eutrophication of the studied lakes. Specific activity of
lipase (i.e. activity calculated per bacterium) was
markedly lower in highly eutrophicated Lake Jagodne
(12 amol cell-’
h-‘) than in mesotrophic
Lake
Kisajno (56 amol cell-’ hP ‘). Because of similar
contributions of lipolytic bacteria to the total bacterial numbers in the studied lakes (data not shown)
we suppose that high content of palmitic acid in
water from Lake Kisajno may regulate lipase synthesis and activity in bacteria. This assumption concedes well with our previous studies on regulation of
bacterial lipase activity where we found that palmitic
acid strongly stimulated activity of this enzyme. We
also found that lipase belongs to the group of most
active ectoenzymes of aquatic bacteria [7]. There are
many examples of competitive inhibition of activity
of microbial ectoenzymes
by the final product of
hydrolysis [24-261 but stimulation of enzyme activity is rarely observed [7,27].
It must be stressed that specific rates of lipase
activity and [ “Hlpalmitic acid uptake were calculated
on the basis of microscopic determination of bacterial cell numbers, Such approach can be misleading
because it does not take into account the fact that
significant
part of the total number of bacteria
counted
under epifluorescence
microscope
after
DAF’I staining do not hold DNA and they are
metabolically
non-active
‘ghost’ cells (HagstrGm,
R.J. Chr&t, A.J. Gajewski/FEMS
personal communication).
Moreover, it was reported
that lipid substrates and products of lipolysis tend to
adsorb at the interfaces (air-water and water-particles) that are colonized by highly active lipolytic
bacteria [28-301. This also means that lipolytic bacteria are not uniformly distributed in the water samples. Because we could not distinguish between activity of free-living and attached bacteria it seems
that our results of bacterial specific activities can be
regarded only as a general potential of microbial
communities
for utilization of lipids in the studied
lakes.
Our studies indicate that turnover time of palmitic
acid in the studied lakes was comparable to turnover
times of other easily utilizable organic compounds,
e.g. glucose, mixture of amino acids [31,32]. Therefore, we postulate that lipid compounds are important substrates for metabolism of aquatic bacteria,
especially in nonpolluted lakes.
Acknowledgements
This work was partially supported by Grant # 6
P205 039 06 from the Committee for Scientific
Research, Poland. We thank Dr. Waldemar Siuda for
his help during sampling and field experiments.
References
[I] Bell, R.T. and Kuparinen, J. (19841 Assessing phytoplankton
[2]
[3]
[4]
[5]
[6]
and bacterioplankton
production during early spring in Lake
Erken, Sweden. Appl. Environ. Microbial. 45, 1709-1721.
Minster, U. and Chrost, R.J. (1990) In: Aquatic Microbial
Ecology -Biochemical
and Molecular Approaches
(Overbeck, J. and Chr6st, R.J., Eds.), pp. 8-46. Springer-Verlag,
New York.
Minster,
U. and Albrecht, D. (1994) Dissolved organic
matter.
Analysis
of composition
and function
by a
molecular-biochemical
approach. In: Microbial Ecology of
Lake PIuDsee (Overbeck,
J. and Chr&t, R.J., Eds.), pp.
24-63. Springer-Verlag,
NY.
Saliot, A., Bouloubassi,
I., Iorre-Boireau,
A., Trichet, J.,
Popupet, P. and Charpy, L. (1994) Lipid composition
of
water and surface sediments in Takapoto atoll lagoon (French
Polynesia). Coral Reefs 13, 243-247.
Jeffrey, L.M. (1966) Lipids in sea water. J. Am. Oil Chem.
Sot. 43, 211-214.
Ogura, N., Ambe, Y., Ogura, K., Ishiwatari, R., Mizutani, T.,
Satoh, Y., Matsushima,
H., Katase, T., Ochiai, M., Ta-
Microbiology
Ecology 18 (1995) 45-50
49
dokoro, K., Takada, T., Sughihara,
K., Matsumoto,
G.,
Nakamoto, N., Funakoshi, M. and Hanya, T. (1975) Chemical composition of organic compounds present in water of
the Tamagawa River. Jap. J. Limnol. 36, 23-30.
[7] Gajewski, A.J., Chrbst, R.J. and Siuda, W. (1993) Bacterial
lipolytic activity in an eutrophic lake. Arch. Hydrobiol. 128,
107-126.
[8] Leatherbarrow,
R.J. (1987) Enzfitter. A Non-Linear Regression Data Analysis Program For the IBM PC. Elsevier-Biosoft, Cambridge.
[9] Forsdyke, D.R. (1971) Application
of the isotopedilution
principle to the analysis of factors affecting the incorporation
of [3H]uridine and [3H]cytidine
into culture lymphocytes.
Biochem. J. 12.5, 721-732.
[lo] Pollard, P.C. and Moriarty, D.J.W. (1984) Validity of the
tritiated thymidine method for estimating bacterial growth
rates: Measurement of isotope dilution during DNA synthesis. Appl. Environ. Microbial. 48, 1076-1083.
[ll] Chrbst, R.J. (1990) Application of the isotope dilution principle to the determination of substrate incorporation by aquatic
bacteria. Arch. Hydrobiol. Beih. Ergebn. Limnol. 34, lll117.
[12] Chr&t, R.J. and Overbeck J. (1989) Application
of the
isotope dilution principle to the determination of [r4C]glucase incorporation by aquatic bacteria. Acta Microbial. Polon.
38, 75-89.
[13] Marker, A.F.H., Crowther, C.A. and Gunn, R.J.M. (1980)
Methanol and acetone as solvents for estimating chlorophyll
and phaeopigments
by spectrophotometry.
Arch. Hydrobiol.
Beih. Ergebn. Limnol. 14, 52-69.
[14] Porter, K.G. and Feig, Y.S. (1980) The use of DAPI for
identifying
and counting
aquatic
microflora.
Limnol.
Oceanogr. 25, 943-948.
1151 Boon, P.J. (1989) Organic matter degradation and nutrient
regeneration in Australian freshwaters: I. Methods for exoenzyme assays in turbid aquatic environments.
Arch. Hydrobiol. 115, 339-359.
1161Reynolds, C.S. (1984) The Ecology of Freshwater Phytoplankton. Cambridge University Press, Cambridge.
[I71 Morris, R.J. (1984) Studies of a spring phytoplankton bloom
in an enclosed experimental ecosystem. II. Changes in the
component fatty acids and sterols. J. Exp. Mar. Biol. Ecol.
75, 59-70.
iI81 Ahlgren, G., Gustafsson, I.-B. and Boberg, M. (1992) Fatty
acid content and chemical composition
of freshwater microalgae. J. Phycol. 28, 37-50.
[191 Reitan, K.I., Rainuzzo, J.R. and Olsen, Y. (1994) Effect of
nutrient limitation on fatty acid and lipid content of marine
microalgae. J. Phycol. 30, 972-979.
ml Bell, J.G., Ghioni, C. and Sargent, J.R. (1994) Fatty acid
compositions of 10 freshwater invertebrates which are natural food organisms of Atlantic salmon parr (Sulmo salar): a
comparison with commercial diets. Aquaculture
128, 301313.
1211Parrish, C.C. (1988) Dissolved and particulate marine lipid
classes: A review. Mar. Chem. 23, 17-40.
D-4 Wainman, B.C. and Lean, R.S. (1990) Seasonal trends in
50
R.J. Chr&t, A.J. Gajewski/
FEMS Microbiology
planktonic lipid content and lipid class. Verh. Intemat. Verein.
Limnol. 24, 416-419.
[=I Slowey, J.F., Jeffrey, L.M. and Hood, D.W. (1962) The
fatty-acid
content of ocean water. Geochimica
et Cosmochimica Acta 26, 607-616.
b41 Chrbst, R.J. (1990) Microbial ectoenzymes in aquatic environments. In: Aquatic Microbial Ecology -Biochemical
and
Molecular Approaches (Overbeck, J. and Chmst, R.J., Eds.),
pp. 47-78. Springer-Verlag,
New York.
[251Chr6st, R.J. (1991) Microbial Enzymes in Aquatic Environments. Springer-Verlag,
NY.
[261Chr6st, R.J. (1994) Microbial enzymatic degradation and
utilization of organic matter. In: Microbial Ecology of Lake
Pludee (Overbeck, J. and Chr&.t, R.J., Eds.), pp. 118-174.
Springer-Verlag,
NY.
1271Chrbst, R.J. and Riemann, B. (1994) Storm-stimulated enzymatic decomposition
of organic matter in benthic/pelagic
coastal mesocosms. Mar. Ecol. Prog. Ser. 108, 185-192.
S. (1977) Distribution
of
[281Kjelleberg, S. and Hikansson,
Ecology 18 (1995) 45-50
lipolytic, proteolytic and amylolytic marine bacteria between
the lipid film and the subsurface water. Mar. Biol. 39,
103-109.
M. and Marshall, K.C. (19851 Utilization of
[291 Hermansson,
surface localized substrate by non-adhesive marine bacteria.
Microb. Ecol. 11, 91-105.
[301Power, K. and Marshall, K.C. (1988) Cellular growth and
reproduction of marine bacteria on surface bound substrate.
Biofouling 1, 163-174.
[311Hobbie, J.E. (1966) Glucose and acetate in freshwater: Concentration and turnover rates. In: Chemical Environment in
the Aquatic Habitat (Golterman,
H.L. and Clymo, R.S.,
Eds.),
pp.
245-251.
Noord-Hollandsche
Uitgevers
Maatschappij, Amsterdam.
of “C-glucose,
r4C-amino
[321 Giide, H. (1988) Incorporation
acids and ‘H-thymidine by different size fractions of aquatic
microorganisms,
Arch. Hydrobiol. Beih. Ergebn. Limnol. 31,
61-69.