The assessment of nutrient availability for the growth of freshwater green
algae Chlorella kessleri by bioassay (Lake Sakadaš, NP Kopački Rit)
Janja Horvatić1, Vesna Peršić1, Željko Popović2
Keywords: enrichment bioassay, N and P limitation, Chlorella kessleri, Lake Sakadaš.
Introduction
In most lake ecosystems, phytoplankton growth may be controlled by the supply of limiting
nutrients, usually nitrogen or phosphorus. However, due to anthropogenic impact the
phytoplankton in freshwaters increasingly tends to be nitrogen limited (Aldridge et al., 1995).
Algal biomass and overall ecosystem productivity may be controlled not only by the type but
as well by the intensity of nutrient availability. The algal growth potential of Chlorella
kessleri measured in bioassay primarily depends on the nutrient concentration in tested waters
(Lukavský, 1992). Based on the concept of algal nutrient limitation, the algal bioassay is also
a responsive test designed to examine algal growth response to nutrient enrichment (Downing
et al., 1999). Therefore, the addition of nitrogen and phosphorus to tested waters causes a
growth response of C. kessleri proportional to the magnitude of limitation of the particular
nutrient (Horvatić et al., 2006). The aim of this paper was to determine the availability of
nutrients and the degree of N and P limitation in the water samples of Lake Sakadaš by a
miniaturized bioassay method.
Methods
Monthly sampling was carried out from March to July 2005 at two sites of Lake Sakadaš.
This lake is the deepest of the lakes in a complex wetland ecosystem of the Nature Park
Kopački Rit located in eastern Croatia (45°36´ N, 18°48´ E). Lake Sakadaš was formed
during the flood wave of the River Danube in 1926, that changed the configuration of
Kopački Rit. The surface area of this oval shaped lake is ca. 0.12 km2. It has relatively steep
slopes and a mean depth of ca. 7 m. The first investigated site was located in the central part
of the lake with a mean depth of 7.54 m and mean transparency of 1.46 m (from 0.84-2.20 m),
during the investigated period. The second site was located near the Kopačevo dam with
mean depth of 6.08 m and transparency 1.26 m (from 0.53-1.69 m). Physical and chemical
parameters (pH, conductivity, dissolved oxygen, total organic carbon, ammonium, nitrates,
Kjeldhal N, total nitrogen, orthophosphates, total phosphorus) were analyzed according to
APHA (1985). Phytoplankton chlorophyll concentration (Chla) was calculated according to
Komárková (1989).
A laboratory miniaturized growth bioassay method according to Lukavský (1992) and
modified after Horvatić et al. (2006) was used to determine the influence of nutrient
availability for the growth of C. kessleri FOTT et NOV. strain LARG/1. C. kessleri was
supplied by Culture Collection Autotrophic Organisms at Třeboň, the Czech Republic. Algae
were cultivated at the Department of Biology, J. J. Strossmayer University in Osijek on the
BBM solid medium (Bischoff & Bold, 1963), exposed to irradiance with fluorescent tubes
(Tungsram 30 W, F 74, daylight, Hungary) by PAR 138 µmol m-2s-1 measured with flat
sensor and temperature 25-30˚C. Due to the prior uptake and possible storage of nutrients, it
was necessary to starve C. kessleri cells before experimental use. Before inoculation the algal
cells were washed out with sterile distilled water from the solid medium and subcultured for 3
1
2
Department of Biology, Josip Juraj Strossmayer University, Gajev trg 6, 31 000 Osijek, Croatia
Faculty of Teacher Education, Josip Juraj Strossmayer University, Lorenza Jägera 9, 31 000 Osijek, Croatia
357
days in sterile distilled water. The algal cell density in this solution was determined using a
Bürker-Türk counting chamber (Karl Hecht KG, Sondheim, Germany) under a light
microscope (Axiovert 25, Carl Zeiss, Inc., Göttingen, Germany). The inoculum solution, used
in the experiment, was diluted with sterile distilled water and the initial cell density of C.
kessleri was 8 x 105 cells cm-3.Water samples from the investigated sites were filtered and
stored at -20 °C. After melting, the samples were filtered through the Whatman GF/C glass
fiber filter to eliminate particles. Bioassay experiments were carried out in polystyrene 96well microplates (Labsystem, Finland) with 9 x 13 cm flat bottom wells of 300 µl. The
miniaturized growth bioassays were conducted with six replicates of original water samples
(water sample from the investigated sites), six replicates of controls (water sample from the
investigated sites diluted with distilled water, 1:1) and six replicates of enriched water
samples (water samples with added N or P). In the enrichment bioassay with triplicate
treatments each nutrient was added single as KNO3 (in final concentrations of 5, 10 and 100
mg N l-1 for N1, N2 and N3), K2HPO4 (in final concentrations of 0.15, 1.5 and 15 mg P l-1 for
P1, P2 and P3) and in combination N3+P3.
The growth of C. kessleri was determined by measuring the optical density at 750 nm every
day. Conversion of OD to dry weight (mg l-1) of C. kessleri was described in detail by
Lukavský (1992). The total biomass of algae during the experiment (up to the 14th day) was
calculated as the area between the growth curves and the horizontal line. The average specific
growth rate (µ) was calculated during exponential growth (up to the 7th day) according to ISO
8692 (1989). Degree of nutrient limitation (∆r) was expressed as the difference in the growth
rate of C. kessleri between enriched (µE) and control water samples (µC) according to
Downing et al. (1999). Variance analysis and t-test were used to determine statistically
significant effects of added nutrients (N or P) to C. kessleri growth rate. Multiple comparisons
of treatments were performed by Tukey’s least significant difference procedure.
Results
The results of the physical and chemical parameters of water samples and phytoplankton Chla
are displayed in Table 1 and Fig. 1. From March to July 2005 water temperature was
reversely related to dissolved oxygen (r = -0.894, P<0.05). The lake showed relatively
constant depth over the period of investigation. The lowest measured depth in June coincided
with the highest measured conductivity, indicating influence of water level fluctuations on
physical and chemical parameters (Table 1).
Table 1. Mean monthly values of physical and chemical parameters and Chla concentrations
of water samples from both investigated sites of Lake Sakadaš.
Variables
Danube water level (m)
pH
Conductivity (µS cm-1)
Water temperature - WT (°C)
Water depth - WD (m)
Secchi depth - SD (m)
Dissolved oxygen - DO (mg l-1)
Total organic carbon - TOC (mg l-1)
Ammonium - NH4 (mg N l-1)
Kjeldhal N (mg l-1)
TN/TP
Chla (µg l-1)
March
5.84
7.76
484.5
15
7.9
1.1
14.786
4.31
0.0109
0.1648
30.88
41.790
April
4.89
8.14
402
16
7.4
1.435
10.763
4.9
0.0168
0.5445
2.73
17.782
May
4.39
7.815
561
19.5
6.1
1.82
10.066
49.16
0.0072
0.1796
6.01
19.878
June
2.06
8.015
740
20
4.4
0.685
6.441
11.76
0.0282
0.1722
7.31
32.295
July
5.68
8.205
404
26
8.4
1.77
6.743
7.63
0.0145
0.1936
3.02
22.496
During the investigated period, concentrations of nitrates (NO3) decreased from 2.8296 mg l-1
in March to 0.7064 mg l-1 in July and were found to follow the same trend as total nitrogen
358
(TN) (Fig. 1A). On the other hand, the concentration of ortho-phosphates (o-PO4) increased
and did not follow the same trend as concentrations of total phosphorus (TP) (Fig. 1B).
300
TB
3
250
2.5
200
2
150
1.5
100
1
50
0.5
0
0
March April May
June
July
o-PO4
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
TP
TB
300
250
200
150
100
T otal biomass of
C. kessleri (mg l-1 )
TN
PO4 , T P concentration (mg l-1 )
NO3
T otal biomass of
C. kessleri (mg l-1 )
NO3 , T N concentration (mg l-1 )
3.5
50
0
March April May June
2005
July
2005
Figure 1. A comparison of total biomass of C. kessleri (mg l-1) and NO3, TN concentrations
(A) as well as of the total biomass of C. kessleri (mg l-1) and PO4, TP concentrations (B).
Columns represent average values of the total biomass and nutrient concentrations from both
sites at Lake Sakadaš, and error bars standard deviation.
A positive correlation was determined between the NO3 concentration and the total biomass
(TB) of C. kessleri (r = 0.969, P<0.01) as well as between the TN and the TB of C. kessleri (r
= 955, P<0.02).
1.4
Control
N3
P2
N1
N+P
P3
Growth rate (d-1 )
1.2
N2
P1
*
1
0.8
0.6
0.4
0.2
0
-0.2
March
April
May
2005
June
1
*
Degree of N, P limitation ( r, d-1 )
1.6
0.8
N1
N3
P1
P3
b
0.6
0.4
N2
N+P
P2
bc
c
June
July
b
a
0.2
0
-0.2
July
-0.4
March
April
May
2005
Figure 2. (A) Average specific growth rate of C. kessleri (d-1) in the control water samples
and samples enriched with N and P concentrations at Lake Sakadaš. * A statistically
significant effect of added N and N+P concentrations. (B) The degree of nutrient limitation
(∆r, d-1). The same letters represent that there was no statistically significant difference
between the degree of N and P limitation (∆r) in the investigated months.
The results of the nutrient enrichment bioassay were summarized in Fig. 2A and B. The
growth of C. kessleri was estimated as the average daily specific growth rate during the
exponential phase of growth. During the enrichment of Lake Sakadaš water samples with N, a
significant increase in the growth rate of C. kessleri was determined only in June and July
(Fig. 2A). P addition had no effect, while combination of N and P supported similar growth as
N added alone. During the investigated period an increase in the degree of N limitation was
observed (Fig. 2B). According to Tukey’s least significant difference test, the response of the
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growth rate of C. kessleri to nutrient enrichment in March was different from the rest of the
investigated period (Fig. 2B) indicating favourable nutrient conditions for phytoplankton
growth.
Discussion
Lake Sakadaš is a relatively small water body, connected with Lake Kopačko and the River
Danube by channels Čonakut and Hulovo. It is also connected with the backwaters of the
Stara Drava Channel through the Kopačevo dam. Therefore, nutrient input in this lake is
highly related to water level fluctuations of the River Danube as well as to the inflow from
surrounding agricultural land through the dam. A positive correlation between TN, NO3
concentrations and the total biomass of C. kessleri indicated N limited conditions in the
investigated waters. The highest values of the total biomass of C. kessleri and phytoplankton
Chla concentration were determined in March and coincided with a high TN/TP ratio as well
as with high inorganic N concentrations (Fig. 1A, Table 1), indicating P deficiency for algae.
However, that was not supported by the results of enrichment experiments (Fig 2A and B).
The problem with the TN/TP ratio is that it can be over-shadowed by nutrient amounts within
the living algae, and is not a good indicator of nutrient deficiency for algae (Kisand et al.,
2001). In June, the availability of P was determining the growth of C. kessleri in untreated
water samples (Fig. 1B). On the other hand, in the enrichment experiment, a significant
influence of added N concentrations in June and July (Fig. 2A) indicated low N availability.
In the same time, the lack of P response indicated that another factor was primarily limiting,
although high pH could cause P to become biologically unavailable. Therefore N was the
controlling factor of C. kessleri growth in June and July. Because inorganic N concentrations
steadily decreased during the investigated period (Fig. 1A), it was expected that the degree of
the bioassay response to N enrichment would increase from March to July 2005, indicating N
limitation. Subsequent decrease of the TN/TP ratio (Table 1), N limited conditions for C.
kessleri growth as well as the improvement of light conditions as the summer progressed,
gave a competitive advantage to cyanobacteria abundance (Nõges et al., 2003).
Summary
In the water of Lake Sakadaš, concentrations of nitrates and total nitrogen decreased from
March to July 2005. In untreated water samples, the availability of nitrogen limited the
growth of C. kessleri. A significant increase in the growth rate of C. kessleri in water samples
enriched with nitrogen indicated N limited conditions in June and July. The increase of the
degree of algal response to N enrichment from March to July 2005 was probably a result of
the decrease of nitrogen availability.
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