Bioresource Technology 131 (2013) 246–249
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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech
Short Communication
Influence of nitrogen sources on biomass productivity of microalgae
Scenedesmus bijugatus
Muthu Arumugam a,⇑, Ankur Agarwal a, Mahesh Chandra Arya a, Zakwan Ahmed b
a
b
Defence Institute of Bio-Energy Research Defence R&D Organization, Ministry of Defence, Govt. of India, Field Station, Pithoragarh 262 501, Uttarakhand, India
Defence Institute of Bio-Energy Research Defence R&D Organization, Ministry of Defence, Govt. of India, Haldwani 263 139, Uttarakhand, India
h i g h l i g h t s
" Scenedesmus growth and biomass production was evaluated.
" Potassium and sodium nitrates (10 mM) performed better.
" Urea also performs better, leading to its applicability for large scale culturing.
a r t i c l e
i n f o
Article history:
Received 4 December 2012
Received in revised form 21 December 2012
Accepted 25 December 2012
Available online 4 January 2013
Keywords:
Biodiesel
Biofuel
Growth medium
Algae and nitrogen source
a b s t r a c t
The influence of different nitrogen source of varying concentrations on biomass production of green algae
Scenedesmus was investigated. The result revealed that there was a significant difference among nitrogen
sources in promoting algal biomass growth at lower concentrations of 5 and 10 mM. Nitrate was found to
be a preferred form of nitrogen source and potassium (0.32 g/L) and sodium nitrates (0.28 g/L) performed
better for biomass growth of Scenedesmus. Among the ammonical forms, urea (0.25 g/L) resulted in
almost equal biomass as nitrates, making it an economical substitute for nitrogen source in large scale
culturing of algae being commercially available.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
Biodiesel is currently receiving much attention due to its potential as a sustainable and environment friendly alternative. Use of
agriculture crops, especially edible oil crops as a feedstock for biofuel has raised concerns of food security. Therefore, algae as a feed
stock has emerged at the fore front of biofuel research due to
increasing awareness of global energy issue in conjunction with
the production limitations of terrestrial crops (Wayer et al.,
2010; Chisti, 2008; Griffiths and Harrison, 2009; John et al.,
2011). Microalgae are the promising source of biofuel due to their
simple cellular structure, higher growth rate and lipid content than
conventional vegetable oligenous crops (Becker, 1994). Interestingly some algal species store 50–60% of their dry cell weight as
storage lipids regularly which was increased by adopting variety
⇑ Corresponding author. Present addresses: Division of Biotechnology, National
Institute for Interdisciplinary Science and Technology (NIIST), CSIR, Industrial Estate
(PO), Trivandrum 695 019, Kerala, India. Tel.: +91 471 2515 279; fax: +91 471 2491
712.
E-mail addresses: [email protected], [email protected] (M. Arumugam).
0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biortech.2012.12.159
of environmental and nutritional stress conditions (Illman et al.,
2000; Hu et al., 2008; Breuer et al., 2012; Lin et al., 2012). However,
it is also noteworthy that stress induced lipid accumulation often corresponds with reduced biomass productivity (Rodolfi et al., 2008). In
general, an algal strain with lower oil content grew faster than the
one restrain higher oil content (Vasudevan and Birggs, 2008). Therefore, the first step in algal biofuel R&D is selection and identification
of a particular algal strain with faster cell growth, maximum rate of
lipid production with an ability to adapt diverse habitats.
Potential application of Scenedesmus for biofuel production has
been reported by various workers (Yoo et al., 2010; Mandal and
Mallick, 2011). This algal species was isolated from locally adapted
algal diversity and showed promising results in terms of adaptability, biomass production and lipid content in our preliminary trials
(Arumugam et al., 2011). One of the major factors that affected the
rate of biomass production and oil productivity in algae was composition of culture medium in open pond as well as in closed photobioreactors (Pruvost et al., 2012; Griffiths and Harrison, 2009).
Culture medium was directly correlated and it often became one
of the major obstacles in microalgal oil production, because it supported the growth and development of algae.
M. Arumugam et al. / Bioresource Technology 131 (2013) 246–249
In the view of above, several studies have been undertaken in
order to optimize suitable growth medium for large scale culturing
with affordable cost to bring algal biofuel production commercially
viable alternative (Li et al., 2008). It has been reported that,
nitrogen was quantitatively most important nutrient affecting
the biomass growth and lipid productivity of various microalgae
(Griffiths and Harrison, 2009). However, very few reports are available on the effect of nitrogen sources on the biomass productivity
of Scenedesmus. Therefore, effect of nitrogen levels and sources on
the growth of the green algae Scenedesmus bijugatus was studied in
the present study to understand its interaction with cell growth
during cultivation and to increase biomass productivity, hence
improving the economics of microalgae-derived biodiesel.
2. Methods
2.1. Algal strain and growth conditions
Algal strain S. bijugatus was isolated from the fresh water source
of Pithoragarh district and taxonomically identified at Center for
Advanced Studies in Botany, University of Madras, Chennai, India
(Arumugam et al., 2011). This strain was grown in 1000 ml
Erlenmeyer flask containing 300 ml modified basal soil extraction
medium with appropriate nitrogen sources (Li et al., 2008). The
culture was grown at ambient temperature with a photoperiod of
14:10 light–dark cycle. The cultures were shuck manually twice a
day to avoid settling and sticking to the surface of the flask. The
cultures were grown for 18 days and no external CO2 was provided
during the experiment.
2.2. Nitrogen sources and concentrations
The experiment was carried out with six different nitrogen
sources like potassium nitrate, sodium nitrate, urea, calcium
nitrate, ammonium nitrate and ammonium chloride. Four different
concentrations viz., 05, 10, 15 and 20 mM were supplemented to
the modified basal soil extraction medium twice during the
experimental period. A control was also maintained with modified
basal soil extraction medium without supplementing any nitrogen
source.
2.3. Growth and biomass estimation
Algal growth was monitored periodically on 3rd, 6th, 10th, 12th
and 18th day by measuring the OD at 540 nm using spectrophotometer (Labomad Inc.) as suggested by Gouveia and Oliveira
(2009). Vortexing of algal sample was done to get homogenous
culture to prevent the settling and erroneous result while reading
the OD.
The second set of experiment was conducted under the same
growth conditions and parameters as of first for OD measurement
with only two concentrations i.e. 5 and 10 mM, which were found
promising during growth measurement by OD. Samples of second
experiment were subjected for determination of biomass growth
(dry cell weight) on the 18th day. The samples were centrifuged
(REMI, CPR-24) at 10,000 rpm for 10 min then subjected to oven
drying. Dried cell mass was determined gravimetrically to express
the growth as dry cell mass (g L 1). The data was recorded and
averages of three independent experiments were plotted as graph
with standard deviation as error bar.
2.4. Experimental design and data analysis
The experiment was designed in complete randomized design
with six different nitrogen sources and five concentrations of each
247
nitrogen source with three replications. Two-factor analysis was
carried out using standard statistical programme (STPR) to study
individual effect (nitrogen source or its concentration) and
interaction of both the factors.
3. Results and discussion
3.1. Effect of nitrogen source and concentration on growth
Present study was aimed at standardizing the appropriate
nitrogen source and its concentration for maximum growth of fresh
water green algae S. bijugatus. The growth data obtained for six
different nitrogen sources over 18 days. Potassium nitrate and
sodium nitrate have shown almost similar trend of growth and performed better, over other nitrogen sources tested (data not shown).
On the contrary, other tested nitrogen sources namely urea, calcium
nitrate, ammonium nitrate and ammonium chloride exhibited
moderate effect on biomass production. The nitrogen sources
responded well in order to enhance the growth but maximum
growth was recorded for potassium nitrate indicating the favorable
nitrogen source for the growth of green alga Scenedesmus.
All nitrogen sources were tested in five different concentrations
viz., 0, 5, 10, 15 and 20 mM in order to standardize the optimum
concentration for algal growth. Interestingly lower concentration
(5 and 10 mM) of other nitrogen sources under study revealed
better biomass growth as evident by Fig. 1. In contrast higher nitrogen (beyond 10 mM) was not much responsive for algal growth as
compared to lower concentration invariably in all the tested nitrogen sources (data not shown). The experiment was also aimed at
examining the influence of culture age (days) to find out the effective harvesting time to obtain maximum biomass in short span of
time. The experimental results revealed that other nitrogen
sources responded well with aging culture. However, higher nitrogen concentration leads to reduce growth after 10 days in majority
of tested nitrogen sources indicating detrimental growth affect.
Whereas lower concentration (5 and 10 mM) showed linear
growth pattern with respect to culture aging and a marginal
increase in growth was recorded beyond 12 days.
Nitrogen supplementation on algal biomass growth and lipid
productivity has been reported for Porphyridium purpureum (Becker,
1994), Scenedesmus dimorphous (Benider et al., 2001), Tetraselmis
suecia, Skeletonema costatum and Thalassiosira pseudonana (Griffiths
and Harrison, 2009; Rodolfi et al., 2008). The results of present study
also revealed that nitrogen was an important nutrient source and
can be supplied in any form to promote algal growth. The results
clearly indicated that nitrate forms (KNO3 and NaNO3) of nitrogen
favored the algal growth over ammonium. Similarly in Neochloris,
highest biomass productivity (0.63 g L 1 day 1) was obtained at
10 mM sodium nitrate with a biomass concentration of 3.2 g L 1
(Li et al., 2008). Dyananda et al. (2006) also found a comparable
result, wherein potassium nitrate was found preferred nitrogen
source over the other sources for biomass growth of Botryococcus
braunii. The most possible reason for the better performance of
KNO3 over other nitrogen sources may be due to the fact that nitrogen and potassium are the two important nutrients for algal growth,
which are available in it.
3.2. Interaction of nitrogen sources and concentrations
The effect of various nitrogen sources and their concentration
along with their interaction on algal biomass growth was analyzed
statistically and presented in Table 1. The results revealed a significant difference among the tested nitrogen sources on growth of
green alga Scenedesmus. Varying nitrogen concentrations also
exhibited significant effect on algae growth during the course of
248
M. Arumugam et al. / Bioresource Technology 131 (2013) 246–249
Biomass growth (OD 540 nm)
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Day 0
Day 3
Day 6
Day 10
Day 12
Day 18
Fig. 1. Biomass growth of Scenedesmus bijugatus grown in media consisting of 10 mM of different nitrogen compounds: potassium nitrate (h), sodium nitrate (j), urea (N),
calcium nitrate (D), ammonium nitrate () and ammonium chloride (s).
Table 1
ANOVA of mean square for the effect of nitrogen source, concentrations and their interactions on Scenedesmus.
Sources
df
Mean sum of square
3rd day
Nitrogen sources (a)
Nitrogen conc. (b)
Interaction (a b)
Error
Total
**
ns
5
4
20
60
89
**
0.0089
0.0008ns
0.0018**
0.0004ns
6th day
**
0.0197
0.0060**
0.0030**
0.0004ns
9th day
**
0.0881
0.0075**
0.0046**
0.0003ns
12th day
**
0.0731
0.0615**
0.0078**
0.0004ns
18th day
0.0993**
0.0851**
0.0088**
0.0002ns
Significant at 1%.
Non significant.
Fig. 2. Effect of various nitrogen sources on biomass (dry weight) recovery of Scenedesmus bijugatus.
study except initial period, but the interaction of nitrogen source
and concentration was significant (Table 1) showing the importance of choosing suitable nitrogen source and concentration for
fast growth of green algae. Fig. 1 also exhibited a considerable
difference in terms of biomass growth when other six nitrogen
sources were compared on a 10 mM concentration. There was
almost twofold enhancement in biomass growth under potassium
nitrate compared to calcium nitrate from 10th day onwards
(Fig. 1).
Higher nitrogen concentration i.e. 15 and 20 mM exhibited poor
algal biomass production, which may be due to deleterious effect
of nitrogen at higher concentrations. Li et al. (2008), in their study
on Neochloris also reported that higher nitrate concentration i.e.
15 and 20 mM resulted in cessation of cell growth. Whereas higher
ammonium-N concentration (>500 mg L 1) exhibited detrimental
effect on Chlorella cell growth, which was attributed to increased
internal ammonia concentration in algal cells as reported by
Kallqvist and Svenson (2003).
M. Arumugam et al. / Bioresource Technology 131 (2013) 246–249
249
3.3. Effect of nitrogen on biomass yield
Acknowledgements
Based on the previous experimental result it was evident that
5 and 10 mM concentrations of nitrogen itself was sufficient to
promote algal growth in green alga S. bijugatus. Therefore, a set
of experiment was conducted to evaluate the effect of different
nitrogen sources in lower concentrations viz., 5 and 10 mM to
standardize the appropriate nitrogen source and optimum concentration. The dried biomass recovery was estimated and results
revealed the significant effect of nitrogen supplementation on algal
biomass recovery over control (Fig. 2).
Out of two nitrogen concentrations tested in the experiment,
5 mM concentration exhibited better results on algal growth in
sodium nitrate and ammonium chloride whereas 10 mM concentration was better in urea, calcium nitrate and ammonium nitrate.
Although both concentrations exhibited similar response on algal
growth over all tested nitrogen sources. The results also revealed
that among the tested nitrogen sources, nitrate was preferred form
of nitrogen by the green alga S. bijugatus (Fig. 2). Urea at lower
concentrations also performed equally better as that of potassium
and sodium nitrates.
Nitrogen is among the quantitatively important factors in
growth medium and also a limiting nutrient and the concentration
at which nitrogen inhibits cell growth depended on culture condition and species. In the present study, urea supplementation at lower concentration as nitrogen sources also exhibited favorable effect
on algal growth. Li et al. (2008) also found that 5 mM urea concentration yielded 2.1 g L 1 dry cell weight of green algae Neochloris
oleoabundans at par with 5 mM sodium nitrate (2.5 g L 1) although
lipid productivity was lower than nitrate but comparatively higher
than ammonium as nitrogen source. In our previous study, favorable effects of urea at lower concentration (0.1%) were observed in
growth medium for Scenedesmus in open pond culturing.
This work was funded by Defence R&D Organization, Ministry
of Defence, Government of India through ‘DRDO-Army Biodiesel
Programme’ S&T-09/DAR-71.
4. Conclusion
Among the various nitrogen sources, nitrate was found to be the
favorable source for growth of Scenedesmus at low concentration i.e.
5–10 mM. Considering the findings of present study, lower concentrations of urea (5 and 10 mM) have also performed equally better
as of nitrates. Therefore, urea can also be used as nitrogen supplement for large-scale culturing of algae. Urea is a commercially available nitrogen source being used in agriculture for cultivation of
crops because of its universal availability and affordability. The
result of this experiment can serve as useful reference for future
work on standardization of nitrogen source and concentrations.
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