Bioresource Technology xxx (2011) xxxxxx

Contents lists available at ScienceDirect

Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Reuse of efuent water from a municipal wastewater treatment plant in microalgae cultivation for biofuel production
Sunja Cho a, Thanh Thao Luong a, Dukhaeng Lee a, You-Kwan Oh b, Taeho Lee a,
a b

Department of Civil and Environmental Engineering, Pusan National University, Busan 609-735, Republic of Korea Bioenergy Research Center, Korea Institute of Energy Research, Daejeon 305-343, Republic of Korea

a r t i c l e

i n f o

a b s t r a c t
This study assessed the usability of efuent water discharged from a secondary municipal wastewater treatment plant for mass cultivation of microalgae for biofuel production. It was observed that bacteria and protozoa in the efuent water exerted a negative impact on the growth of Chlorella sp. 227. To reduce the effect, ltration or UV-radiation were applied on the efuent water as pre-treatment methods. Of all the pretreatment options tested, the ltration (by 0.2 lm) resulted in the highest biomass and lipid productivity. To be comparable with the growth in the autoclaved efuent water, the ltration with a proper pore size lter (less than 0.45 lm) or UV-B radiation of a proper dose (over 1620 mJ cm2) are proposed. These ndings led us to conclude that the utilization can be realized only when bacteria and other microorganisms are greatly reduced or eliminated from the efuent prior to its use. 2011 Elsevier Ltd. All rights reserved.

Article history: Received 9 November 2010 Received in revised form 11 March 2011 Accepted 12 March 2011 Available online xxxx Keywords: Chlorella sp. Efuent water Lipid productivity Nutrients removal Filtration

1. Introduction Fossil fuels generate pollutants and global warming effects, even the energy resources are being exhausted. Therefore, it is essential to develop sustainable and eco-friend (clean) energy sources for supporting modern societies. One of the promising energy sources is bioenergy including biomethane, bioethanol, biobutanol and biodiesel produced from photosynthetic organisms, which has several advantages comparing with petroleum-originated diesel; it is principally a renewable, biodegradable, less-toxic energy, and essentially a sulfur- and aromatics-free energy (Hill et al., 2009). The growth of microalgae is extremely faster than other photosynthetic plants, which indicates high productivity per area. Microalgae can generally double their biomass within 24 h and oil contents in several microalgae exceed over 3080% by weight of dry biomass (Chisti, 2007). These facts have been a driving force in the development of bioenergy for commercialization. To date, great efforts have been concentrating on reducing the costs of producing biodiesel from microalgae as in the application of over-sensing light mechanism for increase of photosynthesis (Wang et al., 2008), increasing the efciency of light utilization by better design of photoreactors (Zijiffers et al., 2008), applying new light sources such as LED (Lee and Palsson, 1994), improving extraction technology in lipid extraction (Lee et al., 2010), and
Corresponding author. Address: School of Civil and Environmental Engineering, Pusan National University, 30 Jangjeon-dong, Geumjeong-gu, Busan 609-735, Republic of Korea. Tel.: +82 (0)51 510 2465; fax: +82 (0)51 514 9574. E-mail address: leeth55@pusan.ac.kr (T. Lee).
0960-8524/$ - see front matter 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.03.037

combining autotrophic and heterotrophic cultivation (Miao and Wu, 2006; Heredia-Arroyo et al., 2010). However, there are still some obstacles to be solved for commercial production of algal-based biodiesels even it has a lot of benets described on the above, for examples, such as limitation of light penetration for mass-cultivation, high cost harvest of algal biomass, and an enormous amount of water demanded by the photosynthetic organisms of the water-dependant culture (GerbensLeenes et al., 2009). Nutrients in wastewater such as nitrogen and phosphorous should be removed before discharging to natural water environments, and they can be removed in photosynthesis of microalgae by uptake as biomass. Therefore, wastewater has already been used for cultivation microalgae, but the main purpose was focused on nutrient removal (Hammouda et al., 1995; Lim et al., 2010; de Godos et al., 2010). Recently, cultivation of microalgae in wastewater is being extended to a process for nutrient removal as well as a solution of water and nutrients demanded in mass-cultivation of microalgae as a feedstock of biodiesel (Yun et al., 1997; Wang et al., 2010; Chinnassamy et al., 2010). To meet huge demands of energy in modern society, bio-energy production based on photosynthesis will require tremendous amounts of water for cultivation of photosynthetic organisms such as microalgae. Fortunately, it is known that microalgae can use water of various sources such as wastewater, sea-water, and fresh-water (NREL report, 1998). However, the reuse-efciency of wastewater in cultivation of microalgae for biodiesel production would be re-estimated by companying with the biomass and lipid productivities in sorts of wastewater. For an example, wastewater

Please cite this article in press as: Cho, S., et al. Reuse of efuent water from a municipal wastewater treatment plant in microalgae cultivation for biofuel production. Bioresour. Technol. (2011), doi:10.1016/j.biortech.2011.03.037

S. Cho et al. / Bioresource Technology xxx (2011) xxxxxx

to reuse generally contains great number of bacteria which can inhibit microalgal growth by competing for nutrients in wastewater and bacterial growth is faster than microalgal growth. UV-radiation has been already used as a pre-treatment method before the efuent is discharged into the natural water environment, and membrane ltration is considered as an alternative efuent purication method in Korea in the near future. Therefore, in this study the effectiveness of ltration and UV-radiation methods were evaluated as the pretreatment methods for application of the efuent discharged from a municipal wastewater treatment plant (WWTP) as a medium to supply nutrients to a microalgal culture for biofuel production. 2. Methods 2.1. Algal strain and culture medium for pre-culture The algal strain used for this study was Chlorella sp. 227 purchased from the Culture Collection of National Institute for Environmental Studies (NIES) in Japan. The Chlorella sp. 227 was cultured under sterile conditions in a soil extract (SE) medium, which consists of the following components; K2HPO43H2O 0.15(g L1), MgSO4 7H2O (0.15 g L1), CaCl22H2O (0.05 g L1), KH2PO4 (0.35 g L1), NaCl (0.05 g L1), H3BO3 (2.86 mg L1), MnCl24H2O (1.81 mg L1), ZnSO47H2O (0.22 mg L1), CuSO45H2O (0.079 mg L1), Na2MoO42H2O (0.0076 mg L1) and NH4Cl (0.535 g L1) (Li et al., 2008). 2.2. Efuent water used for this study To investigate the validity of efuent water from a WWTP as a growth medium for mass-culture of microalgae for biodiesel production, the efuent after secondary treatment (aeration tank) at the Su-young Municipal Wastewater Treatment Facility was used for this study. The facility is located in Busan, Korea and it releases its efuent to the ocean after UV radiation treatment. For this study, the efuent was obtained from the secondary settling tank positioned before UV-radiation treatment. The average efuentwater quality of the WWTPs (from July 2009 to June 2010) was as follows; water temperature (C): 17.6 (5.0), BOD (biological oxygen demand, mg L1): 6.9 (1.3), COD-Mn (chemical oxygen demand, mg L1): 11.2 (3.3), suspended solids (mg L1): 5.0 (1.9), total nitrogen (mg L1): 18.9 (4.1), ammonia-N (mg L1): 10.0 (7.1), nitrate-N (mg L1): 6.6 (4.0), total phosphorus (mg L1): 1.7 (0.3), respectively. 2.3. Pretreatment of the efuent water using for cultivation of Chlorella sp. 227 In the preliminary experiment, the culture with the raw efuent induced algal aggregations, showed a retarded growth of Chlorealla sp. 227. It suspected that bacteria and suspended solids in the efuent might be responsible. Therefore, we attempted to remove microorganisms and suspended solids from the efuent prior to its use as a culture medium by applying two methods, i.e., ltration and UV-B radiation. Then, ltered efuent waters (by 0.20, 0.45 and 1.0 lm lters) and the UV-B radiated efuent waters (by different UV doses of 270, 540 and 1620 mJ cm2 at the distance of 10 cm) were prepared as media for cultivation of Chlorella sp. 227. The autoclaved efuent water (121 C for 15 min) was used as a control. 2.4. Batch experiments Chlorella sp. 227 was cultivated in 1 L Erlenmeyer asks containing 500 ml pretreated efuent waters at a shaking incubator (HB 201 SF-L Hanbaek-Scientic Co.) (25 C, 160 rpm, continuous illumina-

tion, uorescent light of 60 lmol m2 s1). The initial concentration of Chlorella sp. 227 was controlled at the OD660 nm of 0.05, which corresponded to 1.3 106 cells per ml. Batch cultivation was kept for 9 days. The initial pH of culture was adjusted to be 7.2 0.1 by 1.0 M HCl after adding 250 mM NaHCO3 as a carbon source. 2.5. Determination of algal growth and dry cell weight Cell numbers on C-Chip (DHC-N01-2, SKC, Korea) were directly counted using a microscope (Axioskop 2, Carl Zeiss). Cell density was periodically measured on a Spectrophotometer (Spectronic Instruments 20 D+, USA) at the absorbance of 660 nm (A660) and dry cell weight was measured according to the standard methods (APHA/AWWA/WEF, 2005). There was a linear relationship between OD660 nm and dry cell weight (mg L1):

Dry weight mg L1 600:94 OD660 7:5355;

R2 0:9916:

2.6. Fatty acid methyl ester (FAME) analysis The content of fatty acids was analyzed using the modied direct transesterication method (Lepage and Roy, 1984). After cultivation for 9 days, algal cells were harvested by centrifugation (4000g, 10 min), and washed twice with distilled water. The cells were then dried in a freeze-dryer (FD5512, IlShin BioBase Co., Korea) for 4 days. Approximately 10 mg of dry cells were mixed vigorously with 2 ml freshly prepared chloroform/methanol (2:1, v/v) for 10 min in an 11 ml screw-top Pyrex-glass tube. One milliliter of chloroform, containing heptadecanoic acid as an internal standard (500 lg/L), 1 ml methanol and 300 ll sulfuric acid (95%) were added sequentially and mixed for 5 min. Then they were held at 100 C for 10 min, cooled to room temperature and 1 ml of distilled water was added and intensely mixed for 5 min. The mixture was centrifuged at 4000g for 10 min. The lower layer (organic phase) was extracted using a syringe and ltered with a disposable 0.22 lm PVDF syringe lter. Methyl esters of fatty acids were analyzed by GC. The GC was equipped with a ame ionization detector and a 0.32 mm (I.D.) 30 m HP-INNOWax capillary column (Agilent Technologies, USA). Helium was used as a carrier gas at 2.2 ml/min. Injection volume and split ratio were 2 ll and 45:1. The temperatures of the injector and detector were set at 250 and 275 C, respectively. Oven temperature conditions were: 50 C for 1 min, heated to 200 C at 15 C/min, maintained at 200 C for 12 min, heated to 250 C at 2 C/min, and kept at 250 C for 2 min. Mix RM3, Mix RM5, GLC50, GLC70 (Supelco, USA), and heptadecanoic acid (Sigma Chemical Co., USA), were used as standards. Other reagents used were of analytical grade. 2.7. Microbial analysis To determine the total numbers of bacteria in the ltered and UV-radiated efuent waters, a standard plate count method was used (Brown, 2007). Plate count agar medium (Difco) was used for this experiment and the bacterial numbers, which indicated as colony forming unit (CFU) unit, in samples were counted after cultivated at an incubator of 25 C for 48 h. Fluorescence in situ hybridization (FISH) technology was applied to conrm the bacterial distribution in the aggregates. Biomass aggregates were obtained on day 9 from the culture in which no-pretreated efuent (raw efuent) was used, and xed immediately for 23 h with 4% paraformaldehyde. The spatial distribution of bacteria and microalga, Chlorella sp. 227, in the biolm was analyzed using FISH. FISH analysis was carried out as described previously by Okabe et al. (1999). The 16S rRNA-targeted oligonucleotide probe used was EUB 338 (50 - GCTGCCTCCCGTAGGAGT-30 ) probe which hybridizes

Please cite this article in press as: Cho, S., et al. Reuse of efuent water from a municipal wastewater treatment plant in microalgae cultivation for biofuel production. Bioresour. Technol. (2011), doi:10.1016/j.biortech.2011.03.037

S. Cho et al. / Bioresource Technology xxx (2011) xxxxxx

most bacteria (Amann et al., 1990) and labeled with uorescein isothiocyanate (FITC). A model LSM510 confocal laser-scanning microscope (CLSM, Carl Zeiss, Oberkochen, Germany) equipped with an Ar ion laser (488 nm) was used to record all FISH images. In addition, the raw efuent, not pretreated, was observed using a phase-contrast microscope (Axioskop 2, Carl Zeiss) because Chlorella sp. 227 rarely grew in the raw efuent.

2.8. Chemical analysis All samples were ltered through 0.2 lm-pore size membranes (ADVANTEC, Tokyo, Japan) before the analysis. The concentrations of total-nitrogen and total-phosphorus were determined using a TRAACS 2000 (BRAN + LUEBBE). The concentration of total organic carbon (TOC) was measured using a TOC analyzer (TELDDYNE TEKMAR Apollo 9000 combustion TOC Analyzer, USA). Culture pH was measured using a pH electrode (Horiba B-212, Japan).

3. Results and discussion 3.1. Growth of Chlorella sp. 227 in efuent waters with different pretreatments The nutrient composition in an efuent from an aeration tank of municipal WWTPs is generally stable and less-toxic for other organisms compared with an inuent, relatively. The growth of Chlorella sp. 227 increased gradually on cultivation time in all the pretreated efuent waters in which an additional 250 mM of bicarbonate was added as a carbon source. The additional bicarbonate would be altered to carbon dioxide by ue gases in practical application. This indicates that the efuent water itself has valuable compounds required for growth of microalgae without external additions of nutrients. The microalgal growths with the efuent waters, ltered by the lters of 0.20 and 0.45 lm pore-size and radiated by the UV-B dose of 1620 mJ cm2, were quite comparable to the control which was autoclaved for repressing microbial activity (Fig. 1). However, the efuent waters (radiated by the UV-B dose of 270 and 540 mJ cm2) did not support the growth of Chlorella sp. 227 well. There was a signicant difference in biomass productivities between the best (0.2 lm-pore ltration) and the worst efuent

water medium (UV-B dose of 270 mJ cm2), which were 0.074 and 0.024 g L1 d1, respectively. It indicates that the UV-B doses were not enough to inactivate indigenous bacteria and protozoa. The CFUs per ml in the efuent waters pretreated by the methods of ltration and UV-radiation were as follows and the cultivation was done at a incubator of 25 C for 48 h; the average numbers of CFU per ml in the efuents ltered by 0.20, 0.45 and 1.0 lm lters were 0, 2, and 1.0 102, respectively. Those of CFU per ml in the efuents UV-radiated with UV-B dose of 270, 540 and 1620 mJ cm2 were 2.0 103, 7.7 102 and 3.5 10, respectively. The number in the efuent before any pretreatment was 1.0 105. However, the low removal of microorganisms with the UV-B dosed efuent waters can be largely improved when UV-C radiation, the method which to be used generally in WWTPs is having more powerful, rather than UV-B radiation is applied in practical application (Degiorgi et al. 1996; Choi and Choi, 2010). The efuent followed after aeration tanks in municipal WWTPs does not contain enough organic matters to be decomposed easily by bacteria, which allows the existence of unicellular bacteria- and algae-feeding organisms such as protozoa according to ecological succession. Then, the unicellular microalga, Chlorella sp. 227 might not live actively in the environment. Actually, many sorts of algaefeeding organisms including protozoa were observed (data not shown) from the raw efuent and from the efuent water radiated the UV-dose of 270 mJ cm2, which might result in the poor growth of Chlorella sp. 227 there. A great amount of bacterial existence was observed on the surface of the aggregates on the FISH experiment (Supplementary Fig. S1). These bacteria may inhibit the microalgal growth by limiting light penetration as well as nutrients uptake from environments through forming slight or signicant aggregation among biomass because Chlorella sp. 227 did not show active growth the culture with the raw efuent for their living. It can be explained why the more bacterial numbers and populations in the efuent waters resulted in the lower biomass productivity than the others in this study, relatively. Meanwhile, the highest biomass production happened not in the culture with the autoclaved efuent but in the culture ltered by 0.2 lm-pore size lter. It demonstrated that the control still contained some amount of suspended solid, which can cause a problem of light utilization in photosynthesis. Interestingly, since the relationship between microalgae and bacteria is not well understood as regarding cultivation of microalgae in efuent waters for biodiesel production, even some bacteria can be useful to harvest or to grow microalgae in their aquatic cultures (de-Bashan et al., 2004; Lee et al., 2009). Thus, the effect of bacteria on fatty acids production from microalgae should be further investigated. 3.2. Nutrient removal in the different efuent waters Total-nitrogen (T-N) and total-phosphorus (T-P) concentrations in all of the different efuent waters decreased during the culturing time (Fig. 2). The highest efciencies in T-N and T-P removal were achieved from the culture with the efuent water ltered by a 0.2 lm-pore lter. The efciencies were 92% (T-N) and 86% (T-P), and the reduced amounts would be assimilated into biomass production (Fig. 1). In the efuent that passed through an aeration tank in the conventional WWTP, the predominant forms of nitrogen are usually ammonia and nitrate. Both of them are easily utilized by microalgal cells, and nitrate is usually utilized after in vivo transformation to nitrite or ammonia by microalgae through an assimilation process (Perez-Garcia et al., 2011). Nitrogen is an important macro-element in living organisms including microalgae as a constituent of biomass which can range from 1% to more than 10%, while phosphorus is an essential element for

Fig. 1. Growths of Chlorella sp. 227 in the differently pre-treated efuent waters. The numbers of the parentheses indicate pore-sizes (ltered, lm) and UV-B dose amounts (UV-B dosed, mJ cm2).

Please cite this article in press as: Cho, S., et al. Reuse of efuent water from a municipal wastewater treatment plant in microalgae cultivation for biofuel production. Bioresour. Technol. (2011), doi:10.1016/j.biortech.2011.03.037

S. Cho et al. / Bioresource Technology xxx (2011) xxxxxx

Fig. 3. The difference of TOC values before and after the cultivation period of 9 days in the differently pre-treated efuents waters. The numbers of the parentheses indicate pore-sizes (ltered, lm) and UV-B dose amounts (UV-B dosed, mJ cm2). The blacked bars indicate the TOC concentration measured on the initial cultivation, and the gray- and lined-bars on the 9 days of the cultivation.

Fig. 2. Changes of total-nitrogen concentration (a) and total-phosphorus concentration (b), in the differently pre-treated efuent waters during the cultivation period of Chlorella sp. 227. The numbers of the parentheses indicate pore-sizes (ltered, lm) and UV-B dose amounts (UV-B dosed, mJ cm2).

growth and other mechanisms such as energy transfer, biosynthesis of DNAs, etc. (Richmond, 2004). The phosphorus removal mechanism in algalbacterial processes seems to be more complex than nitrogen removal. Phosphorus precipitation at high pH values has also been suggested as a mechanism besides assimilation into biomass and dynamics of intracellular polyphosphate compounds (de Godos et al., 2010). In this study, the average ratio of the removed N to the removed P in the whole culture is 14.1 (from 11.2 to 17.0). Those values are a little over the optimal inorganic N/P ratio for freshwater algal growth suggested to be in the range of 6.810 (Wang et al., 2009), but they were within an average elemental composition for microalgae is given as CH1.7 O0.4 N0.15P 0.0094 (Oswald, 1988) besides a culture among the seven different cultures. Therefore, it seems that there was low possibility of precipitation due to not-enough concentration of P in the efuents. In addition, the rapid decrease (ca. 50% to each total concentration) of T-N and T-P in the cultures supposed to have a leg up on exponential growths, the main mechanism of the T-N and T-P removal assumed to be a biomass uptake. Therefore, the culture which shows a higher growth rate accompanies a high ratio of the removed T-N to the removed T-P (Fig. 2).

Fig. 4. The FAME contents and composition produced by Chlorella sp. 227 in the differently pre-treated efuent waters. The numbers of the parentheses indicate pore-sizes (ltered, lm) and UV-B dose amounts (UV-B dosed, mJ cm2).

3.3. TOC values in the efuent waters The TOC concentrations of before the cultivation in the differently pretreated efuent waters were compared with those of after cultivation for 9 days (Fig. 3). The latter slightly increased as much as from 0.4 (in the efuent water radiated by the UV dose of 270 mJ cm2) to 3.8 mg L1 (in the efuent water ltered by a 0.2 lm pore-sized lter) in all cultures. The TOC increases seem to be proportional to the increase in biomass productivity. Actually, most strains belonging to genus Chlorella are known to enable assimilating organic carbon by heterotrophic ways since there is enough inorganic carbon in the culture solution. However, the organic carbon sources in an efuent discharged from municipal WWTPs have been already degraded by indigenous bacteria in aeration tanks. Thus, carbon matters remaining in the efuent waters are mostly inert, and they are recalcitrant to be utilized easily by microalgae, as suggested Wang et al. (2009). In addition, Chlorella sp. 227 seems to release some amount of extracellular organic com-

Please cite this article in press as: Cho, S., et al. Reuse of efuent water from a municipal wastewater treatment plant in microalgae cultivation for biofuel production. Bioresour. Technol. (2011), doi:10.1016/j.biortech.2011.03.037

Please cite this article in press as: Cho, S., et al. Reuse of efuent water from a municipal wastewater treatment plant in microalgae cultivation for biofuel production. Bioresour. Technol. (2011), doi:10.1016/j.biortech.2011.03.037

Table 1 Summary of the results obtained in algal cultivation for which wastewater was supplied. Wastewater Pre-treatment Cultivation period (day) 21 Light intensity (lmol m2s1) 200 Biomass content (g L1) 1.7 Nutrients and (COD or TOC) (mg L1) NH4N (100) T-Nb(125) T-Pc(18) COD (820) NO3N (19.622.1) PO4P (19.622.1) COD (106183) NH4N (71.8) T-N (131.5) T-P (201.5) COD (2250) NH4N (165) COD (43,210) NH4N (20) T-P (4) COD (320) T-N (19.1) T-P (1.2) COD (15.3) T-N (19.9) T-P (1.3) COD (15.4) T-N (20.0) T-P (1.3) COD (15.6) % Removal ratio nutrients and (COD or TOCa) 100 78.3 71.6 34.3 78.3 82.8 85.6 83.0 35 73 99 95 59 92 86 24.8 85 84 13.8 75 84 13.9 Lipid content g FAMEs per g dry cell weight 0.14 Lipid productivity (mg L1 d1) 11.3 References

Digested dairy manure

Filtration (1.5 lm) after dilution

Wang et al. (2010)

S. Cho et al. / Bioresource Technology xxx (2011) xxxxxx

Treated industrial + sewage wastewater Centrate from sludge centrifuge

Filtration (1.0 lm)

10

4.9d

0.07

Chinnasamy et al. (2010) Wang et al. (2009)

Filtration (1.5 lm) after dilution

200

1.5e

Monosodium glutamate wastewater Articial wastewater

Autoclaved

4000f

1.6

0.14

44.0

Xue et al. (2010)

Autoclaved

14

3000f

1.7

0.33

40.0

Yujie et al. (2011)

Secondary treated wastewater Secondary treated wastewater Secondary treated wastewater

a b c d e f

Filtration (0.2 lm)

60

0.67

0.31

22.9

This study

Filtration (1.0 lm)

60

0.41

0.15

6.9

This study

UV-radiation (3 min)

60

0.5

0.22

13.0

This study

COD (chemical oxygen demand) and TOC (total organic carbon). Total-nitrogen. Total-phosphorus. g m2 d1. This value is produced from the maximum O.D. by the conversion formula in this study. Lux.

S. Cho et al. / Bioresource Technology xxx (2011) xxxxxx

pounds during the cultivation period, but the increase of TOC concentration after the cultivation of microalgae can be an undesirable point for discharging it into natural environments. This indicates that utilization of secondary efuents to supply nutrient without additional costs would be another thing to treat nutrients for satisfying discharge regulation because high productivity of microalgae for biofuel production can release organic compounds more than the COD concentration of discharge regulation. In addition, the organic compound may allow chances for contamination by microorganisms such as bacteria and fungi which grow well with rapid growth rates under heterotrophic environments, and it can result in a decrease of microalgal biomass productivity, relatively. Therefore, it seems difcult to be comparable with getting maximum biomass productivity and treating wastewater using microalgae at the same time. 3.4. FAMEs productivity The content and composition of fatty acids in microalgae are species-specic (Hu et al., 2008). According to the fatty acid results produced in Chlorella sp. 227 (Fig. 4), the microalga produces palmitic acid (C16:0), oleic acid (C18:1), linoleic acid (C18:2) and linolenic acid (C18:3) as the main species of fatty acids, and the sum of the four fatty acids occupies 80% of total fatty acids in the cells (ranging from 79.9% to 87.1% of total fatty acids). Similar results were obtained by Wang et al. (2010) who used Chlorella sp. The FAMEs might be derived from the mixture of microalgae and bacteria because several efuent waters used in this study still contained bacteria. However, regardless of the pretreatment methods, the composition of fatty acids and main FAMEs were similar in all cultures even the FAMEs of the control culture in which used the autoclaved efuent. These results imply that the FAMEs were almost from the microalgae fraction enough to neglect bacteria fraction; the growth of bacteria might be inhibited under the autotrophic condition. The maximum lipid content of bacteria is within 5% (Zhang et al., 2009). The maximum number of bacteria (3.3 103 cells per ml, in the efuent radiated the UV-B dose of 270 mJ cm2) in a culture was so small when it was compared with it of the microalgal cell (1.3 106 cells per ml). The highest lipid productivity was 22.9 mg fatty acids L1 d1 from the culture using efuent water ltered by 0.2 lm pore-size lter. Considering the content per g DCW, it is quite competitive with other results used efuent waters (Table 1). The increase of the lipid content per g DCW on biomass productivity in the cultures was observed even though there was some distortion in the results obtained from the UV-dosed cultures series (Figs. 2 and 4). It reected that effective nutrient uptakes happened where the culture showed high biomass productivity. This leads to be close to nitrogen depletion which promotes lipid production in cells (Li et al., 2008; Chinnassamy et al., 2010). Therefore, nitrogen concentration in secondary efuent waters is promising to promote the lipid accumulation by causing a nitrogen depletion condition. Most of the nitrogen concentration in the efuent water can be consumed for the cultivation period when the cultivation is optimized (Fig. 2). However, in this study the lipid productivity of 6.9 mg L1d1 in the efuent water ltered by 1.0 lm pore sized lter was lower than that obtained by a similar previous research (Wang et al., 2010). This can be explained that this culture was done under phototrophic condition with low intensity of illumination, relatively (Table 1, Liang et al., 2009). 4. Conclusions The efuent waters pre-treated by ltration and UV-radiation were applied to cultivation of Chlorella sp. 227 for biodiesel pro-

duction. The highest lipid productivity obtained from the culture using efuent water ltered by 0.2 lm pore-size lter with the high removal rates of total-nitrogen 92% (T-N) and total-phosphorus 86% (T-P). These ndings suggest that the secondary efuents of municipal WWTPs can be used for mass-cultivation of microalgae for saving the unit cost of production by removing additional nutrients supply. However, a proper pre-treatment method to remove algae-feeding microorganisms and competing microorganisms for nutrient should be applied for effective algae biomass production. Acknowledgement This work is nancially supported by Brain Korea 21 Program. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.biortech.2011.03.037. References
APHA, AWWA, WEF, 2005. Standard Methods for the Examination of Water and Wastewater, 21th ed. APHA, Washington, DC, USA. pp. 258259. Amann, R.I., Binder, B.J., Olson, R.J., Chisholm, S.W., Devereux, R., Stahl, D.A., 1990. Combination of 16S rRNA-targeted oligonucleotide probes with ow cytometry for analyzing mixed microbial populations. Appl. Environ. Microbiol. 56, 1919 1925. de-Bashan, L.E., Hernandez, J.-P., Morey, T., Bashan, Y., 2004. Microalgae growthpromoting bacteria as helpers for microalgae: a novel approach for removing ammonium and phosphorus form municipal wastewater. Water Res. 38, 466 474. Brown, A.E., 2007. Bensons Microbiological Applications: Laboratory Manual in General Microbiology. 10th version, pp.151154. Chinnassamy, S., Bhatnagar, A., Hunt, R.W., Das, K.C., 2010. Microalgae cultivation in a wastewater dominated by carpet mill efuents for biofuel applications. Bioresour. Technol. 101, 30973105. Chisti, Y., 2007. Biodiesel from microalgae. Biotechnol. Adv. 25, 294306. Choi, Y., Choi, Y-j., 2010. The effects of UV disinfection on drinking water quality in distribution systems. Water Res. 44, 115122. Degiorgi, C.F., Fernandez, R.O., Pizarro, R.A., 1996. Ultraviolote-B lethal damage on Pseudomonas aeruginosa. Curr. Microbiol. 33, 141146. de Godos, I., Vargas, V.A., Blanco, S., Gonzalez, B.C., Soto, R., Garcia-Encina, P.A., Becares, E., Munoz, R., 2010. A comparative evaluation of microalgae for the degradation of piggery wastewater under photosynthetic oxygenation. Bioresour. Technol. 101, 51505158. Gerbens-Leenes, W., Hoekstra, A.Y., van der Meer, T.H., 2009. The water footprint of bioenergy. PNAS 106, 1021910223. Hammouda, O., Gaber, A., Abdel-Raouf, A., 1995. Microalgae and wastewater treatment. Ecotox. Environ. Safe. 31, 205210. Heredia-Arroyo, T., Wei, W., Hu, B., 2010. Oil accumulation via heterotrophic/ mixotrophic Chlorella protothecoides. Appl. Biochem. Biotech. doi:10.1007/ s12010-010-8974-4. Hill, J., Polasky, S., Nelson, E., Tilman, D., Huo, H., Ludwig, L., Neumann, J., Zheng, H., Bonta, D., 2009. Climate change and health costs of air emissions from biofuels and gasoline. PNAS 106, 20772082. Hu, Q., Sommerfeld, M., Jarvis, E., Ghirardi, M., Posewitz, M., Seibert, M., Darzins, A., 2008. Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances. Plant J. 54, 621639. Lee, A.K., Lewis, D.M., Ashamn, P.J., 2009. Microabial occulation, a potentially lowcost harvesting technique for marine microalgae for the production of biodiesel. J. Appl. Phycol. 21, 559567. Lee, C., Palsson, B.U., 1994. High-density algal photobioreactors using light-emitting diodes. Biotechnol. Bioeng. 44, 11611167. Lee, J., Yoo, C., Jun, S., Ahn, C., Oh, H., 2010. Comparison of several methods for effective lipid extraction from microalgae. Bioresour. Technol. 101, S75S77. Lepage, G., Roy, C.C., 1984. Improved recovery of fatty acid through direct transesterication without prior extraction or purication. J. Lipid. Res. 25, 13911396. Liang, Y., Sarkany, N., Cui, Y., 2009. Biomass and lipid productivities of Chlorella vulgaris under autotrophic, heterotrophic and mixotriophic growth conditions. Biotechnol. Lett. 31, 10431049. Lim, S., Chu, W., Phang, S., 2010. Use of Chlorella vulgaris for bioremediation of textile wastewater. Bioresour. Technol. 101, 73147322. Li, Y., Horsman, M., Wang, B., Wu, N., Lan, C.Q., 2008. Effects of nitrogen sources on cell growth and lipid accumulation of green alga Neochloris oleoabundans. Appl. Microbiol. Biotechnol. 81, 629636. Miao, X., Wu, Q., 2006. Biodiesel production from heterotrophic microalgal oil. Bioresour. Technol. 97, 841846.

Please cite this article in press as: Cho, S., et al. Reuse of efuent water from a municipal wastewater treatment plant in microalgae cultivation for biofuel production. Bioresour. Technol. (2011), doi:10.1016/j.biortech.2011.03.037

S. Cho et al. / Bioresource Technology xxx (2011) xxxxxx NREL, 1998. A Look Back at the US Department of Energys Aquatic Species Program: Biodiesel from Algae. National Renewable Energy Laboratory report/TP-58024190, p. 217. Okabe, S., Satoh, H., Watanabe, Y., 1999. In situ analysis of nitrifying biolms as determined by in situ hybridization and the use of microelectrodes. Appl. Environ. Microbiol. 65, 31823191. Oswald, W.J., 1988. In: Borowitzda, M.B. (Ed.), Microalgae and Wastewater Treatment. Cambridge University Press, Cambridge, UK, pp. 254260. Perez-Garcia, O., Escalante, F.M., de-Bashan, L.E., Bashan, Y., 2011. Heterotrophic cultures of microalgae: metabolism and potential products. Water Res. 45, 11 36. Richmond, A., 2004. Handbook of Microalgal Culture: Biotechnology and Applied Phycology. Science Ltd. Blackwell. pp. 104105. Wang, L., Li, Y., Chen, P., Min, M., Chen, Y., Zhu, J., Ruan, R.R., 2010. Anaerobic digested dairy manure as a nutrient supplement for cultivation of oil-rich green microalgae Chlorella sp. Bioresour. Technol. 101, 26232628. Wang, L., Min, M., Li, Y., Chen, P., Chen, Y., Liu, Y., Wang, Y., Ruan, R., 2009. Cultivation of green algae Chlorella sp. in different wastewaters from municipal

wastewater treatment plant. Appl. Biochem. Biotech. doi:10.1007/s12010-0098866-7. Wang, Q., Jantaro, S., Lu, B., Majeed, W., Bailey, M., He, Q., 2008. The high lightinducible polypeptides stabilize trimeric photosystem I complex under high light conditions in Synechocystis PCC 6803. Plant Physiol. 147, 12391250. Xue, F., Miao, J., Zhnag, X., Tan, T., 2010. A new strategy for lipid production by mix cultivation of Spirulina plantensis and Rhodotorula glutinis. Appl. Biochem. Biotechnol. 160, 498503. Yujie, F., Li, C., Zhang, D., 2011. Lipid production of Chlorella vulgaris cultured in articial wastewater medium. Bioresour. Technol. 102, 101105. Yun, Y., Lee, S., Park, J., Lee, C., Yang, J., 1997. Carbon dioxide xation by algal cultivation using wastewater nutrients. J. Chem. Technol. Biotechnol. 69, 451 455. Zhang, X., Gillespie, A.L., Sessions, A.L., 2009. Large D/H variations in bacterial lipids reect central metabolic pathways. PNAS 106, 1258012586. Zijiffers, J-W.F., Janssen, M., Tramper, J., Wijffels, R.H., 2008. Design process of an area-efcient photobioreactor. Mar. Biotechnol. 10, 404415.

Please cite this article in press as: Cho, S., et al. Reuse of efuent water from a municipal wastewater treatment plant in microalgae cultivation for biofuel production. Bioresour. Technol. (2011), doi:10.1016/j.biortech.2011.03.037