Appl Biochem Biotechnol

DOI 10.1007/s12010-015-1663-6

Implication of Industrial Waste for Biomass and Lipid

Production in Chlorella minutissima Under Autotrophic,
Heterotrophic, and Mixotrophic Grown Conditions

Kashyap Kumar Dubey 1 & Sudhir Kumar 1 &

Deepak Dixit 1 & Punit Kumar 1 & Dhirendra Kumar 1 &
Arshad Jawed 2 & Shafiul Haque 2,3

Received: 9 March 2015 / Accepted: 7 May 2015

# Springer Science+Business Media New York 2015

Abstract Following the diminishing hopes from the first and second generation biofuels, mainly
due to the limitations of land availability, feed stock requirements, and complicated pre-treatments,
third generation biofuels from microalgae are becoming a priority in the current scenario. The
present study focuses on comparison and optimization of lipid accumulation efficiency in algal
strain Chlorella minutissima grown under autotrophic, heterotrophic, and mixotrophic modes of
nutrition, employing various carbon sources obtained from cheap industrial wastes such as glucose,
acetate, and glycerol. Other pertinent factors such as the effect of various nitrogen sources, effect of
salinity on the cell growth, and lipid accumulations in the algal cells were also studied. The results
suggested that C. minutissima can grow efficiently under autotrophic, heterotrophic, and
mixotrophic modes of nutrition. C. minutissima cells were capable of utilizing other non-popular
carbon sources such as glycerol and acetate collected as waste products from different industries
along with commonly used glucose. The maximum biomass concentration (8.9 g/L) and lipid
content (36.19 %) were found in heterotrophic mode of nutrition. Our findings indicated that
C. minutissima can efficiently utilize these cheaper carbon sources from industrial waste products
for its growth and the production cost of various bioenergy sources can be reduced significantly.

Keywords Chlorella minutissima . Bioenergy . Lipid accumulation . Industrial waste

* Shafiul Haque
shafiul.haque@hotmail.com

1
Microbial Biotechnology Laboratory, University Institute of Engineering and Technology, M.D.
University, Rohtak 124001 Haryana, India
2
Research & Scientific Studies Unit, College of Nursing and Allied Health Sciences, Jazan University,
Jazan 45142, Saudi Arabia
3
Department of Biosciences, Faculty of Natural Sciences, Jamia Millia Islamia (A Central University),
New Delhi 110025, India
 Appl Biochem Biotechnol

Introduction

Constant economic growth combined with rising population has led to a steady increase in
global energy demands [1]. Fossil fuels have become indispensable for economic development
[2, 3]. Biofuel is one of the prime candidates for the replacement of fossil fuels. A plethora of
research has been done on first and second generation biofuels, but no significant outcomes
have been achieved for its production at process level or industrial scale [4]. A prime candidate
for the production of biofuels is microalgae, which are an easy and convenient resource to
produce biofuels with lower impact on the environment and on the world’s food supply when
compared to conventional biofuel production. Microalgae are a group of diverse marine and
freshwater microscopic organisms having the capability of carrying out photosynthesis. The
presence of water, CO2, and other nutrients in their habitat makes them efficient in converting
solar energy into biomass [5, 6]. Their simple structure and absence of supporting structures
make them a good candidate for aquaculture [7]. Microalgal biomass possesses a high caloric
value that makes microalgae more suitable for biofuel than any plant (lignocellulosic)-based
materials [8]. Their inherently high-lipid content, semi-steady state production, sustainability,
and suitability in different culture conditions make them the right candidate for the production
of biofuel [9]. A unique aspect of microalgae is the number of amenable species available for
biofuel production [10]. Different species may be selected for producing biofuels with
variations in the content of fatty acids present [11].
In spite of the interest in the technique, the cultivation of algae on a large scale and
processes for their utilization have successfully been developed in only few countries recently,
while attempts to design an economically viable process are in full swing in some of the
developing countries [12]. Extensive research on different fundamental and applied aspects of
microalgae has been carried out and demonstrated that algal biomass can be used for various
applications such as animal feed, bio-fertilizer, soil conditioner, and as a feed in aquaculture.
Having high efficiency for intracellular accumulation of lipids, microalgae have emerged as
a potent candidate for research in the field of bioenergy production. Microalgae can be easily
grown in a culture in which required essential nutrients such as nitrogen and phosphorus can
be obtained from different sources, e.g., a waste water source, that can keep the production cost
at its minimum and also prove to be environment friendly. Also, microalgae need less space to
grow [13] and have high productivity in contrast with other sources of bioenergy [9]. Earlier
reports indicate that under heterotrophic mode of nutrition, microalgae stimulate elevated
production rate of biomass as well as a conspicuous lipid accumulation intracellularly [14].
Microalgal species such as Chlorella protothecoides can accumulate 18–25 % (w/v) and
55.2 % (w/v) lipid content under autotrophic and heterotrophic mode of cultivation, respec-
tively [15]. Microalgae can be grown under mixotrophic mode of cultivation where the
presence of light and a suitable organic carbon source, such as glucose, molasses, glycerol,
acetate, biomass hydrolysate, etc., is necessary [16].
In order to produce biofuel, a reaction of triglycerides with methanol is needed, which is
popularly known as transesterification reaction. Transesterification process produces methyl
esters of fatty acids, which belong to the category of biodiesel, and glycerol as a byproduct.
The conversion reaction is a multistep reaction where triglycerides are converted into diglyc-
erides as a first step; afterwards, it converts into monoglycerides, and at the end, monoglyc-
erides are converted into glycerol [17]. Biodiesel can be directly used or it can be blended with
fossil fuels in a particular ratio for its utilization as a fuel. Biodiesel with a name B99 has 1 %
fossil fuel, which represses the growth of molds. In addition to the production of biodiesel,
Appl Biochem Biotechnol

microalgae can also provide various types of renewable biofuels [1]. These renewable biofuels
include methane produced by anaerobic digestion of the algal biomass biodiesel derived from
microalgal oil and photo-biologically produced bio-hydrogen. Another advantage of using
microalgae for biodiesel production is that it does not affect the existing product yield from
crops.
The present study focuses on comparison and optimization of the efficiency of lipid
accumulation in algal strain Chlorella minutissima grown under autotrophic, heterotrophic,
and mixotrophic modes of nutrition using different carbon sources such as glucose, acetate,
and glycerol from cheaper industrial wastes. As glycerol and acetate are the waste products of
various industrial processes, these waste products can be used as potent substrates for
utilization in bioenergy production [18, 19]. Also, other relevant factors such as nitrogen
source and salt concentration that affect the growth and accumulation of lipid content in the
microalgal cells have been studied.

Materials and Methods

Microalgal Strains

A strain of unicellular green microalgae C. minutissima was obtained from the national facility
for blue-green algal collections, i.e., Centre for Conservation and Utilization of Blue Green
Algae (CCUBGA), Indian Agricultural Research Institute, New Delhi, India.

Culture Media

Various industrial wastes were used for the growth of microalgae. The industrial wastes were
collected from various industries available in nearby areas of New Delhi, India, viz. sugar
industry, Rohtak (Haryana), India; sugar industry, Panipat (Haryana), India; soap industry,
Rohtak (Haryana), India; Indian Oil Corporation Limited, Faridabad (Haryana), India; and
Panipat refinery, Panipat (Haryana), India. The industrial wastes were clarified and added to
the culture medium at a final concentration of 20 % (v/v). Concentrations of glucose, acetate,
glycerol, and other components were estimated and are presented in Table 1. The effluent
emerging from the final drain line was collected and employed for the study. The effluents
were analyzed for physicochemical properties (Table 1).
The culture media for this study was modified in order to optimize the growth of
microalgae. The culture media was supplemented with different carbon sources, namely,
glucose, glycerol, acetate, and their combinations, according to the experimental conditions
from 0.5 to 10 % (w/v). Yeast extract was used as nitrogen source and adequate concentrations
of inorganic supplements such as KNO3 (1.103 g/L), KH2PO4 (0.075 g/L), K2HPO4 (0.1 g/L),
MgSO4·7H2O (0.5 g/L), Ca(NO3)3·4H2O (0.0675 g/L), FeSO4·7H2O (0.01 g/L), H3BO3
(0.00286 g/L), MnCl2·4H2O (0.00181 g/L), ZnCl2 (0.000105 g/L), Na2MoO4·2H2O
(0.0000030 g/L), CuSO4·5H2O (0.000079 g/L), and CoCl2 (0.000030 g/L) were also used in
the culture media [20]. Various nitrogen sources such as yeast extract (2 % w/v), soya peptone
(2 % w/v), urea (0.3005 g/L), ammonium chloride (0.535 g/L), and potassium nitrate (KNO3,
1.103 g/L) were used for the study of microalgal growth and its efficiency of lipid accumu-
lation. Glycerol assay kit (MAK 117) and acetate colorimetric assay kit (MAK 086) utilized in
the study were procured from Sigma Aldrich, USA. All analytical reagents and other
 Appl Biochem Biotechnol

Table 1 Physicochemical properties of effluents used in this study

Serial Parameters Sugar refinery Soap industry Oil refinery effluent

number effluent effluent

1 Color Dark brown Colorless Pale yellow

2 pH 4.29 7.81 7.06
3 Glucose concentration (w/v) 15.4 % NA NA
4 Acetate concentration (w/v) 0.6 % NA 0.04 %
5 Glycerol concentration (w/v) 0.3 % 0.6 % NA
6 BOD; COD 943 mg/L; 3205 mg/L NA; 3.9 mg/L 11.3 mg/L; 87.3 mg/L
7 Total dissolved solids (TDS) 1380 mg/L 16 mg/L 140 mg/L
8 Sulfates 274 mg/L 350 mg/L 0.4 mg/L
9 Dissolved oxygen 4.8 mg/L 7.2 mg/L NA
10 Oil and grease 12 mg/L NA 4.4 mg/L

NA Not available

chemicals and assay kits utilized in this study were purchased from HiMedia Laboratories,
Mumbai (MS), India and Sigma-Aldrich (USA).

Analytical Methods and Statistical Analysis

The relative concentration of C. minutissima biomass was estimated with a UV-visible

spectrophotometer (Lab India3000+) by measuring the optical density (OD) at 540 nm [19].
Dry cell weight (DCW) of C. minutissima culture broth was estimated by centrifuging at
8000 rpm for 10 min followed by washing the pellet in double distilled water twice and then
drying the pellet at 90 °C temperature until the attainment of constant weight. For the
estimation of glucose concentration, a method of dinitrosalicylic acid assay was employed
[21]; however, acetate and glycerol present in the industrial effluents were estimated using
commercial assay kits procured from Sigma-Aldrich, USA. Lipid concentration was deter-
mined by using the solvent extraction method given by Bligh and Dyer [22]. The statistical
analysis was performed using one-way ANOVA along with Tukey’s method and Student’s t
test. The statistical significance level was maintained as p value <0.05 during the entire analysis.
All experiments in the present study were performed in triplicates and repeated two times.

Results and Discussion

Microalgal Growth Under Different Culture Conditions

In the present study, an alternate method was employed for the production of lipids to be used
as biofuels using cheaper substrates (i.e., medium ingredients like industrial waste products
obtained from various industries—glucose syrup, acetate, and glycerol—for the growth of
microalgae). Figure 1 depicts an integrated process for microalgal growth for various process-
es, e.g., biodiesel production, animal feed, etc.
In order to establish the conditions for the production of lipids, shake flask level study
under autotrophic, mixotrophic, and heterotrophic modes of nutrition was performed in
Appl Biochem Biotechnol

Fig. 1 Integrated process for biofuel production using cheaper substrates (i.e., medium ingredients like industrial
waste products—glucose syrup, acetate, and glycerol—for the growth of microalgae)

500-mL flasks in batch mode conditions. Maximum microalgal growth (1.02 g/L) was
achieved under heterotrophic mode of nutrition, and minimum growth (0.45 g/L) was achieved
under autotrophic mode of nutrition after 72 h. However, mixotrophic growth (0.89 g/L) was
found to be higher than autotrophic but lower than heterotrophic mode of nutrition (Table 2).
The initial concentration of glucose was fixed at 5 g/L for heterotrophic and mixotrophic
modes of nutrition in subsequent experiments (Fig. 2).
Autotrophic cultivation of microalgae was found to be less productive with slow growth
and lower lipid accumulation (20.23 %) capacity. Fermentation of microalgae in heterotrophic
(1.02 g/L) and mixotrophic (0.89 g/L) modes produced higher concentration of biomass as
compared with autotrophic mode (0.45 g/L). Higher productivity was achieved because of the
presence of glucose as an organic carbon source that metabolizes quickly and provides instant
energy to the cell, which could be utilized for the growth and maintenance of cell metabolism.
The highest biomass concentration and productivity was found in heterotrophic cultures.
Glucose was consumed at a faster rate under heterotrophic mode of cultivation in the absence
of light with noticeable growth. However, the lowest biomass was observed in autotrophic
cultures. Lipid content was found to be highest (36.19 %) in heterotrophic cultures of

Table 2 Biomass production and lipid accumulation under autotrophic, heterotrophic, and mixotrophic modes
of nutrition of Chlorella minutissima after 72 h of growth using 4 g/L glucose and 0.5 g/L yeast extract

Product Autotrophic Heterotrophic Mixotrophic

Biomass (g/L) 0.45±0.11 1.02±0.17 0.89±0.14

Lipid content (%) 20.23±04 36.19±02 24.33±03
 Appl Biochem Biotechnol

Fig. 2 Dry cell weight (DCW) (g/L) produced by algae and residual sugar concentration (%) utilized versus
incubation time

microalgae mainly due to limited chlorophyll formation and initiation of lipid accumulation
process. The finding was supported by the color of the culture as it turned into light yellow as
the growth progressed. The biomass concentration and the lipid content of mixotrophic
cultures were observed in between the range of autotrophic and heterotrophic cultures
(24.33 %) as it contained the properties of both autotrophic and heterotrophic cultivations.
During mixotrophic cultivation, cells of microalgae contained chlorophyll at 4.99±1.2 mg/g of
dry weight of biomass, which was lower than that observed under autotrophic mode (28.7±
1.9 mg/g). A similar observation has also been reported in the past [23]. Owing to this reason,
the culture looked light green with respect to the autotrophic culture which was dark green.
Hence, heterotrophic and mixotrophic conditions were preferred over autotrophic conditions
for further studies. A comparative table showing lipid content and lipid and biomass produc-
tivity from various microalgae is listed in Table 3 [1–77].

Effect of Glucose Concentration on Growth and Lipid Accumulation

Glucose is the best carbon source because it is directly used by the cell for cellular metabolism.
It enters in the tricarboxylic acid (TCA) or Krebs cycle directly while other carbon sources are
primarily converted into glucose before entering in the TCA cycle. Glucose provides energy in
very short duration due to its direct breakdown in the cell, which makes glucose as a potential
source of energy. Glucose was used at four different concentrations as carbon source with
concentrations of 0.5, 1.0, 5.0, and 10 % in heterotrophic cultivation of microalgae. At 0.5 %
glucose, it consumed up to 88 % within the first 2 days and the substrate limitation was
observed where no growth took place. At 1 % (w/v) glucose, the consumption of glucose was
Appl Biochem Biotechnol

Table 3 Lipid and biomass productivity of different microalgae [1–77]

Marine and freshwater Lipid content (% dry Lipid productivity Volumetric productivity
microalgae species weight biomass) (mg/L/day) of biomass (g/L/day)

Ankistrodesmus sp. 24.0–31.0 NA NA

Botryococcus braunii 25.0–75.0 NA 0.02
Chaetoceros muelleri 33.6 21.8 0.07
Chaetoceros calcitrans 14.6–16.4/39.8 17.6 0.04
Chlorella emersonii 25.0–63.0 10.3–50.0 0.036–0.041
Chlorella minutissima 28.0–32.0 NA NA
Chlorella minutissima (from this study) 32.46–36.19 NA 1.22–1.38
Chlorella protothecoides 14.6–57.8 12–14 2.00–7.70
Chlorella sorokiniana 19.0–22.0 44.7 0.23–1.47
Chlorella vulgaris 5.0–58.0 11.2–40.0 0.02–0.20
Chlorella sp. 10.0–48.0 42.1 0.02–2.5
Chlorella pyrenoidosa 2 NA 2.90–3.64
Chlorococcum sp. 19.3 53.7 0.28
Crypthecodinium cohnii 20.0–51.1 NA 10
Dunaliella salina 6.0–25.0 116 0.22–0.34
Dunaliella primolecta 23.1 NA 0.09
Dunaliella tertiolecta 16.7–71.0 NA 0.12
Dunaliella sp. 17.5–67.0 33.5 NA
Ellipsoidion sp. 27.4 47.3 0.17
Euglena gracilis 14.0–20.0 NA 7.7
Haematococcus pluvialis 25 NA 0.05–0.06
Isochrysis galbana 7.0–40.0 NA 0.32–1.60
Isochrysis sp. 7.1–33 37.8 0.08–0.17
Monodus subterraneus 16 30.4 0.19
Monallanthus salina 20.0–22.0 NA 0.08
Nannochloris sp. 20.0–56.0 60.9–76.5 0.17–0.51
Nannochloropsis oculata 22.7–29.7 84.0–142.0 0.37–0.48
Nannochloropsis sp. 12.0–53.0 37.6–90.0 0.17–1.43
Neochloris oleoabundans 29.0–65.0 90.0–134.0 NA
Nitzschia sp. 16.0–47.0 NA NA
Oocystis pusilla 10.5 NA NA
Pavlova salina 30.9 49.4 0.16
Pavlova lutheri 35.5 40.2 0.14
Phaeodactylum tricornutum 18.0–57.0 44.8 0.003–1.9
Porphyridium cruentum 9.0–18.8/60.7 34.8 0.36–1.50
Scenedesmus obliquus 11.0–55.0 NA 0.004–0.74
Scenedesmus quadricauda 1.9–18.4 35.1 0.19
Scenedesmus sp. 19.6–21.1 40.8–53.9 0.03–0.26
Skeletonema sp. 13.3–31.8 27.3 0.09
Skeletonema costatum 13.5–51.3 17.4 0.08
Spirulina platensis 4.0–16.6 NA 0.06–4.3
Spirulina maxima 4.0–9.0 NA 0.21–0.25
 Appl Biochem Biotechnol

Table 3 (continued)

Marine and freshwater Lipid content (% dry Lipid productivity Volumetric productivity
microalgae species weight biomass) (mg/L/day) of biomass (g/L/day)

Thalassiosira pseudonana 20.6 17.4 0.08

Tetraselmis suecica 8.5–23.0 27.0–36.4 0.12–0.32

NA Not available

up to 95 % within 5 days, and no further growth was observed due to substrate limitation. The
best growth was observed at 5 % glucose, as a profound amount of carbon source was
available to the cells. Algal cells were capable of utilizing up to 70 % of available glucose
within 7 days; afterwards, the growth became retarded (Fig. 2).
At 10 % (w/v) glucose, the growth was not as much as it was observed in the
culture with 5 % glucose as a carbon source. It was possibly due to catabolite
repression by glucose that hampered the growth of algal cells at higher concentrations.
The utilization of substrate was up to 32 % only. In comparison with the mixotrophic
cultivation, the heterotrophic mode of cultivation had higher efficiency at 5 g/L
concentration of glucose. The dry cell weight of microalgae at different concentrations
of glucose is presented in Fig. 2. Similarly, the carbon source utilization by the
microalgae with respect to time is depicted in Fig. 2.
The quantification of lipids accumulated in microalgal cultures at various concentrations of
glucose indicated that the lipid accumulation process was directly proportional to the glucose
concentration. At lower concentrations of glucose (i.e., 0.5 and 1.0 %) in the culture media, the
accumulated lipid was less (33.2 and 33.4 %, respectively) in amount as compared to the
higher concentrations of glucose. The lipid content accumulated in cultures of microalgae
under various concentrations of glucose is depicted in Fig. 3. The maximum biomass
production was found at 5 % glucose concentration (Figs. 1 and 4).

Fig. 3 Lipid content (%) accumulated under different glucose concentrations (%)
Appl Biochem Biotechnol

Fig. 4 DCW (g/L) produced by algae and lipid content (%) accumulated in heterotrophic and mixotrophic
modes under different carbon sources and their combinations

Effect of Glycerol on Growth and Lipid Accumulation

Glycerol was used as a carbon source at two different concentrations, i.e., 0.5 % glycerol and
glycerol along with glucose in a 1:4 ratio at a final concentration of 0.5 %, and both were
compared with same concentrations of glucose. Microalgae were cultivated under both
heterotrophic and mixotrophic modes of nutrition. The biomass produced using glycerol as
a carbon source is shown in Fig. 4. The low biomass production using glycerol as a carbon
source is possibly due to the inhibitory effect of glycerol [1] on the growth of microalgae,
while the lipid content of the microalgae was increased probably due to adverse conditions due
to diminution in anabolism of photosynthetic pigments of cells [23]. The highest biomass
concentration in both heterotrophic and mixotrophic modes of nutrition was observed in
conditions of glucose/glycerol (1:4) as a carbon source, while lipid concentration was maxi-
mum when glucose was used as a carbon source in both heterotrophic and mixotrophic modes
of nutrition. Figure 6a shows the lipid content using various concentrations of various carbon
sources.

Effect of Acetate on Growth and Lipid Accumulation

Acetate was also tested as a potential carbon source at two different concentrations, i.e., 0.5 %
acetate and acetate along with glucose (1:4) with a final concentration of 0.5 %, and compared
with the same concentration of glucose. Microalgae were cultivated in both heterotrophic and
mixotrophic mode of nutrition as performed earlier (Fig. 4). The highest biomass was obtained
under glucose/sodium acetate (1:4) carbon source conditions in both heterotrophic and
mixotrophic modes of nutrition, but the lipid content was found to be highest in the presence
 Appl Biochem Biotechnol

of glucose. The lipid content accumulated from the culture grown under various concentrations
of acetate is shown in Fig. 4. Interestingly, sodium acetate was found better than glycerol for
biomass production; however, the lipid content was a little higher under glycerol carbon
source. Hence, our results suggest that acetate is a better substrate for growth of microalgae
than glycerol.

Effect of Different Nitrogen Sources on Growth and Lipid Accumulation

C. minutissima was grown under conditions of different nitrogen sources such as potassium
nitrate (KNO3− 1.103 g/L), ammonium chloride (0.535 g/L), urea (0.3005 g/L), yeast extract
(2 % w/v), and soya peptone (2 % w/v) with 5 % glucose as a carbon source (Fig. 5). The
cultivation of microalgae was performed under heterotrophic mode of nutrition with fixed
concentration of other nutrients. The highest biomass (~9.7 g/L) was obtained using yeast
extract, and the lowest biomass (8.54 g/L) was found with ammonium chloride. However, the
highest lipid content (36.19 %) was obtained using soya peptone, and the lowest lipid content
(32.46 %) was obtained using ammonium chloride. The best nitrogen source was found to be
soya peptone as it gave the highest lipid and biomass (9.6 g/L), and this was slightly less than
the biomass produced using yeast extract (9.7 g/L). Overall lipid accumulation in the cultures
using soya peptone was higher (3.48 g/L) than the culture with yeast extract (3.42 g/L).

Effect of Salinity on Growth and Lipid Accumulation in Microalgae

Salinity also plays an important role in biomass production and lipid accumulation in the algal
cells. C. minutissima is also affected with salinity as it is a marine microalgae and has the
ability to grow under saline conditions. In this experiment, the algal cells were cultivated under
1.5 % NaCl in the culture media and another media having all the components in equivalent
amount except NaCl. The experiment was performed under the heterotrophic and mixotrophic
modes of nutrition using various carbon sources such as glucose, glycerol, and sodium acetate.

Fig. 5 DCW (g/L) produced by algae and lipid content (%) accumulated in the presence of different nitrogen
sources
Appl Biochem Biotechnol

Fig. 6 a, b DCW of microalgae and percent lipid content accumulation, respectively, in heterotrophic and
mixotrophic mode of nutrition under various carbon sources having different concentrations with 1.5 % NaCl and
without NaCl

Glucose, glycerol, and sodium acetate were used at 0.5 % concentration, and glucose with
glycerol and sodium acetate were used in the ratio of 1:4.
The highest biomass concentration (1.5 g/L) was found when microalgae were grown under
heterotrophic mode of nutrition using 1.5 % NaCl and glucose along with sodium acetate (1:4)
with a final concentration of 5.0 g/L (Fig. 6a); however, the highest lipid content was found
using glucose with 1.5 % NaCl in the media (Fig. 6b).

Conclusion

In conclusion, C. minutissima can grow in autotrophic, heterotrophic, and mixotrophic modes

of nutrition, and it has the capacity to accumulate lipid in its cells. Heterotrophic mode of
nutrition was found to be the best cultivation condition for biomass production and lipid
accumulation, whereas autotrophic mode of nutrition showed least growth and resulted in
 Appl Biochem Biotechnol

lower biomass and lipid accumulation capacity inside the algal cells. Biomass productivity and
lipid accumulation were lower under mixotrophic mode of nutrition than heterotrophic mode;
however, both were higher under mixotrophic mode of nutrition than autotrophic mode of
nutrition. Although mixotrophic mode of nutrition is less efficient than heterotrophic mode, it
proves more beneficial because CO2 produced during respiration is utilized by the cells for
photosynthesis. In this study, the best carbon source was found to be glucose in comparison to
glycerol and acetate as it is directly utilized by the cells. The use of glucose syrup does not
produce higher lipid content and thus reduces the cost effectiveness and economic feasibility if
the process is to be taken to a larger scale. In order to avoid higher production cost, cheap
carbon sources, such as glycerol and acetate, can be used as glycerol is the waste product of
various industrial processes and acetate is obtained during anaerobic digestion of organic
matter. Although the productivity is low, these carbon sources prove cheaper than glucose and
are cost effective. Also, the use of minor concentration of glucose along with glycerol and
acetate as chief carbon sources can give enhanced production of biomass and higher accumu-
lation of lipid.
Many reports have been published on the work dealing with microalgae for biofuel
production, including C. minutissima, but most of them were focused on commonly developed
conditions and media components. Here, in this study, we have used cheaper sources of carbon
from industrial wastes and recovered competent yields. Also, here we have used media
supplemented with saline with a vision to utilize the strain and the culture conditions in waste
water laden with high salts.

Acknowledgment The authors sincerely acknowledge the Microbial Biotechnology Laboratory, University
Institute of Engineering and Technology, M.D. University, Rohtak, Haryana, India for providing the laboratory
facility for this research study.

Ethical Statement The authors confirm that the present manuscript complies with the ethical rules applicable
for this journal.

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