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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 dairy wastewater (Shekhawat et al. ), swine manure
over consumption of fossil fuels in the 21st century. wastewater (Zhou et al. ) and secondary effluent (Li
Recently, developing biodiesel using microalgae as one et al. a). However, there still exists a lot of technological
most promising substitute for fossil fuels has been a hot barriers that seriously obstruct the economic feasibility and
topic due to its advantages of having a higher photosynthetic competitiveness of the coupled system in technical appli-
efficiency (Chisti ), occupying less arable land resource cation. Among those, the relationship between microalgae
(Deng et al. ), transforming greenhouse gas (CO2) into and other microorganisms, always co-existing with microal-
neutral fuel production (Schenk et al. ), producing gae in aquatic environments, is critical. In recent years,
non-toxic and highly biodegradable biofuels (Schenk et al. numerous symbiotic interactions existing between microor-
), being directly used into the vehicle engine (Deng ganisms and algae were certified by some researchers. To
et al. ), and so on. Currently, the huge consumption of further save the overall cost, a microalgae-bacteria-based
inorganic nutrients and water resources for microalgae cul- system was utilized for the dual purpose of biofuels pro-
tivation is costly, which limits the development of biodiesel duction, chemical products and wastewater treatment
production worldwide. (Olguin ). Currently, it was used to treat different types
So far, a lot of efforts have been put into developing bio- of wastewater, such as swine slurry (Gonzalez-Fernandez
diesel from microalgae. Notably, coupling the biodiesel et al. ) and agro-industrial wastewater (Hernandez et al.
production with wastewater treatment based on microalgae ). But few studies have been systemically conducted on
became a viable way to lighten the burden on the high cost the characteristics of algal growth, lipid accumulation and
(Hu et al. ). Some researchers have certified that microal- nutrient removal properties when the algae co-existed with
gae could be successfully cultivated in wastewaters, such as other microorganisms. Some researches only reported the
doi: 10.2166/wst.2013.748
 574 Q. Zhang & Y. Hong | Lipid production and nutrient removal before/after sterilization Water Science & Technology | 69.3 | 2014

above characteristics partially, e.g. the algal growth of Chlor- Experimental set-up
ella ellipsoidea co-cultured with bacterial contaminants was
boosted 0.5–3 times more than that of the algae alone (Park Part of the two types of water samples filtered through
et al. ); the biomass of Chlorella vulgaris increased in 0.45 μm membranes was sterilized at 121 C for 30 min
W

non-sterile batch cultures (Pisman et al. ). Conversely, (named as X1, Q1) and the other part was non-sterile
the algal lipid content and productivity of Chlorella pyrenoi- (named as X2, Q2). The Chlorella sp. HQ was cultivated
dosa were lightly reduced when co-existing with in 300 mL sterile and non-sterile secondary effluents in
microorganisms (Zhang et al. ). 500 mL conical flasks. The algal initial inoculation density
Hence, realizing the relationship between microalgae was 2 × 105 cells mL
1. The flasks were set-up in an artificial
W
and other microorganisms in the wastewater and confirming climate chamber (HPG-280H, HDL, China) with 25 C of
the potential interference for algal growth were of great temperature, 60 μmol photons · (m2 s)
1 of light intensity,
importance for selecting proper microalgae for the coupled light/dark cycle of 14/10. All the experiments were con-
system of biodiesel production and wastewater treatment. A ducted in triplicate.
freshwater microalga Chlorella sp. HQ isolated previously The algal density was tested every two days during the cul-
has been proved as an algae strain with high lipid content tivation process. After 15 days of cultivation, the dry weight of
in a low-nutrient environment. In this case, this study aims algal biomass (mg L
1), lipid content per algal biomass (%)
to compare the differences in its characteristics of growth, and triacylglycerol (TAG) content per lipid (%) were analyzed,
lipid accumulation and nutrient removal when cultivated respectively. Simultaneously, the concentrations of total nitro-
in actual secondary effluents sampled from two municipal gen (TN) and total phosphorus (TP) from algal cultures filtered
wastewater treatment plants in Beijing (China) under sterile by 0.45 μm membranes were determined.
and non-sterile conditions.
Growth analysis

MATERIALS AND METHODS Algal density (N, cells · mL
1) was measured by counting
cell numbers under an optical microscope (XSZ-HS3,
Microalga and algal cultures COIC, China) with a haemacytometer every 48 h. The dry
weight of algal biomass was determined by drying the
The microalga Chlorella sp. HQ (Collection No. algal cells for 24 h in an oven (MOV-112F, SANYO,
W

GCMCC7601 in China General Microbiological Culture Japan) at 110 C according to the Monitoring Method of
Collection Center) used in this study was reported pre- Water and Wastewater (State Environmental Protection
viously (Zhang & Hong ). The secondary effluents Administration ).
were sampled from two municipal wastewater treatment The algal specific growth rate (r, d
1) can be obtained by
plants in Beijing (China), which used a biological aerated logistic curve fitting with the data series of N and t, as shown
filter (BAF) and anaerobic-anoxic-oxic (A2O) processes, in Equation (1). When N equals half of K, the population
respectively. Hence, the two types of secondary effluents growth rate (R) reaches its maximal value Rmax
were named as X and Q, respectively in this study. All the (cells·mL
1·d
1), which can be calculated in Equation (2).
samples were immediately delivered to the laboratory, fil- Where N is the algal cell density at time t (d), K (cells · mL
1)
tered through 0.45 μm membranes to remove suspended is the maximal algal cell density in the growth medium, and
solids and stored in the laboratory for some days. Then the a is a constant.
water quality was determined and the result is exhibited in
 
Table 1. Each data was measured from three independent K
ln
1 ¼ a
rt (1)
cultures. N

Table 1 | Water quality of secondary effluents used in the experiment

Secondary effluents Treatment process TN (mg L
1) TP (mg L
1) pH

X BAF 10.84 ± 0.18 0.93 ± 0.00 8.53 ± 0.01

2
Q A O 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 via competition for the energy sources and carbons in waste-
accumulation of Chlorella sp. HQ in X and Q under sterile water with abundant nutrients (Zhang et al. ).
and non-sterile conditions are presented in Figures 2, 3 and Additionally, it is clear that the algal biomass of Chlor-
4. As shown in Figure 2, the dry weight of algal biomass in X ella sp. HQ in X with 10.8 mg L
1 TN and 0.93 mg L
1 TP
under sterile conditions was obtained at 280 mg L
1, which under both sterile and non-sterile conditions were larger
is quite low compared to that co-existing with microorganisms, than that in Q with 10.2 mg L
1 TN and 0.06 mg L
1 TP,
reaching up to 460 mg L
1. However, in Q, the algal biomass which may be caused by the P-deprivation in Q. As reported,
of Chlorella sp. HQ cultured under sterile conditions Scenedesmus sp. LX1 achieved the highest algal biomass at
(180 mg L
1) was significantly larger than that under non- 110 mg L
1 in domestic secondary effluents (Li et al. b),
sterile conditions (110 mg L
1). Concisely, in this study, the which was significantly lower than that of Chlorella sp. HQ
biomass accumulation of Chlorella sp. HQ cultured in X was with a peak of 460 mg L
1. The results imply that Chlorella
boosted to a great extent by non-sterilization. As reported, sp. HQ had great potential in secondary effluents to obtain
the algal biomass production of Botrococcus braunii was absolutely more dry weight of algal biomass.
enhanced to a certain degree cultivated in the presence of As shown in Figure 3, the lipid contents per algal biomass
Pseudomonas sp. and Rhizobium sp. (Rivas et al. ). of Chlorella sp. HQ cultured in X under sterile and non-sterile
Based on the above, it can be reasonably speculated that conditions were nearly equal, approximately 7%. However,
microalgae could inhibit the growth of other microorganisms 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 with other microorganisms, and 38.9% under sterilization.
may be attributed to the great difference in algal biomass. In However, due to the opposite difference in algal biomass,
Q, the lipid content of Chlorella sp. HQ was achieved as the TAG yields under sterilization and non-sterilization
8.8% under sterile conditions and as 9.9% under non-sterile were almost equal, around 5.5 mg L
1.
conditions. Being influenced by the algal biomass and the A common phenomenon, which can be found in both X
lipid content synchronously, the lipid yields obtained under and Q was that the TAG content of Chlorella sp. HQ was
both conditions varied by a small difference, reaching boosted under sterile conditions. Seemingly it may be
14.2 mg L
1 and 12.8 mg L
1, respectively. Similarly, the caused by the longer stationary phase being maintained
algal lipid content and lipid production rate of Chlorella under non-sterile conditions. Virtually, the reason is the
pyrenoidosa were reported to decrease slightly when it was time for Chlorella sp. HQ cultivated under nutrient-
co-cultured with microorganisms (Zhang et al. ). deficiency was longer, which can be certified by a common
The TAG contents per lipid of Chlorella sp. HQ cultured conclusion that the accumulation of TAGs could be acceler-
in secondary effluents under sterile and non-sterile con- ated under adverse conditions, especially N-deficiency
ditions were obtained, as shown in Figure 4. It is obvious (Rodolfi et al. ). In addition, the result that the TAG con-
that the TAG content of Chlorella sp. HQ in X co-existing tent of Chlorella sp. HQ in Q (up to 50.2%) was much higher
with other microorganisms was as much as 2.1 times that than that in X (up to 31%) also implies the nutrient-deficiency
under sterilization, reaching 31.0% and 14.7%, respectively. was an efficient approach to promote the TAG accumulation
Moreover, the TAG yield in non-sterile X was nearly 5.7 of this alga.
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 Nitrogen and phosphorus removal capacities in sterile
Q, the TAGs content reached as high as 50.2% co-existing 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 Water Science & Technology | 69.3 | 2014

In this study, the nutrient removal characteristics of assistance of Prof. Hou Yang-Long, Prof. Qiang Zhi-Min,
Chlorella sp. HQ in secondary effluents after sterilization Dr Wu Yin-Hu and Dr Yu Yin for paper revision. This
and non-sterilization were evaluated. The changes in the con- study was supported by Beijing Nova Program (No.
centrations of TN and TP with the cultivation time and the 2010B019) and the Fundamental Research Funds for the
nutrient removal efficiencies are illustrated in Table 3. It is Central Universities (No. YX2010-27).
clear that nearly 100% of the TP was removed by Chlorella
sp. HQ, both in X and Q under sterile and non-sterile con-
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First received 23 June 2013; accepted in revised form 4 November 2013. Available online 18 November 2013

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