Algal Research 16 (2016) 216223

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Algal Research

journal homepage: www.elsevier.com/locate/algal

Performance evaluation of microalgae for concomitant wastewater

bioremediation, CO2 bioxation and lipid biosynthesis for
biodiesel application
Manoranjan Nayak, Ankush Karemore, Ramkrishna Sen
Department of Biotechnology, Indian Institute of Technology Kharagpur, West Bengal 721302, India

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

Article history: This study was directed towards evaluating the potential of microalgae for simultaneous wastewater treatment,
Received 3 January 2016 CO2 bioxation and lipid biosynthesis for biofuel application. The cultivation potential of various microalgal
Received in revised form 17 February 2016 species in domestic wastewater (DWW) was studied in shake-asks. The microalga, Scenedesmus sp. showed
Accepted 12 March 2016
superior results in terms of the maximum specic growth rate of 0.44 d1, biomass yield of 0.43 g L1, biomass
Available online xxxx
productivity of 61.4 mg L1 d1 and total lipid content of 23.1%. Subsequently, the performance evaluation
Keywords:
of Scenedesmus sp. with respect to biomass growth, lipid accumulation, CO2 bioxation rate and nutrient uptake
Microalgal biomass was carried out at different CO2 concentrations in a photobioreactor. The culture supplemented with 2.5% (v/v)
Domestic wastewater CO2 was found most suitable and resulted in highest biomass productivity, total lipid content, lipid productivity,
Nutrient removal and CO2 consumption rate of 196 mg L1 d1, 33.3%, 65.17 mg L1 d1 and 368 mg L1 d1, respectively. The
CO2 xation microalga could bioremediate ammonium, nitrate, phosphate and chemical oxygen demand (COD) efciently
Lipid from the wastewater to the extent of 7098%. The FAME composition of the microalgal lipid was found encour-
Fatty acid aging for biodiesel application as saturated: unsaturated fatty acid ratio was favorable to about 1.2:1. The study
indicates that Scenedesmus sp. can efciently utilize DWW for its growth instead of using synthetic culture
medium and can produce a signicant amount of biomass at 2.5% CO2 for biofuel application.
2016 Elsevier B.V. All rights reserved.

1. Introduction environmental complications [5]. This generated efuent has created

The human society is facing many environmental challenges like en-
ergy crisis, water pollution, air pollution, global warming, etc. [1]. The
energy crisis is considered as one of the most critical problems in this
new millennium. The depletion of petroleum oil reserves coupled
with the increasing global energy demand made it necessary to develop
alternative energy sources that should be sustainable, renewable and
eco-friendly in nature [1,2]. Also, the continual usage of fossil fuels
emits large amounts of greenhouse gasses (GHGs) mainly CO2 into the
atmosphere, thereby causing global warming and climatic changes [3].
Reported literature suggests that because of the excessive utilization
of fossil fuels, the concentration of atmospheric CO2 is on the continual
rise and has reached an alarming level of 400 ppm [4]. On the other
hand, due to rapidly increasing human population in the cities, subur-
ban and rural areas, large amounts of fresh water have been extensively
used, and their consequent released of domestic wastewater (DWW)
discharge causes severe pollution of surface and ground water [2,5].
The DWW contains sufcient amount of carbon, nitrogen, phosphorus
and other minerals, if left untreated, causes eutrophication and other
Corresponding author.
E-mail addresses: rksen@hijli.iitkgp.ernet.in, rksen@yahoo.com (R. Sen).
immense pressure on wastewater treatment facilities. So there is an ur-
gent need to reduce greenhouse gas emission and wastewater disposal.
Thus, the principal concern is to nd a greener solution for cleaner
water and air [6]. Various efforts are undertaken for wastewater treat-
ment and CO2 mitigation through different physical, chemical and
biological approaches [7]. However, these treatment systems are not
found to be cost-effective for wastewater remediation and CO2 seques-
tration process [5]. Despite several current wastewater treatment and
CO2 mitigation technologies, biological treatment process remains the
very efcient approach because it requires no additional energy [2].
Among all, the microalgae-based biological method is drawing in-
creasing interest, as the DWW contains sufcient amounts of various
nutrients, which can be used as a cheap substrate for the algal cultiva-
tion along with DWW treatment [8]. Also, the microalgae have the capa-
bility to use CO2 as the carbon source in the process of photosynthesis,
and thereby helps sequester greater amounts of CO2 [9,10]. Studies
show that approximately 1.8 kg of CO2 can be xed through photosyn-
thesis for the production of 1 kg biomass of microalgae [3]. Moreover,
microalgae appear to be one of the most capable renewable energy
feedstocks for biodiesel production due to their outstanding advantages
over other traditional energy crops of having faster growth rates, high
lipid content, high photosynthetic efciency, and require much less

http://dx.doi.org/10.1016/j.algal.2016.03.020
2211-9264/ 2016 Elsevier B.V. All rights reserved.
 M. Nayak et al. / Algal Research 16 (2016) 216223
land area for the cultivation [3,11]. Also, microalgae could fulll multiple
purposes where the algal biomass could be converted into many useful
products including food, feeds and biofuels [12].
However, when resource consumption is taken into consideration in
the life cycle analysis of microalgal biofuel, the environmental impacts
of microalgal biofuel may not be as favorable as anticipated and the
net energy obtained from the biofuel production process could even
be negative [13]. In this assessment, nutrients such as nitrogen, phos-
phorous, trace elements and a large volume of water were required,
and thus found to be the most critical resources for commercial scale
production of microalgal biofuels [11,14]. According to Yang et al.'s anal-
ysis, 3.7-ton water, 330 g nitrogen, and 710 g phosphorus are necessary
to produce 1 kg biodiesel without water reuse [14].
This enormous demand for nutrients and freshwater resources
for microalgae biomass production can be solved through coupling
microalgae cultivation with DWW treatment [1114]. If certain
microalgae strains could grow in DWW, the organic and inorganic nutri-
ents present in wastewater could be utilized for microalgae cultivation
in a cost effective manner. Also, DWW is readily available in large quan-
tities which can be one of the most feasible alternatives for large-scale
production of microalgal biofuel. Thus, this microalga-based bioremedi-
ation can be an economical and eco-friendly approach for DWW treat-
ment along with microalgal biomass production [5]. The CO2 can be
used as carbon source for biomass growth and can simultaneously re-
duce CO2 emission to the atmosphere. Thus, coupling DWW with CO2
for microalgal cultivation might give collective benets for biomass pro-
duction as biodiesel feedstocks, nutrient removal and CO2 sequestra-
tion, which may be the key to solving above problems [15].
Therefore, this work aims to develop a sustainable and eco-friendly
strategy to the current energy and environmental challenges. In the
present study, the feasibility of four different green microalgae in
DWW was studied for biomass and lipid production. Subsequently,
the performance evaluation of the most appropriate microalga was car-
ried out in various CO2 concentrations for biomass production, CO2
bioxation, wastewater remediation and lipid production for biodiesel
application.
Fig. 1. Shows microscopic view of (1) Chlorella vulgaris, (2) Chlorella minutissima, (3) Scenedesmus sp., (4) Chlorococcum sp. (scale bar: Figs. 14 = 5 m).
217
2. Materials and methods
2.1. Wastewater collection and analysis
The domestic wastewater (DWW) used in the study was collected
from wastewater sewage pump, IIT Kharagpur campus (22.3302N,
87.3237E). The wastewater sample was collected in the month of
March and immediately stored at 4 C until use. The wastewater was l-
tered through a Whatman No.1 lter paper to remove large solids and
debris and subsequently the wastewater was sterilized by autoclaving
at 121 C for 15 min before being used as the culture medium.
The DWW was characterized by different methods described in
subsequent section, the major nutrient concentrations in DWW were
as follows: NNH4 = 38.6 1.5 mg L1, NNO3 = 17.1
0.5 mg L 1, NNO2 = 0.34 0.02 mg L1, PPO4 = 9.24
0.3 mg L 1, BOD: 39.3 2.1 mg L 1 and COD: 142.2 5.2 mg L1.
The pH of the DWW was 8.1 0.05.
2.2. Microalgae strains, culture conditions and inoculum preparation
In this study, four microalgae of Chlorophyceae were employed for
their ability to grow in wastewater and as possible candidates for sus-
tainable energy production (Fig. 1). The microalgae strains were pro-
cured from Indian Agricultural Research Institute, New Delhi, and are
of freshwater origin namely Chlorella vulgaris, Chlorella minutissima,
Scenedesmus sp. and Chlorococcum sp.
The microalgae cultures were separately inoculated in a 500 mL
Erlenmeyer ask containing 250 mL modied Bold's Basal Medium
(BBM) at 10% concentration (Vinoculum/Vmedia) [16]. The initial pH of
the culture medium (BBM) was adjusted to 6.66.8. The microalgal
cells were cultivated at 25 2 C under constant illumination of
60 mol m2 s1 provided by cool white uorescent light. The light in-
tensity was measured by a Lux-meter (HTC, India; Model no. 102). For
inoculation, the log phase grown culture was taken and harvested
through centrifugation at 3000 rpm for 15 min at 4 C. Five milliliters
of microalgae was used as initial inoculums to reach the cell density of
218 M. Nayak et al. / Algal Research 16 (2016) 216223
0.04 g L 1 for wastewater experiments. The microalgae were hand
shaken thrice per day to prevent microalgal settling. All the experiments
were carried out in triplicate, and the average values were reported in
the gures. Microalgal inoculum was taken during the log phase culture
algal cells for inoculation and was harvested.
2.3. Experimental setup
The experiments were carried out in two phases (Fig. 2). In the rst
phase, selection of suitable microalgae species was carried out in shake-
ask using DWW on the basis of highest biomass and lipid productivity.
In the second phase, the selected microalga was studied in a bubble col-
umn photobioreactor using DWW supplement with different concen-
trations of CO2.
The microalgae were cultivated in 250 mL Erlenmeyer asks con-
taining 100 mL of wastewater. All experiments were operated at
25 2 C, with the light intensity of 60 mol m2 s1 under a photope-
riod of 14:10 h (light/dark) using white uorescent tubes (Philips,
36 W). The light intensity was measured at the surface of the ask
using the lux meter (HTC, India; Model no. 102).
The selected microalga was cultured in the bubble column
photobioreactor (30 cm length, 8 cm diameter) with working volume
of 0.5 L. The continuous aeration with different concentrations of CO2
(0.03%, 1%, 2.5%, 5% and 10%) was fed into the culture medium having
DWW at the rate of 0.5 vvm, volume gas per volume medium per min-
ute. All experiments were operated in a culture room at above-
described condition (Section 2.2) for 7 days. Sampling was done at the
interval of 24 h during the microalgal cultivation to analyze biomass
growth and other biochemical parameters. All the batch experiments
were carried out in triplicate.
2.4. Microalgae growth and CO2 consumption rate
The microalgae growth was measured every 24 h, and the dry cell
weight of biomass concentration (g L1) was determined by measuring
the absorbance at 750 nm (OD750) using a UVvis spectrophotometer
(Agilent Cary 60). Microalgae culture with high biomass density was di-
luted by appropriate ratios to ensure that absorbance values were in the
Fig. 2. Flow diagram of experimental processes involved in this present study of
microalgae cultivation in domestic wastewater.
range of 0.11.0. Microalgal cells at different concentrations were col-
lected by centrifugation (3500 rpm for 10 min) at 4 C and the algal
cell pellets were washed twice with distilled water, and subjected to
oven dry at 65 C for 24 h for estimation of dry cell weight (DCW)
[17]. Further, a calibration curve of OD750 vs. biomass (g L1) was
established for all strains to convert OD750 values to biomass concentra-
tion (DCW) in g L1 and the correlation equations shown in the follow-
ing equations (Eqs. 14) [17].
   
Chlorellavulgaris; biomass g L1 O:D750  0:442 R2 0:991 1
   
Chlorellaminutissima; biomass g L1 O:D750  0:43 R2 0:997 2
   
Chlorococcum sp:; biomass g L1 O:D750  0:568 R2 0:993 3
   
Scenedesmus sp:; biomass g L1 O:D750  0:425 R2 0:994 : 4
The specic growth rate (, d1), of microalgae dened as the in-
crease in the biomass concentration per unit time, was calculated
using the following equation:
ln W1 =W0 =t 5
where, W1 and W0 are the biomass concentration (g L1) at the end and
the beginning of a batch run, respectively. t is the cultivation time in
days [18].
The doubling time (TD) is the time required to achieve a doubling of
the number of viable cells and was calculated based on the specic
growth rate by using the following equation [19]:
TD ln 2= 0:6931=: 6
The biomass productivity during the incubation, PB (mg L1 d1)
was calculated by using the following equation:
PB CB  1000=t: 7
Where CB (g L 1) is the biomass concentration at the end of the
batch run and t is the duration of the cultivation.
The CO2 xation efciency was calculated in terms of CO2 consumed
by the microalga. Moreover, the carbon dioxide consumption rate (PCO2,
mg l 1 d1) was derived by using the following equation: [16]
PCO2 1:88  PB 8
where PB denotes the biomass productivity (mg l1 d1).
2.5. Nutrient removal analysis
To check the nutrient removal efciencies of the microalga,
Scenedesmus sp., ammonium NH4, nitrate NO3, phosphate PO4
and COD were measured on the daily basis to study the potential of
microalgae for secondary as well as tertiary stage of wastewater treat-
ment. The microalgal culture was collected every 24 h and centrifuged
at 12,000 rpm for 15 min. Then the supernatants were ltered using a
0.45 m nylon membrane lter. Then, the ltrates were appropriately
diluted and analyzed for ammonium, nitrate, phosphate and chemical
oxygen demand (COD) concentration.
2.5.1. Ammonium
Ammonium content was quantied by using Nessler's reagent as re-
ported by Herbert et al. [20]. In this colorimetric method, 0.3 ml of
Nessler's reagent was added to 1 ml of wastewater and the resulting
orange-red color was measured in the UV/Vis spectrophotometer at
 M. Nayak et al. / Algal Research 16 (2016) 216223 219

420 nm. The standard curve was prepared from NH4Cl to calculated am-
monium content.
2.5.2. Nitrogen
NO 3 was determined according to the modied method reported by
Collos et al., [21]. Nitrate content was determined at a wavelength of
220 nm using the UVvis spectrophotometer. Sodium nitrate at a con-
centration range of 030 mg L1 was used as the standard.
2.5.3. Nitrite
Nitrite was measured spectrophotometrically at 540 nm by follow-
ing the method of Lowe and Evans [22]. Nitrite was determined by
adding 0.5 ml of wastewater sample, 1 ml of sulfanilamide solution
(1 g of sulfanilamide in 100 ml of 1:4 (HCl:water)) and1 ml of -(N-
1)-naphthyl ethylene diamine dihydrochloride (NEDD) (0.2% (w/v) in
millipore water). Finally, the absorbance of the pink color was measured
at 540 nm. The standard curve was prepared from NaNO2 and expressed
as mg L1.
2.5.4. Phosphorus
PO4 was determined by stannous chloride method [23]. In detail, to
water sample (1 mL), ammonium molybdate reagent (80 L) and stan-
nous chloride reagent (10 L) were added. Then the sample was kept
for1012 min of incubation and the absorbance was recorded at
690 nm (OD690) using the spectrophotometer. The concentration of
phosphorus PO4 in the sample was measured using a standard
graph. Authentic mono-potassium phosphate at a concentration of 0
10 mg L1 was used as the standard.
2.5.5. Chemical oxygen demand
COD of the waste samples was measured by closed reux titrimetric
method following the procedure 5220 C, American public health associ-
ation (APHA) [24].
The nutrient removal percentage was calculated using the following
equation:
Nutrient removal% 100  Co Ci =Co :
Where, Co and Ci are the mean values of nutrient concentration at
initial (to) and nal (ti) time respectively.
The rate of nutrient removal was calculated using the following
equation:
 
1 ame ionization detector (FID) and a split injector was used. A BPX 70
Removal rate mg d L1 Co Ci =t:
Where, Co and Ci are the mean values of nutrient concentration at
initial (to) and nal (ti) time respectively. t is the cultivation time in
days.
2.6. Analysis of total lipids
At the end of cultivation, the biomass was harvested by means of
centrifugation at 3500 rpm for 10 min at 4 C. The centrifuged biomass
was then washed with distilled water and then freeze-dried under
80 C. The dried microalgae biomass was then kept in an empty con-
tainer for total lipids and fatty acid methyl ester (FAME) analysis.
A modied method of Bligh and Dyer [25] was applied for extraction
of total lipid from microalgal biomass. The algal lipids were extracted
with chloroform and methanol (2:1, v/v), and separated into two layers
of chloroform and aqueous methanol layer, by the addition of methanol
and water to give a nal solvent ratio of 2:2:1 of chloroform: methanol:
water. The chloroform layer containing lipid was washed with 1% NaCl
solution. It was collected and evaporated to dry using a rotary evapora-
tor. The lipid content was determined gravimetrically. Each analysis was
carried out in triplicate, and the values were reported as mean SD or
with error bars.
The lipid productivity PL (mg L1 d1) was calculated by using the
following equation:
PL CL =t: 11
Where CL (mg L1) is the concentration of lipid at the end of the
batch run and t is the duration of the cultivation.
The lipid yield of microalgae YL (%) was calculated using the follow-
ing equation:
YL 100  WL =WDA : 12
Where WL is the weight of the total lipid and WDA is the weight of
the dry algae biomass.
2.7. Analysis of fatty acid methyl esters
The content and composition of microalgal FAME were determined
by transesterication method adapted from Nayak et al. [17]. Microalgal
lipid was taken in a 50 ml round bottom ask and to this 15 ml of 2% sul-
furic acid in methanol solution was added, and the contents were
reuxed for 4 h at 70 C. On completion of the reaction, the mixture
was cooled down to room temperature, after which the contents were
9 diluted with 5 ml of distilled water and 10 ml of ethyl acetate. Now
upper phase containing fatty acid methyl ester (FAME) was thoroughly
washed with water and dried over anhydrous sodium sulfate. Ethyl ac-
etate was evaporated on the rotary evaporator to recover fatty acid
methyl ester.
The fatty acid composition was carried out in a gas chromatograph
(Thermo Fisher Scientic-Chemito Ceres 800 plus) equipped with a
10
capillary column (30 m 0.25 mm i.d, 0.25 m lm thickness) was
used for the analysis of FAME composition. The temperature of the
oven starting at 70 C for 1 min, and increasing from 5 C min1 to
180 C for 10 min and 6 C min1 to 220 C for 11 min. The injector tem-
perature and detector temperature were set at 250 C and 280 C re-
spectively. The injection volume of 1 L and split ratio of 1:25 were
used for analysis. Nitrogen was used as a carrier gas with a ow rate
of 1.2 mL min1. The identication of chromatographic peaks was per-
formed by comparing their retention times and fragmentation patterns
with standard mixture (FAME Mix 37, Supelco Inc., Bellefonte, PA). The
results of all the experiments were conducted in triplicate, and the

Table 1
Specic growth rate, doubling time, biomass yield, biomass productivity potential, lipid productivity and total lipid content of four microalgal species cultivated in domestic wastewater
during 7 days of cultivation time.

Microalgae Specic growth rate Doubling time Biomass concentration Biomass productivity Total lipid yield Lipid productivity
1 1 1 1
, d TD, h CB, g L PB, mg L d YL, % PL, mg L1 d1

Chlorella vulgaris 0.42 0.013 40 1.1 0.38 0.012 54.3 1.6 21.5 0.6 11.7 0.4
Chlorella minutissima 0.41 0.014 41 1.3 0.34 0.011 48.6 1.4 22.9 0.7 11.1 0.3
Chlorococcum sp. 0.39 0.011 42 1.3 0.32 0.012 45.7 1.2 14.7 0.4 6.7 0.2
Scenedesmus sp. 0.44 0.011 38 1.2 0.43 0.008 61.4 1.8 23.1 0.7 14.2 0.4
220 M. Nayak et al. / Algal Research 16 (2016) 216223

result shows that all the algae successfully adapted to the DWW. The
biomass yield of four microalgal species grown in the wastewater
ranged from 0.32 to 0.43 g L1 (Table 1). The highest biomass yield, bio-
mass productivity, specic growth rate and minimum doubling time
were observed, in the case of Scenedesmus sp., and found to be
0.43 g L1, 61.4 mg L1 d1, 0.44 d1 and 38 h, respectively, when cul-
tivated in shake ask for 7 days. This biomass yield was relatively lower
compared to the yield obtained when cultivated in standard medium
(data not shown here), this is due to the presence of lower nutrients
concentration in the DWW. Reported literature suggests similar
biomass yield and even could be as low as 0.11 g L 1 in municipal
wastewater [26].

3.1.2. Lipid content

The total lipid contents of different microalgae cultivated in DWW
were in the range from 14.7% to 23.1%. Maximum total lipid content of
23.1% and the minimum of 14.7% were found in microalga, Scenedesmus
sp. and Chlorococcum sp. respectively. The lipid productivity was also
found to be highest (14.2 mg L1 d1) in microalga Scenedesmus sp.
in comparisons to other microalgal strains. The higher accumulation of
lipid in Scenedesmus sp. may be due to limiting condition of nutrients
in the DWW medium [2729].

3.2. CO2 sequestration study by Scenedesmus sp. in photobioreactor

3.2.1. Effect of CO2 concentration on biomass growth

Fig. 3. Biomass growth (A) and pH change (B) of microalga, Scenedesmus sp. aerated with
air (0.03%), 1%, 2.5%, 5% and 10% CO2, respectively cultivated in the domestic wastewater
during 7 days of cultivation time.
results are presented as means of the three replicates expressed as
mean error bars.
3. Results and discussion
3.1. Screening of microalgae in shake ask
The characteristics of domestic wastewater (DWW) indicated the
presence of major nutrients namely; NNH4, NNO3, NNO2, PPO4
and COD. The ratios of C/N and inorganic N/P were found to be 2.5
and 6.1, respectively. These ratios may vary with the characteristics of
wastewater depending on the location and time of collection even with-
in a DWW treatment plant. Particularly, inorganic N/P ratio is consid-
ered important for microalgal growth, while a range of 6.810 is
considered optimal for fresh water algae [26]. The N/P value of DWW
nearly close to the optimal range indicates to be used as growth medi-
um for microalgal cultivation.
3.1.1. Microalgal growth in domestic wastewater
The growth characteristics of all four microalgae (Chlorella vulgaris,
Chlorella minutissima, Scenedesmus sp. and Chlorococcum sp.) cultivated
in DWW have been presented in Table 1 after incubation of 7 days. The
In this study, the selected microalga, Scenedesmus sp. was cultivated
in the photobioreactor using DWW, supplemented with different con-
centration of CO2; 0.03%, 1%, 2.5%, 5%, and 10%. The growth proles of
Scenedesmus sp. in various CO2 concentrations with the incubation
time of 7 days are shown in Fig. 3.
The biomass yield of Scenedesmus sp. grown in different concentra-
tions CO2 ranged from 0.89 to 1.37 g L1 (Table 2). The most suitable
CO2 concentration for Scenedesmus sp. was found to be 2.5%. The growth
of Scenedesmus sp. in 2.5% CO2 resulted in the highest biomass yield of
1.37 g L1, biomass productivity of 196 mg L1 d1 and specic growth
rate of 0.51 d1 on 7 days of cultivation, whereas the lowest biomass
yield values were found in control experiment containing CO2 of 0.03%
concentration. With 2.5% CO2 supply nearly 1.54 fold increase in the bio-
mass yield was observed when compared to the control experiment.
This indicates the importance of feeding CO2 as the carbon source. The
result indicated that aeration with higher CO2 concentration did not
drastically affect the biomass yield and was found tolerant to the higher
concentration of CO2 (510%) under the present experimental
conditions.
To evaluate the potential of Scenedesmus sp. to x CO2, the CO2 xa-
tion rate of the microalgae was calculated. The CO2 xation rate of
Scenedesmus sp. when aerated with more than 1% CO2 was in the
range of 279368 mg L1 d1, as shown in Table 2. The highest CO2
consumption rate of 368 mg L1 d1 was obtained with 2.5% CO2 aera-
tion, whereas the minimum was obtained in air aeration (0.03% CO2) of
239 mg L1 d1. The maximum CO2 consumption rate recorded under
2.5% CO2 conditions was higher than the values earlier reported by Tang
et al., for S. obliquus with 10% CO2 was 288 mg L1 d1, indicating the

Table 2
Specic growth rate, doubling time, biomass yield, biomass productivity potential and CO2 consumption rate of microalga, Scenedesmus sp. cultivated in domestic wastewater under dif-
ferent CO2 concentrations during 7 days of cultivation time.

CO2 concentration Specic growth rate Doubling time Biomass concentration Biomass productivity CO2 consumption rate
pH
(%) , d1 TD, h CB, g L1 PB, mg L1 d1 PCO2, mg L1 d1

0.03 0.44 0.013 37.4 1.2 0.89 0.03 127 4.1 239 7.2 8.4 0.31
1 0.46 0.014 35.8 1.1 1.04 0.03 148 4.5 279 8.4 7.3 0.35
2.5 0.51 0.015 32.8 0.7 1.37 0.04 196 5.1 368 10.9 6.8 0.39
5 0.49 0.015 33.8 1.3 1.26 0.01 180 4.9 338 10.1 6.3 0.31
10 0.48 0.012 34.8 1.1 1.14 0.02 163 4.8 306 11.6 6.1 0.32
 M. Nayak et al. / Algal Research 16 (2016) 216223 221

Table 3
Comparative results of various reported studies with respect to carbon dioxide.

Microalgae CO2% Culture medium Culture type Growth Biomass Biomass CO2 consumption Reference
rate concentration productivity rate

, d1 CB, g L1 PB, mg L1 d1 PCO2, mg L1 d1

Chlorella vulgaris 10% Modied Bristol Medium BioFlo fermentor 0.29 1.94 129 251.64 [31]
(CO2 enriched gas)
Scenedesmus obliquus 12% MC medium Column 0.22 1.8 140 263 [32]
(CO2 enriched gas) photobioreactors
Scenedesmus obliquus 10% Modied BG11 Erlenmeyer 0.887 1.84 155 288 [30]
ask
Chlorella sp. 2% Modied f/2 medium Column 0.492 1.211 151.4 284 [18]
photobioreactors
Chlorella sorokiniana 4% Modied TAP Photobioreactor (airlift) 0.44 1.1 150 251 [33]
(Flue gas)
Scenedesmus sp. 2.5% Domestic wastewater Photobioreactor (airlift) 0.51 1.37 196 368 This study
(CO2 enriched gas)

great potential of microalga, Scenedesmus sp. for the capture of CO2 from
a gas stream [30]. Further, biomass productivity and growth rate of
Scenedesmus sp. were better comparative to the previously reported
studies shown in Table 3 [18,3033].
The change in pH values of the media when aerated with various
concentrations of CO2 namely 0.03%, 1%, 2.5%, 5% and 10% CO2 was
found to be 8.4, 7.2, 6.8, 6.4 and 6.2, respectively (Table 2). The highest
biomass yield and CO2 consumption rate at 2.5% CO2 aeration may be
due to the maintenance of the favorable pH value of 6.8 in the culture
medium, which increases the activity of the extracellular carbonic
anhydrase enzyme of microalgae mainly responsible for the carbon con-
centration mechanism [30].
3.2.2. Effect of CO2 concentration on nutrient removal
The integration of wastewater with CO2 sequestration and biomass
production is encouraging for higher growth rates of microalgae. More-
over, the use of wastewater will minimize the requirement for freshwa-
ter and nutrients which eventually reduces the cost of production for
large-scale application [34]. Optimal growth of microalgae requires es-
sential nutrients such as carbon, nitrogen and phosphorous. Therefore,
the major composition of DWW was determined and found to be
comprised of ammonium (NH4) of 38.6 1.5 mg L1, nitrate (NO3) of
17.1 0.5 mg L1, phosphate (PO4) of 9.24 0.3 mg L1, and COD of
142.2 5.2 mg L1.
Table 4 shows the removal efciency of ammonium (NH4), nitrate
(NO3), phosphate (PO4) and COD by the Scenedesmus sp. at different
CO2 concentrations using DWW efuent. The concentration of CO2
was varied from 0.03 to 10%, and their effect on nutrient removal was
assessed. The wastewater treatment efciency was determined by mea-
suring the overall nutrient removal percentage and nutrient removal
rate as shown in Table 4. The overall nutrient removal efciency was
ranged from 61 to 98% in all culture conditions (Table 4). The nutrient
removal capacity improved with the increase in CO2 concentration.
The highest nutrient removal efciency was obtained in 2.5% CO2 sys-
tem, where the removal efciency for NH4, NO3, PO4, and COD was
found to be 98%, 70.2%, 78.9% and 95.9% respectively. This result corrob-
orated with the biomass yield and growth rate data of Scenedesmus sp.,
where highest values were also observed with 2.5% CO2. Thus, the
nutrient removal efciency was directly related to the growth of
microalgae. It was clearly observed that all the nutrient concentrations
were decreased due to the assimilation capacity of microalgal cells. It
is also reported that nutrient assimilation is proportional to biomass
concentration of the culture. A similar type of observations was ob-
served by Ruiz et al. [35]. The COD value, an indicator of organic carbon
concentration, decreased to 69.195.9% levels at the end of growth, sug-
gesting that the carbon was assimilated due to the photo-heterotrophic
mechanism of microalgal growth [36].
3.2.3. Effect of CO2 concentration on the total lipids content
Total lipid contents and lipid productivity ranged from 16.6% to 33.3%
and from 24.03 mg L1 d1 to 65.17 mg L1 d1, respectively (Fig. 4).
Culture aerated with 2.5% CO2 was found to result in maximum total
lipid contents and lipid productivity of 33.3% and 65.17 mg L1 d1.
Overall lipid content in 2.5% CO2 was enhanced by 44% when compared
with the shake-ask study. Minimum lipid content of 16.6% was found
in microalga Scenedesmus sp. inux with 10% CO2, and the minimum
lipid productivity of 24.03 mg L1 d1 was found when the system was
inux with 0.03% CO2 aeration. Thus, the microalga, Scenedesmus sp.
exposed to a CO2 concentration of 2.5% exhibited the best lipid accumula-
tion and biomass production. Our results corroborated with Hu et al.
where the lipid production increased with the increase of CO2 up to
2.5% [36]. The study carried out by Elvira-Antonio et al. showed similar
kind of result, where the lipid accumulation declined with the increase
in CO2 concentration, [37].
3.2.4. Effect of CO2 concentration on fatty acid composition
The fatty acid methyl ester proles of microalgae cultivated under
different CO2 concentrations were analyzed by GCMS and were
presented in Fig. 5. The fatty acid prole measurement in microalgal
lipid is important for evaluating the quality and suitability of lipids for
biodiesel conversion. The FAME composition of Scenedesmus sp. supple-
mented with different concentrations of CO2 showed a dominance of
palmitic acid (C16:0), accounted for approximately 16.2% to 32.9% of
the total fatty acids in the cells as shown in Fig. 5a. Other predominant
fatty acids of microalga, Scenedesmus sp. were 18:1, 18:2 and 18:3. All
FAME proles showed a predominance of C16C18 chain fatty acids

Table 4
Nutrient removal (%) and removal rate of microalga, Scenedesmus sp. inuxed with different CO2% over 7 days of cultivation time in domestic wastewater.

CO2 concentration Nutrient removal (%) Nutrient removal rate (mg/L/d)

(%)
NH4 NO3 PO4 COD NH4 NO3 PO4 COD

0.03 95.3 3.1 61.1 2.4 81.9 2.4 69.1 2.4 5.26 0.21 1.49 0.05 1.08 0.04 14 0.4
1 96.3 3.5 62.3 2.5 80.9 3.1 75.1 3.1 5.31 0.2 1.52 0.04 1.07 0.01 15.2 0.6
2.5 98.3 3.8 70.2 2.3 78.9 2.8 95.9 3.5 5.42 0.22 1.71 0.06 1.04 0.03 19.5 0.5
5 96.9 2.2 65.5 2.1 72.8 2.6 92.9 3.2 5.34 0.18 1.6 0.02 0.96 0.03 18.9 0.7
10 93.5 2.9 56.7 2.6 74.3 2.1 86.6 3.7 5.16 0.23 1.38 0.04 0.98 0.02 17.6 0.7
222 M. Nayak et al. / Algal Research 16 (2016) 216223
Fig. 4. Lipid contents and lipid productivity of microalga, Scenedesmus sp. cultivated in domestic wastewater on day 7 under different CO2 concentrations.
ranging from 78.2 to 81.9% indicating good quality of biodiesel (Fig. 5b)
and could be considered as ideal feedstock for conversion to biodiesel
through transesterication process [30]. And the amounts of saturated
fatty acid (SFA) and unsaturated fatty acids (UFA) accounted were
50.6 3.9% and 49.4 3.8% of the total fatty acids, respectively
(Fig. 5b). The percentage ratio of SFA and UFA showed the similar
trend with the reported literature for high-quality biodiesel, which
helps to maintain high oxidative stability and low-temperature proper-
ty [38]. The most suitable ratio of saturated to unsaturated fatty acids
was observed in 2.5% CO2 of 54.3 to 45.7, and with a low percentage
Fig. 5. Fatty acid prole (% of total FAME) of microalga, Scenedesmus sp. grown under
different CO2 concentrations with domestic wastewater.
of polyunsaturated fatty acids (16.9%). This may have a great potential
for the production of good quality biodiesel.
4. Conclusion
The present study was aimed at developing an integrated approach
towards treating wastewater by microalgae with concomitant CO2 se-
questration and lipid production to ensure sustainability and eco-
friendliness of the process. The DWW collected from IIT Kharagpur cam-
pus supported the growth of microalgae and got remediated in the pro-
cess to produce reusable water and biomass feedstock for biofuel. The
growth of Scenedesmus sp. was found to be most feasible in DWW
among four different microalgae cultures. Further, the growth of
Scenedesmus sp. was up-scaled in the bubble column photobioreactor
with supplementation of different concentrations of CO2 ranging from
0.03 to 10%. The CO2 concentration of 2.5% was found to be most favor-
able and resulted in an increase in the biomass yield and lipid content.
For nutrients removal capacity from DWW, ammonium, nitrate, phos-
phate and COD removal efciency was evaluated and found to be rang-
ing from 70 to 98%. Thus, the DWW generated in huge amount could be
used as a promising culture medium and possible cheap nutrient substi-
tute for microalgae cultivation. FAME analysis showed an optimal ratio
of saturated and unsaturated fatty acid with the predominance of C16
and C18 fatty acids. The overall fatty acid composition of algal lipid sug-
gests the use for high-quality biodiesel. Integration of various processes
such as wastewater remediation, CO2 sequestration and biomass pro-
duction for biodiesel application could make the whole process environ-
mentally and economically sustainable. Further scale-up studies in
outdoor condition are required.
Acknowledgments
Authors acknowledge the nancial support received from the
Department of Science & Technology (DST), Govt. of India, for the pro-
ject (No.: DST/IS-STAC/CO2-SR-160/13(G); Date: 08.07.2013).
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