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Lipid production ofChlorella vulgariscultured in artificial wastewater medium

Yujie Feng a,*, Chao Li a, Dawei Zhang b

a State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, No 73 Huanghe Road, Nangang District, Harbin 150090, PR Chinab Department of Environmental Science & Engineering, Harbin Institute of Technology (Weihai), Weihai 264200, China

a r t i c l e i n f o

Article history:

Received 31 March 2010

Received in revised form 1 June 2010

Accepted 2 June 2010

Keywords:

Chlorella vulgaris

Lipid content

Artificial wastewater

Energy and cost analyses

a b s t r a c t

Chlorella vulgariswas used to study algal lipid production with wastewater treatment. Artificial wastewa-

ter was used to cultivate C. vulgaris in a column aeration photobioreactor (CAP) under batch and semi-continuous cultivation with various daily culture replacements (0.5 l1.5 l per 2 l reactor). The cell den-

sity was decreased from 0.89 g/l with the daily replacement of 0.5 l to 0.28 g/l with 1.5 l replacement.

However, C. vulgaris culture achieved the highest lipid content (42%, average value of the phase) and

the lipid productivity (147 mg/l d1) with daily replacement of 1.0 l. And then the nutrient removal effi-

ciency were 86% (COD), 97% (NH4 ) and 96% (TP), respectively. Analyses of energy efficiency showed that

the net energy ratio (NER) for lipid production with daily replacement of 1.0 l (1.25) was higher than the

other volume replacement protocols. And cost analyses showed that the algal biomass can be competitive

with petroleum at US$ 63.97 per barrel with the potential credit for wastewater treatment. According to

the above results, it is concluded that the present research will lead to an economical technology of algal

lipid production.

2010 Elsevier Ltd. All rights reserved.

1. Introduction

Global demand for food is expected to double within 50 years,

and the demand for transportation fuels is expected to increase

even more rapidly (Hill et al., 2006). Diversion of food crops to bio-

fuels would not be right approach to solve the problems because

they compete with food production for high-grade arable land

(Rittmann, 2008). There is a great need for renewable energy sup-

plies that do not cause significant environmental harm and not

competed with food supply. Because of their higher photosynthetic

efficiency, higher biomass production and faster growth compared

with other energy crops, microalgae have been receiving attentions

as candidates for fuel production (Minowa et al., 1995). Microalgae

can be used to produce various forms of biofuel including biodiesel

(Converti et al., 2009; Gao et al., 2010), ethanol (Shirai et al., 1998),

bioelectricity (Powell et al., 2009), hydrogen (Ghirardi, 2006;

Hemschemeier et al., 2009), and methane (Stucki et al., 2009).

Biodiesel is produced from plant oils or animal fats, and biodie-

sel industries are expanding rapidly both in the United States and

in Europe with soybean or rapeseed oils as the feedstock. However,

the potential market for biodiesel far surpasses the availability of

plant oils, waste cooking oil and animal fats. Therefore, microalgae

have been studied as alternative feedstock for biodiesel production

recently. Use of microalgae to produce biodiesel would not com-

promise production of food, fodder and other products derived

from crops (Chisti, 2007). Many microalgae accumulate lipids as

storage materials and their accumulation is stimulated under envi-

ronment stress, such as nutrient deficiency (Dunahay et al., 1996)

or salt stress (Takagi et al., 2006).Widjaja et al. (2009) reported

that maximum lipid content ofChlorella vulgariswas only 26% un-

der normal nutrition medium with nitrogen (NaNO3) content of

70.02 mg/l. However, after normal nutrition cultivation, the med-

ium was changed into nitrogen depletion(0.02 mg/l) continued

for 7 d and 17 d, and the lipid contents were 36% and 43%, respec-

tively. Furthermore, according to the results obtained by Converti

et al. (2009), a threefold increase (from 5.9% to 15.3%) in lipid con-

tent took place with NaNO3 concentration decrease from 1.5 to

0.375 g/l.Hsieh et al. (2009)used urea as the nitrogen source at

concentrations of 0.025, 0.050, 0.100, 0.150, and 0.200 g/l. After

6 days of cultivation, the lipid contents of Chlorella sp. were 66%,

60%, 52%, 37%, and 33% respectively.

Microalgal biomass can be produced through autotrophic culti-

vation in open ponds or photobioreactors by using solar energy

and fixing carbon dioxide. Alternatively they are cultivated hetero-

trophically or mixotrophically using organic compounds as energy

and carbon sources. Due to the reduction in light penetration

(Chaumont, 1993) in autotrophic culture, the cell density is usually

less than 1 g/l (Borowitzka, 1994). So far as we know, there is

no effective cultivated method to increase cell density in the

autotrophic cultivation processes. Therefore, the downstream

0960-8524/$ - see front matter 2010 Elsevier Ltd. All rights reserved.doi:10.1016/j.biortech.2010.06.016

* Corresponding author. Tel.: +86 451 86283068; mobile: +13069891017; fax:

+86 451 87162150.

E-mail address:yujief@hit.edu.cn(Y. Feng).

Bioresource Technology 102 (2011) 101105

Contents lists available at ScienceDirect

Bioresource Technology

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / b i o r t e c h

http://dx.doi.org/10.1016/j.biortech.2010.06.016mailto:yujief@hit.edu.cnhttp://dx.doi.org/10.1016/j.biortech.2010.06.016http://www.sciencedirect.com/science/journal/09608524http://www.elsevier.com/locate/biortechhttp://www.elsevier.com/locate/biortechhttp://www.sciencedirect.com/science/journal/09608524http://dx.doi.org/10.1016/j.biortech.2010.06.016mailto:yujief@hit.edu.cnhttp://dx.doi.org/10.1016/j.biortech.2010.06.016

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processing costs are relatively high (Shi et al., 1997). For the het-

erotrophic or mixotrophic cultivation, organic carbon compounds

such as glucose are responsible for higher production costs.

Glucose used in this process comprises about 80% of the total costs

(Li et al., 2007).

In this work C. vulgaris was used to produce algal lipid using

wastewater as medium. In order to simply investigate the factors

related with algal lipid production during wastewater treatment,

preliminary results were obtained here using synthetic wastewater

instead of real wastewater. The use of wastewater as feedstock for

algal lipid is economically attractive since the production costs can

be reduced with credits for wastewater treatment as well as with

reduction in the greenhouse gas emission.

2. Methods

2.1. Algal strain and culture medium

C. vulgaris (FACHB1068) was purchased from Freshwater Algae

Culture Collection, Institute of Hydrobiology, Chinese Academy of

Sciences (Wuhan, China). The strain was preserved in the BG11

medium containing following chemicals: NaNO3 (1.5 g/l),K2HPO43H2O (0.04 g/l), MgSO47H2O (0.075g/l), CaCl22H2O

(0.036 g/l), Na2CO3 (0.02 g/l), citric acid (0.006 g/l), Ferric ammo-

nium citrate (0.006 g/l), EDTA (0.001 g/l), and A5 + Co solution

(1 ml/l) that consists of H3BO3 (2.86 g/l), MnCl2H2O (1.81 g/l),

ZnSO47H2O (0.222 g/l), CuSO45H2O (0.079 g/l), Na2MoO42H2O

(0.390 g/l) and Co(NO3)26H2O (0.049 g/l). C. vulgaris was inocu-

lated at 20% (v/v) in 250 ml Erlenmeyer flasks containing 100 ml

BG11 medium. The flasks were incubated under stationary condi-

tion at 30 C with 3000 lx continuous cool-white fluorescent light

illumination, and were hand shaken three to five times daily to

avoid sticking. The algal cells which just reached the stationary

phase were used to inoculate the column aeration photobioreactor

(CAP).

2.2. Artificial wastewater

The artificial wastewater was prepared dissolving following

chemicals; glucose (0.4125 g/l), NH4Cl (0.078 g/l), KH2PO4(0.018 g/l), MgSO47H2O (0.013 g/l), CaCl22H2O (0.043 g/l), FeS-

O7H2O (0.005 g/l), and A5 + Co solution (1 ml/l). The initial pH

was adjusted to 7.08.0 and sterilized at 121 C for 20 min before

inoculation. The initial N-NH4+, total phosphate (TP), and COD con-

centration were 20, 4, and 400 mg/l, respectively.

2.3. Reactor design and its operation

Four 2.2 l CAPs were consructed with 2 l effective volume

(10 cm diameter and 25 cm height) using polymethyl methacrylate(PMMA). The CAPs containing 1.5 l sterilized artificial wastewater

were inoculated with 0.5 l flask culture ofC. vulgaris. The culture

pH decreased due to NH4+ assimilation, which was observed in

our previous study (data not shown). Therefore, the pH of culture

was maintained between 8 and 10 during Day 2 to 14. All the

experiments were carried out at 30 C and 3000 lx continuous

cool-white fluorescent light illumination. The reactors were aer-

ated with sterilized air at 0.5 vvm (volumes of air per total volume

of bioreactor per minute) to provide mixing and CO2, as well as O2to the algae.

Before the cultures reach stationary phase various volume was

replaced daily with fresh medium to operate the reactors in the

semi-continuous mode. When the cell density reached about

0.8 g/l on Day 4 after the inoculation, the culture was operated inthe first phase of semi-continuous cultivation for 3 d by replacing

0.5 l of the culture with fresh artificial wastewater everyday. After

that, the culture was operated in the second and third phases of

semi-continuous mode by replacing 1.0 and 1.5 l of the culture

with fresh medium everyday, respectively. Both these phases were

maintained for 4 days each. The batch culture under the same con-

dition except for medium replacement was used as positive con-

trol. It was operated for 14 days. All the experiments were

carried out in duplicate and average values are reported.

2.4. Lipid extraction

Algal cells were harvested by centrifugation at 10,000 rpm, 4 C

for 10 min. Supernatant was decanted and cell pellets were washed

with distilled water and then freeze-dried under 80 C. Thereaf-

ter, the total lipids were extracted from microalgal biomass using

a modified method ofBligh and Dyer (1959). Fifty mg of lyophi-

lized microalgal biomass was placed into a 15 ml test tube and

1.6 ml water, 4.0 ml methanol and 2.0 ml chloroform were added.

The solution was mixed for 30 s. Thereafter, an additional 2.0 ml of

chloroform and 2.0 ml water were added and the content of the

test tube was mixed for 30 s. The test tubes were centrifuged at

5000 rpm for 10 min. The upper layer was withdrawn by using apipette and the lower chloroform phase containing the extracted

lipids was transferred into a 30-ml culture tube. The solid material

left at the bottom of extraction tube was extracted with the same

procedure two more times and the chloroform phases were mixed

together and then evaporated in a nitrogen evaporator until

obtaining dry lipid. Thereafter, the total lipids were measured

gravimetrically, and then lipid content and lipid yields were

calculated.

2.5. Analyses

Samples were taken from CAPs each day for analyses. Optical

density (OD) of the algae culture at 658 nm was measured daily

as the cell density indicator using a spectrophotometer (752 Grat-

ing Spectrophotometer, Shandong Gaomi Caihong Analytical

Instrument Factory, China). A linear relationship between OD658and dry weight (DW, g/L) of algal biomass was determined previ-

ously for this strain:

Dry weight g=l 0:4818 OD658; R2 0:9962 1

Samples were centrifuged at 10,000 rpm for 10 min to determine

NH4 , TP and COD concentrations in the supernatants. COD was

determined by a Multi-Function Reactor (ET3150B Multi-Function

Reactor, Euro Tech, China), Nash reagent photometry was used for

measuring NH4 concentration. TP was determined by molybde-

numantimony anti-spectrophotometric method.

For the energy analysis, the values of lipid content, cell den-sity, and hydraulic retention time were obtained from the results

of the semi-continuous cultivation. According to Jorqueras

(2010) research, a production scale of 100 ton of algal biomass

per year was set as the basis to calculate energy balance. And

the energy consumption term included only the energy required

for air pumping that was used to maintain appropriate culture

mixing and liquid/gas mass transfer. Thereafter, volumetric pro-

ductivity, reactor volume required for a biomass production of

100 ton/year, net lipid yield, energy consumption required for a

biomass production of 100 ton/year, total energy consumption,

energy produced as lipid, and NER for lipid production were cal-

culated. Because the structure and the operational mechanism of

CAP were similar to flat-plate photobioreactor, the energy con-

sumption of CAP was assumed equal to flat-plate photobioreactor(53 W/m3).

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3. Results and discussion

3.1. Algal growth and lipid content

CAPs inoculated by the test organism were operated for 14 days

in a batch mode monitoring algal growth and lipid content (Fig. 1).

As shown inFig. 1, the alga grew up to 7 days of cultivation to the

cell density of 1.581.72 g/l with a lag phase of one day. On theother hand the lipid content increased to 37% from 15% in Day 2,

and decreased to 7.8% during the growth phase. However, the lipid

content increased to 34.1% in day 10 but did not increase further at

the stationary phase.

The high lipid content at Day 2 is probably because the

culture was under heterotrophic/mixotrophic conditions. Organic

carbon in the culture was quickly consumed and was exhausted

on Day 2 (see Section 3.2), and then C. vulgaris switched from

mixotrophic metabolism to autotrophic metabolism. It had been

reported (Miao and Wu, 2004) that the autotrophic microalgae

had low lipid content in comparison with those under heterotro-

phic and mixotrophic conditions. Therefore, the switch of metab-

olism on Day 2 resulted in the significantly decrease of lipid

content.

The lipid content in the stationary phase was up to 37%.Li et al.

(2008)reported that the nitrogen deficiency would result in more

metabolic flux generated from photosynthesis to be turned to lipid

accumulation in Neochloris oleoabundans. The reason may be that

under nitrogen deficiency or limitations the synthetic rate of

essential cell structures including proteins and nucleic acids be-

comes low. Therefore, the major part of carbon fixed is converted

into carbohydrate or lipid (Richardson et al., 1969). The artificial

wastewater used in these experiments contains only 20 mg/l nitro-

gen in form of N NH4 . Although N NH4 was depleted on Day 3

(seeFig. 2c), the growth rate of algal cell was not limited signifi-

cantly. The reason may be that nitrate has been introduced to

the culture at inoculation (The initial N NO3 concentration in

BG11 medium is 247 mg/l). Only ammonium is utilized when cul-

tures containing both nitrate and ammonium (Ahmad and Helle-bust, 1990). Therefore, ammonium was depleted first in the

culture, and then algal cell grew with nitrate as nitrogen source un-

til nitrate was depleted. Thus, the lipid accumulation was not en-

hanced up due to most metabolic flux generated from

photosynthesis was still used for cell synthesis from Day 3 to 8.

After Day 8, the growth of algae was nearly ceased, thus resulting

in the increased lipid synthesis. The relatively high lipid content at

Day 1 is believed due to the fact that cells in this phase are similar

to those in the stationary phase culture in BG11 medium which

was used as the inoculums.

CAPs was operated in semi-continuous mode at Day 4 by

replacing different volume of the culture with fresh medium every

day; 0.5 l (first phase), 1.0 l (second phase) and 1.5 l (third phase).

The cell density and lipid content were determined, and the lipid

productivity was calculated based on the results. As shown in

Table 1, cell density decreased from 0.89 g/l in the first phase to

0.28 g/l in the third phase with the increase in daily changed cul-

ture volume during the semi-continuous cultivation. As for the li-

pid content, it increased significantly from 20% in the first phase

to 42% in the second phase, and then decreased slightly to 38% in

the third phase. According to the results of cell density and lipid

content, lipid productivity was calculated. The highest lipid pro-

ductivity (147 mg/l d1) was achieved during the second phase

compared with the first (44 mg/l d1) and the third (79 mg/l d1)

phases.

The main reason for the reduction of cell density is due to the

reduced algal cell retention time in the reactor with the increaseddaily changed volume. As discussed earlier, the lipid content is

dependent on the nitrogen limitations and on the trophic condi-

tions. Higher lipid content is expected in the cells facing nitrogen

limitation under mixotrophic conditions. In a semi-continuous

0

0.4

0.8

1.2

1.6

2

0 2 4 6 8 10 12 14 16

Days of cultivation

Celldensity(g/l)

0

10

20

30

40

Lipidcontent(%)

cell density

lipid content

Fig. 1. Cell density and lipid content ofC. vulgaris culture in the batch cultivation.

0

100

200

300

400

0 2 4 6 8 10 12 14 16Days of cultivation

C

ODconcentrations

(mg/l)

0

20

40

60

80

100

Removalefficiency(%)

semi-continuous

batch

removal efficiency in semi-continuous

0

1

2

3

4

0 2 4 6 8 10 12 14 16

Days of cultivation

TPconcentrations

(mgP/l)

0

20

40

60

80

100

Removalefficiency(%)

semi-continuous

batch

removal efficiency

in semi-continuous

0

5

10

15

20

25

0 2 4 6 8 10 12 14 16Days of cultivation

NH4+concentrations

(mgN/l)

0

20

40

60

80

100

Removalefficiency(%)

semi-continuous

batch

removal efficiency

in semi-continuous

A

B

C

Fig. 2. Removal efficiency and mean concentration of nutrients for C. vulgaris

growing in the batch and semi-continuous cultivation. (A) COD; (B) TP; (C) NH4 .

Table 1

Cell density and lipid content ofC. vulgarisin different phases of the semi-continuous

cultivation.

First Second Third

Daily change medium (l/2 l reactor) 0.5 1.0 1.5

Cell density (g/l) 0.89 0.69 0.28

Lipid content (%) 20 42 38

Lipid productivity (mg/l d1) 44 147 79

*Cell density, lipid content and productivity were average value in the phase.

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culture system the nitrogen availability and trophic conditions are

determined by the volume of daily changed medium. The lipid

content was low in the first phase with low volume change. This

is believed due to the fact that organic carbon was not enough to

supportC. vulgarisgrown in mixotrophic metabolism for long time

to maintain a high lipid synthesis rate. And nitrate may have been

introduced to the culture at inoculation, thus resulting in abundant

nitrogen in culture in the first phase. On the other hand the highest

lipid content of 42% was achieved during the second phase of the

semi-continuous cultivation. This result suggests that the culture

was supplied with enough organic carbon to maintain mixotrophic

metabolism with nitrogen limitation. However, further increase in

daily changed volume during the third phase caused a slight

decrease in lipid content (38%). These results suggest that the

culture had abundant nutrient during the third phase with high

changed volume, therefore, the algae grew vigorously and more

assimilated organic carbon was used for cell growth. Thus, the lipid

content obtained a slight decrease.

3.2. Nutrients removal efficiency

The culture supernatant was analyzed for COD, TP, and NH4+ to

determine the process performance of the system (Fig. 2). As ex-

pected the nutrients removal efficiency was poor at the beginning

of the reactor operation (Day 01) due to lowcell density (0.05 g/l).

It is expected that the higher the cell density, the better the nutri-

ent removal efficiency (Lau et al., 1995). Thereafter, the removal

efficiency of nutrient achieved higher level during the growth

phase, due to the higher cell density and vigorous growth. On

Day 2 the removal efficiencies of COD, TP, and NH4+ were 87%,

94%, and 90%, respectively. It was interesting to note that COD in

the supernatant prepared from the batch cultivation was higher

than those from the semi-continuous cultivation during Days 2

to 14, although the higher cell density and longer HRT in batch

cultivation. The possible reason for this result is thatC. vulgaris

secretes extra cellular substances during the growth process (Babel

et al., 2002; Paralkar and Edzwald, 1996), which was hardlydegradable by the alga. In the semi-continuous cultivation, the

extra cellular substances were removed by medium replacement.

Therefore, the removal efficiency of COD in semi-continuous

cultivation increased from 85% to 88% along with the increasing

daily changed medium. As for TP and NH4+, the batch cultivation

had good removal efficiency which was 96% and 97%, respectively.

The removal efficiency of TP in the third phase of semi-continuous

cultivation was 92% that was lower than that of batch cultivation.

This might be due to low cell growth. The removal efficiency of

NH4 was high (97%) in the whole semi-continuous processes.

3.3. Energy and cost analysis

The net energy ratio (NER) for lipid production was defined asthe ratio of the energy produced as lipid over the total energy con-

sumption. As shown in Table 2, the second phase not only achieved

the highest value of energy produced as lipid, but also the lowest

total energy consumption among the three phases. Therefore, the

NER for lipid production in the second phase (1.25) was higher

than the others. It suggests that the semi-continuous in the second

phase was the most efficiency energy production system among

others.

A large part of the production cost of algal lipid is downstream

processing costs including cell harvest and lipid extraction costs

that are dependent considerably on the cell density and lipid con-

tent (Li et al., 2008). High cell density reduces the cell harvesting

cost, so does high lipid content to lipid extraction cost. Therefore,

the downstream processing costs of algal lipid can be calculatedbased on the following equation:

z xDy=DL 2

where z, downstream processing cost of algal lipid ($/g); x, algae

harvesting cost ($/l), y, lipid extraction cost ($/g); D, cell density

(g/l);L , lipid content (%). It was assumed that the harvesting cost

is dependent on the volume of cultures, and that the extraction cost

dependent on the weight of algae.

Cell density and lipid content of each phase were used to calcu-

late the downstream processing cost according to Equation (2). The

downstream processing costs of algal lipid were 5.6x+ 5y,

3.4x+ 2.4y, and 9.5x+ 2.6y for the first, second, and third phases,

respectively. Hence, the downstream processing of the secondphase was the lowest among three phases.

According to Chistis (2008) research, the algal biomass (lipid

content of 42%) with the production costs of US$ 217.22/ton be-

comes competitive with petroleum at US$ 60.00 per barrel. Using

a value of US$ 0.22/kWh for the energy consumption, the produc-

tion cost of 1 ton algal biomass in the second phase was estimated

to be US$ 808.79. To produce 1 ton algal biomass, 1443 m3 of

wastewater is treated. If the credit for wastewater treatment at

US$ 0.4/m3 is counted, the price of 1 ton of biomass would be re-

duced to US$ 231.59. This figure shows that algal biomass can be

competitive when supposing the price of petroleum is US$ 63.97

per barrel. However, since the energy and cost analyses were car-

ried out based on synthetic wastewater, the results could be differ-

ent when using different real wastewater.

4. Conclusions

The results in this study showed that microalgae cultivation

with wastewater as medium is a promising method to produce al-

gal lipid. The highest lipid content (42%) and productivity (147 mg/

l d1) were achieved in the semi-continuous cultivation with daily

replacement of 1.0 l of the 2.0 l culture. And then the nutrient re-

moval efficiencies were 86% (COD), 97% NH4 and 96% (TP),

respectively. These results were used to analyze the energy effi-

ciency. The NER for lipid production (1.25) was greater than unity.

And cost analysis exhibited that the algal biomass can be compet-

itive with petroleum at US$ 63.97 per barrel with the potential

credit for wastewater treatment. Furthermore, this process also re-duces the greenhouse gas emission in wastewater treatment.

Table 2

Comparative analyses of algal biomass and lipid production in different phases of the

semi-continuous cultivation.

Variable First Second Third

Cell density (g/l) 0.89 0.69 0.28

Hydraulic retention time (d) 4 2 1.3

Volumetric productivity (g/l d1) 0.223 0.346 0.21

Reactor volume required for a biomass

production of 100 ton/yeara (m3)

1246 803 1323

Lipid content (%) 20 42 38

Net lipid yieldb (m3/year) 22 47 42

Energy consumption (W/m3) 53 53 53

Energy consumption required for a biomass

production of 100 ton/yearc (W)

66,038 42,550 70,119

Total energy consumptiond (GJ/year) 2054.05 1323.48 2180.99

Energy produced as lipide (GJ/year) 772.93 1651.27 1475.60

NER for lipid production 0.38 1.25 0.68

a Determined by dividing the annual biomass production by the volumetric

productivity.b Determined by dividing the product of annual biomass and lipid content by the

density of lipid (assumed to be 0.9 kg/l).c Determined by multiplying the energy consumption by the reactor volume

required.d Determined by multiplying the energy consumption by the number of hours of

air pumping (it was 24 h of one day).e Determined by multiplying the net lipid yield by energy content of lipid

(assumed value of 35, 133.33 kJ/l).

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Acknowledgements

The research is supported by the Scientific Research Foundation

for the Returned Overseas Chinese Scholars, State Education Minis-

try, China. The authors also acknowledge the support of the Na-

tional Creative Research Groups of China (50821002) and the

technical and financial support of the State Key Laboratory of Ur-

ban Water Resource and Environment (2010TS08), HIT, China. Prof.Byung Hong Kim (Water Environment and Remediation Research

Center, Korea Institute of Science and Technology) is gratefully

thanked for his efforts on this paper.

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