Research Article
Open Access
Understanding Urea Assimilation and its
Effect on Lipid Production and Fatty Acid
Composition of Scenedesmus Sp
Saumya Dhup,1* Dheeban C. Kannan2 and Vibha Dhawan2
1Centre for Bioresources and Biotechnology, TERI University, 10, Institutional Area, Vasant Kunj, New Delhi 110070, India
2Biotechnology and Management of Bioresources Division, the Energy and Resources Institute (TERI), India Habitat
Centre, Lodhi Road, New Delhi 110003, India
*Corresponding author: Saumya Dhup, Centre for Bio resources and Biotechnology, TERI University, 10, Institutional Area, Vasant Kunj, New
Delhi 110070, India, Tel : +91- 8860262363; E-mail:
@
Received: 9 May, 2016; Accepted: 24 May, 2016; Published: 28 May, 2016
Citation: Saumya D, Kannan DC, Dhawan V (2016) Understanding Urea Assimilation and its Effect on Lipid Production and Fatty
Acid Composition of Scenedesmus Sp. SOJ Biochem 2(1), 7. DOI:
http://dx.doi.org/10.15226/2376-4589/2/1/00112
Abstract
Much advancement has been made in reducing the cost of largescale
production and harvesting of bio fuels. Nutrients contribute
to one of the major cost components in algal production; hence, it
is essential to understand the importance of minimizing costs at
the nutrient level. In the current study, evaluation of urea as a lowcost
and efficient source of nitrogen was investigated. The effect
of its uptake mechanisms on growth, change in lipid productivity
and fatty acid composition of Scenedesmus sp. was explored. Total
Disappearance Rate (TDR) and total uptake rate were used to study
the efficiency of urea as a nitrogen source. It was found that the
nutrient uptake efficiency of urea was higher than that of nitrate.
In addition, urea showed an increase in biomass productivity and
lipid productivity by 26% and 45%, respectively, when compared
to control. Results also demonstrated degradation of urea into
ammonia upon uptake by algal cells, which is then diffused out of the
cells into the medium. Ammonia present in the medium is converted
into ammonium ion with simultaneous degradation into nitrate.
Eventually, both ammonia and nitrate are absorbed by cells in the
final growth phase. These results suggest that urea can be effectively
used as an alternative nitrogen source.
Keywords: Scenedesmus sp; Urea uptake mechanism; Nutrient
uptake efficiency; Lipid productivity; Fatty acid composition
Introduction
Microalgae are known for their remarkable potential as a
feedstock for biofuels and proteins due to their environmentfriendly
conversion and enhanced yields. However, the
challenges faced while using microalgae at every step of
production add to the cost of the end F product. Thus, realizing
the importance of overcoming these challenges, constant efforts
are being made to develop efficient methods for reducing the
cost. High lipid productivity of dominant, fast-growing algae is a
major prerequisite for the commercial production of microalgal
biodiesel.
Biomass generation by photosynthetic microalgae varies
depending on a number of environmental factors, including nitrogen concentration, temperature and light intensity. Nitrogen
is one of the essential nutrients critical for the cultivation of
algae due to its role in growth and regulation of metabolism [1].
It is well documented that there are three different sources of
nitrogen such as nitrate, ammonia and urea, which are absorbed
by microalgae [2,3]. Urea is known to contain approximately
46.7% nitrogen content, that is, 0.467 Kg of urea–N and hence it
is considered as a low-cost and efficient form of nitrogen when
compared to other sources [4,5]. Under typical water conditions,
urea gives rise to nutrients such as N, P and C (100%) and thus
is supplied as a chemical fertilizer to the culture medium for
algal uptake [4]. Conversely, nitrate (sodium or potassium salt)
provides only 16% of nitrogen, 100% of which is readily available
for the culture due to its efficient water-soluble property [5].
The above-mentioned increase in the availability of nitrogen
source through urea as compared to other sources of nitrogen
is observed due to the presence of two nitrogen atoms per mole
of urea. Urea also proves to be an efficient alternative source to
other nitrate forms as its cost of production and price per kg of
N available is much lower (price per kg of sodium nitrate is 3.1
times that of urea; Table 1).
The mechanism of uptake of each nitrogen source varies,
eventually changing its pathway for metabolism. Previous
studies suggest that the nitrate and ammonium sources have
the simplest uptake mechanisms, as they are taken up as nitrate
and ammonium ion, respectively, from the medium under
sufficient sunlight during photosynthesis; however, much less
is known about urea uptake [6]. In addition, urea, as a nitrogen
Table 1: Comparison of cost per kg of urea and sodium nitrate with
respect to availability of nitrogen (N)23,24
|
Cost (Rs/ kg) |
N available (%) |
Cost of N available (Rs/ kg) |
Urea |
5–9 |
46.7 |
11.56–19.26 |
Sodium nitrate |
31–37 |
16.5 |
187.8–224.2 |
source, cannot be directly assimilated, thus involving a complex
mechanism [7]. Suggested that carbon dioxide and ammonia
released upon urea degradation are eventually absorbed by
microalgae [7]. Conversion of urea, its removal and uptake from
the culture have seldom been reported.
NH2–CO–NH2 + H2O CO2 + 2NH3
NH3 + H2O
NH4++ OH−
Both ammonia-based chemical fertilizers and urea
accumulate ammonia in water in an unionized form. Therefore, in
order to study the effect of an alternative source of nitrogen such
as urea on the algal cell growth and understand the mechanism of
conversion of urea into other nitrogen sources and their uptake,
experiments were performed in a semi-continuous culture
system of Scenedesmus sp. The effect of urea on growth, lipid
productivity and fatty acid composition were studied to evaluate
its efficacy as a major nitrogen source.
Materials and Methods
Microalgal strain and culture conditions
The freshwater microalgae Scenedesmus sp. (TX3) was
procured from Hauz Khas lake park, Hauz Khas, India. The
culture was maintained in Bold's basal medium (BBM) which
consisted of the following components (g/l): KH2PO4 (0.175),
CaCl2.2H2O (0.025), MgSO4 (0.075), K2HPO4 (0.075), NaCl (0.025),
H3BO3 (0.115), NaNO3 (0.25), Na2EDTA (0.001), KOH (6.2), FeSO4
(4.98) and conc. H2SO4 (1 ml) [8]. The pH of the medium was
adjusted to 6.8. The medium was sterilized at 121°C for 15 min.
The axenic cultures were maintained in 250-ml glass jars as stock
and incubated at 25 ± 2°C.
Experimental design
Experiments were designed with four biological replicates
in two similar batches – experimental and control in a semicontinuous
mode. The factors differentiating the batches
were based on alternate sources of nitrogen such as urea in
experimental batch and nitrate in control batch. Duplicates for
each biological replicate were taken for measurement at every
step. Sodium nitrate has already been reported to be a typical
nitrogen source for BBM; therefore, it was considered as control
[8]. A pre-incubation step was carried out in which sodium
nitrate was replaced with urea for acclimatization of the strain
in medium with nutrient urea. This culture was then used as an
inoculum and added into a 1-L Erlenmeyer flask containing a
working volume of 800 ml. An initial inoculum of the strain was added at a proportion of 10% (v/v) into the solution. As shown
in Figure 1, the experiment was conducted for three passages.
The results of the third passage were recorded for analysis. The
inoculum used for the third phase was 50% (v/v) of the previous
passage. Therefore, the culture for the final phase was inoculated
at a proportion of 50% (v/v). The culture flasks were maintained
under continuous agitation at 160 rpm under 16:8 h light–dark
photoperiod conditions. Removal of different sources of nitrogen
from the medium including ammonium, nitrate and urea was
evaluated. The obtained data were analysed statistically using
one-way analysis of variance (ANOVA).
Analytical method
Growth Measurements: Culture growth was observed
by measuring the optical density at 680 nm using a Shimadzu
spectrophotometer. Sampling (3 ml) was done every 4 days for
growth kinetics and in the late exponential phase for lipid and
fatty acid methyl ester (FAME) analyses. The dry cell weight
was determined by filtering 3 ml of the cell culture through a
0.22-mm-pore-size glass fibre filter (oleic acid). The filters with
the biomass were washed with water and dried in a hot air oven
at 100°C. The increase in weight of the dry filter was measured.
Nutrient Analysis: Culture samples (3 ml) were taken at
an interval of 4 days until they were harvested for nutrient
measurements. The samples collected were filtered through
glass fibre filters for analysis. Nutrients were analysed photo
metrically using the standard methods developed by the American
Public Health Association (APHA). The total disappearance
rate (TDR) was calculated by summation of the disappearance
rates of all nitrogen forms in a particular medium. The nutrient
disappearance rate (NDR, mg-Nutrient/L/day) was calculated as
follows:
Where Co and Ct are the nutrient concentrations (mg/ l) at the
beginning and end of the experiment, respectively, and t is the
time interval (days).
Figure 1: Optimization of two different concentrations of urea, graph
represents the biomass growth for sodium nitrate and urea at two different
concentrations.
Figure 2: Periodic fluctuation of growth in semi-continuous culture: L
represents pre-culture period; P1, P2 and P3 are nutrient recharge and
biomass removal intervals.
The percentage of stored nitrogen reserves within an algal
cell was calculated using a CHN elemental analyser (Thermo
Finnigan, San Jose, CA, USA). The nutrient uptake rate (NUR, mg-
Nutrient/L/day) refers to the amount of nutrient taken up by
unit mass of algae and it was calculated on the basis of dry cell
weight as follows:
Where Df and Di are the final and initial dry cell weight in
mg/l, respectively, and Ni and Nf denote the percentage of the
initial and final concentration of nitrogen, respectively, present
in the algal biomass.
Lipid extraction, esterification and fatty acid analysis
The two batches of samples were harvested by continuous
centrifugation (8000 rpm for 5 min). The concentrated algal
samples were frozen overnight at −80°C and freeze-dried under
vacuum. The algal mass was accurately weighed, and lipid
extraction was carried out according to the method adopted by
[9].
Lipid content (%) and lipid productivity (g/L/day) were
calculated as follows:
Lipid content (Clipid) = (wt. of lipid / wt. of sample) × 100
Lipid productivity = (Clipid × DCW) / t
Where, Clipid is the lipid content (%), DCW the dry cell weight
(g/l) and t the time interval (days).
Lipid extracts were converted to methyl esters with
methanolic HCl and hexane. FAMEs were prepared by adding 1
ml of concentrated HCl along with 5 ml methanol to the methyl
esters. The mixture was heated at 80-90°C in a water bath for 30
min. Then, 1 ml of hexane was added to the vial after methylation.
The top hexane layer containing the methyl esters was placed
into gas chromatography (GC) vials for subsequent GC analysis
(Agilent 6890N, Agilent Technologies, Palo Alto, CA, USA) using
a DB-5 column (0.2mm ID, 30m, 0.25mm film thickness; Agilent
Technologies, Palo Alto, CA, USA) equipped with a flame ionization
detector. The temperature programme consisted of an initial
temperature of 2°C and followed by an increase of 50°C min−1 up to 250°C. The peaks were integrated using the Chemstation
and identified by comparing the retention times with the pure
standard (Sigma). The system's performance was checked with
blanks and standard samples prior to analysis. Concentrations
were expressed in mg/ml and then converted to percentage. All
the tests were performed in triplicates.
Results and Discussion
Effect of urea on growth kinetics
BBM, best known for culturing green algae, provides an
optimal environment for a large variety of microalgal species. It is
essential to understand that each algal species has specific culture
conditions, and thus its optimization is very crucial. As nutrients
are one of the major cost components in algal production, they
have to be optimized efficiently for a higher productivity. An
optimal nutrient condition is attributed to the combined effect
of both concentration and source of nutrient [10]. Conducted a
study on S. bijugata and reported the significance of selecting a
suitable nitrogen source and concentration for rapid growth [10].
The authors also stated that the association between the source
of nitrogen and concentration is significant. In this study, an
alternative source of nitrogen was considered because nitrogen,
being one of the major basic elements, often limits growth and
accumulation of lipids. The effect of urea on growth and lipid
productivity of Scenedesmus sp. was investigated. The study was
carried out to evaluate the use of a cost-efficient alternative of
nitrogen in the medium. The amount of urea and sodium nitrate
added in the medium is indicated in Table 2. The concentration
of urea in the medium was optimized previously for growth
and lipid productivity. In an earlier study, two different molar
concentrations of urea were tested: 1.45 mM (equiatomic to
sodium nitrate) and 2.5 mM (equivalent weight basis: 17.6 mM). As the urea concentration (2.5 mM) in the medium was higher
than the equiatomic concentration of sodium nitrate, it resulted
in higher biomass growth (Figure 1) and lipid productivity (12.4
mg/l/day). The lipid productivity with urea concentration of
1.45 mM and sodium nitrate was 7.3 mg/l/day and 8.8 mg/l/day
respectively. It can be inferred that an increased concentration
of urea provides an increased amount of nitrogen which leads to
higher growth and productivity [10]. Observed that an optimal
higher concentration of urea results in better biomass growth
[10]. Therefore, the concentration of nitrogen available after
conversion of urea is observed to be optimum, thereby enhancing
lipid productivity. Urea is also known to enhance growth rate as
it acts as a complementary source of organic carbon. Thus, the
combined effect of nitrogen and carbon simultaneously leads to an increase in the growth of Scenedesmus sp.
Table 2: Growth parameters and productivity of Scenedesmus sp.
N Source |
Urea |
Sodium Nitrate |
Amount (mM) |
2.5 |
2.9 |
Biomass productivity (g/l/day) |
0.048 |
0.038 |
Specific growth rate (day-1) |
0.027 |
0.018 |
Lipid content (%) |
8.7 ± 0.4 |
7.6 ± 0.6 |
Lipid productivity (mg/l/day) |
4.176 |
2.88 |
Figure 3: Disappearance of nitrogen forms from the medium containing urea (experimental) versus sodium nitrate (control).
Figures A, B and C show concentrations of urea, ammonium and nitrate at disappearance, respectively
E, F and G show the disappearance rate of urea, ammonium and nitrate, respectively.
The concentration of the nutrient in the medium varies
according to the nutrient source used. A change in the nutrient
source might lead to an increased concentration in the
medium above the tolerance level of the algal cells; such high
concentrations are toxic and inhibit cell growth [11]. But an
appropriate amount of nitrogen concentration is known to
increase the microalgal growth rate [12]. In this study, it was
observed that the difference in the available forms of the nitrogen
for intake leads to a change in the nutrient concentration in the
medium and thus causes variations in the assimilation rate.
These alterations subsequently affect the growth rate of the algal
species.
Growth curves for Scenedesmus sp. grown in two nitrogen
sources are presented in Figure 2. In the medium containing urea,
a higher exponential increase in the growth curve is observed with
a specific growth rate of 0.06 day−1. However, in the medium with
sodium nitrate, specific growth rate was 0.05 day−1. Therefore, it
is evident from Table 2 that the specific growth rate of this species
is significantly affected by the nitrogen source used (P < 0.05), as
the growth rate with urea in the medium is 1.4 times higher than
with nitrate. These results suggest that urea is more preferred
as a nitrogen source in the medium [13]. Reported a similar
growth pattern in the Chlorella sp. with urea and sodium nitrate
as the two nitrogen sources and their specific growth rates being
0.071 day−1 and 0.047 day−1, respectively [10,13]. In their study
on Scenedesmus sp. also reported that the amount of biomass
obtained with urea in the medium was equivalent to that with nitrate [10]. Previous reports on phytoplankton species suggest
that urea is degraded into ammonia inside the cells [13]. In this
study, ammonia, a product of urea degradation in the medium
with urea, is being preferred over nitrate in the batch having
sodium nitrate as the nitrogen source [14]. Observed a similar
result wherein some microalgal species prefer ammonium ion
over nitrate [14,15]. Reported that Scendesmus sp. grew faster in
the stable phase of growth when the ammonium form of nitrogen
was provided in the medium [15]. They stated that in accordance
with the specific growth rate, the preference of nitrogen source
is in the order of ammonium < urea < nitrate. Ammonia is
considered as a preferred source for intake by the cells because
it is directly absorbed into the cell and converted into amino
acids which are eventually used for metabolic activities. When
nitrate as a nitrogen source is absorbed by the cells, it reaches
the cytosol where it gets reduced to nitrite by nitrate reductase.
Nitrite produced is transported immediately to the chloroplast
where it is further reduced to ammonium ion by nitrite reductase
[16]. Therefore, the algal cells prefer a source which is directly
assimilated and used for metabolic activities. The results suggest
that ammonium ion is a much preferred nitrogen source for
Scenedesmus sp. Therefore, the production of ammonium ions
during urea degradation makes this nutrient more suitable for
algal growth than sodium nitrate.
The above-mentioned results show a variation in the growth
characteristics based on two different nitrogen sources, and a
change in the biomass concentration is also expected. As shown
in Table 2, a difference between the biomass productivity of
urea and sodium nitrate was observed. The medium with urea
Table 3: Nutrient disappearance and uptake efficiency for Scenedesmus sp. in medium with urea and sodium nitrate.
|
Total Disappearance Rate (mg/l/day)
|
Total Uptake Rate (mg/l/day)
|
Total Disappearance Rate
|
Nutrient Uptake Rate
|
Nutrient Uptake Efficiency (%) |
Days |
0–4 |
4–9 |
9–17 |
0–4 |
4–9 |
9–17 |
0–17 |
Urea |
1.22 |
1.11 |
1.58 |
1.11 |
1.1 |
1.54 |
1.3636 |
1.3117 |
96.00% |
Sodium Nitrate |
1.26 |
1.47 |
0.96 |
1.12 |
1.45 |
0.9 |
1.1845 |
1.1182 |
94.00% |
showed an increase in biomass productivity by 26% in 17
days with a biomass concentration of 0.832 g/l. Similar results
with an enhanced biomass dry weight were reported with
urea in Scenedesmus sp [17]. They reported that Scendesmus
sp. performed better in a medium with urea, resulting in a
significantly higher biomass concentration and lipid productivity
when compared to nitrate. It was also observed that the
percentage of nitrogen (calculated using an elemental analyser;
Thermo Finnigan, San Jose, CA, USA) inside the cells was higher in
the medium with urea (8.179% in medium with urea and 7.219%
with nitrate). This suggests that using urea as the nutrient source
leads to a higher nitrogen uptake efficiency, thereby increasing
the amount of nitrogen in vivo and also resulting higher biomass
concentration.
Uptake mechanism of urea and sodium nitrate
This study focuses on urea as an alternative nitrogen
source and its impact on growth, lipid production and fatty acid
composition. The impact is observed largely due to the difference
in the uptake mechanisms of the nitrogen sources such as urea
and sodium nitrate. Studies related to the uptake mechanism
of urea by algal species are limited. In 1988, Price and Harrison
conducted a study proposing a model of urea uptake and its
assimilation by a diatom, Thalassiosira pseudonana in nitratesufficient
and nitrate-starved cells [13]. They have explained the
diffusion of urea through an ammonia conversion route using
radioactive isotopes. They proposed that upon cellular uptake,
urea gets degraded into ammonia which is released. The ammonia
reacts with water and is converted into ammonium ion, thereby
releasing hydroxide ion in the medium. This mechanism can be
further explored in other species in order to assess and confirm
the consumption of urea by other phytoplankton communities. In
this study, a similar trend was observed using different analytical
methods for uptake and assimilation. It has been previously
studied that ammonia-based chemical fertilizers such as urea
accumulate a unionized form of ammonia in the medium. The
unionized form of ammonia is taken up biologically by the algal
culture during photosynthesis in sufficient light intensity before
it gets oxidized to nitrate, thus maintaining the culture alkalinity
[18]. But it was also observed that a small quantity of ammonia
which was diffused out from the cells was converted into
ammonium ion and nitrate simultaneously [18]. The nitrate and
ammonium ion were eventually reabsorbed by the cells. Thus,
based on this study, it can be assumed that under light and dark conditions both nitrogen sources, ammonium ion and nitrate,
respectively, are simultaneously available in a medium with urea.
The rate of urea disappearance from the medium after 4 days
averaged 1.14 mg/l/day. But the maximum disappearance rate
of urea from the medium was recorded as 1.52 mg/l/day for an
interval of 4-9 days. With the disappearance of urea, there was an
exponential increase in the reappearance of ammonium ion in the
medium (Figure. 3E and F). This suggests that urea absorbed by
the cells was degraded in vivo into ammonia. Ammonia was then
diffused out into the medium. Degradation of urea inside the cell
was carried out in the presence of urease enzyme present in most
algal cells [19]. The ammonia diffused out was eventually taken
up as ammonium ion, as during the final interval of cell growth,
the rate of disappearance of ammonium ion from the medium
was observed to be 1.13 mg/l/day (Figure 3F). While ammonia
disappeared during the final phase, simultaneous reappearance
of nitrate in the medium was observed at a rate of 0.1 mg/l/day
(Figure 3G).
When considering the rate of uptake, the overall
disappearance of nitrogen source from the medium was taken
into account over the period. The uptake rate in the medium
with urea for an interval of 0-4 days was observed to be more
than that for a period of 4-9 days (1.11 mg/l/day and 1.10 mg/l/
day, respectively). The TDR was also observed to be higher for
the interval of 0-4 days (Table 3). In the final phase of growth,
the maximum disappearance rate was observed due to the
simultaneous uptake of both ammonium ion and nitrate nitrogen
from the medium with urea. Thus, an increase in the rate of
uptake of the nitrogen source was observed. In the medium
with nitrate, the maximum disappearance and uptake rates was
observed for an interval from 4 to 9 days. It was also observed
that the nutrient uptake efficiency of medium with urea was 96%,
while that of medium with sodium nitrate was 94% (Table 3).
Thus, the results indicated an enhanced uptake of nutrient from
the medium with urea in comparison to the medium with sodium
nitrate. It can be concluded that urea is a better nitrogen source
as it is being efficiently absorbed and utilized for metabolic
functions leading to enhanced growth rate and lipid productivity.
Effect of urea on lipid productivity and fatty acid
composition
Biomass productivity is considered as an imperative
parameter for determining the effect of a condition. Lipid content
is another parameter which is essential for the evaluation of the
overall performance of the strain. In the current study, the lipid
content and biomass were considered as the twin effects for
evaluating the overall lipid productivity under different nutrient sources.
Table 4: Fatty acid profile of Scenedesmus sp. in medium with urea and
sodium nitrate.
|
Urea (%) |
Sodium nitrate (%) |
C14 |
0.36 |
0.76 |
C14:1 |
0.13 |
0.39 |
C15 |
0.41 |
0.44 |
C15:1 |
1.86 |
2.78 |
C16 |
5.40 |
9.00 |
C16:1 |
3.49 |
17.70 |
C17 |
0.73 |
0.75 |
C17:1 |
1.58 |
2.51 |
C18 |
0.00 |
0.00 |
C18:1 |
14.16 |
8.99 |
C18:2 TRANS |
1.05 |
5.44 |
C18:3 |
61.04 |
37.84 |
C18:3 (6) |
2.55 |
4.13 |
C18:3 (3) |
2.79 |
3.97 |
C20 |
2.60 |
0.96 |
C22:2 |
0.58 |
2.03 |
C20:2 |
0.60 |
0.80 |
C21:1 |
0.52 |
0.71 |
C24:1 |
0.16 |
0.40 |
SFA |
9.50 |
11.90 |
MUFA |
21.90 |
33.47 |
PUFA |
68.60 |
54.23 |
We proceeded to analyse the total lipid content of the strain
in both media (experimental and control) by the end of the cycle
where minimal nutrients were left in the medium. Harvesting in
the late exponential phase leads to a decrease in the nitrogen and
phosphorous concentrations but an increase in the lipid content
[12]. Table 2 shows the total lipid content and productivity of
the two media. It was observed that the total lipid content of the
batch with urea (8.7%) was higher than that of sodium nitrate
(7.6). Due to a 14% increase in the lipid content of the strain in the
medium with urea, a difference between the lipid productivity of
the two media was observed. The lipid productivity in the batch
with urea was found to be 1.5 times higher than that with sodium
nitrate. It was inferred that the lipid content increases with an
increase in biomass productivity. Hence, the lipid productivity of
the cells in the batch with urea increased. Similar results were
also indicated in previous studies carried out by [20]. Thus,
the results listed in Table 2 shows that the lipid content and
productivity were significantly dependent on the nitrogen source
provided (P > 0.05). These results indicate that nitrogen in the
medium with urea should be provided in sufficient amount for
the cells to grow and store lipids efficiently. More importantly,
in the medium with urea, the presence of ammonia makes it
possible for the cells to absorb it as a direct nitrogen source for assimilation. Direct assimilation of ammonia facilitates rapid cell
metabolism, thereby increasing the lipid productivity.
Fatty acid composition is another key parameter that
determines the potential of an algal strain as a biodiesel candidate.
In this particular study, fatty acid analysis of the species in the two
different media gave interesting insights with a prominent shift in
the fatty acid composition. The major saturated, monosaturated
and polyunsaturated fatty acids were found to be C16:0, C16:1
and C18:1 and C18:3, respectively. According to the previous
studies conducted on Scenedesmus sp., stearic acid was absent in
both media [21]. As shown in Table 4, there is a significant visible
shift of the fatty acid percentage from C16:0 and C16:1 to C18:1
and C18:3 in the medium with urea; the percentage of the latter
increased by 5 and 23, respectively [22] (Table 4). Observed a
similar shift from palmitic acid to oleic acid in other feedstock
soybean, but stated that such an increase in oleic acid was not due
to environment influences [22]. But, herein, the shift to oleic acid
with a significant increase in the level of an essential omega-3
fatty acid (alpha-linolenic acid) was observed due to the change
in nitrogen source. An increase in oleic acid makes the strain
desirable as a potential source for biodiesel, as monounsaturated
fatty acids are most suitable when considering cloud point and
oxidative stability. In addition, an increased amount of saturated
fatty acids poses problems with gelling of fuel in cold weather.
Due to the presence of a high amount of omega-3 fatty acids,
this strain is an important source of essential nutrients and
also presents health benefits apart from biodiesel production;
however, it negatively influences the oxidative stability.
Therefore, the percentage of essential fatty acids of the total algal
lipids in urea was 94% (significant percentages of α-linolenic
acid and oleic acid were 61% and 14%, respectively), whereas
in the medium with sodium nitrate, it was 74%. Hence, it can be
concluded that with a change in the nitrogen source from nitrate
to urea, there is an increase in lipid productivity with a positive
shift in the fatty acid composition from non-essential to essential
fatty acids.
Conclusion
The growth and lipid productivity of green algae Scenedesmus
sp. was tested in BBM medium containing a low-cost nitrogen
source. Our findings shed light on the use of urea as a nitrogen
source, as it proved to be more efficient in terms of growth,
lipid productivity and fatty acid composition when compared to
sodium nitrate. With regard to the nutrient uptake mechanism
and fate of urea in Scenedesmus sp., it was observed that the
results are in compliance with previous studies conducted on
other phytoplankton species, with a reasonably higher nutrient
uptake efficiency than nitrate. Although sodium nitrate is a
readily available source of nitrate ion, the ammonium ion
obtained after urea degradation is a much-preferred source
of nitrogen for uptake. In this study, the presence of nitrate as
an additional nitrogen source along with ammonium ion was
observed. The nitrogen obtained upon degradation of ammonia
was also observed to be absorbed by the cells. Hence, urea can
be effectively used as an alternative economical substitute of nitrogen for growth and lipid production of Scenedesmus sp. at
a large scale.
Highlights
• Urea is considered as a low-cost alternative source of
nitrogen.
• A higher uptake efficiency of urea (two nitrogen sources
including ammonium ion and nitrate) is observed when
compared to sodium nitrate.
• A change in the fatty acid composition is observed upon
urea uptake.
Acknowledgement
We sincerely thank the Department of Biotechnology,
Government of India for funding the research. We also thank
the Director General, TERI, for providing the necessary
infrastructural services and facilities for this study as well as Late
Dr. Prem Dureja, Ms V Devi and Ms Swati Patel for their valuable
guidance during the research.
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- Market price. http://www.needsinfo.com/globalinquirymagazine/market_price.htm
- Chemical prices remain steady. Buisness standards. http://www.business-standard.com/article/markets/chemical-prices-remain-steady-111020800160_1.html