Bioresource Technology 243 (2017) 163–168

Contents lists available at ScienceDirect

Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Application of agar liquid-gel transition in cultivation and harvesting of

microalgae for biodiesel production
Vinod Kumar a,⇑, Manisha Nanda b, Monu Verma a,c
a
Dept. of Chemistry, Uttaranchal University, Dehradun, India
b
Dept. of Biotechnology, Dolphin (PG) Institute of Biomedical and Natural Sciences, Dehradun, India
c
Dept. of Chemistry, Indian Institute of Technology, Roorkee, India

h i g h l i g h t s g r a p h i c a l a b s t r a c t

Microalgal cells are seeded in the agar

medium in four different
concentrations.

No difference in growth rate and
biochemical contents of agar and
control media.

Microalgae grow within the agar gel
in clusters rather than individual
cells.

Microalgal clusters gravimetrically
settle at the bottom in 2 h.

In this method agar can be reused.

a r t i c l e i n f o a b s t r a c t

Article history: In order to increase microalgal biomass productivity efficient cultivation and harvesting methods are
Received 27 April 2017 needed against the available traditional methods. The present study focuses on the same by harvesting
Received in revised form 13 June 2017 microalgae using agar gel. Agar medium containing bold’s basal medium (BBM) undergoes a thermore-
Accepted 14 June 2017
versible gel transition. As compared to the traditional protocols, this gel is used to cultivate microalgae
Available online 17 June 2017
without even affecting the total productivity. To develop the gel for microalgae cultivation, agar was
boiled in BBM. Then the agar was cooled to 35 °C and microalgae culture was added to it. After seeding
Keywords:
the microalgae the temperature of the agar was further decreased by 10 °C to induce gelation. Instead of
Chlorella sorokiniana
Agar gel
isolated cells microalgae were grown in clusters within the agar gel. Microalgal clusters gravimetrically
Cultivation settle at the bottom within 2 h. In this method agar can be reused.
Harvesting Ó 2017 Elsevier Ltd. All rights reserved.

1. Introduction
Rapidly increasing fossil fuel demands across the globe is
increasing fossil fuel depletion and carbon emissions (Liam et al.,
2016). This has led to the discovery of the alternative fuels.
Microalgae are currently attracting wide interests for alternative
energy production. By photoautotrophic mechanism microalgae
convert CO2 into biomass, lipid (fatty acid) and protein
⇑ Corresponding author.
E-mail address: vinodkdhatwalia@gmail.com (V. Kumar).
(Ravindran et al., 2016). The total lipid content in microalgae varies
from 10 to 70% of dry algae biomass from species to species and
has 20–40 times more productivity than oil crops (Mata et al.,
2010; Li et al., 2011; Spolaore et al., 2006). The most tedious area
of industrial algal biofuel production is harvesting of algal biomass
(Greenwell et al., 2010). It is also a major factor that limits the
commercial use of micro-algae (Olguı’n, 2003. Harvesting con-
tributes to approximately 20–30% of the total cost of biofuel pro-
duction (Mata et al., 2010; Molina et al., 2003; Verma et al.,
2010). Thus, Industrial production of microalgae needs cost

http://dx.doi.org/10.1016/j.biortech.2017.06.080
0960-8524/Ó 2017 Elsevier Ltd. All rights reserved.
164 V. Kumar et al. / Bioresource Technology 243 (2017) 163–168
effective methods to cultivate, harvest, and separate biomass from
growth media (Drexler et al., 2014).
There are number of methods available for harvesting microal-
gae viz., sedimentation, flocculation, flotation, Tris-Acetate-
Phosphate-Pluronic (TAPP) gel, centrifugation and filtration. A
combination of any of these can also be used (Ana et al., 2015;
Estime et al., 2017). Recent studies have suggested various cost
effective methods for the recovery of microalgae against the avail-
able conventional methods. Xu et al. (2011) harvested two
microalgae species using nanoparticles. Vandamme et al. (2012)
have used auto-flocculation, where increasing the pH value up to
11 caused flocculation due to magnesium precipitation. Fayad
et al. (2017) harvested Chlorella vulgaris using electro-coagula
tion-flocculation.
Keeping in view the above mentioned facts, the present study
focuses on the following: the first is to develop and grow microal-
gae culture on agar gel. Next is to characterize parameters for har-
vesting microalgal biomass from the agar gel. Finally, the potential
of the harvested algal biomass to produce lipid and biodiesel was
determined.
2. Materials and methods
2.1. Materials
Chlorella singularis UUIND5 (GenBank accession number:
KY745895) isolated earlier by our group from fresh water was used
in this study. Chemicals used for the preparation of agar and sol-
vents were acquired from Himedia, India. All solvents and reagents
used in this study were of HPLC grade. Microalgae was grown in
sterilized 250 ml conical flasks.
2.2. Preparation of agar media, culture conditions and harvesting
For determining biomass productivity after every 2 days, 7 dif-
ferent sets of each of the four concentrations i.e., 0.2%, 0.3%, 0.5%
and 1% of agar were prepared in Bold’s Basal Medium (BBM).
BBM was prepared according to the composition given by
Guarnieri et al. (2013). After adding the agar to BBM, boiled the
solution for 5 min at 100 °C. 0.5 g L1 sodium bicarbonate as car-
bon source was added to agar and control medium. Further, it
was allowed to cool up to 35–40 °C. Inoculated microalgae to the
above agar solution and decreased the temperature by 10 °C to
induce gelation. All cultures were incubated at photoperiod of
24 h: 0 h (light:dark cycle) with 200 lmol photons m2 s1 (illumi-
nated externally) for a period of 14 days. Temperature was main-
tained at 25 °C. The control was maintained by inoculating
microalgae into BBM alone (i.e., without agar) and incubated under
same conditions. After 14 days of cultivation, the resulting agar-
microalgae matrix was used for biomass and biodiesel production
analysis.
Harvesting was done by gravimetrical and centrifugation meth-
ods. Microalgal clusters gravimetrically settle at the bottom within
2hrs on heating the gel for 5 min at 100 °C to bring it in liquid form
(Fig. 1). Biomass samples were also hravested from agar gel by cen-
trifugation at 1500 rpm for 5 min. The harvested cells were vac-
uum dried at 90 °C overnight. Dry biomass thus obtained was
then measured.
2.3. Determination of cell size, biomass productivity, lipid content and
lipid productivity
Cell diameter (major axis recorded at 50 lm scale bar) of
approximately 100 cells was estimated using ‘Image J 1.49 a’
software.
The Cell Dry Weight (CDW) (mg/L/day) was calculated accord-
ing to the Eq.
ðCDW x  CDW 1 Þ
P¼
tx  t1
where CDWx and CDW1 are the cell dry weight at time tx and t1 (the
time recorded after lag phase). Accumulation of lipid droplets was
monitored by rapidly using Nile red method (Arora et al., 2016)
by staining the cells at regular intervals and visualizing under a flu-
orescent microscope (EVOS-FL, AMG, USA).
Lipids were extracted from fresh microalgal biomass using a
modified method of Bligh and Dyer (1959). Cell disruption was
done using liquid nitrogen. The disrupted cells were treated with
chloroform:methanol (2:1; v/v) and stirred at 180 rpm at room
temperature for 3 h. After incubation, the suspension was cen-
trifuged. The supernatant was then collected in a screw cap glass
tube. Further 0.034% MgCl2 was added to the supernatant followed
by centrifugation at 5000 rpm for 10 min. The lower phase was
then separated in a glass tube. To the lower phase 2 N KCl was
added followed by centrifugation at 5000 rpm for 10 min. Lastly,
lower phase thus obtained was mixed with artificial organic layer
(chloroform:methanol:water; 3:47:48; v/v/v). The mixture was
again centrifuged at 5000 rpm for 5 min. The lower phase so
obtained was vacuum dried to obtain total lipids (Patel et al.,
2015). The weight of oily extract was weighed and the total lipids
obtained were measured gravimetrically and then lipid content (%)
was calculated by the following equations:
Lipid contentð%Þ ¼ total lipidsðgÞ=dry biomassðgÞ
2.4. Determination of biochemical composition
Total protein isolation and estimation was done by the method
given by Slocombe et al. (2013) with some modification.
In this method 5 mg of freeze-dried micro-algae material was
taken. The Sample was vortexed in either 10 ml of 24% (w/v)
TCA. The homogenate was incubated in a water bath at 95 °C, for
15 min, in screw-capped micro-centrifuge tube. After incubation
the sample was allowed to cool to room temperature. Then added
1 ml water to it and further centrifuged 15,000 rpm for 20 min at
4 °C. The supernatant was discarded. The precipitate was resus-
pended in phosphate buffer pH 7.4. Protein quantification followed
the method of Lowry et al. (1951).
Total carbohydrate content in lipid extracted algal biomass
(10 mg) was hydrolyzed by 5 ml of 5% H2SO4 then autoclaved at
121 °C for 1 h. Supernatant obtained was used for total sugar esti-
mation by phenol sulphuric acid method (Dubois et al., 1956) tak-
ing D-glucose as standard.
2.5. Biodiesel production – acid catalyzed transesterification
The total extracted lipids were transesterified into fatty acid
methyl esters (FAMEs) by methanolic sulphuric acid (6%) (Arora
et al., 2016). Treating extracted lipids with methanolic sulphuric
acid (6%) at 90 °C for 1 h. FAMEs were recovered by mixing with
hexane and then washing with distilled water (2:1; v/v). The sus-
pension was then centrifuged at 5000 rpm for 5 min. Hexane con-
taining FAMEs were then transferred into new beaker. The FAMEs
were analyzed using gas chromatography–mass spectroscopy (GC–
MS; Agilent technologies, USA). 1 ll of sample was injected and
GC–MS analysis was done as described by Patel et al. (2015).
2.6. Statistical analysis
The statistical analysis was carried out by analyzing the tripli-
cate (n = 3) results for each culture. These results have been
 V. Kumar et al. / Bioresource Technology 243 (2017) 163–168
Fig. 1. Microalgae cells are added in the agar medium in liquid phase at 35 °C. Then, the temperature is decreased to 25 °C for gel formation and microalgal cultivation. After
the cultivation gel is boiled to 2 min and gel comes in liquid form. Allowing microalgal clusters to gravimetrically settle at the bottom in 2 h. Agar can be used reused for new
cultivation.
reported as mean ± SD. The data was further validated by One-way
ANOVA using Graph Pad Prism software (version 6.0f) with
p < 0.05.
3. Results and discussion
3.1. Biomass and biochemical productivity analysis
Four different concentrations of agar were used to cultivate the
microalgae. To correlate the change in cell morphology and size on
exposure to agar medium, cell diameter (major axis for ellipsoid)
was measured using both FE-SEM and light microscopic images.
No statistical difference in cell size was recorded when grown in
0.2% agar and control. Cell size decreases with increasing concen-
tration of agar in media (Fig. 2A). Amongst these 0.2% agar dis-
played the maximum biomass productivity followed by 0.3, 0.4
and 0.5%. Microalgal biomass concentrations, after 14 days cultiva-
tion period, were found to be 3.2 ± 0.1 g/l, 2.9 ± 0.3 g/l, 2.7 ± 0.2 g/l,
2.3 ± 0.1 g/l with 0.2%, 0.3%, 0.4% and 0.5% agar concentrations
respectively. Dry Cell Mass (DCM) were recorded to be approxi-
mately equal in control 3.3 ± 0.1 g/l and 0.2% agar (3.2 ± 0.1 g/l)
(Fig. 2B). No statistical difference was recorded in the lipid, carbo-
hydrates and protein productivity of microalgae grown in control
and 0.2% agar (Fig. 3A). Estime et al. (2017) have also reported
the growth of microalgae Chlamydomonas reinhardtii with similar
165
biomass productivity, lipid and carbohydrate composition in Tris-
Acetate-Phosphate-Pluronic (TAPP) gel, as obtained from cultiva-
tion in a traditional method.
In this study continuous supply of CO2 was not possible in agar
gel medium, thus 0.5 g L1 sodium bicarbonate as carbon source
was added to both agar and control medium.
Microalgae utilize bicarbonate as the external source of carbon
for photosynthesis. It derives CO2 via the action of carbonic anhy-
drase (Bozzo et al., 2000). NaHCO3 can be used as an alternative
inorganic carbon source (Hsueh et al., 2007) for microalgal cultiva-
tion as it has greater solubility than CO2. Addition of bicarbonate
increases inorganic carbon uptake which increases the productiv-
ity of algal biomass (Keyuri et al., 2016). Bicarbonate salts can
easily be stored and transported to algal facilities as compared to
gaseous CO2 which is costly to store and transport.
FAME profile (Fig. 3B) showed that palmitic acid (C16:0), 7,10-
hexadecadienoic acid methyl ester (C16:2), stearic acid (C18:0),
oleic acid (C18:1) and linoleic acid (C18:2) were present in maxi-
mum proportions. Myristic acid (C14:0), linoleic acid (C18:2) and
C19:2 were present in small amount.
3.2. Characterization of microalgae on the basis of cellular morphology
The effect of agar on the microalgae morphology was deter-
mined by FE-SEM. The cell surfaces of microalgae grown in agar
medium appeared smooth and contained some traces of agar gel.
166 V. Kumar et al. / Bioresource Technology 243 (2017) 163–168

A
6
5

Cell size (µm)

4
3
2
1
0
Control 0.20%
0.30% 0.40%
0.50%

B
3.3
Microalgal biomass

3.2

3.1

3
(g/l)

2.9 CDW

2.8

2.7

2.6
Control 0.20% 0.30% 0.40% 0.50%

Fig. 2. (A) Average diameter of microalgal cells growing in control, 0.2%, 0.3%, 4% and 0.5%. (B) Percentage of microalgal biomass recovery through gravimetrically settling.
The data are mean ± S.D. for triplicate (n = 3) results (p < 0.05).

3.3. Harvesting, energy usage, and recultivation of microalgae using
thermoreversible liquid-gel transition
The main reasons for high harvesting costs of microalgae are its
small size and its concentration in growth media (Danquah et al.,
2009; Molina et al., 2003). One of the major advantages of using
agar medium is the potential for a time reducing and efficient
microalgae harvesting process than the traditional method.
Growth of microalgae cells within the agar gel is economical
method for harvesting the algal biomass. Microalgae cell grow in
clusters in agar medium. Estime et al. (2017) also reported the
growth of microalgae in clusters using Tris-Acetate-Phosphate-
Pluronic (TAPP) medium.
The morphology of the algae cells affects the settling speed dur-
ing the gravimetric separation. Settling velocity of a particle
depends on fluid density, particle density, size, shape and concen-
tration (Liyanage et al., 2016; Jang and Choi, 2007). Grijspeerdt and
Verstraete (1997) reported that spherical shaped clusters settle
faster than anisotropic shape. Smith and Friedrichs (2011) reported
the correlation between the size and the settling velocity. Accord-
ing to Stokes’s law, settling velocity depends upon the size of algae
cell/cluster in medium (Bai et al., 2006).
Microalgae clusters were harvested by gravimetric separation
method in this study. On boiling agar gel at 100 °C for 2 min it
turned into solution phase. This allows the gravimetric separation
of microalgae which settles down at the bottom within 2 h at 40 °C.
The agar used for cultivating microalgae was reused thrice by add-
ing BBM after the harvesting process. Microalgal clusters were also
harvested by centrifugation method. The growth of microalgae in
clusters made the harvesting approximately ten times faster than
centrifugal harvesting of microalgae grown as isolated cells.
Microalgae clusters were separated in 5 min at 1500 rpm as com-
pared to microalgae grown as isolated cells which were separated
in 50 min at 1500 rpm. Commercial harvesting of microalgae using
centrifugation is time consuming and costly. Combination of agar
gel method with centrifugation can significantly reduce process
costs of microalgae (Girma et al., 2003; Chen et al., 2014).
Schlesinger et al. (2012) reported that the combination of floccula
tion–sedimentation with centrifugation can reduce harvesting
costs of microalgae. Comparative study of different harvesting
methods, effectiveness and energy requirements given in Table 1.
Electricity required to increases the temperature of 1 L
of agar medium from 25 °C to 100 °C during the harvesting of
microalgae.
Q ¼ m:cðDTÞ;
Where Q = the heat energy transferred (joule, J), m = the mass of the
liquid being heated (grams, g), c = the specific heat capacity of the
liquid (joule per gram degree Celsius, J/g °C), DT = the change in
temperature of the liquid (degree Celsius, °C)
 V. Kumar et al. / Bioresource Technology 243 (2017) 163–168 167

A
80

Relative (%) of proteins,

carbohydrate and lipids
70

60

50 Carbohydrate
40 Protein
30 Lipid
20

10

0
Control 0.20% 0.30% 0.40% 0.50%

70
C16:2
Composion (%)

60
C19:2
Fay Acid

50
C18:1
40 C16:1
30 C18

20 C16
C14
10

0
Control 0.20%

Fig. 3. (A) Weight percentage of protein, lipid and carbohydrate in microalgal biomass. The data are mean ± S.D. for triplicate (n = 3) results (p < 0.05). (B) Fatty acid methyl
ester (FAME) profile of microalgae control and 0.2 agar.

Table 1
Harvesting methods, effectiveness and energy requirements.

Harvesting process Advantages Disadvantages Energy References

Centrifugation
Gravity sedimentation
Filtration (natural)
Filtration (pressurized)
Polymer flocculation
Electro-flotation
Agar liquid-gel transition
requirement
(kWh)
Rapid, easy, efficient Very high energy input, extra pumping cost 8 Girma et al. (2003)
Low cost, potential for water recycling Slow process 0.1 Shelef et al. (1984)
Less shear stress Slow process, scale up potentially has problems 0.4 Semerjian and Ayoub (2003)
Less shear stress Scale up potentially has problems 0.88 Semerjian and Ayoub (2003)
High efficiency, no damage to cells No water reuse, higher energy input 14 Danquah et al. (2009)
High efficiency High energy input 5.0 Azarian et al. (2007)
Rapid, easy, efficient, potential for Energy input 0.9 (for 100 L) Current method
water recycling, no pumping cost

Q ¼ 1000  4:2  ð100  25  CÞ ity as compared to that of individual cells. This allowed for an
energy efficient gravimetric separation of the algae biomass from
315 KJ ¼ 298:56 BTU ¼ 0:09 kWh the culture medium. Microalgal clusters gravimetrically settle at
the bottom within 2 h. Moreover, experimental data showed that
agar medium did not affect biomass and biochemical contents. In
4. Conclusions this method agar can be reused.

In this study, cultivation and harvesting of microalgae using Acknowledgements

agar gel was investigated. The growth of microalgae in agar gel
was observed as large clusters as compared to control media where This research was supported by a grant from Uttaranchal
it was grown as isolated cells. Clusters increase the settling veloc- University under Project UU-0005-2016.
168 V. Kumar et al. / Bioresource Technology 243 (2017) 163–168
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.biortech.2017.06.
080.
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