Asian Journal of Aquatic Sciences, December 2022.

Vol 5, Issue (3) 315-321 e-issn: 2716-4608

p-issn: 2655-366X

GROWTH RATE OF Nannochloropsis sp ACCORDING TO ADDITING

CONCENTRATION OF ZINC (ZN) AND COOPER (CU)

Herlina Meria Uli Sagala1*, Chrisoetanto P Pattirane1, Roni Sewiko1,

Ully Wulandari1, Beta Indi Sulistyowati1
1
Polytechnic of Marine and Fisheries Karawang
Tanjungpura-Klari, Karangpawitan, West Karawang, Karawang, 41315
*[email protected]

ABSTRACT

Zinc and copper are micronutrients that are the factors that support microalgae growth
rate besides macronutrients, CO2, temperature, salinity, and pH. Growth of Nannochlopsis sp
was observed to determine the influence of the addition of Zn and Cu concentration.
Microalgae cultivation was located outdoors on the semi-mass scale using 800 L medium on
mesophilic condition in 3 treatments with the addition of 1 ppm, 3 ppm, and 5 ppm
concentrations of Zn and Cu metals. Cells density was calculated using a hemocytometer, and
the absorbed metal content was measured by AAS. The study aimed to test the growth
response of Nannochloropsis sp cells to Zn and Cu metals. The growth rate of microalgae
was observed for the addition of Zn and Cu concentration in the cultivation medium. The
result showed the highest Nannochloropsis sp growth rate for Zn addition was 0.053/day and
for Cu addition was 0.279/day. Between Zn and Cu addition, the highest growth rate was
observed in the addition of 1 ppm Cu metal (Cu1) which was equal to 0.279/day. This shows
that adding micronutrients are not automatically will increase the growth rate of microalgae
as well.

Keywords: Growth Rate, Micronutrient, Zinc, Copper, Nannochloropsis sp.

I. INTRODUCTION Nannochloropsis sp. reproduces

Nannochloropsis sp. is an alga asexually by dividing. The multiplication of
measuring 2-4 m, greenish in color, not Nannocholropsis sp. cells occurs very
motile, and not flagellated. The cells are quickly. This is due to adequate nutrient
spherical in shape and small. sources and supportive environmental
Nannochlropsis sp. can live at a factors such as pH, salinity, temperature,
temperature of 25-35ºC, 25-35 ppt salinity, and light. Nutrient factors are one of the
and pH 8-9.5 [1]. Nannochloropsis sp. is important factors that need to be considered
better known as marine chlorella because it in the growth of microalgae. The nutrients
has very high nutritional value, is easy to needed by microalgae consist of
cultivate in bulk, does not cause toxins, macronutrients and micronutrients. Zinc
contains antibiotics, grows relatively fast, (Zn) and copper (Cu) are some of the
and has good accumulation ability. The micronutrients needed by microalgae for
relatively fast growth process of their growth [1].
Nannochloropsis and its good accumulating Zinc (Zn) acts as an enzyme activator
ability allows it to be a biosorbent of heavy and constituent of chlorophyll and
metals [2]. increases CO2 fixation [3] while copper
(Cu) acts as part of the phenolase, lactase,

Received : 6 September 2022 ajoas.ejournal.unri.ac.id

Accepted : 16 November 2022
Asian Journal of Aquatic Sciences, December 2022. Vol 5, Issue (3) 315-321 e-issn: 2716-4608
p-issn: 2655-366X

and ascorbate oxidase enzymes, constituent microalgae by increasing the density of

of plastocyanin which plays a role in microalgae. It should be noted and
electron transport in the process of remembered that micronutrients such as Zn
photosynthesis [4], forming chlorophyll and Cu are essential metals, so it is
and plays a role in microalgae cells necessary to know how much concentration
division. Zn and Cu besides being needed of micronutrients Nannochloropsis sp.
as micronutrients are essential heavy metals needs.
that are only needed by aquatic organisms
in certain concentrations. If the 2. RESEARCH METHOD
concentration of these metals exceeds the Time and Place
permissible threshold, it can pose a hazard The research was carried out at the
to aquatic organisms. These heavy metals Ecology Laboratory, Polytechnic KP
can interfere with the growth of organisms Karawang, West Java, and the Integrated
in water, one of which is phytoplankton [5]. Chemistry Laboratory, IPB Baranangsiang
Threshold values have been set for Zn 0.05 Campus, Bogor, West Java. The research
ppm and Cu 0.008 ppm [6]. period starts from March to June 2022.
The addition of growth nutrients into
the microalgae cultivation media is Research Methods
considered an important aspect and the Microalgae Cultivation
most influential on the quantity and quality Research on the growth of microalgae
of biomass and microalgae growth. Several was carried out on a semi-mass scale. The
research results on the effect of the addition microalgae used were from the
of Zn and Cu micronutrients on microalgae Eutigmatophyceae class, namely
noted that the addition of certain Nannochloropsis sp. The microalgae were
concentrations of Zn and Cu metal ions in cultivated in 16 L containers that had been
cultivation media could inhibit and increase given WALNE fertilizer as a source of
the growth of microalgae cells. The nutrients and placed in the laboratory under
interaction of Cu ions with Chaetoceros controlled conditions for 7 days intended
calcitrans showed a trend of decreasing for the restocking of the inoculum used in
population, which was directly proportional the study. The microalgae were then
to the increase in Cu concentration in the cultivated in an 800 L container that had
cultivation medium up to a concentration of been given TSP, ZA, and Urea fertilizers as
40 ppm [7]. The same was noted in a source of nutrients and placed outdoors
Scenedesmus incrassatlus [8]. It was noted for 17 days. The density of
that S.obliquus and Desnodesmus Nannochloropsis sp at initial cultivation
pleiomorphus showed varied trends in the was 700 x 104 cells/mL. The generalization
addition of Zn between 1-45 ppm [9]. of the initial density of cultivated
Soeprobowati and Hariyati [10] noted that microalgae is intended as a research
the addition of Cu metal concentration in standard because each cell has different
Spirulina platensis was different, 1 ppm Cu characteristics, survival abilities, and
increased growth, 3 ppm inhibited cells growth rates.
growth and 5 ppm was toxic to S. platensis.
The growth of Cyomodecea nodosa was Cells Density Observation
inhibited by Zn and Cu ions in the Phytoplankton density observations
cultivation medium [11]. were carried out every day from the
The effect of increasing the beginning to the end of the study.
concentration of Zn and Cu on microalgae Calculations were carried out using a
was determined by the growth rate of haemacytometer and to facilitate the

Growth Rate of Nannoropsis sp According to Additing (Sagala et al.) 316

Asian Journal of Aquatic Sciences, December 2022. Vol 5, Issue (3) 315-321 e-issn: 2716-4608
p-issn: 2655-366X

calculations, a hand tally counter was used. treatment with the addition of Zn and Cu
Microalgae samples were dripped using a metal ion concentrations to the control
dropper as much as 1 drop (0.05 mL) on a treatment was calculated by equation (9):
hemocytometer and observed under a
microscope with a magnification of 100 or % Cells inhibition =
-
x 100 %
400 times. According to [13] the density of
N. oculata cells for each mL with a
hemocytometer can be calculated using the DC is the optimum cells density of
following formula: the control treatment (x cells/mL) and
DT is the optimum cells density of the
treatment (x104 cells/mL).
N = ni xx 104 (i),
Statistical analysis
where N is the cells density and ni is the The effect of different concentrations
number of cells counted. of Zn and Cu on the growth of microalgae
Nannochloropsis sp was tested using
Research Procedure Analysis of Variety (ANOVA) through
Treatment of Addition of Zn and Cu. SPSS Version 22 with a 95% confidence
Metal Concentrations interval.
The metals or micronutrients used in
this study were Zn and Cu. There are 3 3. RESULT
treatments, namely control, the addition of Effect of Addition of Zn Metal
Zn metal, and the addition of Cu metal. The Concentration.
concentrations of metal ions and Cu with The results of the observation of the
three different concentrations of 1 ppm, 3 effect of increasing the concentration of
ppm, and 5 ppm were added to each pond zinc metal ion (Zn) on the cultivation
without any mixing between the two metals medium are presented in Table 1. The
or a single metal system. The addition of highest density observed in the treatment of
metal ion concentrations of Zn and Cu was increasing the concentration of Zn metal in
carried out at the beginning of cultivation. the cultivation medium on the growth of
Nannochloropsis sp. There is a Zn
Data analysis concentration of 3 ppm (Zn3) with an
Growth rate optimum density of 1410 x 104 cells/mL
The growth rate of microalgae (k) with a growth rate of 0.053/day. The lowest
was calculated using equation [12;13]: density occurred in the treatment with the
addition of 5 ppm. Zn metal concentration
(Zn5) with an optimum density of 1290 x
104 cells/mL with a growth rate of
0.045/day.
k is the specific growth rate (day-1),
Nt is the density of microalgae cells at time Effect of Addition of Cu Metal
t, No is the initial density of microalgae Concentration
cells, Tt is the initial time of cultivation, The results of observations of the
and To is the end time of cultivation. effect of increasing the concentration of
copper metal ions (Cu) on the cultivation
Percentage of Nannochloropsis sp Cells medium are presented in Table 2. The
Growth Inhibition highest density observed in the treatment of
The percentage of growth inhibition increasing the concentration of Zn metal in
of Nannochloropsis sp. cells in each the cultivation medium on the growth of

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Asian Journal of Aquatic Sciences, December 2022. Vol 5, Issue (3) 315-321 e-issn: 2716-4608
p-issn: 2655-366X

Nannochloropsis sp. There is a Cu addition of 5 ppm Cu metal concentration

concentration of 1 ppm (Cu1) with an (Cu5) with the optimum density reaching
optimum density of 1320 x 104 cells/mL 1100 x 104 cells/mL with a growth rate of
with a growth rate of 0.279/day. The lowest 0.061/day.
density occurred in the treatment with the

Table 1. The growth rate of Nannochloropsis sp. in the addition of Zn metal

Growth Rate (k)
Days to-
K Zn1 Zn3 Zn5
1 0 0 0 0
2 0.035 0.108 0.24 0.396
3 -0.014 0.187 0.091 0.052
4 0.028 0.293 0.252 0.045
5 0.097 0.073 0.016 0.115
6 0.138 -0.004 -0.056 -0.073
7 0.154 -0.3 0.087 -0.078
8 0.105 -0.446 -0.023 -0.167
9 0.121 -0.09 0.053 -0.452
10 0 0.025 0.029
11 -0.045 0.007
12 -0.027 -0.074
13 0.008 -0.08
14 -0.044 -0.087
15 0.048
16 0.019
16 0.019
Description: K: Control; Zn1: Addition of 1 ppm Zn metal; Zn3: Addition of 3 ppm Zn metal; Zn5:
Addition of 5 ppm Zn metal.

Table 2. Growth Rate of Nannochloropsis sp in Cu. Metal Addition Treatment

Growth Rate (k)
Day -
K Cu1 Cu3 Cu5
1 0 0 0 0
2 0.035 0.049 0.164 0.182
3 -0.014 0.040 0.03 0.118
4 0.028 0.094 0.211 0.156
5 0.097 0.058 -0.121 0.061
6 0.138 -0.100 -0.044 -0.084
7 0.154 0.112 0.207 -0.205
8 0.105 0.279 -0.359 -0.265
9 0.121 0.100 -0.013 0.132
10 0 -0.015 0.052 -0.154
11 -0.045 0.004 0.025 -0.063
12 -0.027 -0.068 -0.09 -0.024
13 0.008 -0.104 -0.216
14 -0.044 0.040
15 0.048 -0.097
16 0.019 -0.245
Description: K: Control; Cu1: Addition of 1 ppm Cu metal; Cu3: Addition of 3 ppm metal Cu; Cu5:
Addition of 5 ppm Cu metal

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Asian Journal of Aquatic Sciences, December 2022. Vol 5, Issue (3) 315-321 e-issn: 2716-4608
p-issn: 2655-366X

Percentage of Cells Growth Inhibition of addition of copper (Cu) concentration

Nannochloropsis sp. treatment, the growth inhibition of
Table 3 shows the percentage of Nannochloropsis sp. cells reached 14.838-
growth inhibition of Nannochloropsis sp. 29.0322%. From the results of this
cells due to the addition of heavy metal calculation, it can be seen that the
concentrations of zinc (Zn) and copper (Cu) percentage of growth inhibition of
in the cultivation medium. The addition of Nannochloropsis sp. by copper (Cu) is
zinc metal concentration (Zn) in cultivation higher than the addition of zinc (Zn)
media inhibited about 9.032 -16.774% concentration in the cultivation media of
growth of Nannochloropsis sp. In the Nannochloropsis sp.

Table 3. Percentage of Growth Inhibition of Nannochloropsis sp.

Microalgae Metal Treatment % Cells Inhibition
Zn1 12,25806
Zn Zn3 9.032258
Zn5 16,77419
Nannnochloropsissp
Cu1 14.83871
Cu Cu3 29,03226
Cu5 23.87097
Description: K: Control; Zn1: Addition of 1 ppm Zn metal; Zn3: Addition of 3 ppm Zn metal; Zn5:
Addition of 5 ppm Zn metal; Cu1: Addition of 1 ppm Cu metal; Cu3: Addition of 3 ppm
Cu metal; Cu5: Addition of 5 ppm Cu metal

4. DISCUSSION microalgae cells on the cultivation medium

The density of Nannochloropsis sp. at must be proportional to the nutrients [15].
each addition of metal concentrations of The growth rate of Nannochloropsis
both Zn and Cu continued to increase until sp. in the control treatment in this study
the fourth day. It is estimated that the was obtained at 0.121/day, this result was
nutrient content is still sufficient for the higher than the research conducted [12],
growth of Nannochloropsis sp. [1] and the which was 0.05/day. On an outdoor scale,
ability of Nannochloropsis sp. cells to this result is higher than the research
absorb metal concentrations. The specific conducted by [16], which achieved a
growth rate (k) is the growth rate of algae growth rate of 0.048/day.
during the exponential phase. The highest growth rate of
The growth rate (k) describes the Nannochloropsis sp. obtained in the
speed of microalgae cells growth per unit treatment of the addition of Zn metal in this
of time to determine the carrying capacity study reached 0.029/day, this result is lower
of the medium or nutrients for the growth than the results of the study [17] that
and division of microalgae cells. The reached 0.665/day. In the treatment of the
growth rate (k) can be used as a benchmark addition of Cu cells of Nannochloropsis
to determine the effect of adding metal ion sp., the highest growth rate was 0.205/day,
concentrations of Zn and Cu to the which was lower than the study [17], which
cultivation media on the growth of was 0.7469/day. In several similar studies
Nannochloropsis sp. cells. One of the the results shown are not much different
factors that affect the growth rate is the from the results obtained in this study, [18]
nutrient content or nutrients contained in noted that Chlorella vulgaris achieved a
the cultivation media for cells activity growth rate of 0.295/day and S. platensis
[Myers in 15]. The growth rate of reached 0.105/day on cultivation media
added with Cu and Cr metals and [19]

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Asian Journal of Aquatic Sciences, December 2022. Vol 5, Issue (3) 315-321 e-issn: 2716-4608
p-issn: 2655-366X

noted that the growth rate of S.costatum The results of statistical tests using
reached 0.31/day. analysis of variance (ANOVA) at a 95%
The results of the percentage of confidence interval showed that the
growth inhibition in the study were lower addition of metal ion concentrations of Zn
than the research conducted by [9] where and Cu to the cultivation media of
the addition of Zn inhibited the growth of Nannochloropsis sp. had a significant effect
D.pleiomorphus by about 74.0 ± 1.5% and on the growth of microalgae
in S.obliquus around 97.1 ± 2.0%. When Nannochloropsis sp. (p > 0.05), while in
compared with the results of research research conducted by [22] showed that the
conducted [20] it was higher where the addition of different Zn heavy metals in
addition of 0.5 ppm concentration of Cu cultivation media did not significantly
metal inhibited the growth of Chlorella affect the growth of microalgae, especially
vulgaris by about 1.59%. However, [20] Dunaliella salina.
also noted that if the microalgae cultivation
medium was added to a combination of 1.5 5. CONCLUSSION
ppm Cu metal with 2.0 ppm Cd metal ion, The addition of metal ion
it could inhibit the growth of Chlorella concentrations of Zn and Cu to the
vulgaris up to 78.55%. The difference in cultivation media of Nannochloropsis sp.
growth rate and the percentage of growth had a significant effect on the cultivation of
inhibition of microalgae obtained in this Nannochloropsis sp. The addition of a Cu
study was due to differences in concentration of 1 ppm (Cu1) gave a
environmental factors that affect the growth significant effect on the growth rate of
of microalgae. Internal factors can also Nannochloropsis sp. that was characterized
affect differences in the growth rate of by the achievement of the highest density
microalgae because the strains or species of and growth rate. To see a more significant
Nannochloropsis sp used in each study are effect of Zn and Cu on Nannochloropsis sp.
different. Several studies showed that cells, it is necessary to analyze the
microalgae death occurred at a maximum chlorophyll content, photosynthetic
metal content of Zn 10 mg/L and Cu 18 pigment content, and oxygen exchange of
mg/L [21]. these microalgae.

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