Appl Biochem Biotechnol

DOI 10.1007/s12010-014-1041-9

Low-Cost Production of Green Microalga Botryococcus

braunii Biomass with High Lipid Content
Through Mixotrophic and Photoautotrophic Cultivation

Chittra Yeesang & Benjamas Cheirsilp

Received: 1 May 2014 / Accepted: 26 June 2014

# Springer Science+Business Media New York 2014

Abstract Botryococcus braunii is a microalga that is regarded as a potential source of

renewable fuel because of its ability to produce large amounts of lipid that can be converted
into biodiesel. Agro-industrial by-products and wastes are of great interest as cultivation
medium for microorganisms because of their low cost, renewable nature, and abundance. In
this study, two strategies for low-cost production of B. braunii biomass with high lipid content
were performed: (i) the mixotrophic cultivation using molasses, a cheap by-product from the
sugar cane plant as a carbon source, and (ii) the photoautotrophic cultivation using nitrate-rich
wastewater supplemented with CO2 as a carbon source. The mixotrophic cultivation added
with 15 g L−1 molasses produced a high amount of biomass of 3.05 g L−1 with a high lipid
content of 36.9 %. The photoautotrophic cultivation in nitrate-rich wastewater supplemented
with 2.0 % CO2 produced a biomass of 2.26 g L−1 and a lipid content of 30.3 %. The benefits
of this photoautotrophic cultivation are that this cultivation would help to reduce accumulation
of atmospheric carbon dioxide and more than 90 % of the nitrate could be removed from the
wastewater. When this cultivation was scaled up in a stirred tank photobioreactor and run with
semi-continuous cultivation regime, the highest microalgal biomass of 5.16 g L−1 with a
comparable lipid content of 32.2 % was achieved. These two strategies could be promising
ways for producing cheap lipid-rich microalgal biomass that can be used as biofuel feedstocks
and animal feeds.

Keywords Botryococcus braunii . Lipid . Mixotrophic . Photoautotrophic . Wastewater

Introduction

Botryococcus braunii is a microalga that is regarded as a potential source of renewable fuel

because of its ability to produce large amounts of lipid >75 % of dry cell weight. Today, the
common procedure for the cultivation of microalgae is photoautotrophic culture. In the
photoautotrophic culture, the cells harvest light energy and use CO2 as a carbon source. Such

C. Yeesang : B. Cheirsilp (*)

Department of Industrial Biotechnology, Faculty of Agro-Industry, Prince of Songkla University,
Hat-Yai 90112, Thailand
e-mail: [email protected]
 Appl Biochem Biotechnol

an approach could contribute to solving a number of environmental problems. These include

reducing atmospheric CO2, and their high lipid content might help solve any future crises due
to the shortage of energy sources [1]. However, the problem dealing with the photoautotrophic
culture is the limited biomass production due to cellular self-shading that hinders light
availability. The low biomass concentration obtained by this photoautotrophic culture in-
creases the biomass harvesting cost. An alternative culture that would give higher biomass
production is a heterotrophic culture in which organic carbons such as sugars and organic acids
are used in the absence of light. This heterotrophic culture can be performed in conventional
microbial bioreactors. Thus, it is much easier to alter conditions to improve the yield of
biomass and reduce the cost of microalgal biomass production. Mixotrophic cultivation, a
variant of the heterotrophic cultivation, is another strategy to improve the efficient use of light
or eliminate its requirement by cells. In the mixotrophic culture, CO2 and organic carbons are
simultaneously assimilated and both respiratory and photosynthetic metabolism operates
concurrently, thus leading to an additive or synergistic effect of the two processes [2].
In addition to conventional microalgae culture systems, the ability of microalgae to assim-
ilate inorganic nitrogen into their biomass also allows their potential use in nitrogen removal
from the wastewater [3]. In primary wastewater treatment, the settled and floating materials are
removed. However, wastewater may still contain high levels of nitrogen and phosphorus when
it is discharged in secondary treatment. On the one hand, these compounds need to be removed,
and on the other hand, they are suitable and cost-effective for microalgae cultivation.
Microalgae are highly effective in wastewater treatment for two reasons. They could use the
inorganic nitrogen and phosphorus in the wastewater for their growth, and they also purify the
wastewater by producing oxygen and removing heavy metals and xenobiotic substances [4].
Since the major barrier to the commercialization of algae-derived biofuels is the high production
cost, there is a need to develop the low-cost production process for microalgae cultivation. The
incorporation of such microalgal systems into conventional wastewater treatment would offer
the combined advantages of treating the wastewaters and simultaneously producing microalgal
biomass that could be applied to the production of biofuel [5, 6] and animal feeds [7].
A B. braunii TRG strain with high lipid content has been previously isolated from a lake in
Trang province, Thailand [8]. The present study focused on evaluating the production of
biomass and lipid by this TRG strain under photoautotrophic, heterotrophic, and mixotrophic
conditions. Two strategies for economical production of microalgal biomass and lipid were
performed: the mixotrophic cultivation using molasses, a cheap by-product from the sugar
cane plant as a carbon source, and the photoautotrophic cultivation using nitrate-rich waste-
water supplemented with CO2 as a carbon source. Finally, batch and semi-continuous culti-
vation of this microalga was scaled up in a stirred tank photobioreactor.

Materials and Methods

Microalgae

A B. braunii green microalga TRG strain with a high lipid content was isolated from lake in
Trang province, Thailand, in 2008 [8].

Medium Preparation

The medium used in this study was a modified Chu 13 medium which contained 0.2 g KNO3,
0.04 g K2HPO4, 0.1 g MgSO4 ·7H2O, 0.054 g CaCl2 ·2H2O, 0.01 g Fe citrate, 0.1 g citric acid,
Appl Biochem Biotechnol

0.036 g NaHCO3, and 1 mL of microelement solution per liter, at pH 6.7. For the microele-
ment solution, 1 L contained 2.85 g H3BO3, 1.8 g MnCl2 ·4H2O, 0.02 g ZnSO4 ·7H2O, 0.08 g
CuSO4 ·5H2O, 0.08 g CoCl2 ·6H2O, and 0.05 g Na2MoO4 ·2H2O [9]. The molasses used in
this study were kindly provided from a sugar cane plant in Songkhla province (Thailand). It
contained 62 % of total sugar. All chemicals and solvents used were reagent grade and
obtained from various suppliers.

Wastewater from Seafood Processing Plant

The wastewater used in this study was the secondarily pretreated wastewater from activated
sludge pond in a CP seafood processing plant (Songkhla, Thailand). The secondarily
pretreated wastewater was filtered through a mesh for discharging suspension solids and pH
adjusted at 6.7 before sterilization in an autoclave at 121 °C for 15 min.

Culture Conditions

The microalgae was inoculated in 100 mL of modified Chu 13 medium and incubated at 25 °C
with agitation at 125 rpm under an optimum intense illumination of 49.5 μmol photon m−2 s−1
with a 16:8-h light and dark cycle for the photoautotrophic condition. For the mixotrophic and
heterotrophic condition, glucose was initially added as a carbon source at a concentration of
5 g L−1 with and without light, respectively. The effect of sugar concentration was studied in
mixotrophic cultivation by varying its concentration in the range of 3–15 g L−1 for glucose and
5–20 g L−1 for molasses. The effect of carbon dioxide concentration was investigated using a
two-tier culture vessel consisting of two 250-mL small-neck Erlenmeyer flasks. The lower
compartment of the flask contained 100 mL of a 3-M buffer mixture (KHCO3/K2CO3) at
specific ratios which generated a specific CO2 partial pressure in the two-tier flask. The
concentration of buffer mixture was changed to obtain a CO2 partial pressure of 0.5, 1.0,
and 2.0 % (v/v) as provided by Tripathi et al. [10].
The batch and semi-continuous cultivation was performed in a 2-L stirred tank
photobioreactor with 1.5-L working volume. The cultures were incubated at 25 °C and agitated
at 125 rpm under the optimum intense illumination of 49.5 μmol photon m−2 s−1 with a 16:8-h
light and dark cycle. The pH of the culture was controlled at 6.7 by the injection of CO2 in
response to signals from a pH sensor. The biomass concentration and lipid content were
estimated daily. In the semi-continuous cultivation, 500 mL of medium was withdrawn and
replaced with the same volume of fresh medium at 4- or 6-day intervals.

Analytical Methods

The secondarily pretreated wastewater from the seafood processing plant was characterized
based on pH, chemical oxygen demand (COD), ammonia (phenate method), total nitrogen
(Kjeldahl method), and nitrate (cadmium reduction method) according to the standard methods
[11]. The total sugar and reducing sugar in the broth were determined by the anthrone method
and DNS method [12], respectively.
The cells were harvested by centrifugation at 1,585×g for 15 min. The cell pellets were
freeze-dried by freeze-drier (Type Labvista, FTS Duradry Freeze Dryer System, Triad Scien-
tific Ltd., USA). Freeze-dried alga (100 mg) was extracted with 50 mL of n-hexane and
sonicated with sonicator (Model Transsonic 460/H, εlma Ltd, Germany) at room temperature
[13]. The extraction process was repeated two times. The combined suspensions were filtered
through Whatman No. 40 filter paper and the filtrate was placed in a preweighed glass vial.
 Appl Biochem Biotechnol

The hexane solution was evaporated to dryness at 30 °C under vacuum. The lipid content was
measured gravimetrically and expressed as a dry weight percentage. The specific growth rate
(μ) was calculated from the slope of the following equation:
C
Ln ¼ μdt ð1Þ
C0
where C0 is the initial biomass concentration (g L−1) and C is the biomass concentration
(g L−1) at any time t [14].
The data are presented as a mean value with standard deviations (mean±SD) from at least
duplicate trials. The statistical significance of the results was evaluated by one-way analysis of
variance (ANOVA) and Duncan’s multiple range tests (P<0.05) using SPSS 10 software.

Results and Discussion

Biomass and Lipid Production by B. braunii TRG Strain Under Various Culture Conditions

The biomass and lipid production by B. braunii TRG strain was studied under photoautotro-
phic, heterotrophic, and mixotrophic conditions (Fig. 1). The glucose was used as a carbon
source for the heterotrophic and mixotrophic conditions. Among the three culture conditions
tested, the mixotrophic condition gave the fastest growth with the highest specific growth rate
of 0.195 day−1, followed by the heterotrophic condition (0.115 day−1) (Table 1). TRG strain
grew slowly under the photoautotrophic condition with the lowest specific growth rate
(0.093 day−1). The highest biomass of 2.46 g L−1 with the highest lipid content of 37.5 %
was obtained under the mixotrophic condition. The lipid productivity during this mixotrophic
growth was then highest at 64.5 mg L−1 day−1 which was 2.7-fold and 1.4-fold of those during
the photoautotrophic and heterotrophic growth, respectively (Table 1). This indicated that the
organic carbon source added into the culture did positively affect lipid accumulation of TRG
strain. In the absence of light under the heterotrophic condition, the use of a suitable carbon
source is crucial for attaining a high biomass yield in algal culture. Among organic and
inorganic carbon sources, glucose and acetate have been most commonly used for the
heterotrophic cultivation of green microalgae such as Chlorella zofingiensis [15] and
Haematococcus pluvialis [16]. A culture of Chlorella protothecoides grown heterotrophically
produced a 3.4 times higher yield than that grown photoautotrophically [17]. Oh et al. [18] also
indicated that it is necessary to apply an organic carbon source to enable the heterotrophic
growth of Porphyridium cruentum under the complete dark conditions.
It has been reported that a Botryococcus strain does utilize an exogenous carbon source for
improved growth and lipid production. Various carbon sources, including C1-C6 compounds
and disaccharide (lactose, sucrose), were screened in attempts to decrease the mass doubling
time of this alga [19]. Zhang et al. [20] reported that B. braunii grew faster in the mixotrophic
cultivation with various organic carbon sources than it did in the photoautotrophic mode. The
results in this study are also in agreement with those using other microalgae strains [12]. Under
mixotrophic condition, the presence of organic substrates means that the cell growth is not
strictly dependent on photosynthesis, and hence, light is no longer an indispensable growth
factor. It has been reported that the microalgae growth in mixotrophic culture was stimulated
during the light phase in media supplemented with glucose, and there was also less biomass
loss during dark period [21–23].
In the present study, TRG strain produced the highest biomass and lipid when both light
and organic carbon simultaneously present as energy sources in the mixotrophic culture.
Appl Biochem Biotechnol

4.0
Mixotrophic
Heterotrophic
Photoautotrophic

Dry cell weight (g/L)

3.0
Aa Aa
Ab

2.0 Aa Ba
Ac
Bab
Ba
Bb Cab
1.0 Bb
Bc

0.0

50
Mixotrophic
Heterotrophic
40 Photoautotrophic
Ab Aa
Lipid content (%)

Ac Ba
30 Ba
Ad
Bb Cab Ca

20 Bc
Bb
Bc
10

0
0 5 10 15 20 25
Time (days)

Fig. 1 Growth and lipid content of TRG strain cultivated under photoautotrophic, heterotrophic, and
mixotrophic conditions. Different uppercase and lowercase letters on the symbols indicate significant differences
between treatments and cultivation time, respectively

However, they formed less chlorophyll content resulting in yellowish cells in comparison to
the photoautotrophic culture. Heterotrophic and photosynthetic growths have been reported to
occur simultaneously and independently in mixotrophic cultures. However, the presence of
organic carbon could alter the photosynthetic metabolism and decrease the production of
photosynthetic pigments [22–25]. Similarly, Xu et al. [26] showed that the lipid-soluble
compounds from photoautotrophic cells appeared as blackish green cells with chlorophyll
and carotenoid as the major components. On the other hand, the lipid-soluble compounds from
the heterotrophic cells appeared as light yellow grease.

Mixotrophic Cultivation of TRG Strain Using Glucose and Molasses as Carbon Sources

The results from the previous experiment showed that TRG strain grew best under mixotrophic
condition using glucose as a carbon source (Fig. 1). Therefore, the effect of initial glucose
 Appl Biochem Biotechnol

Table 1 Effect of culture conditions on the production of biomass and lipid by TRG strain

Culture condition Specific Maximum dry Maximum Lipid Residual sugar

growth rate cell weight lipid content productivity (g L−1)/nitrate
(day−1) (g L−1) (%) (mg L−1 day−1) (mg L−1)

Effect of various culture conditions

Photoautotrophic 0.093 1.14 25.1 24.1 –
Heterotrophic 0.115 1.75 29.3 46.7 2.29
Mixotrophic 0.195 2.46 37.5 64.5 1.17
Effect of initial glucose concentration (g L−1) in mixotrophic culture
3 0.136 1.17 32.7 54.1 0.00
5 0.195 2.46 37.5 64.5 1.17
10 0.221 2.76 27.6 59.9 2.58
15 0.107 1.57 27.7 34.4 10.5
Effect of initial molasses concentration (g L−1)a in mixotrophic culture
5 0.111 1.83 29.8 32.5 5.50
10 0.124 2.02 30.8 47.6 5.71
15 0.226 3.05 36.9 67.7 10.3
20 0.123 2.10 35.1 45.9 18.1
Effect of initial nitrate concentration (mg L−1) in the effluent
6.0 0.022 0.84 5.8 4.61 0.00
12.0 0.033 0.87 8.2 7.96 0.72
18.0 0.046 0.95 16.2 10.7 3.38
24.1 0.059 1.16 17.6 15.7 4.21
Effect of CO2 concentration (%) supplemented in the effluent
0.03 0.059 1.16 17.6 15.7 4.21
0.5 0.078 1.67 20.7 17.8 3.31
1.0 0.114 1.76 23.8 25.4 2.64
2.0 0.122 2.26 30.3 45.5 2.17
Effect of culture mode in a photobioreactor
Batch 0.189 2.66 31.0 69.1 4.32
Semi-continuousb 0.366 5.16 32.2 118.5 11.8–15.1c
a
The values are the initial sugar concentrations
b
The values are the data during the 19th–23rd day
c
The residual nitrate concentration before replacing with fresh effluent medium at 4–6 days of intervals

concentration on growth and lipid content of TRG strain under the mixotrophic condition was
further investigated (Fig. 2). The growth of TRG strain increased when the concentration of
glucose increased from 3 to 10 g L−1 (Fig. 2a–c). A further increase in glucose concentration
did not encourage the growth of this strain. At 10 g L−1 glucose, the microalgae grew best with
the highest specific growth rate of 0.221 day−1 and reached the maximum biomass concen-
tration of 2.76 g L−1. The lipid content also increased from 32.7 to 37.5 % when the glucose
concentration increased from 3 to 5 g L−1, but decreased to 27.6 % with further increase of
glucose concentration of up to 10 g L−1 (Fig. 2 and Table 1). Although the biomass at 5 g L−1
glucose (2.46 g L−1) was slightly lower than that at 10 g L−1 glucose (2.76 g L−1), its higher
lipid content (37.5 %) resulted in higher lipid productivity (64.5 mg L−1 day−1) compared to
Appl Biochem Biotechnol

20 4 80
(A) Glucose 3 g/L (B) Glucose 5 g/L
15 Dry cell weight (g/L) 3 60

Lipid content (%)

Aa Aa
Sugar (g/L)

Bb
10 Aa 40
2 Ba Ba Aa
Cc
Cb Cb
Ca Ca Ca Ca
5 1 Dc 20
Dc

0 0 0

20 4 80
(C) Glucose 10 g/L (D) GlucoseTime (days)
15 g/L
Dry cell weight (g/L)

15 3 Aa 60

Lipid content (%)

Bc ABb
Sugar (g/L)

10 2 Cd 40
Ca Ca Ca
Ca Ca Ca
Db Ca Ca
Db
5 1 Db 20
Db

0 0 0
0 0 5 10 15 20 25 0 5 10 15 20 25
Time (days) Time (days)

Fig. 2 Effect of initial glucose concentration on growth (open circle), lipid content (filled circle), and glucose
consumption (open triangle) of TRG strain under mixotrophic condition. The initial glucose concentrations were
3 g L−1 (a), 5 g L−1 (b), 10 g L−1 (c), and 15 g L−1 (d). Different uppercase and lowercase letters on the symbols
indicate significant differences between initial glucose concentrations and cultivation time, respectively. The
statistical significance of the changes in sugar concentration was not evaluated because of using different initial
sugar concentrations

that at 10 g L−1 glucose (59.9 mg L−1 day−1). At the highest glucose concentration tested
(15 g L−1), the glucose remained at about 5 g L−1. The biomass and lipid productivity were
lowest at 1.57 g L−1 and 34.4 mg L−1 day−1, respectively (Fig. 2d and Table 1). The possible
reason for this phenomenon would be because a high concentration of glucose could result in a
high osmotic pressure, and this had a negative effect on cell growth and lipid production of the
microalgae. Mallick et al. [27] found that the optimal glucose concentration for mixotrophic
cultivation of Chlorella vulgaris was 0.7 % (7 g L−1), and at higher glucose concentration, the
biomass became lower.
In order to reduce the production cost of lipid, molasses, a cheaper source of sugar than
glucose, was then used as a carbon source in the mixotrophic cultivation of TRG strain. The
molasses was diluted to vary the initial sugar concentration in the range of 5–20 g L−1. Figure 3
illustrates the influence of the molasses concentration on the growth and lipid content of TRG
strain. The biomass and lipid content increased markedly with increasing sugar concentration
from 5 to 15 g L−1 and tended to level off at a sugar concentration higher than 15 g L−1. At
15 g L−1 sugar concentration, the highest biomass of 3.05 g L−1 with a high lipid content of
36.9 % was achieved, and this gave the highest lipid productivity of 67.7 mg L−1 day−1
(Table 1). An increase in sugar concentration of up to 20 g L−1 decreased the cell growth, lipid
 Appl Biochem Biotechnol

content, and hence the lipid productivity of this strain. This might be due to the inhibition
effect of substrate at high concentration. In addition, due to the dark color of the molasses
medium, the light may have hardly penetrated and its intensity attenuated drastically, espe-
cially when the molasses concentration was increased. At this point, it was possible that the
growth in the medium containing molasses was largely due to the heterotrophic metabolism of
sugar rather than the photoautotrophic metabolism.
It should be noted that the consumption of glucose (Fig. 2) by TRG strain was faster than
that of molasses (Fig. 3). This could be because glucose is one of the final products of
photosynthesis; it may be assumed that any photosynthetic microorganism must be able to
incorporate it in its metabolism. In this study, it was obvious that the supplementation of
glucose or molasses led to a significant improvement not only in the cell growth but also in the
lipid content of the TRG strain. It has been reported that molasses contains trace amounts of
vitamins and significant amounts of several minerals such as calcium, magnesium, potassium,
and iron. Chen and Chou [28] reported that molasses contained a high amount of iron at
2.39 mg g−1 by dry weight. The positive effect of iron on the lipid content of the microalgae
has been previously reported [8, 29]. Therefore, it can be concluded that molasses is a good
candidate for being used as an alternative cheap carbon and mineral sources for the
mixotrophic cultivation of the microalgae.

20 4 80
(A) Molasses 5 g/L (B) Molasses 10 g/L
Dry cell weight (g/L)

15 3 60

Lipid content (%)

Sugar (g/L)

Ca
10 2 CDa CDa CDb 40
CDb
Db Ec Ba
Ec Ba Ba
Ba Ba
Cb
5 1 Cb 20
Dc

0 0 0

20 4 80
(C) Molasses 15 g/L (D) Molasses
Time20(days)
g/L
Aa
Dry cell weight (g/L)

15 3 Bb Bb 60
Lipid content (%)

Ca
Sugar (g/L)

CDa Ca
10 2 Aa 40
Aa Aa Db Aa
Dc
Bb

Cb BCbc
5 1 20
Dd

0 0 0
0 0 5 10 15 20 25 0 5 10 15 20 25
Time (days) Time (days)

Fig. 3 Effect of initial molasses concentration on growth (open circle), lipid content (filled circle), and sugar
consumption (open triangle) of TRG strain under mixotrophic condition. The initial sugar concentrations were
5 g L−1 (a), 10 g L−1 (b), 15 g L−1 (c), and 20 g L−1 (d). Different uppercase and lowercase letters on the symbols
indicate significant differences between initial molasses concentrations and cultivation time, respectively. The
statistical significance of the changes in sugar concentration was not evaluated because of using different initial
sugar concentrations
Appl Biochem Biotechnol

Photoautotrophic Cultivation of TRG Strain Using Nitrate-Rich Wastewater from Seafood

Processing Plant

Another important advantages of microalgae is that they can grow photosynthetically. Thus, no
organic carbon source is required for growth, and any carbon dioxide released on combustion
could be fixed by their photoautotrophic ability. This process then greatly contributes to a
reduction in greenhouse gases and global warming. In addition, microalgae could also remove
inorganic compounds, mainly nitrogen and phosphorus, in the wastewater. Therefore, the
feasibility of cultivating TRG strain in the secondarily pretreated wastewater from the seafood
processing plant was studied. Carbon dioxide in the air (normally about 0.03 %) and the
complex organic carbon in the wastewater might be used as a carbon source. The character-
istics of the secondarily pretreated wastewater were as follows: chemical oxygen demand
(COD) 740 mg L−1, NO3−-N 24.1 mg L−1, NH4+-N 4.6 mg L−1, and NO2−-N 0.04 mg L−1. It
can be seen that the amount of nitrite and ammonium ion in the pretreated wastewater was very
low. Therefore, only nitrate would be used as the main nitrogen source for the microalgae
growth. To assess this, its concentration was monitored during the cultivation process.
The secondarily pretreated wastewater was diluted to obtain different levels of the initial
nitrate concentration. The results were compared with that using undiluted wastewater (the
initial concentration of nitrate was 24.1 mg L−1) (Fig. 4). The undiluted wastewater gave the
highest biomass concentration of 1.16 g L−1 with a lipid content of 17.6 % (Fig. 4d and
Table 1). The dilution of the wastewater reduced the growth and lipid content of TRG strain.
This could be due to the fact that a lower nitrate concentration in the wastewater would induce
a lower growth rate of the microalgae. When the wastewater was diluted, the lipid content was
also as low at 5–8 % and tended to decrease during cultivation likely due to the utilization of
storage lipid as reserve energy. It has been reported that it is common for oleaginous
microorganisms to reserve lipids during the growth phase and to degrade them under nutrient
starvation phase [30]. Thus, the supply of a sufficient level of nitrate is required for high cell
density and lipid production. It should be noted that the biomass concentration of TRG strain
in the wastewater was as low as that obtained in the photoautotrophic culture (Fig. 1). This
indicated that the cell growth of TRG strain in the wastewater was likely due to the
photoautotrophic growth. Only the CO2 in the air was used as a carbon source, but not the
contents of the wastewater.
At an initial nitrate concentration of less than 12 mg L−1 (Fig. 4a, b), the nitrate concen-
tration gradually decreased after 8 days of cultivation and was almost completely depleted at
the end of cultivation. At an initial nitrate concentration higher than 12 mg L−1 (Fig. 4c, d), the
nitrate concentration rapidly decreased during the growth phase of the microalgae. From then,
it slowly decreased and finally remained in the wastewater at a concentration of 4.21 mg L−1.
The results indicated that more than 80 % of the initial nitrate was removed by the growth of
TRG strain. In a similar way, An et al. [3] demonstrated that 80 % of the initial nitrate in a
secondarily treated piggery wastewater was removed by the green alga B. braunii UTEX 572.
Tansakul et al. [9] also reported that B. braunii grew well in an adjusted wastewater from a
seafood processing plant and reduced nitrate and phosphate concentration in the wastewater by
90 and 56 %, respectively.
To promote the biomass and lipid production by TRG strain, the availability of CO2 was
increased by performing the cultivation in a two-tier flask supplemented with CO2 (Fig. 5).
TRG strain grew better after being supplemented with CO2 (0.5–2 %) compared with using
normal air (0.03 % CO2 concentration). Without the supplementation of additional CO2, the
specific growth rate was lowest at 0.059 day−1, and this was significantly increased up to 0.114
and 0.122 day−1 by supplementation with CO2 up to 1 and 2 %, respectively (Table 1). The
 Appl Biochem Biotechnol

40 1.50 40
(A) Nitrate 6 mg/L (B) Nitrate 12 mg/L
Dry cell weight (g/L)
30 1.25 30

Lipid content (%)

Nitrate (mg/L)

20 1.00 20
Ca Ca Ca Ca Ca Ca
CDab Ca Ca
Db
10 0.75 Ba Ba 10
Ca Cb
Db Db Db Db Dc Dc

0 0.50 0

40 1.50 40
(C) Nitrate 18 mg/L (D) Nitrate 24.1
Timemg/L
(days)
Dry cell weight (g/L)

30 1.25 Aa 30
Nitrate (mg/L)

Lipid content (%)

Bb Bb Bb
BCa
20 1.00 Ca Ca Ca Cc Aa 20
Aa Aa Aa

Aa CDa
Bb ABab
10 0.75 Bb Bb Bb 10

0 0.50 0
0 5 10 15 20 25 0 5 10 15 20 25
Time (days) Time (days)

Fig. 4 Effect of initial nitrate concentration on growth (open circle), lipid content (filled circle), and nitrate
consumption (open triangle) of TRG strain cultivated in the secondarily pretreated wastewater from seafood
processing plant. The initial nitrate concentrations were 6.0 mg L−1 (a), 12.0 mg L−1 (b), 18.0 mg L−1 (c), and
24.1 mg L−1 (d). Different uppercase and lowercase letters on the symbols indicate significant differences
between initial nitrate concentrations and cultivation time, respectively. The statistical significance of the changes
in nitrate concentration was not evaluated because of using different initial nitrate concentrations

biomass was also increased up to 1.76 and 2.26 g L−1, respectively. This enhancement was due
to the enrichment of available CO2 as a carbon source. This result was in agreement with the
previous studies reporting that the cultivation of microalgae supplemented with CO2 resulted
in a high cell density [31, 32]. Rao et al. [33] also found that CO2 at a level of 2.0 % in a two-
tier flask enhanced the growth and carotenoid contents of all B. braunii strains tested. In the
present study, it was found that the color of the cells was a darker green, likely due to an
increase in the chlorophyll content when the CO2 was increased up to 2 %.
The lipid content of TRG strain also increased with an increased CO2 concentration. The
maximum lipid content of 30.3 % was obtained at 2 % CO2 followed by 1 % CO2 (23.8 %)
and 0.5 % CO2 (20.7 %). The highest lipid productivity of 45.5 mg L−1 day−1 was also
obtained at 2 % CO2 (Table 1). It has been reported that photosynthetic cultures of
Botryococcus require CO2 for improved growth and lipid production [19]. However, the
results in this study are not consistent with the previous studies of Chiu et al. [31] who found
that the lipid content of Chlorella sp. did not respond to the CO2 concentration. Such divergent
results for the lipid content of microalgae cultured under CO2 aeration are most likely due to
differences in the microalgal species.
The nitrate removal rate also increased with an increase in the concentration of CO2
(Fig. 5). The nitrate concentration remaining at the end of cultivation was 4.21, 3.31, 2.64,
Appl Biochem Biotechnol

40 4 80
(A) CO2 0.03% (B) CO2 0.5%
Dry cell weight (g/L)
30 3 60
Nitrate (mg/L)

Lipid content (%)

20 2 Aa 40
Ca
Ba Ca
Db
Bb Ea Ea
Fb Ea Ca
10 1 Cb Db 20
Da Da Da Ec
Eb Dc Db
Ed Ec Dc
Fd
0 0 0
40 4 80
(C) CO2 1.0% (D) CO2 2.0%
Time (days)
Dry cell weight (g/L)

30 3 60
Nitrate (mg/L)

Lipid content (%)

Aa
Bb
20 2 Ca Ca Cc 40
Db Aa Aa
Ca Ed Aa
Ba
Ec Cb Ba Ca
10 1 Db 20
Ec CDb Db
Dc Fd Fc Fc
0 0 0
0 5 10 15 20 25 0 5 10 15 20 25
Time (days) Time (days)

Fig. 5 Effect of CO2 concentration on growth (open circle), lipid content (filled circle), and nitrate consumption
(open triangle) of TRG strain cultivated in the secondarily pretreated wastewater from seafood processing plant. The
CO2 concentrations were 0.03 % (a), 0.5 % (b), 1.0 % (c), and 2.0 % (d). Different uppercase and lowercase letters
on the symbols indicate significant differences between CO2 concentrations and cultivation time, respectively

and 2.17 mg L−1 for the cultures supplemented with CO2 at 0.03, 0.5, 1, and 2 %, respectively.
The efficiency of nitrate reduction was highest at 91 % in the culture supplemented with 2.0 %
CO2. The final pH values of the cultures were only slightly reduced from 6.7 to about 6.43,
6.4, 6.28, and 6.05 for the cultures supplemented with 0.03, 0.5, 1.0, and 2.0 % CO2,
respectively. This could be due to a good buffering capacity of the wastewater medium. It
has been reported that bubbling of CO2 at higher levels (>2 %) or continuous bubbling at a 2 %
level resulted in significant decrease in the pH of the culture medium having poor buffering
capacity or in which the salt strength was low [33]. Similar results were found in the culture of
Chlorella sp. supplemented with 2–15 % CO2 [31]. The marine Dunaliella tertiolecta was able
to grow sufficiently without controlling the pH, even during flushing with 24 % CO2-enriched
air [34]. This ability of algae might be related to osmoregulation which may be achieved
biochemically by synthesis or dissimilation of intracellular glycerol. This adjustment mecha-
nism would be of significance if it was cultivated using flue gas containing a high content of
CO2 [35].

Batch and Semi-continuous Cultivation in a Stirred Tank Photobioreactor

To produce a large amount of microalgal biomass and lipid, the photoautotrophic cultivation of
TRG strain was scaled up in a 2-L stirred tank photobioreactor with a working volume of
 Appl Biochem Biotechnol

1.5 L. The process was done under batch and semi-continuous mode. It should be noted that a
sufficient supply of CO2 is necessary for optimal microalgal growth. However, if the CO2 is
supplied in excessive amounts, the pH would drop rapidly and this unsuitable pH level would
result in low biomass productivity. Hence, the pH of the culture was controlled at 6.7 by the
injection of CO2 in response to signals from a pH sensor. Through this strategy, the CO2 could
be fed sufficiently without a drop in the pH. The growth, lipid content, and nitrate removal are
shown in Fig. 6. The cell growth in the batch culture reached the highest value of 2.66 g L−1 at
day 11 (Fig. 6a) with a specific growth rate of 0.189 day−1 (Table 1). Afterward, the biomass
concentration gradually decreased.
The lipid content showed a trend to increase in parallel with the biomass and reached its
maximum of 31 % at day 12. A slight decrease of the lipid content was observed after 12 days
of cultivation likely due to the internal degradation of storage lipid. It should be noted that the
lipid productivity in a stirred tank photobioreactor (69.1 mg L−1 day−1) was almost 1.5 times of
that in a shake flask supplemented with 2 % CO2 (45.5 mg L−1 day−1) (Table 1). This was
probably due to the sufficient supply of CO2 using the pH-stat control system. The nitrate
concentration in the wastewater sharply decreased, and more than 82 % of the initial nitrate
was removed by TRG strain. The rate of nitrate removal decreased with a prolonged cultiva-
tion time. The maximum removal rate of nitrate was obtained at the beginning of cultivation
(first day) at 3.5 mg L−1 day−1. The nitrate removal efficiencies achieved in this study was
similar to the nitrate removal rate of Chlorella pyrenoidosa [36].
To achieve a higher cell density of TRG strain, semi-continuous cultivation mode was
attempted (Fig. 6b). An advantage of this type of cultivation is that the supplementation with
fresh wastewater could provide an additional nitrogen source that could be efficiently used for
cell growth. This would then result in a higher cell density compared to the batch culture. In
addition, the microalgae in the log phase which is generally of higher lipid content than the old
microalgae can be harvested. As shown in Fig. 6b, the growth rate of TRG strain continually
increased when the fresh wastewater medium was added. The highest biomass concentration
was achieved at day 23 (5.16 g L−1) with the highest specific growth rate of 0.366 day−1
(Table 1). Up to and during 21 days of cultivation, the lipid accumulation continually increased
along with the cell growth of TRG strain. The lipid content of TRG strain reached its
maximum level (32 %) at day 21 and remained constant until the end of cultivation. It should
be noted that the maximum biomass concentration in semi-continuous culture was about

7 70
(A) Batch (B) Semi - continuous
Lipid content (%), Nitrate (mg/L)

6 60
Dry cell weight (g/L)

5 50

4 40

3 30

2 20

5 10

0 0
0 5 10 15 0 5 10 15 20 25 30
Time (days) Time (days)

Fig. 6 Batch and semi-continuous cultivation of TRG strain in the undiluted secondarily pretreated wastewater
from seafood processing plant. Growth (open circle), lipid content (filled circle), and nitrate consumption (open
triangle). The cultures were carried out in 2-L photobioreactor with 125 rpm agitation at 25 °C at 49.5 μmol
photon m−2 s−1 light intensity with 16:8-h light and dark cycle. The pH was controlled at pH 6.7 by automatically
injecting CO2, as activated by the signal from pH sensor. In semi-continuous mode, 500 mL of medium was
withdrawn and replaced with fresh wastewater medium in the same volume at 4- or 6-day intervals
Appl Biochem Biotechnol

double that in the batch culture. The enhanced lipid productivity by semi-continuous culture
was more obvious as it was 1.7-fold of that by the batch culture. This was because the semi-
continuous culture gave a higher biomass with higher lipid content.
The pattern of the decline of nitrate in batch culture decreased over time. In semi-
continuous mode, the nitrate showed a dominant peak at the day when the fresh wastewater
medium was replaced in the culture which resulted in an intermittent peak. The maximum rate
of nitrate removal in the semi-continuous mode (7.24 mg L−1 day−1) was almost two times that
of the batch mode (3.4 mg L−1 day−1). It is thought that the development of a tubular
photobioreactor is a potential technology for the application of outdoor cultivation in tropical
countries with plenty of sunlight. Furthermore, the microalgae cultivation using combustible
CO2 from the factory would be another step for further reducing the production cost of lipid
and contributing to the reduction of atmospheric carbon dioxide. Since it has been reported that
microalgae synthesize photosynthetic pigments such as chlorophylls and carotenoids [37], the
concomitant production of these valuable biochemical products together with the lipid should
also be considered to offset the production cost of lipid.

Conclusion

B. braunii TRG strain grew and accumulated lipid better under the heterotrophic and
mixotrophic conditions than under the photoautotrophic condition. The supplementation of
either glucose or molasses as organic carbon sources in the mixotrophic cultivation effectively
enhanced the biomass and lipid production by this microalga. This study has also shown that it
is possible to produce the high cell density of microalgae economically using industrial
wastewater supplemented with carbon dioxide. In addition, a semi-continuous cultivation
mode was shown to be an effective method for achieving a high production of microalgal
biomass with high lipid content. These successful strategies for microalgae cultivation using
industrial wastes would contribute greatly to the industrialized lipid production by the
microalgae and may have even more benefit in reducing accumulation of atmospheric carbon
dioxide and treating the wastes from industry.

Acknowledgments This research was financial supported by the International Foundation for Science and Thai
Research Fund under Grant MRG5280211. The first author was supported by a grant funded under the program
Strategic Scholarships for Frontier Research Network for the Ph.D. Program Thai Doctoral degree from the
Office of the Higher Education Commission and Prince of Songkla University.

References

1. Chisti, Y. (2007). Biotechnology Advances, 25, 294–306.

2. Lee, Y. K. (2004). In A. Richmond (Ed.), Handbook of Microalgal Culture, Biotechnology and Applied
Phycology (p. 116). Oxford: Blackwell Publishing.
3. An, J. Y., Sim, S. J., Lee, J. S., & Kim, B. W. (2003). Journal of Applied Phycology, 15, 185–191.
4. Martinez, M. E., Jimenez, J. M., & Yousfi, F. E. (1999). Bioresource Technology, 67, 233–240.
5. Ramos-Suárez, J. L., & Carreras, N. (2014). Chemical Engineering Journal, 242, 86–95.
6. Alzate, M. E., Muñoz, R., Rogalla, F., Fdz-Polanco, F., & Pérez-Elvira, S. I. (2014). Chemical Engineering
Journal, 243, 405–410.
7. Li, S., Xu, J., Chen, J., Chen, J., Zhou, C., & Yan, X. (2014). Aquaculture, 428–429, 104–110.
8. Yeesang, C., & Cheirsilp, B. (2011). Bioresource Technology, 102, 3034–3040.
9. Tansakul, P., Savaddiraksa, Y., Prasertsan, P., & Tongurai, C. (2005). Thai Journal of Agricultural Science,
38, 71–76.
 Appl Biochem Biotechnol

10. Tripathi, U., Sarada, R., & Ravishankar, G. A. (2001). World Journal of Microbiology and Biotechnology,
17, 325–329.
11. A.P.H.A., A.W.W.A., W.P.C.F. (1995). Standard methods for the examination of water and wastewater (19th
ed.). Washington: American Public Health Association.
12. Miller, G. L. (1959). Analytical Chemistry, 31, 426–429.
13. Mexwell, J. R., Douglas, A. G., Eglinton, G., & McCormick, A. (1968). Phytochemistry, 7, 2157–2171.
14. Cerón García, M. C., Sánchez Mirón, A., Fernández Sevilla, J. M., Molina, E., & Grima García Camacho, F.
(2005). Process Biochemistry, 40, 297–305.
15. Ip, P.-F., Wong, K.-H., & Chen, F. (2004). Process Biochemistry, 39, 1761–1766.
16. Jeon, Y. C., Cho, C.-W., & Yun, Y.-S. (2006). Enzyme and Microbial Technology, 39, 490–495.
17. Shi, X. M., Liu, H. J., Zhang, X. W., & Chen, F. (1999). Process Biochemistry, 34, 341–347.
18. Oh, S. H., Han, J. G., Kim, Y., Ha, J. H., Kim, S. S., Jeong, M. H., Jeong, H. S., Kim, N. Y., Cho, J. S., Yoon,
W. B., Lee, S. Y., Kang, D. H., & Lee, H. Y. (2009). Journal of Bioscience and Bioengineering, 108, 429–434.
19. Banerjee, A., Sharma, R., Chisti, Y., & Banerjee, U. C. (2002). Critical Reviews in Biotechnology, 22, 245–279.
20. Zhang, H., Wang, W., Li, Y., Yang, W., & Shen, G. (2011). Biomass and Bioenergy, 35, 1710–1715.
21. Chojnacka, K., & Noworyta, A. (2004). Enzyme and Microbial Technology, 34, 461–465.
22. Marques, F. J., Sasaki, K., Kakizano, T., Nishio, N., & Nagai, S. (1993). Journal of Fermentation and
Bioengineering, 5, 408–410.
23. Torzillo, G., Sacchi, A., & Materassi, R. (1991). Bioresource Technology, 38, 95–100.
24. Obgonna, J. C., & Tanaka, H. C. (1998). Bioresource Technology, 65, 62–72.
25. Villarejo, A., Orus, M. I., & Martinez, F. (1995). Plant Physiology, 94, 680–686.
26. Xu, H., Miao, X., & Wu, Q. (2006). Journal of Biotechnology, 126, 499–507.
27. Mallick, N., Mandal, S., Singh, A. K., Bishai, M., & Dash, A. (2012). Journal of Chemical Technology and
Biotechnology, 87, 137–145.
28. Chen, J. C. P., Chou, C. C. (1993) Cane Sugar Handbook. Wiley and Sons.
29. Liu, Z. Y., Wang, G. C., & Zhou, B. C. (2008). Bioresource Technology, 99, 4717–4722.
30. Fakas, S., Galiotou-Panayotou, M., Papanikolaou, S., Komaitis, M., & Aggelis, G. (2007). Enzyme and
Microbial Technology, 40, 1321–1327.
31. Chiu, S.Y., Kao, C.Y., Chen, C.H., Kuan, T.C., Ong, S.C., Lin, C.S. (2008). Bioresource Technology, 99,
3389-3396.
32. Lee, J. S., Kim, D. K., Lee, J. P., Park, S. C., Koh, J. H., Cho, H. S., & Kim, S. W. (2002). Bioresource
Technology, 82, 1–4.
33. Rao, A. R., Dayananda, C., Sarada, R., Shamala, T. R., & Ravishankar, G. A. (2007). Bioresource
Technology, 98, 560–564.
34. Suzuki, T., Matsuo, T., Ohtaguchi, K., & Koide, K. (1995). Journal of Chemical Technology and
Biotechnology, 62, 351–358.
35. Goyal, A., & Gimmler, H. (1989). Archives of Microbiology, 152, 138–142.
36. Tam, N. F. Y., & Wong, Y. S. (2000). Environmental Pollution, 107, 145–151.
37. Borowitzka, M. A. (2013). Journal of Applied Phycology, 25, 743–756.