Renewable Energy 98 (2016) 64e71

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Renewable Energy
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Critical parametric influence on microalgae cultivation towards

maximizing biomass growth with simultaneous lipid productivity
P. Chiranjeevi a, b, S. Venkata Mohan a, b, *
a
Bioengineering and Environmental Sciences (BEES), CSIR-Indian Institute of Chemical Technology (CSIR e IICT), Hyderabad 500 007, India
b
Academy of Scientific and Innovative Research (AcSIR), India

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

Article history: Enhancing microalgae biomass productivity through different abiotic and environmental factors opti-
Received 31 December 2015 mization is crucial. Design of experimental (DOE) methodology using Taguchi orthogonal array (OA) was
Received in revised form studied to evaluate the specific influence of eight important factors (light, pH, temperature, carbon
16 March 2016
concentration, nitrates, phosphates, magnesium ion concentration and carbon source) on the biomass
Accepted 19 March 2016
Available online 7 April 2016
production using three levels of factor (21
37) variation with experimental matrix [L18-18 experimental
trails]. All the factors were assigned with three levels except light illumination (21). Substantial influence
on biomass productivity is observed with carbon concentration contributing 16.8%, followed by nitrates
Keywords:
Mixotrophic
12.8% and light 9.3%. Experimental setup eight (Light, pH-8.5, Temperature 25 C, Carbon concentration
CO2 sequestration 10 g/l, nitrates 1.5 g/l, phosphates 0 g/l, magnesium 150 mg/l, Carbon source (glucose)) showed
Hydrothermal liquefaction (HTL) maximum biomass growth (5.26 g/l) and good substrate degradation (63%, COD removal efficiency)
Wastewater treatment contributing to carbohydrate production (257 mg/g biomass) which is further converted to lipids (20%
Lipid synthesis Total lipid and 10% Neutral lipids). Chlorophyll (a, b), carbohydrates composition, FAME analysis for lipid
Taguchi design of experimental (DOE) percentage were monitored during process operation. Elemental analysis reveals that the carbon to
methodology hydrogen and oxygen ratio present in dried algal biomass can be hydrothermally liquefied (HTL) to
produce biocrude.
© 2016 Elsevier Ltd. All rights reserved.

1. Introduction environments as they can adapt their metabolism according to

Microalgae is touted as an attractive alternative to traditional
forms of biomass for biofuels production, due to high productivity
per unit area, usage of non-arable lands for cultivation, utilization
of wastewater as a substrate and CO2 sequestration. Direct lipid
extraction/Thermochemical conversion methods are widely being
used to transform algal biomass into fuels along with high valued
products [1]. Optimization of different factors such as nutrient
stress, light, temperature, CO2, salinity, etc. have been explored by
several researchers to enhance lipid accumulation in microalgae
rather than biomass [2]. The cell growth and microalgal biomass
production will be influenced by sufficient supply of macro and
micronutrients present in the medium and environmental factors
(light, pH and temperature) [3]. Microalgae can survive in extreme
* Corresponding author. Bioengineering and Environmental Sciences (BEES),
CSIR-Indian Institute of Chemical Technology (CSIR e IICT), Hyderabad 500 007,
India.
E-mail address: vmohan_s@yahoo.com (S.V. Mohan).
altering environmental conditions. Usually, microalgal biomass can
be produced through autotrophic cultivation in open ponds or a
photobioreactor using solar energy for fixing CO2 [4,5]. Alterna-
tively, they are also cultivated in heterotrophic mode of nutrition
using organic compounds as energy and carbon sources [6e8].
Among the different modes of cultivation, mixotrophic operation
offers several advantages like low-cost for biomass harvesting and
substrate degradation [9]. Moreover, in mixotrophic operation,
both cell growth and biosynthesis of products are significantly
influenced by the nutrients present in the medium and by the
environmental factors. The syntrophic association between
microalgae and its existing microenvironment facilitates higher
biomass production and lipid accumulation along with significant
substrate degradation. Mixotrophic cultivation of microalgae pro-
vides a sustainable and viable route for the possible utilization/
mitigation of CO2 and organic waste present in wastewater for
biofuel production [10].
Enhancing the biomass productivity with simultaneous lipid
synthesis by optimizing influencial factors employing design of

http://dx.doi.org/10.1016/j.renene.2016.03.063
0960-1481/© 2016 Elsevier Ltd. All rights reserved.
 P. Chiranjeevi, S.V. Mohan / Renewable Energy 98 (2016) 64e71
experiment (DOE) methodology has been followed in this
communication. DOE methodology by Taguchi orthogonal array
(OA) is a factorial based approach which merges statistical and
engineering techniques [11e13]. Analysis of the experimental data
using ANOVA (analysis of variance) provides information about
statistically significant factors and their optimum levels for the
design of experimental parameters. Taguchi method utilizes OAs to
study a large number of variables with a small number of experi-
ments, saving both time and cost [11e14]. The specific objective of
this investigation is to study the methodological application of
Taguchi orthogonal array (OA) design of experiment (DOE) to
optimize various process parameters viz., Environmental factors
(light, pH, temperature), abiotic factors (carbon concentration, ni-
trates, phosphates, magnesium ion concentration and carbon
(glucose/acetate)) that influence the microalgae biomass and lipid
productivity along with reducing sugars synthesis. The increased
biomass can also be used in HTL for biocrude production.
2. Experimental methodology
2.1. Collection and cultivation of microalgae
Microalgal cultures collected from the outlet channel of
Nacharam Lake (lentic water body), Hyderabad in pre-monsoon
season was used as parent inoculum [8]. Prior to experimenta-
tion, the culture was washed twice with water and pelletized by
centrifugation (3000 rpm; 10 min at 30 C) to remove associated
debris. Microalgae was stored in rectangular plastic tubs
(36 cm
24 cm
12 cm) exposed to sunlight. Domestic sewage
(DS- pH, 7.8; COD, 220 mg/l; VFA, 165 mg/l; BOD, 120 mg/l; total
alkalinity, 140 mg/l; chlorides, 175 mg/l; nitrates, 115 mg/l; color,
200 Hazen units) was used for algal cultivation as carbon and
nutrient source. During initial period of culture maintenance, the
biomass was mixed periodically to avoid settling and allow uniform
distribution of sunlight to the cells. After consistent amount of
biomass and cell density was achieved, this culture was used as
inoculum for the experimental study.
2.2. Design of experimental (DOE) methodology
Taguchi's DOE methodology was employed. Selection/identifi-
cation of important factors whose variation had a critical effect on
the biomass growth and lipid production was primary step. Eight
factors viz., light, pH, temperature, nutrient stress, salinity, nitro-
gen, phosphorous and trace metals were selected for optimization
due to their significant role on photosynthetic machinery in context
of biomass growth and synthesis of lipids (Table 1). Subsequently,
an experimental matrix was designed considering three levels of
factor orthogonal array (OA) layout, 21
37 with experimentation
size by symbolic arrays of matrix [L18-18 experimental trails]
(Table 2). Except for nutrient stress operation (21), all other factors
were assigned with three levels. In the designed OA, each column
Table 1
Selected factors and assigned levels.
S.No Factor
1 Light (74 mmol m2 s1)
2 pH
3 Temperature (0C)
4 Carbon Concentration (mg COD/l)
5 Nitrates (NaNO3) (g/l)
6 Phosphates (mg/l)
7 Magnesium (mg/l)
8 Carbon Source
65
consisted of a number of conditions depending on the levels
assigned to each factor and the diversity of factors can be studied by
crossing OA of control factors.
2.3. Microalgal cultivation studies
Batch experiments for microalgal cultivation were conducted by
employing 18 selected experimental variations (Table 2) in com-
bination with seven factors at 3 levels and one factor at 2 levels
(Table 1). According to the designed experiments, sterilized (auto-
claved for 20 min at 121 C and 1.05 kg/cm steam pressure prior to
the inoculation to avoid the bacterial contamination) 250 ml of
modified growth medium (as per design, Table 1) was inoculated
with microalgae inoculums (10% v/v; OD, 0.1). Growth phase (GP)
was operated for a period of 8 days to accelerate algal biomass
growth and to avoid bacterial contamination further antibiotic
(Ampicillin: 0.2 g/l) was added to the each experimental setup for
every alternate day of the operation. Total experimental setup was
kept in a temperature controlled shaking incubator (120 rpm).
During GP operation, biomass, cell density, pigment analysis
(chlorophyll a and b) along with total cellular carbohydrate con-
centrations were estimated once in every alternate day. Lipid
analysis was done at initial (before the GP) and end of GP. All the
experiments were carried out in triplicates and the results pre-
sented here represent an average of three independent operations.
2.4. Lipid extraction and derivatization of FAME
After GP, the biomass was separated by centrifugation
(5000 rpm; 5 min at 28 C) and the algal biomass pellet was sub-
jected to solar drying followed by blending in to powder form. The
blended powder was further disrupted using sonicator (20 kHz) for
30 min (Power Sonic 410) and the total lipids were extracted by
modified Bligh and Dyer method using chloroform and methanol
(2:1) as solvents and hexane was used for neutral lipid extraction
[8]. Further followed by centrifugation were solvent: lipid layer was
transferred into pre-weighed round bottom flask and the total and
neutral lipids were determined gravimetrically in terms of per-
centage dry cell weight. Lipid productivity (%) was calculated based
on the ratio of total lipid extracted to dry weight of algae as shown
below
Lipid productivity ¼ g of oil/dry weight of biomass
The resulted lipid was transesterified (using methanol-
sulphuric acid mixture) to FAME and was analyzed using GC-FID.
After conversion of fatty acids to methyl esters, the concentrated
sample was used for the detection of FAME composition by GC with
FID (Nucon-5765) through capillary column [Valcobond (VB)
30 mm (0.25 mm
0.25 lm)] using nitrogen as carrier gas (1 ml/
min). The temperature of the oven was initially maintained at
140 C (for 5 min), later increased to 240 C at a ramp of 4 C/min for
Level 1 Level 2 Level 3
Yes No e
6 7.5 8.5
20 25 30
0 5000 10,000
0 1.5 2.5
0 40 80
0 75 150
Glucose (G) Acetate (A) Glucose þ Acetate (G þ A) (50% each)
66 P. Chiranjeevi, S.V. Mohan / Renewable Energy 98 (2016) 64e71

Table 2
Orthogonal array [L18 (21
37)] of designed experiments with experimental output.

Experiment number Factors Biomass productivity (g/l) Mode of nutrition

1 2 3 4 5 6 7 8

1 1 1 1 1 1 1 1 1 1.26 Autotrophic
2 1 1 2 2 2 2 2 2 1.44 Mixotrophic
3 1 1 3 3 3 3 3 3 1.46 Mixotrophic
4 1 2 1 1 2 2 3 3 1.27 Autotrophic
5 1 2 2 2 3 3 1 1 1.92 Mixotrophic
6 1 2 3 3 1 1 2 2 1.32 Mixotrophic
7 1 3 1 2 1 3 2 3 1.32 Mixotrophic
8 1 3 2 3 2 1 3 1 5.26 Mixotrophic
9 1 3 3 1 3 2 1 2 1.23 Autotrophic
10 2 1 1 3 3 2 2 1 1.36 Heterotrophic
11 2 1 2 1 1 3 3 2 1.30 Heterotrophic
12 2 1 3 2 2 1 1 3 1.29 Heterotrophic
13 2 2 1 2 3 1 3 1 1.39 Heterotrophic
14 2 2 2 3 1 2 1 3 1.35 Heterotrophic
15 2 2 3 1 2 3 2 1 1.21 Heterotrophic
16 2 3 1 3 2 3 1 2 1.43 Heterotrophic
17 2 3 2 1 3 1 2 3 1.25 Heterotrophic
18 2 3 3 2 1 2 3 1 1.33 Heterotrophic

The bold value represents the maximum value of biomass of all the experiments.

10 min. The injector and detector temperatures were maintained at
280 and 300 C respectively with a split ratio of 1:10. FAME
composition was compared with the standard FAME mix (C8eC22;
LB66766, SUPELCO).
2.5. Analysis
Algal biomass growth was monitored by measuring OD at
600 nm and changes in pigment concentration (chlorophyll a and
b) were measured at 647 and 664 nm respectively. Total soluble
carbohydrate content (TSCC) in the algal cells was determined by
Anthrone method. Bioprocess monitoring in terms of COD, pH, ni-
trates and phosphates were performed according to the standard
methods [15]. The C, H, N and S content was estimated using a
CHNS Analyzer (Vario Micro Cube).
2.6. Data analysis
The data derived from the experiments was processed through a
software (Qualitek-4; Nutek Inc) to analyze the output using ‘bigger
is better’ performance characteristics.
3. Results and discussion
3.1. Biomass production- factors influence
The designed experiments and the selected conditions showed a
significant variation in the microalgal biomass production (Table 2).
The difference between values at levels 2 and 1 (L2-L1) of each
factor indicate the relative influence of the effect on the process (Fig
1). The larger the difference, the stronger is the influence. The
negative value was ignored to assess the main effect as the place-
ment order of levels assigns either positive or negative. The relative
influence of the factors on biomass production is as follows.
Carbon load > Nitrates > Light > Temperature > pH > Carbon
sources > Magnesium > Phosphates
Among the selected factors, carbon concentration/load showed
a stronger influence on the biomass production while phosphates
showed the least influence. High carbon concentration (10 g/l
glucose) documented higher biomass production (5.26 g/l) with
productivity rate of 0.66 g/l/day in experimental condition eight
(Fig 2). Excess carbon in the form of high glucose concentration
favors biomass growth as it is important for cell division and for
carrying out the physiological activities in a cell. The organic carbon
in microalgae can assimilate via multistep pathways such as aerobic
glycolysis through the Embden-Meyerhof Pathway (EMP) and
Pentose Phosphate Pathway (PPP), and tricarboxylic acid cycle
(TCA). In EMP pathway glucose is directly absorbed into the cell by
simple diffusion via hexose/Hþ symport system that is easy and less
energy consuming when compared to CO2 uptake by photosyn-
thesis [16]. The diffused glucose directly involves in glycolysis and
increases the rate of metabolism. The increase in the concentration
of glucose increases biomass because more energy is released in the
form of ATP and NADPH when glucose gets broken down. Among
photoautotrophic and heterotrophic modes, mixotrophic cultiva-
tion of microalgae offers several advantages including CO2 miti-
gation, good control over cultivation process by natural sunlight,
higher growth and easy biomass harvesting [17].
Nitrates concentration of 1.5 g/l has shown high biomass pro-
duction. Nitrogen is required in good amounts as an essential
component of peptides, proteins, enzymes, chlorophylls, energy-
transfer molecules (ATP, ADP), genetic materials (RNA, DNA), and
other cellular constituents [18]. In algae, inorganic nitrogen viz.,
nitrate (NOþ2 þ2 þ
3 ), nitrite (NO2 ), and ammonium (NH4 ) are directly
assimilable in metabolic activity. Nitrate is the most thermody-
namically stable form and gets oxidized in aquatic environments.
Following translocation across the plasmalemma (which is an
energy-dependent process), the assimilation of NOþ2 3 require
chemical reduction to NHþ 4 . This process is mediated by two en-
zymes viz. nitrate reductase and nitrite reductase [19]. Nitrate
reductase located in the cytosol uses NADPH to catalyze the bi-
electron transfer. In algae nitrate reductase is coupled to the
oxidation of pyridine nucleotide [20]. The nitrite by nitrate reduc-
tase is reduced in a six-electron transfer reaction. Nitrite reductase
utilizes ferredoxin in algae; in the latter, the enzyme is localized in
the chloroplasts. In algae, photosynthetic electron flow is an
important source of reduced ferredoxin for nitrite reduction. The
incorporation of ammonium into amino acids is primarily brought
about by the sequential action of glutamine synthetase (GS) and
glutamine 2-oxoglutarate aminotransferase (GOGAT). Ammonium
assimilation by GS requires glutamate as substrate and ATP and
catalyzes the irreversible reaction. The amino nitrogen of glutamine
 P. Chiranjeevi, S.V. Mohan / Renewable Energy 98 (2016) 64e71 67

Light 1100
1200 pH
1000
1000
900
800 800
Factors perform ance
1050 Temperature 1200 Carbon concentration

1000
900
800
750 600

1200 Nitrates 1050 Phosphates

1000
900
800

Magnesium Carbon Source

1000 1000

800 800
Level 1 Level 2 Level 3 Level 1 Level 2 Level 3

Fig. 1. Individual factors performance at different levels.

(74 mmol m2 s1) microalgae showed higher biomass productivity

than in the absence of light. In the mixotrophic mode, the acetyl-
5
CoA pool will be maintained from both the carbon source i.e. CO2
fixation (Calvin cycle) and extracellular organic carbon. However,
both light and glucose are sources for the ATP production in the
4
mixotrophic culture [22]. Since glyceraldehyde- 3-phosphate
Biomass (g/l)

generated by photosynthesis partly enters the glycolytic pathway

3 and TCA cycle, both light and glucose account for the production of
2 phosphorylation. Here, light is the major source for ATP production
1 limitation, light saturation and light inhibition [23,24]. The light
0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Experimental Variation Studied
Fig. 2. Biomass productivity of all the eighteen experimental setup performed by DOE
Taguchi.
is subsequently transferred to 2-oxoglutarate, and reduced, form-
ing 2 mol of glutamate. Both GS and GOGAT are found in chloro-
plasts, although isoenzymes (multiple forms of an enzyme with the
same substrate specificity, but genetic differences in their primary
structures) of both enzymes may also localize in the cytosol. The
enzyme glutamate synthase is exported from the chloroplast to the
cytosol where transamination reaction can proceed, thereby facil-
itating the synthesis of other cell building blocks (amino acids) [20].
After nitrogen, light showed strong influence on microalgae
biomass production. In mixotrophic mode of operation, microalgae
utilize organic carbon and therefore light energy is a crucial factor
for the biomass growth [21]. In the presence of light (12 L/12 L)
NADH and FADH2 in the TCA cycle. Therefore, the ATP produced is
from light and glucose via photophosphorylation and oxidative
in the early phase of mixotrophic cultivation. Microalgae broadly
undergo three different phases under light exposure such as light
utilization efficiency and biomass productivity proportionally in-
crease with the light intensity till the cells become light saturated.
The light saturation value of algae is in the range of
74e185 mmol m2 s1 (Yeesang, 2011). At low light intensity,
photosynthesis becomes limiting. Lee and Pirt (1981) reported an
increase in the cellular maintenance requirements during long time
exposure of dark, which causes a reduction in the biomass pro-
ductivity. At high light intensity, photosynthesis is disrupted due to
photoinhibition. The excess light energy is degenerated as fluo-
rescence or heat through non-photochemical quenching (NPQ)
[24]. The photoinhibition disrupts the synthesis and degradation
cycle (D1/D2 proteins) of the light-harvesting complex at PSII,
eventually leading to inhibition of photosynthesis [23]. Moreover
microalgae always try to remain on the surface of the water to catch
maximum light intensity for this it uses many techniques like a
synthesis of gas vacuoles, accumulation of the fat and mucilage
synthesis, which help reduce the density of the algal cell and pre-
vent it from sinking. In many ecological water bodies, the homeo-
static structure and function of a living system supported by
chemical, physical and organic activity of the biota balances the
ecological status [25]. Light shielding also occurs when cell density
increases considerably. The ecological water bodies contaminated
68 P. Chiranjeevi, S.V. Mohan / Renewable Energy 98 (2016) 64e71
with wastewater consists of many suspended particles, which may
lead to the shielding effect. The color and transparency of the
wastewater also control light penetration [26,27].
Analysis of the experimental output documented the influence
of temperature on the biomass productivity of microalgae. The
temperature at level 2 (25 C), showed high biomass production
than level 1 (20 C) and level 3 (30 C). The temperature of the algal
media/wastewater is an important parameter which determines
gas solubility (O2 and CO2), pH, ionic equilibrium, etc. of the algal
media/wastewater. Temperature also affects the cellular enzymatic
reactions, including the cytosolic pH of the microalgae. Most of the
microalgal species can grow over a wide range of temperature from
15 C to 30 C and carry out photosynthesis [28]. The growth of the
microalgae is more sensitive to high temperature compared to low
temperature. A little raise over the optimum temperature may
sternly affect the algal growth. Even temperatures lower than op-
timum reduce the cellular activities such as limiting the electron
transport chain (ETC), the activity of carbonic anhydrase (CA), etc.
[29]. The high temperature shows inhibitory effect on the cellular
physiology and denaturing of essential proteins/enzymes that
affect CO2 fixation. For example, photosystem II is very thermola-
bile and prone to denaturation at a higher temperatures. Besides,
an increase in photorespiration, a decrease in RuBisCO affinity for
CO2 and a decrease in CO2 to O2 solubility ratio are the other vital
phenomena negatively affecting the CO2 fixation at a higher tem-
peratures [30]. Further, the temperature exposed to algal cells de-
pends upon the time of light exposure and seasonal variations.
pH is one of the important factor that influences microalgal
biomass production. Alkaline pH (8.5) showed higher algal biomass
productivity than neutral (pH, 7.5) and acidic (pH, 6) condition. The
algae have neutral or slightly alkaline cytosolic pH as cellular en-
zymes are pH sensitive and become inactive at acidic conditions
[31]. Therefore, the optimal pH of most of the algal species has been
found in the range of 7e9. The extreme pH negatively affects the
growth of microalgae and CO2 fixation by disrupting cellular pro-
cesses. CO2 dissolves in water by forming carbonaceous species
(aqueous CO2, carbonic acids, bicarbonate, and carbonate) and their
availability to the algal cells depends upon the pH of the culture as
the equilibrium shifts among them on the change in the pH. Along
with the growth of the algae, pH increases due to consumption of
CO2 due to the release of hydroxide ions outside the cell at the
expense of capture of Hþ ions inside the thylakoid membranes
during the activation of CCM (carbon capture mechanism) [32]. The
presence of nitrogen source in the medium also influences the pH.
For example, the consumption of nitrate from the medium helps in
the increase of alkalinity via OH production [33]. Contrary to this,
uptake of NH4þ by the microalgae leads to Hþ production [34].
Higher algal biomass production observed with glucose as a
carbon source might be due to the involvement of some metabolic
reactions and participation of some enzymes towards the biomass
production. Glucose (carbon source) at level 1 with maximum
concentration (10 g/l) showed higher biomass production compare
to level 2 (Acetate) and level 3 (Glucose þ Acetate). In this study, we
are using mixed microalgae culture as biocatalyst, so, 10 g/l of
glucose concentration showed higher biomass production. Next to
carbon source, magnesium showed influence on biomass produc-
tion. Magnesium concentration of 150 mg/l at level 3 showed
increment in algal biomass production. Magnesium has an impor-
tant role in photosynthesis because it forms the central atom of
chlorophyll [35]. Magnesium was found to be essential for growth
of the microalgae as it is required for ribosome stability, functioning
of chlorophyll, helps in activation of enzymes related to photo-
synthesis and respiration. Magnesium is a necessary activator for
many critical enzymes, including ribulose biphosphate carboxylase
(RuBisCO) and phosphoenolpyruvate carboxylase (PEPC), both
essential enzymes in carbon fixation. It also assists in DNA and RNA
synthesis helping in cell multiplication. Low amounts of Mg lead to
a low turnout in photosynthesis and enzymatic activity within the
microalgae. Even though phosphates plays an important role in the
energy transfer of the algal cells as well as in the synthesis of
phospholipids and nucleic acids, low concentration of phosphates
at level 1 (0 mg/l) showed higher biomass production. Phosphorus
deficiency promoted the accumulation of lipids in certain algae.
Phosphorus, rather than nitrogen, is the primary limiting nutrient
for microalgae in many natural environments. Individually carbon
concentration (10 g/l) along with pH (8.5) and magnesium ion
concentration (150 mg/l) showed the highest effect at level 3.
Whereas, temperature (25 C) and nitrates (1.5 g/l) exhibited higher
effects at level 2. On the contrary, light (74e185 mmol m2 s1),
carbon source (glucose) and phosphates (0 mg/l) at minimal energy
inputs (level 1) showed positive influence on the mixotrophic
mixed microalgae biomass production.
3.1.1. Factor interaction
Optimization of mixed microalgal biomass productivity requires
an understanding not only of how various factors individually affect
it but also of the multiple interactions that adds to the complexity
of the whole process (Table 3). In this way, the individual parameter
interactions, called as severity index (SI), of different factors
derived from data analysis helps to understand the influence of the
two individual factors at various levels of interaction. Carbon con-
centration with nitrates showed highest SI (85.13%) followed by
temperature and light (60.4%) and pH with magnesium (SI 59.3%).
The results showed significant interactions among the factors, with
the least SI between carbon concentration and light (32.6%). Carbon
concentration and nitrates proved to be the most influential factors
at interactive levels and individually also has a significant influence
on microalgae biomass productivity. Also, the presence of magne-
sium ion concentration has shown major influence on interactive
roles rather than individual components. Phosphates and carbon
source have showed negligible influence on interaction level as
their role comes into play in the growth phase. The interactive
parameters viz. Carbon concentration, nitrates, light, temperature,
pH, carbon source and magnesium have a direct influence on
biomass growth. The SI analysis suggested that the light, temper-
ature, carbon source and magnesium which were of least impact
factors at individual levels maximize the total lipid productivity in
combination.
3.1.2. Analysis of variance (ANOVA)
ANOVA with the percentage of contribution of each factor with
interaction is shown in Table 4. The level of factors to produce the
best results can observe from Table 5. F-ratios suggest that all the
factors and interactions considered in the experimental design had
statistically significant effects at a 48.4% confidence limit. Experi-
mental degree of freedom (DOF) is 17 while factorseDOF is 2. The
percentage contribution was calculated for each factor by the ratio
of the pure sum to the total sum of the squares. Among the selected
factors, ANOVA indicates that the most influential factor was car-
bon concentration, accounting for 16.8% of the overall variance
followed by nitrates (12.8%) and light (9.3%). pH, phosphates, and
carbon source showed zero influence on the biomass production on
variance. Altogether, light, carbon concentration and nitrates
contributed a majority of 38.9% at their individual levels on the
mixed microalgal biomass production indicating that these factors
played a critical role in the optimization.
3.1.3. Optimum parameters
Optimum conditions to achieve higher biomass productivity
from mixed microalgae by the selected factors contribution is
 P. Chiranjeevi, S.V. Mohan / Renewable Energy 98 (2016) 64e71 69

Table 3
Automated test for presence of interaction factors.

S. No Interacting factor pairs (based on SI) Columns SI (%) Reserved column Optimum levels

1 Carbon concentration
Nitrates 4
5 85.13 8 [3,2]

2 Temperature
light 3
1 60.45
3 pH
Magnesium 2
7 59.38
4 Phosphates
Magnesium 6
7 51.45
5 Light
Nitrates 1
5 48.34
6 Light
Carbon concentration 1
4 34.32
7 pH
Phosphates 2
6 36.1
8 Light
Magnesium 1
7 44.6
9 Phosphates
Carbon concentration 6
4 32.84
10 Carbon concentration
Magnesium 4
7 32.8
11 Carbon concentration
Light 4
1 32.62
5 [2,1]
3 [1,3]
1 [2,1]
4 [1,3]
6 [1,3]
4 [2,2]
11 [1,3]
17 [1,1]
9 [1,1]
15 [1,2]

Table 4
Analysis of variance ANOVA.

S No Factor DOF Sum of squares Variance F-ratio Pure sum Percent

1 Light (74 mmol m2 s1) 1 227.3 113.6 3.3 159.0 9.3
2 pH 2 11.6 5.8 0.1 0 0
3 Temperature ( C) 2 111.0 55.5 1.6 42.7 4.5
4 Carbon Concentration (mg COD/l) 2 190.2 95.1 2.7 122.0 16.8
5 Nitrates (NaNO3) (g/l) 2 122.8 122.8 3,5 88.7 12.8
6 Phosphates (mg/l) 2 25.0 12.5 0.3 0 0
7 Magnesium (mg/l) 2 143.8 71.9 2.1 75.6 7.9
8 Carbon Source 2 46.0 23.0 0.6 0 0
Other/Error 2 68.244 34.122 48.41
Total 17 946.358 100%

Table 5
Optimum conditions and their contributions.

S.No Factor Level description Level Contribution

1 Light (74 mmol m2 s1) Yes 1 3.75

2 pH 8.5 3 1.60
3 Temperature ( C) 25 2 2.60
4 Carbon Concentration (mg COD/l) 10000 3 4.30
5 Nitrates (NaNO3) (g/l) 1.5 2 3.32
6 Phosphates (mg/l) 0 1 1.11
7 Magnesium (mg/l) 150 3 2.61
8 Carbon Source Glucose 1 2.15

Total contribution from all factors 21.478

Current grand average of performance 20.876
Expected result at optimum condition 42.354

shown in Table 5. Carbon source was found to be the most signif-
icant factor influencing biomass production. Light, nitrates, and
magnesium were the next most important factors followed by
temperature, carbon source, pH, and phosphate concentration. A
total contribution of factors at optimized conditions was 21.4% for
biomass production with a maximum contribution of 4.3% from
carbon concentration. The optimum operating conditions showed
the requirement of light (74e185 mmol m2 s1) with pH of 8.5,
temperature 25 C, 10 g/l carbon supplementation as glucose, 1.5 g/l
of nitrates, 150 mg/l of and magnesium ion concentration were
derived from the data analysis resulting in the increment of mixed
microalgae biomass production from 21.4% to 42.3%.
3.2. Lipid synthesis during growth phase
Carbohydrate assimilation by the photosynthetic microalgae
through CO2 sequestration and direct assimilation in the mixo-
trophic mode of cultivation plays a major role in redirecting the
carbohydrate content towards lipid synthesis [10,17]. The experi-
mental condition eight showed carbohydrate concentration of
257 mg/g of biomass at the end of growth phase (Fig 3). Carbohy-
drate profile determined the presence of 30% of xylose and 15% of
arabinose.
Lipid percent of the dried biomass at the end of the growth
phase was observed to be total lipids (TL) (20.33%)/neutral lipids
(NL) (10.7%) with lipids productivity of 0.203 kg/kg of biomass (TL);
0.107 kg/kg of biomass (NL) of the same experimental condition
(Fig 4). Fatty acid composition was also monitored to the similar
condition to enumerate the composition (Fig 5). Fatty acid methyl
ester (FAME) composition showed total eighteen fatty acids
comprising of ten saturated fatty acids (SFA) and eight unsaturated
fatty acids (USFA) (Fig 5). Among the SFAs detected, the presence of
Lauric acid (C12:0), Myristic acid (C14:0), Myristoleic acid (C14:1)
and Pentadecylic acid (C15:0), which are used in the preparation of
antibacterial agents and antioxidants [36] while Margaric acid
(C17:0) (C17:1) can readily use in diesel engines [37]. The presence
of Palmitic acid (C16:0), Palmitoleic acid (C16:1), Hexadecanoic acid
(C16:2), which can be used in the preparation of biofuels and
lubricating oils [36]. Among the USFAs, the presence of Stearic acid
(C18:0), oleic acid (C18:1), linoleic acid (C18:2), and linolenic acid
70 P. Chiranjeevi, S.V. Mohan / Renewable Energy 98 (2016) 64e71

(C18:3) which could use as emulsifiers, solubilisers, lubricators and

5
in engine combustions.

3.3. Influence of nutrition mode on biomass productivity and

4
Carbohydrates (mg/gm)

simultaneous lipid synthesis

Based on the light, nutrient and carbon availability all the

3 eighteen experimental setups were categorized into different
nutritional modes (Autotrophic, Mixotrophic and Heterotrophic)
(Table 2). Among all the experimental conditions experiment setup
2 eight showed maximum biomass and lipid productivity which is
following mixotrophic mode of nutrition. Mixotrophic growth can
take advantage of both photosynthetic and respiratory metabo-
1 lisms which showed significant increase in the biomass and lipid
productivity. Higher partition of carbon from glycolytic pathway
can lead in mixotrophic nutrition to higher metabolic activity and
0 higher growth rates when compared to CO2 utilization through
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 photosynthetic pathways (Autotrophic mode of nutrition) [38].
Experimental Variation Studied Many microalgae have demonstrated that glucose enables superior
growth rates and activation of respiratory metabolism than other
Fig. 3. Variation in carbohydrates concentration of all the experimental conditions. carbon sources, sugar phosphates, organic acids, and monohydric
alcohols [39]. The pathways reported for glucose catabolism in
algae are the Embden-Meyerhof pathway (EMP) and the Pentose
50 Phosphate pathway (PPP) which is extensively used for glucose
Lipid Productivity (mg/Kg biomass)

Total Lipid
Neutral Lipid 1000
assimilation and producing high cell density cultures in mixo-
Lipid Productivity trophic cultivation of mixed microalgae leading to high lipid pro-
40
ductivity [40].
800

30 3.4. Parametric evaluation of biomass productivity

Lipid (%)

600
Biomass growth increased linearly during all the experimental
20
400 variations studied. Among the experimental variations operated,
the eighth conditions showed maximum biomass growth (5.26 g/l).
10 The ability of microalgae to assimilate available organic com-
200
pounds, as well as atmospheric CO2 as a carbon source under the
mixotrophic mode of nutrition, was attributed [36,10]. The
0 0 elemental (C, H, N, S) analysis for the dried mixed microalgal
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
biomass showed the following composition: 38.97% carbon, 5.84%
Experimental Variation Studied hydrogen, 4.37% nitrogen, and 0.25% sulfur. Increase in the biomass
was observed with a high concentration of glucose supplementa-
Fig. 4. Total and nuetral lipid percent and lipid productivity of eighteen experimental
setup performed by DOE Taguchi.
tion while a corresponding increment in the pigments (Chlorophyll
a and b). Chlorophyll is considered as a growth parameter as it
correlates to the amount of CO2 fixed using sunlight as the energy
60 source and transformation of that energy into biomass. Chlorophyll
is one of the cellular compounds used for estimating the biomass
Total Lipids
Fatty acid methyl ester (FAME) (%)

Neutral Lipids and high glucose concentration has a significant impact on the
50 chlorophyll pigment contents and productivities in mixotrophic
microalgae (Chl a: 6.389 mg/mg; Chl b: 3.479 mg/mg) (SFig 1).
Substrate degradation (expressed as COD removal efficiency (%))
40
was evaluated to determine the efficient utilization of carbon by
microalgae. Maximum substrate degradation was noticed in the
mixotrophic mode of operation (63.64%) (SFig 2) which might be
30
due to the syntrophic association occurring between the culture
and the microenvironment [41,8].
20
4. Conclusions

10 The experimental methodology illustrated its application for

0
C12:0 C14:0 C14:1 C15:0 C16:0 C16:1 C16:2 C17:0 C17:1 C18:0 C18:1 C18:2 C18:3
Fig. 5. Fatty acid methyl ester (FAME) composition of experimental condition eighth.
process optimization based on selected critical factors of mixed
microalgae biomass productivity, reducing sugars and lipid syn-
thesis. The methodology also facilitated to understand the specific
functional role of eight factors involved in the microalgae
biochemistry for the biosynthesis of carbohydrates and lipids.
Among the eight factors, carbon concentration individually showed
significant influence on the biomass and lipid productivity along
 P. Chiranjeevi, S.V. Mohan / Renewable Energy 98 (2016) 64e71
with reducing sugars synthesis, followed by Nitrates, light, tem-
perature, pH, carbon source, Magnesium, and Phosphates. In
combination carbon concentration and nitrates, temperature and
light and pH with magnesium showed positive influence on the
process performance. The FAME derived from lipids documented
good biodiesel properties with a higher number of saturated fatty
acids. The optimized conditions from experimental output reveal
that mixotrophic mode of cultivation with mixed microalgae
showed enhanced process performance. Bioprocess monitoring
and wastewater treatment showed good agreement with biomass
production. The produced algal biomass can be transformed further
into biocrude along with high valued products by employing HTL
conversion methods which have significant practical relevance.
Acknowledgments
The authors wish to thank Director, CSIR-IICT Hyderabad for
encouragement. Authors acknowledge funding from Council for
Scientific and Industrial Research (CSIR) (BioEn; CSC-0116). PC ac-
knowledges CSIR for providing research fellowship.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.renene.2016.03.063.
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