Nature Environment and Pollution Technology
An International Quarterly Scientific Journal
Original Research Paper
p-ISSN: 0972-6268
(Print copies up to 2016)
Vol. 19
No. 2
pp. 809-813
2020
e-ISSN: 2395-3454
https://doi.org/10.46488/NEPT.2020.v19i02.038
Original
Research Paper
Open Access Journal
Growth and Removal of Nitrogen and Phosphorus by a Macroalgae Cladophora
glomerata Under Different Nitrate Concentrations
Aulia Ulfah Farahdiba*, Euis Nurul Hidayah*†, Gina Aprilliana Asmar* and Yadanar Win Myint**
*Department of Environmental Engineering, University of Pembangunan Nasional Veteran Jawa Timur,
Surabaya, Indonesia
**Nanotechnology Research Department, Department of Research and Innovation Yangon,
Ministry of Education, Myanmar
†Corresponding author: Euis Nurul Hidayah; euisnh@gmail.com
Nat. Env. & Poll. Tech.
Website: www.neptjournal.com
Received: 18-07-2019
Accepted: 05-10-2019
Key Words:
Macroalgae;
Cladophora glomerata;
Nitrate; Phosphate;
Kinetics
ABSTRACT
Effectiveness of macroalgae was investigated for enhancing wastewater treatment processes.
Bioremediation using macroalgae could remove nitrate and phosphate contaminants in the water where
algae assimilate nitrogen and phosphorus and convert them to biomass. This study evaluates the
effects of high nitrate concentration on the kinetics of cell growth during nitrate and phosphate removal
by a macroalga Cladophora glomerata. The algal growth and nitrate removal from media containing
initial nitrate concentrations of 5mg/L to 400 mg/L were monitored in batch growth, whereas control
media has no additional nitrate. Light exposure was kept for 12 and 20 hours. The purpose of this
research was to find out the effect of various nitrate concentrations on nitrate and phosphate removal
with macroalgal growth. Maximum growth kinetic reaches µ=0.075/day in 20 hours light exposure with
100 mg/L initial nitrate concentration. Nitrate and phosphate reach about 90% removal rates on the
fifth day. Nitrate concentration was not significantly affected by biomass growth (Pearson correlation:
0.295). But, phosphate concentration has a moderate correlation with macroalgae biomass (Pearson
correlation: 0.533).
INTRODUCTION
In recent years, microalgae and macroalgae have been used
in many environmental applications. Bioremediation using
algal technology is believed to be a promising alternative
technology. Several macroalgae have been suggested for
the treatment of wastewaters with high nitrogen concentration (Cole et al. 2016, Ge & Champagne 2017). Moreover,
previous studies have shown that Cladophora glomerata
has significant potential of bioremediation for wastewater
treatment (Whitton 1970b).
Nitrogen in biogeological cycles produces compounds
with different oxidation states like nitrate, nitrite, ammonium,
organic nitrogen including amino acids, urea and proteins
that are available to phytoplankton. Furthermore, high nitrate
concentration will enhance the possibility of eutrophication.
According to a study conducted by Lee et al. (2015), the nutrients with the complement of substrates greatly affected the
optimization of macroalgae growth. Furthermore, if nutrient
concentration is in excess in the water, macroalgal growth
will be inhibited (Han et al. 2016).
Recently, C. glomerata became the most prodigal algae
in the water streams. Eutrophication, caused by high nutrient
content, will have a high influence of C. glomerata biomass
with the optimal growth conditions of 0.07 mg/L phosphorous (P), 0.6 mg/L nitrate-nitrogen (NO3-N) and 0.2 mg/L
ammonium nitrogen (NH4-N) (Whitton 1970a).
Algae can use nitrate (NO3-), nitrite (NO2-) or ammonium
(NH4 +) as a nitrogen source. Nitrate (NO3-) is the main form of
nitrogen in natural waters as well as a major nutrient for plant
growth and algae (Putra & Farahdiba 2018). The main sources
of nitrogen in the water are nitrate and ammonium ions.
Phosphate is a form of phosphorus that can be used by
plants. Phosphorus is also an essential nutrient for higher
plants and algae and becomes a limiting factor for plants
and algae (Selvaratnam et al. 2015). Orthophosphate (PO43
) is an inorganic phosphorus source which is important
for algal growth and can be produced by various forms of
phosphorus-containing organic matter (Han et al. 2016).
Macroalgal growth will also be affected by the duration of
light exposure. Furthermore, among many environmental
conditions, it was hypothesized that the macroalgae-bacteria
system would respond to different photoperiod conditions in
terms of an increase or decrease in the algal population and
nitrogen concentration (Lee et al. 2015).
810
Aulia Ulfah Farahdiba et al.
Therefore, controlling the concentration of nitrate in
water resources light exposure is required. Currently, there
is limited data available on the toxicity of high concentration
of nitrate and its influence on macroalgal biomass with N
and P removal. In this study, the effects of high nitrate concentration and nitrate as the nitrogen source for the growth
of Cladophora glomerata and nitrate and phosphate removal
in the growth media have been investigated.
MATERIALS AND METHODS
The experiment was conducted at the Research Laboratory,
Environmental Engineering UPNV. The study took place
from the beginning of February to the end of May 2019, from
the preparation stage to the analysis results. USEPA (1996)
was followed to find nitrate concentration within the range
finding test (RFT). Nitrate concentration used in this study
was 100-1000mg/L. This preliminary study found that the
critical macroalgae could live in nitrate concentration of 100400 mg/L (Farahdiba et al. 2019). When nitrate concentration
was higher than 400mg/L, macroalgae became withered and
yellow immediately.
Batch scale experiment was conducted with 300 mL of
laundry wastewater sample in a glass jar within 5 days with
5 different nitrate concentrations. Each reactor was spiked
with macroalgae C. glomerata to remove high nitrate and
phosphate concentration. This research was conducted for
5 days with additional light from a LED lamp of 20 watts
(or 3600 lux) for 12 and 20 hours illumination. Nitrate and
phosphate were determined according to Standard Methods
(González-Camejo et al. 2018).
Nitrate concentration used from RFT test was 0-400
mg/L. Macroalgae spiked with the water laden with 5 different concentrations: a) 5 mg/L (control reactor, without
additional nitrate); b) 100 mg/L; c) 200 mg/L; d) 300 mg/L,
and e) 400 mg/L.
Initial macroalgae biomass was measured as the average
of 10 samples weighing from fresh algal sample to dry weight
of macroalgae biomass at 105°C for 4 hours (Horwitz &
Chemists 2000). The preliminary test was to determine the
initial macroalgae biomass value obtained from drying 10
macroalgae samples with the same weight. The dry weight
results of the 10 macroalgae samples were averaged and the
initial biomass yield was 1332.9 mg/L (Ge & Champagne
2017). This data becomes the baseline of the algae biomass
within sampling on the day.
In this study, the calculated specific growth rate µ/day in
the exponential phase of algal growth was measured by using
Eq. 1 (Issarapayup et al. 2009, Zhu et al. 2013).
where N1 and N2 are defined as dry biomass (mg/L) at
time t1 and t2, respectively.
The biomass productivity (P) was calculated according
to the formula given in Eq. 2.
…(2)
Where, DWi and DW0 are dry biomass (mg/L) at time ti
and t0 (initial time), respectively.
100
100
80
80
Nitrate Percentage Removal (%)
Nitrate Percentage Removal (%)
…(1)
µ(/day) = ln(N2 – N1)/(t2 – t1)
60
40
±5 mg/L(12h)
±100 mg/L(12 h)
±200 mg/L(12 h)
±300 mg/L(12 h)
±400 mg/L(12 h)
20
0
60
40
±5 mg/L(20h)
±100 mg/L(20 h)
±200 mg/L(20 h)
±300 mg/L(20 h)
±400 mg/L(20 h)
20
0
0
1
2
3
4
5
6
0
1
2
3
4
5
6
Days
Days
Fig. 1: Nitrate removal with 12h light exposure by macroal
Fig. 2: Nitrate removal with 24h light exposure by macroal
Fig. 1: Nitrate removal with 12h light exposure by macroalgae under
five nitrate concentration levels within five days.
Vol. 19, No. 2, 2020 · Nature Environment and Pollution Technology
Fig. 2: Nitrate removal with 24h light exposure by macroalgae under
five nitrate concentration levels within five days.
811
GROWTH AND NUTRIENT REMOVAL BY A MACROALGAE
Data analysis was performed with EXCEL (Microsoft
Office Enterprise 2010) and Minitab 2016 for Windows and
correlation was determined wherever applicable.
RESULT AND DISCUSSION
Effect of Nitrate Concentration and Light Exposure on
Nitrate Removal
Fig. 1 shows that the nitrate dramatically decreased within
5 days in all the experiments. Furthermore, the removal in
the treatment with an initial nitrate concentration of 5 mg/L
(control) was slowed down as compared to other nitrate
concentrations. However, the control reactor has a different
downward trend. Other reactors showed an increase slightly
from day 1 to 4 and a reduction on the fifth day.
In the first day’s observation, the maximum nitrate reduction was 98.26% with the initial nitrate concentration of
200mg/L. When the test came to the fourth day, 94.32%,
96.89%, 98.21%, 74.53% and 81.97% were accordingly removed from 5, 100, 200, 300 and 400 mg/L nitrate cultures.
Nitrate in 0, 100 and 200 mg/L increased on the first day
and dropped on the fifth day; while in 300 and 400 mg/L,
it increased on the first day and stabilized until the end of
the experiment.
Nitrate removal efficiency in 20-hour irradiation reactor
could be seen in Fig. 2, which has a similar trend with 12
hours of exposure. Moreover, in 5 mg/L nitrate concentration
with 20 hours, the faster removal efficiency was reached
than the 12 hours exposure (on the third day); while in the
following days it reduced significantly.
and began to fall on the fifth day. Increased level of nitrate
reduction is because the algae have well adapted to the day
and night period. This has increased the dissolved organic
carbon in water through active photosynthesis which is
strongly correlated with the bacterial system (Kouzuma
& Watanabe 2015, Lee et al. 2015, Unnithan et al. 2014).
Throughout the experiment, pH condition is in the normal
range, which indicates that nitrification and denitrification
were not the main processes responsible for nitrogen removal.
Since the mean pH in the reactor was less than 8.5, ammonia
volatilization through the surface might have been limited
(Derabe-Maobe 2014).
In addition, increased nitrate concentration could be
caused by the release of cellular nutrients by microalgae.
Macroalgae lysis during the death phase can also increase
nitrates (Ma et al. 2014).
The average nutrient removal rates were not significantly different among the two photoperiod conditions during
the experiment as the 12 or 20 hours time exposure could
be partially attributed to the high adaptability of algae in
controlling carbon assimilation and respiration (Ma et al.
2014).
Effect of Nitrate Concentration and Light Exposure
Variation on Phosphate Removal
In the four days of observation, the maximum nitrate
reduction of 98.18%, 98.51%, 74.26%, and 81.07% was
accordingly removed from 100, 200, 300 and 400 mg/L.
The phosphate removal with 12 hours light exposure in this
study was measured and shown in Fig. 3. On the first day until
fourth day phosphate reduction was relatively stable. Four
days were assumed to be the optimum time for macroalgae
to remove phosphate. On the last day of the main study (day
5), it was found that the percentage of phosphate reduction
began to decline, the condition was considered to be the point
or time of saturation of macroalgae in remaking phosphate.
The significantly high removal efficiency of nitrate was
achieved among all the treatments (Figs. 1 and 2). Based
on Fig. 1 and Fig. 2, the nitrate level in the test reactor is
sufficient to reach a very high removal efficiency in nitrate
on days 1 to 4 and decreases on the last day. Whereas, in
the control reactor it gradually increases from days 1 to 4
Furthermore, this trend is similar to the 20 hours trend.
The phosphate removal is not significantly directly affected
by light illumination. From the two irradiation periods, it
can be seen that the highest per cent reduction is achieved
by macroalgae on the fourth day, which is considered as the
optimum macroalgae time in absorbing phosphate.
Table 1: Growth parameters of macroalgae under five nitrate concentrations and light exposure in 5 days.
Initial nitrate concentration (mg/L nitrate)
Specific growth rate µ (/day)
Biomass increase (mg/L)
Biomass productivity (mg/L.day)
12 h
24h
12 h
24h
12 h
24h
5 (Control)
0.009
0.014
61.433
96.767
18.937
30.912
100
0.056
0.075
431.433
604.767
44.321
60.888
200
0.035
0.0262
259.766
186.767
35.165
36.679
300
0.027
0.0261
194.767
185.433
31.176
31.570
400
0.011
0.0264
77.767
187.767
20.558
29.547
Nature Environment and Pollution Technology · Vol. 19, No. 2, 2020
812
Aulia Ulfah Farahdiba et al.
The four days observation on both the hours’ exposure,
showed that the maximum phosphate reduction of 92.72%,
92.82%, 92.72%, 92.57%, 92.77% was accordingly achieved
from 5, 100, 200, 300 and 400 mg/L of nitrate concentration.
Reduced phosphate levels in microalgae media are
caused by the use of phosphate as a nutrient for microbial
growth (Fig. 3). Phosphate functions are energy metabolism,
protein synthesis, regulation of starch and starch production,
formation of proteins, carbohydrates, cell structures and
cell membrane stabilizers (Grover & Mar 2008). Decreasing in phosphate levels is due to the increasing number of
macroalgae which increases the requirement for phosphate
(Tang et al. 2016).
The efficiency of the resulting phosphate reduction
varies depending on the composition of the media and environmental conditions such as initial nutrient concentration,
irradiation time, and the ratio of nitrate:phosphate, light or
dark. Moreover, the process of decreasing of pollutants in
wastewater using aquatic plants is a collaboration between
plants and microbes associated with these plants (Lee et al.
2015).
Macroalgae Growth Kinetics
Table 1 shows the growth kinetics and biomass productivity in this experiment with 12 hours and 20 hours of light
exposure. Biomass testing can be used as a reference to
determine the growth of macroalgae by dry weight biomass. Macroalgae growth can be observed by determining
the growth phase which is divided into four phases which
include the lag, exponential, stationary and lysis (Zhu et
al. 2013, Cahyonugroho et al. 2020). The results in Fig. 4
show the macroalgae growth obtained from the five different
nitrate concentrations. All reactors have increased biomass
growth on the first day. The results of biomass growth in
the reactor with 20:4 irradiation were found to have similar
trends. Furthermore, the specific growth rate in 12 and 24
hours has a high correlation, with the Pearson correlation
coefficient of 0.879.
The macroalgae, cultivated in the media with the nitrate
concentration of 5, 100, 200, 300 and 400 mg/L had an
exponential stage and then fluctuate in the stationary phase
with 12 to 20 hours light exposure (Fig. 4).
Statistical analysis was conducted to determine the
correlation between biomass concentration and N, P concentrations using Pearson correlation value. Biomass is greatly
affected by phosphate concentration (Pearson value: 0.533
in 12 and 20 hour). Nevertheless, nitrate concentration has a
lower correlation value (0.295 in 12 and 20 hour).
However, the exponential growth in the culture with all
the reactors lasted for one to two days, following a predicted
lag phase, which lasted for about 4 days. In five days, algal
cells reduce in all the nitrate concentrations; in order with
similar trends with a lower removal efficiency of N and P. It
was predicted that on the fifth day, macroalgae would be in
the stationary-lysis stage. In this study, the lack of a visible
lysis phase was because the cultivation period was short.
Research by Taziki et al. (2016) showed that the completed
algal life stage would appear after about 12 days. However,
the algal life stage is dependent on the algal species, nutrient
and environmental condition.
100
80
1500
60
Dry Weight (mg/L)
Phosphate Percentage Removal (%)
2000
40
±5 mg/L
±100 mg/L
±200 mg/L
±300 mg/L
±400 mg/L
20
1
2
3
4
5
±5 mg/L
±100 mg/L
±200 mg/L
±300 mg/L
±400 mg/L
500
0
0
1000
6
Days
Fig. 3: Phosphate removal with 12h and 20h light exposure by
Fig. 3: Phosphate removal with 12h and 20h light exposure by macroalgae under five nitrate concentration levels in five days.
Vol. 19, No. 2, 2020 · Nature Environment and Pollution Technology
0
0
1
2
3
4
5
6
Days
Fig. 4: Growth curves for macroalgae (Cladophora glomera
Fig. 4: Growth curves for macroalgae (Cladophora glomerata) grown
under five nitrate concentration levels in five days.
GROWTH AND NUTRIENT REMOVAL BY A MACROALGAE
The specific growth rate (µ) of macroalgae in 5, 100,
200, 300 and 400 mg/L nitrate concentration was 0.024,
0.075, 0.0262, 0.0261 and 0.0264 day/L respectively. The
final biomass productivity increase significantly at 100 mg/L
which was the highest, reaching to 60.88 mg/L.day, while the
culture in 5 and 400 mg/L nitrate showed the lowest biomass
increase (30 and 29 mg/L.day, respectively). The biomass
productivity in this study was lower than that in the research
by Zhu et al. (2013).
CONCLUSION
The macroalgae Cladophora glomerata has high removal
efficiency to reduce nitrate and phosphate concentration in
high nitrate concentration, but macroalgae biomass was not
directly influenced by nitrate concentration. Moreover, the
reactor has low growth kinetics and biomass productivity.
This is probably the complication in the algae reactor because
of various factors which affect the macroalgae performance
to remove contaminants in the water.
ACKNOWLEDGEMENT
This study was a part of funding supported by grants from
Directorate of Research and Community Service, Directorate
General of Research Development, Ministry of Research
Technology and Higher Education of Indonesia. This research was carried out under contract No. 201/SP2H/LT/
DRPM/2019.
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