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Comparison of growth and lipid accumulation properties of two oleaginous

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 573 © IWA Publishing 2014 Water Science & Technology | 69.3 | 2014

Comparison in growth, lipid accumulation, and nutrient

removal capacities of Chlorella sp. in secondary effluents
under sterile and non-sterile conditions
Qiao Zhang and Yu Hong

ABSTRACT
Qiao Zhang
The growth, lipid accumulation and nutrient removal characteristics of an oleaginous microalga
Yu Hong (corresponding author)
Chlorella sp. HQ in two types of secondary effluents (named as X and Q) before/after sterilization College of Environmental Science and Engineering,
Beijing Forestry University,
were evaluated. The results show that the algal growth rates under sterilization were higher than Beijing 100083,
China
those under non-sterilization. However, sterilization caused a significant decrease in algal lipid and E-mail: yuhong829908@gmail.com
triacylglycerol (TAG) contents in both X and Q. And the lipid and TAG yields in non-sterile X were as
much as 2.7 and 7.7 times higher than those in sterile X, reaching up to 51.3 and 16.1 mg L
1,
respectively. However, the sterilization caused algal biomass increase in sample Q. Sterilization or
not had almost no effect on the total phosphorus (TP) removal ability of Chlorella sp. HQ and it was
found to have similar abilities to remove almost 100.0% TP from samples X and Q. While the total
nitrogen (TN) removal efficiencies were promoted slightly under non-sterilization, increasing from
88.5 to 89.7% in X and from 13.3 to 17.2% in Q. Hence, non-sterile circumstances are basically
beneficial for Chlorella sp. HQ to accumulate its lipid (TAGs) and remove nutrients from wastewater.
Key words | Chlorella sp., lipid accumulation, nutrient removal, sterile or non-sterile secondary
effluent, triacylglycerols

INTRODUCTION

The energy crisis and global warming are intensified by the
over consumption of fossil fuels in the 21st century.
Recently, developing biodiesel using microalgae as one
most promising substitute for fossil fuels has been a hot
topic due to its advantages of having a higher photosynthetic
efficiency (Chisti ), occupying less arable land resource
(Deng et al. ), transforming greenhouse gas (CO2) into
neutral fuel production (Schenk et al. ), producing
non-toxic and highly biodegradable biofuels (Schenk et al.
), being directly used into the vehicle engine (Deng
et al. ), and so on. Currently, the huge consumption of
inorganic nutrients and water resources for microalgae cul-
tivation is costly, which limits the development of biodiesel
production worldwide.
So far, a lot of efforts have been put into developing bio-
diesel from microalgae. Notably, coupling the biodiesel
production with wastewater treatment based on microalgae
became a viable way to lighten the burden on the high cost
(Hu et al. ). Some researchers have certified that microal-
gae could be successfully cultivated in wastewaters, such as
doi: 10.2166/wst.2013.748
dairy wastewater (Shekhawat et al. ), swine manure
wastewater (Zhou et al. ) and secondary effluent (Li
et al. a). However, there still exists a lot of technological
barriers that seriously obstruct the economic feasibility and
competitiveness of the coupled system in technical appli-
cation. Among those, the relationship between microalgae
and other microorganisms, always co-existing with microal-
gae in aquatic environments, is critical. In recent years,
numerous symbiotic interactions existing between microor-
ganisms and algae were certified by some researchers. To
further save the overall cost, a microalgae-bacteria-based
system was utilized for the dual purpose of biofuels pro-
duction, chemical products and wastewater treatment
(Olguin ). Currently, it was used to treat different types
of wastewater, such as swine slurry (Gonzalez-Fernandez
et al. ) and agro-industrial wastewater (Hernandez et al.
). But few studies have been systemically conducted on
the characteristics of algal growth, lipid accumulation and
nutrient removal properties when the algae co-existed with
other microorganisms. Some researches only reported the
 574 Q. Zhang & Y. Hong | Lipid production and nutrient removal before/after sterilization
above characteristics partially, e.g. the algal growth of Chlor-
ella ellipsoidea co-cultured with bacterial contaminants was
boosted 0.5–3 times more than that of the algae alone (Park
et al. ); the biomass of Chlorella vulgaris increased in
non-sterile batch cultures (Pisman et al. ). Conversely,
the algal lipid content and productivity of Chlorella pyrenoi-
dosa were lightly reduced when co-existing with
microorganisms (Zhang et al. ).
Hence, realizing the relationship between microalgae
and other microorganisms in the wastewater and confirming
the potential interference for algal growth were of great
importance for selecting proper microalgae for the coupled
system of biodiesel production and wastewater treatment. A
freshwater microalga Chlorella sp. HQ isolated previously
has been proved as an algae strain with high lipid content
in a low-nutrient environment. In this case, this study aims
to compare the differences in its characteristics of growth,
lipid accumulation and nutrient removal when cultivated
in actual secondary effluents sampled from two municipal
wastewater treatment plants in Beijing (China) under sterile
and non-sterile conditions.
MATERIALS AND METHODS
Microalga and algal cultures
The microalga Chlorella sp. HQ (Collection No.
GCMCC7601 in China General Microbiological Culture
Collection Center) used in this study was reported pre-
viously (Zhang & Hong ). The secondary effluents
were sampled from two municipal wastewater treatment
plants in Beijing (China), which used a biological aerated
filter (BAF) and anaerobic-anoxic-oxic (A2O) processes,
respectively. Hence, the two types of secondary effluents
were named as X and Q, respectively in this study. All the
samples were immediately delivered to the laboratory, fil-
tered through 0.45 μm membranes to remove suspended
solids and stored in the laboratory for some days. Then the
water quality was determined and the result is exhibited in
Table 1. Each data was measured from three independent
cultures.
Table 1 | Water quality of secondary effluents used in the experiment
Secondary effluents Treatment process
X BAF
2
Q A O
Water Science & Technology | 69.3 | 2014
Experimental set-up
Part of the two types of water samples filtered through
0.45 μm membranes was sterilized at 121 C for 30 min
W
(named as X1, Q1) and the other part was non-sterile
(named as X2, Q2). The Chlorella sp. HQ was cultivated
in 300 mL sterile and non-sterile secondary effluents in
500 mL conical flasks. The algal initial inoculation density
was 2 × 105 cells mL
1. The flasks were set-up in an artificial
W
climate chamber (HPG-280H, HDL, China) with 25 C of
temperature, 60 μmol photons · (m2 s)
1 of light intensity,
light/dark cycle of 14/10. All the experiments were con-
ducted in triplicate.
The algal density was tested every two days during the cul-
tivation process. After 15 days of cultivation, the dry weight of
algal biomass (mg L
1), lipid content per algal biomass (%)
and triacylglycerol (TAG) content per lipid (%) were analyzed,
respectively. Simultaneously, the concentrations of total nitro-
gen (TN) and total phosphorus (TP) from algal cultures filtered
by 0.45 μm membranes were determined.
Growth analysis
Algal density (N, cells · mL
1) was measured by counting
cell numbers under an optical microscope (XSZ-HS3,
COIC, China) with a haemacytometer every 48 h. The dry
weight of algal biomass was determined by drying the
algal cells for 24 h in an oven (MOV-112F, SANYO,
W
Japan) at 110 C according to the Monitoring Method of
Water and Wastewater (State Environmental Protection
Administration ).
The algal specific growth rate (r, d
1) can be obtained by
logistic curve fitting with the data series of N and t, as shown
in Equation (1). When N equals half of K, the population
growth rate (R) reaches its maximal value Rmax
(cells·mL
1·d
1), which can be calculated in Equation (2).
Where N is the algal cell density at time t (d), K (cells · mL
1)
is the maximal algal cell density in the growth medium, and
a is a constant.
 
K
ln
1 ¼ a
rt (1)
N
TN (mg L
1) TP (mg L
1) pH
10.84 ± 0.18 0.93 ± 0.00 8.53 ± 0.01
10.22 ± 0.17 0.063 ± 0.01 8.44 ± 0.02
 575 Q. Zhang & Y. Hong | Lipid production and nutrient removal before/after sterilization Water Science & Technology | 69.3 | 2014

another clean tube. Subsequently, 8 mL sample was used

rK
Rmax ¼ (2) for the determination of TN using a total organic carbon
4
analyzer (TOC-VCPH, SHIMADZU, Japan). A sample of
Lipid and TAG analysis algal culture 1 mL was digested by adding into 3 mL distilled
water and 1 mL potassium persulfate (5%) in the digestion
W

The algal cells were harvested by centrifugation at apparatus (DRB 200, HACH, USA) at 130 C for 30 min,
W
12,000 rpm, 4 C (high-speed freezing centrifuge, HITACHI and the ammonium molybdate spectrophotometric method
CR22G, Japan) for 10 min. The total lipid content was then was used to determine TP using a spectrophotometer
extracted with 5 mL chloroform/methanol/high purity (DR 5000, HACH, USA) (State Environmental Protection
water (2/2/1, v/v/v) and then the extracted lipid was separ- Administration ).
ated into three layers by centrifugation at 4,000 rpm for
10 min. The chloroform layer (bottom layer) with lipid was
collected and the methanol layer including water was RESULTS AND DISCUSSION
removed concomitantly. Then the chloroform in mixed
extracts was blown away using a nitrogen evaporator Growth properties in sterile and non-sterile secondary
(DC-12, ANPEL, China) to obtain the total algal lipid, and effluents
finally quantified gravimetrically (Bligh & Dyer ).
After the determination of total lipid, 0.4 mL isopropa- The growth curves of Chlorella sp. HQ in sterile and non-
nol was added into the dry algal lipid and mixed well, and sterile secondary effluents are shown in Figure 1 and the
then the TAGs were tested by the enzymatic method values of K, r and Rmax are presented in Table 2. It is clear
(Zhang & Hong ). that Chlorella sp. HQ showed a good ability to grow in sec-
ondary effluents. The characteristics of algal growth in X
under different conditions were of a significant difference,
Water quality analysis
e.g. the algal cells turned into the stationary phase at
the twelfth day generally under sterile conditions, and on
The algal culture collected was filtered through 0.45 μm
the third day under non-sterile conditions. But after the cul-
membranes. The filtered supernatant was transferred to
tivation, the maximal algal densities under both conditions
were approximate, reaching around 107 cells mL
1. When
the alga was cultivated in Q, the cells turned into the station-
ary phase almost since the second day under both
conditions. However, the specific growth rate (r) and
maximal algal density varied by significant differences,
e.g. both the r and the maximum of algal density were
(0.19 ± 0.01 d
1; 5.3 × 106 cells mL
1) remarkably higher
under sterilization than that under non-sterilization
(0.07 ± 0.06 d
1; 1.6 × 106 cells mL
1). The result is
consistent with the conclusion that the population of a
freshwater microalga Chlorella sorokiniana and a plant
growth-promoting bacterium Azospirillum brasilense were
significantly lower as free suspensions in non-sterile
Figure 1 | Growth curves of Chlorella sp. HQ in sterile and non-sterile X and Q. municipal wastewater for tertiary wastewater treatment in

Table 2 | The logistic parameters of Chlorella sp. HQ cultured in sterile and non-sterile X and Q

Logistic parameters X1 (sterile) X2 (non-sterile) Q1 (sterile) Q2 (non-sterile)

7
1
K (10 cells mL ) 1.01 ± 0.04 1.12 ± 0.04 0.53 ± 0.55 0.16 ± 0.06
r (d
1) 0.47 ± 0.05 0.39 ± 0.04 0.19 ± 0.01 0.07 ± 0.06
Rmax (107 cells · (mL d)
1) 0.12 ± 0.02 0.11 ± 0.01 0.03 ± 0.03 0.00 ± 0.00
 576 Q. Zhang & Y. Hong | Lipid production and nutrient removal before/after sterilization Water Science & Technology | 69.3 | 2014

comparison with their populations in sterile wastewater

(Covarrubias et al. ).
Notably, the peak of algal density cultured in X with
relatively more nutrients was 1.01 × 107 cells mL
1, which
was rather higher than that in Q of maximum of 5.3 × 106
cells mL
1. It can be reasonably explained by the rule that
the concentrations of nutrients in wastewater mainly
restricted the r and the maximal algal density (Zhila et al.
; Li et al. a). Moreover, the above results indicate
that the influence of non-sterilization on the growth charac-
teristics of Chlorella sp. HQ cultivated in different types of
wastewater was distinctly variable. It was reported that
Azospirillum brasilense strain Cd could significantly
enhance the growth of both Chlorella species when the co-
immobilized microorganisms were grown in wastewater Figure 2 | Comparison of algal biomass in sterile and non-sterile X and Q.

(de-Bashan et al. ). In this study, the growth of Chlorella

sp. HQ in X was hardly affected, attributing to the fact that
microalgae and bacteria could promote the growth of each
other in circumstances with sufficient nutrients, leading to
a relatively balanced living environment (Kazamia et al.
); in Q, the algal growth was obviously inhibited by
other microorganisms existing in the water, which may be
owing to the following reasons: (1) bacteria could utilize
phosphate more efficiently ( Jansson ), yet in Q the phos-
phate was deficient, so bacteria had the opportunity to
utilize phosphate over microalgae; (2) some proteins or
enzymes secreted by bacteria had adverse effects on the
algal growth.

Algal biomass and lipid accumulation in sterile and non- Figure 3 | Comparison of lipid content and yield in sterile and non-sterile X and Q.

sterile secondary effluents

The characteristics of algal biomass production and lipid
accumulation of Chlorella sp. HQ in X and Q under sterile
and non-sterile conditions are presented in Figures 2, 3 and
4. As shown in Figure 2, the dry weight of algal biomass in X
under sterile conditions was obtained at 280 mg L
1, which
is quite low compared to that co-existing with microorganisms,
reaching up to 460 mg L
1. However, in Q, the algal biomass
of Chlorella sp. HQ cultured under sterile conditions
(180 mg L
1) was significantly larger than that under non-
sterile conditions (110 mg L
1). Concisely, in this study, the
biomass accumulation of Chlorella sp. HQ cultured in X was
boosted to a great extent by non-sterilization. As reported,
the algal biomass production of Botrococcus braunii was
enhanced to a certain degree cultivated in the presence of
Pseudomonas sp. and Rhizobium sp. (Rivas et al. ).
Based on the above, it can be reasonably speculated that
microalgae could inhibit the growth of other microorganisms
via competition for the energy sources and carbons in waste-
water with abundant nutrients (Zhang et al. ).
Additionally, it is clear that the algal biomass of Chlor-
ella sp. HQ in X with 10.8 mg L
1 TN and 0.93 mg L
1 TP
under both sterile and non-sterile conditions were larger
than that in Q with 10.2 mg L
1 TN and 0.06 mg L
1 TP,
which may be caused by the P-deprivation in Q. As reported,
Scenedesmus sp. LX1 achieved the highest algal biomass at
110 mg L
1 in domestic secondary effluents (Li et al. b),
which was significantly lower than that of Chlorella sp. HQ
with a peak of 460 mg L
1. The results imply that Chlorella
sp. HQ had great potential in secondary effluents to obtain
absolutely more dry weight of algal biomass.
As shown in Figure 3, the lipid contents per algal biomass
of Chlorella sp. HQ cultured in X under sterile and non-sterile
conditions were nearly equal, approximately 7%. However,
the lipid yield of Chlorella sp. HQ was 2.7 times higher
 577 Q. Zhang & Y. Hong | Lipid production and nutrient removal before/after sterilization Water Science & Technology | 69.3 | 2014

under sterile conditions, reaching up to 51.3 mg L
1, which
may be attributed to the great difference in algal biomass. In
Q, the lipid content of Chlorella sp. HQ was achieved as
8.8% under sterile conditions and as 9.9% under non-sterile
conditions. Being influenced by the algal biomass and the
lipid content synchronously, the lipid yields obtained under
both conditions varied by a small difference, reaching
14.2 mg L
1 and 12.8 mg L
1, respectively. Similarly, the
algal lipid content and lipid production rate of Chlorella
pyrenoidosa were reported to decrease slightly when it was
co-cultured with microorganisms (Zhang et al. ).
The TAG contents per lipid of Chlorella sp. HQ cultured
in secondary effluents under sterile and non-sterile con-
ditions were obtained, as shown in Figure 4. It is obvious
that the TAG content of Chlorella sp. HQ in X co-existing
with other microorganisms was as much as 2.1 times that
under sterilization, reaching 31.0% and 14.7%, respectively.
Moreover, the TAG yield in non-sterile X was nearly 5.7
times that under sterile conditions, that is 16.1 mg L
1 and
2.8 mg L
1, respectively. When the alga was cultivated in
Q, the TAGs content reached as high as 50.2% co-existing
with other microorganisms, and 38.9% under sterilization.
However, due to the opposite difference in algal biomass,
the TAG yields under sterilization and non-sterilization
were almost equal, around 5.5 mg L
1.
A common phenomenon, which can be found in both X
and Q was that the TAG content of Chlorella sp. HQ was
boosted under sterile conditions. Seemingly it may be
caused by the longer stationary phase being maintained
under non-sterile conditions. Virtually, the reason is the
time for Chlorella sp. HQ cultivated under nutrient-
deficiency was longer, which can be certified by a common
conclusion that the accumulation of TAGs could be acceler-
ated under adverse conditions, especially N-deficiency
(Rodolfi et al. ). In addition, the result that the TAG con-
tent of Chlorella sp. HQ in Q (up to 50.2%) was much higher
than that in X (up to 31%) also implies the nutrient-deficiency
was an efficient approach to promote the TAG accumulation
of this alga.
Nitrogen and phosphorus removal capacities in sterile
and non-sterile secondary effluents

The concentrations of TN and TP are still high in waste-

water treated by the common technology, which will
increase the chance of eutrophication. The concept of utiliz-
ing microalgae to remove nutrients from wastewater has
been proposed for half a century and been widely used
over the decades. However, the nutrient removal efficiency
can be affected by some factors, such as the strains of micro-
algae, N/P ratios (Li et al. a), the form of algal cells
existing in the wastewater (Shi et al. ), the microorgan-
isms’ influence (de-Bashan et al. ; Olguin ), and so
on. Recently, the system of microalgae-bacteria used to
treat wastewater has aroused significant concern. Hence,
disclosing the effect of microorganisms on the nutrient
Figure 4 | Comparison of TAG content and yield in sterile and non-sterile X and Q. removal ability of microalgae is of great importance.

Table 3 | Changes in the concentrations of TN and TP with the cultivation time and their final removal efficiencies of Chlorella sp. HQ in sterile and non-sterile X and Q

X1 (sterile) X2 (non-sterile) Q1 (sterile) Q2 (non-sterile)

TN TP TN TP TN TP TN TP
Cultivation time (d) (mg L
1) (mg L
1) (mg L
1) (mg L
1)

0 10.84 0.93 10.84 0.93 10.22 0.063 10.22 0.063

5 7.55 0.35 1.83 0 9.50 0 8.91 0
10 3.58 0 1.36 0 9.03 0 8.50 0
15 1.25 0 1.12 0 8.86 0 8.47 0
TN removal efficiency (%) 88.5 ± 3.5 89.7 ± 1 13.3 ± 3.0 17.2 ± 4.4
TP removal efficiency (%) 100 ± 0 100 ± 0 100 ± 0 100 ± 0
 578 Q. Zhang & Y. Hong | Lipid production and nutrient removal before/after sterilization
In this study, the nutrient removal characteristics of
Chlorella sp. HQ in secondary effluents after sterilization
and non-sterilization were evaluated. The changes in the con-
centrations of TN and TP with the cultivation time and the
nutrient removal efficiencies are illustrated in Table 3. It is
clear that nearly 100% of the TP was removed by Chlorella
sp. HQ, both in X and Q under sterile and non-sterile con-
ditions, but the TN removal efficiency was significantly
different. Notably, the TN removal efficiencies of Chlorella
sp. HQ in non-sterile cultures were slightly higher than
those in sterile conditions, e.g. in Q, 13.3% of TN was
removed under sterile conditions and 17.2% under non-sterile
conditions; in X, the TN removal efficiencies under sterile and
non-sterile conditions were 88.5 and 89.7%, respectively. A
similar phenomenon was observed in the other research
that the co-immobilization of a microalgae Chlorella vulgaris
and a bacterium Azospirillum brasilense strain Cd was more
efficient to remove nutrients than the microalgae alone, reach-
ing removal of up to 100% ammonium, 15% nitrate, and 36%
P, in comparison to 75% ammonium, 6% nitrate, and 19% P
by the microalgae alone (de-Bashan et al. ).
Apparently, the TP removal efficiency was not signifi-
cantly different after sterilization and non-sterilization, while
the TN was removed a little more in non-sterile secondary
effluents. It may attributed to the different volume ratios of
microorganisms in the microalgae–bacteria system, which
can significantly affect the metabolic capacity of the system.
CONCLUSIONS
A previously isolated microalga Chlorella sp. HQ could grow
well and show a good capacity of lipid accumulation in actual
secondary effluents. Additionally, the TN (up to 88.5%) and
TP (almost 100%) could be also removed efficiently. Promis-
ingly, non-sterile circumstances are basically beneficial for
lipid (TAG) accumulation and nutrient removal of Chlorella
sp. HQ, e.g. the algal lipid and TAG yields under non-
sterilization in X were as much as 2.7 and 7.7 times those under
sterile conditions. The results indicate that Chlorella sp. HQ
had advantages, to produce lipid and remove nutrients from
wastewater under non-sterile conditions, over sterilization.
ACKNOWLEDGEMENTS
We gratefully acknowledge the assistance of Prof. HU Hong-
Ying for the dedicated technical assistance and the
Water Science & Technology | 69.3 | 2014
assistance of Prof. Hou Yang-Long, Prof. Qiang Zhi-Min,
Dr Wu Yin-Hu and Dr Yu Yin for paper revision. This
study was supported by Beijing Nova Program (No.
2010B019) and the Fundamental Research Funds for the
Central Universities (No. YX2010-27).
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