Bioresource Technology xxx (2014) xxx–xxx

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Bioresource Technology

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Effects of pH control and concentration on microbial oil productionfrom Chlorella vulgaris cultivated in the effluent of a low-cost organicwaste fermentation system producing volatile fatty acids

http://dx.doi.org/10.1016/j.biortech.2014.09.0690960-8524/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author at: Division of Advanced Nuclear Engineering, PohangUniversity of Science and Technology, Hyoja-dong, Nam-Gu, Pohang, Kyungbuk790-784, Republic of Korea. Tel.: +82 54 279 2275; fax: +82 54 279 8659.

E-mail address: jmpark@postech.ac.kr (J.M. Park).

Please cite this article in press as: Cho, H.U., et al. Effects of pH control and concentration on microbial oil production from Chlorella vulgaris cultivthe effluent of a low-cost organic waste fermentation system producing volatile fatty acids. Bioresour. Technol. (2014), http://dx.doi.org/1j.biortech.2014.09.069

Hyun Uk Cho a, Young Mo Kim d, Yun-Nam Choi b, Xu Xu a, Dong Yun Shin a, Jong Moon Park a,b,c,⇑a School of Environmental Science and Engineering, Pohang University of Science and Technology, Hyoja-dong, Nam-Gu, Pohang, Kyungbuk 790-784, Republic of Koreab Department of Chemical Engineering, Pohang University of Science and Technology, Hyoja-dong, Nam-Gu, Pohang, Kyungbuk 790-784, Republic of Koreac Division of Advanced Nuclear Engineering, Pohang University of Science and Technology, Hyoja-dong, Nam-Gu, Pohang, Kyungbuk 790-784, Republic of Koread Department of Civil Engineering, Dong-A University, Nakdong-daero, Saha-gu, Busan 604-714, Republic of Korea

h i g h l i g h t s

� A sewage sludge fermentation systemproducing volatile fatty acids (SSFV)was operated.� The VFAs produced from the SSFV

were well assimilated by the Chlorellavulgaris.� The pH would be an important factor

that can promote increased biomassconcentration.� Fatty acids from the biomass

complied with the requirement ofhigh-quality biodiesel.

g r a p h i c a l a b s t r a c t

a r t i c l e i n f o

Article history:Received 4 August 2014Received in revised form 14 September2014Accepted 15 September 2014Available online xxxx

Keywords:Volatile fatty acids (VFAs)Sewage sludgeMicrobial lipidsChlorella vulgarisFatty acids

a b s t r a c t

The objective of this study was to investigate the feasibility of applying volatile fatty acids (VFAs) pro-duced from low-cost organic waste to the major carbon sources of microalgae cultivation for highly effi-cient biofuel production. An integrated process that consists of a sewage sludge fermentation systemproducing VFAs (SSFV) and mixotrophic cultivation of Chlorella vulgaris (C. vulgaris) was operated to pro-duce microbial lipids economically. The effluents from the SSFV diluted to different concentrations at thelevel of 100%, 50%, and 15% were prepared for the C. vulgaris cultivation and the highest biomass produc-tivity (433 ± 11.9 mg/L/d) was achieved in the 100% culture controlling pH at 7.0. The harvested biomassincluded lipid contents ranging from 12.87% to 20.01% under the three different effluent concentrationswith and without pH control. The composition of fatty acids from C. vulgaris grown on the effluents fromthe SSFV complied with the requirements of high-quality biodiesel. These results demonstrated that VFAsproduced from the SSFV are favorable carbon sources for cultivating C. vulgaris.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction warming derived from greenhouse gases and atmospheric pollu-

Biodiesel, which can be acquired from rapeseeds, canolas, andcorns, has been received attention in that it does not cause global

tion (Alreda et al., 2011). However, the use of biodiesel based onagricultural commodities encounters problems such as limitedsupply and competition with food crops. In this regard, muchattention has been focused on microbial lipids produced fromoleaginous microalgae due to less land requirement for theirgrowth, high lipid productivity compared to conventional crops,and photosynthetic efficiency (Chisti, 2007).

ated in0.1016/

Table 1Characteristics of soluble metabolites in the feed sludge and effluent used in thisexperiment.

Feed sludge Effluent from the SSFV

pH 6.75 ± 0.27 6.5 ± 0.10SCOD (g/L) 10.15 ± 0.64 17.25 ± 1.25Total VFAs (g COD/L) 2.01 13.73

Acetic acid 0.70 ± 0.18 5.36 ± 0.33Propionic acid 0.66 ± 0.24 2.14 ± 0.19Butyric acid 0.09 ± 0.03 2.00 ± 0.38Iso-butyric acid N.D 1.11 ± 0.27Iso-valeric acid 0.55 ± 0.12 2.51 ± 0.35Valeric acid N.D 0.61 ± 0.22

NH4+ (mg/L N) 531 ± 80.45 1657 ± 142.24

NO2� (mg/L N) N.D N.D

NO3� (mg/L N) N.D N.D

PO4�3 (mg/L P) 237 ± 62.63 144 ± 38.28

Note: ‘‘N.D’’ means the data is not detected.

2 H.U. Cho et al. / Bioresource Technology xxx (2014) xxx–xxx

It is well known that microalgae utilize autotrophic, heterotro-phic, or mixotrophic mode for their growth and converting theirgrowth modes is affected by the environmental conditions. Amongthem, heterotrophic and mixotrophic modes have distinct advan-tages over the autotrophic mode since higher biomass and lipidproductivities can be achieved and the requirement of light canbe reduced (Brennan and Owende, 2010). Researches on the effectsof different organic carbon sources such as glucose, acetic acid,glycerol, and ethanol on the growth of microalgae have been con-ducted by many researchers and the results supported thesuperiority of heterotrophic and mixotrophic culture conditions(Perez-Garcia et al., 2011; Liang et al., 2009; Li et al., 2007; Miaoand Wu, 2006). However, most studies on microbial lipidproduction dealing with organic carbon sources used syntheticmedia or wastewaters with added commercial chemicals as theorganic carbon sources. The main impediment to cultivatingmicroalgae heterotrophically to produce biodiesel is the high costof feedstock, constituting 40–80% of overall biodiesel productioncost (Li et al., 2007). Therefore, an alternative inexpensive feed-stock is required.

Recently in Korea, the generation of sewage sludge has becomea prominent environmental problem due to the increase in thenumber of wastewater treatment facilities (Cho et al., 2013). Vola-tile fatty acids (VFAs), which are favorable carbon sources for lipidproduction by microalgae, can be produced by anaerobic fermenta-tion of organic wastes. Therefore, combining a sewage sludge fer-mentation system that produces VFAs (SSFV) with microalgaecultivation can be an ideal alternative to producing biodiesel eco-nomically by utilizing these VFAs as the organic carbon sourcesfor cultivating the microalgae. Because ammonium (NH4

+-N) andphosphate (PO4

�3-P) are also released during the anaerobic fermen-tation of sewage sludge, the resulting effluent can be regarded as agood substrate for cultivation of microalgae.

Although a number of studies have revealed the feasibility ofcultivating microalgae autotrophically with real wastewaters,mixotrophic cultivation of microalgae with those including organiccarbon sources provides more benefits to biofuel production andnutrient removal, resulting in high commercial potential (Parket al., 2011; Ji et al., 2013). Therefore, an integrated process thatcombines microalgae cultivation with the SSFV was tested in thisstudy to determine whether the process can produce lipids stablyand economically. The objectives of this study were to investigatewhether VFAs produced biologically from the low-cost organicwaste are feasible for use as the substrate for microalgae cultiva-tion, and to quantify the influences of culture conditions such asnutrient concentration and pH on lipid production.

2. Methods

2.1. The SSFV operation and monitoring

A continuous stirred tank reactor (CSTR) with a working volumeof 3 L was operated at a solids retention time (SRT) of 6 days and atemperature of 35 �C. The pH was automatically controlled at6.5 ± 0.1 using 3.0 N sodium hydroxide (NaOH) and hydrochloricacid (HCl) during the operation. Anaerobic sludge obtained froman anaerobic digester at a regional wastewater treatment plant inDaegu, Korea was used as the inoculant for the fermentation process.Secondary sludge taken from a secondary sedimentation tank at amunicipal wastewater treatment plant in Daejeon, Korea was usedas the feed sludge which was 50.86 ± 3.7 g/L and 35.86 ± 2.5 g/L oftotal solids (TS) and volatile solids (VS), respectively. The reactorperformance was periodically monitored and the characteristicsof the feed sludge and effluent from the SSFV were measured(Table 1).

Please cite this article in press as: Cho, H.U., et al. Effects of pH control and conthe effluent of a low-cost organic waste fermentation system producing vj.biortech.2014.09.069

2.2. Microalgal strain

Chlorella vulgaris (C. vulgaris) was obtained from Korean Collec-tion for Type Cultures (Daejeon, Korea) and its spherical shape,which is a typical morphology of the species, was identified by ascanning electron microscope (SEM). The strain was grown in amodified-BG11 medium containing the following per liter of dis-tilled water: NaNO3 1.5 g, K2HPO4 0.04 g, MgSO4�7H2O 0.075 g,CaCl2�2H2O 0.036 g, Citric acid 0.006 g, Ferric ammonium citrate0.006 g, EDTA (disodium salt) 0.001 g, Na2CO3 0.02 g, Acetic acid2 g, and 1 mL of trace metals solution consisting of H3BO3

(2.86 g/L), MnCl2�4H2O (1.81 g/L), ZnSO4�7H2O (0.22 g/L), Na2

MoO4�2H2O (0.39 g/L), CuSO4�5H2O (0.079 g/L), and Co(NO3)2�6H2O(49.4 mg/L). C. vulgaris was cultivated in Erlenmeyer flasks con-taining 400 mL of the autoclaved modified-BG11 medium. The cul-ture was conducted in a light incubator (HB-201MS-2R, HanbaekScientific Co., Korea) with a light intensity of 100 lmol/m2/s, tem-perature of 25 �C ± 1.0, agitation of 120 rpm, and light/dark cycle of12 h light/12 h dark.

2.3. Experimental design

The effluent from the SSFV was used as a substrate to cultivateC. vulgaris. It was collected and centrifuged at 6000 rpm and 4 �Cfor 20 min. The centrifuged supernatant was filtered through0.45 lm-pore membrane filters and autoclaved for 10 min at121 �C. Batch experiments were conducted in Erlenmeyer flaskswith working volumes of 600 mL. To find the influence of VFAand nutrient concentrations on the cultivation of C. vulgaris, theeffluent was diluted with distilled water to three different concen-trations at the level of 100% (undiluted effluent), 50%, and 15%before being autoclaved. The effect of pH control on C. vulgarisgrowth was also investigated by cultivating it in the three effluentconcentrations with pH controlled at 7.0 (pH 100%, pH 50%, pH15%) or allowed to vary naturally (100%, 50%, 15%). Culture condi-tions are labeled with the codes for the treatment factors. Threedifferent effluent concentrations are prefixed with pH (i.e., pH con-trolled at 7.0 periodically) or without pH (i.e., uncontrolled pH) toindicate the culture conditions. The pH in the flasks of one group(pH 100%, pH 50%, pH 15%) was adjusted every 48 h to 7.0 ± 0.02by adding 0.5 N NaOH or HCl except on the first day of the test.These experiments were conducted in a light incubator (HB-201MS-2R, Hanbaek Scientific Co., Korea) with a light intensity of100 lmol/m2/s, temperature of 25 �C ± 1.0, agitation of 120 rpm,and diel cycle of 12 h light: 12 h dark. A 10% (v/v) inoculum grownin the medium (Section 2.2) was used for every batch test.

centration on microbial oil production from Chlorella vulgaris cultivated inolatile fatty acids. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/

Fig. 1. Growth of C. vulgaris in different concentrations of effluent from the SSFV(‘‘pH’’ means the pH was controlled at 7.0 periodically).

H.U. Cho et al. / Bioresource Technology xxx (2014) xxx–xxx 3

2.4. Analytical methods

Microalgal biomass concentration was indicated by dry cellweight (DCW) determined by drying the cells at 105 �C for 24 hafter filtering those through pre-dried and pre-weighed GF/C filters(Whatman, UK). Total solids (TS), volatile solids (VS), solublechemical oxygen demand (SCODcr), and NH4

+-N were measuredaccording to standard methods (Clesceri et al., 1998). Every solublesample was filtered through syringe filters with a pore size of0.45 lm (Millipore, USA). NO2

�, NO3�, and PO4

�3 concentrations weredetermined using an ion chromatography (ICS-1000, Dionex, USA)after filtration through syringe filters with a pore size of 0.2 lm(Millipore, USA). The amount of VFAs was quantified using a highperformance liquid chromatography (HPLC-1100, Agilent Technol-ogy, USA) equipped with a refractive index detector (RID), diodearray detector (DAD), and column (Aminex HPX-87H, Biorad Inc.,USA) (Cho et al., 2013). The pH values of broths were analyzedusing a pH measuring instrument equipped with a pH electrode(Mettler Toledo, Switzerland). Quantification of lipid contentswas performed conforming to Bligh and Dyer with modified meth-ods as reported by Bourque and Titorenko (2009) (Xu et al., 2014).The compositions of fatty acid methyl esters (FAME), producedfrom the extracted lipid through transesterification, were analyzedusing a gas chromatography (6890N, Agilent Technology, USA)equipped with a flame ionized detector (FID) and INNOWAX capil-lary column (Agilent Technology, USA). The temperature of the col-umn was controlled based on a previously described method (Xuet al., 2014). The initial temperature of 100 �C was maintainedfor 5 min, then increased to 250 �C at 10 �C/min and maintainedat 250 �C for 30 min. Both the detector and injector were operatedat 250 �C. FAME components were classified and quantified aftercomparing obtained peaks with those of standard solutions.

3. Results and discussion

3.1. The SSFV performance

The characteristics of soluble compounds in the feed sludge andeffluent from the SSFV are represented in Table 1. Sludge hydroly-sis can be indicated by the variations of SCOD concentrations(Andreasen et al., 1997; Cho et al., 2013). An increase in the SCODconcentration of the effluent proved that organic particulates inthe sewage sludge were solubilized efficiently. The effluent con-tained considerable VFAs, which are the principal products of aci-dogenic fermentation of sewage sludge and those were composedof acetate (39.0%), propionate (15.6%), iso-butyrate (8.1%), butyrate(14.6%), iso-valerate (18.3%), and valerate (4.4%) during the steady-state operation of the reactor. The three main components of VFAswere acetate, propionate, and iso-valerate, comprised of 72.9% oftotal VFAs. This result is consistent with previous studies dealingwith waste activate sludge as a substrate (Wang et al., 1999). Ingeneral, the amounts of released NH4

+-N and PO4�3-P are directly

proportional to the degree of fermentation (Yuan et al., 2011).The fermentation of sludge led to producing NH4

+-N significantlyby biological decomposition of protein (Table 1). NH4

+-N can inhibitgrowth of anaerobic microorganisms, but did not seem to have anegative effect on the anaerobic process. In contrast, the PO4

�3-P

Table 2Biomass productivities in the growth phase of C. vulgaris grown in different concentration

100% pH 100%

Cultivation days 9 9Biomass productivity (mg/L/d) 296 ± 6.7 433 ± 11.9

Note: ‘‘pH’’ means the pH was controlled at 7.0 periodically.

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concentration was lower in the effluent than in the feed sludge.Possibly the released PO4

�3-P and heavy metals during the anaero-bic fermentation of sludge reacted with each other, resulting in achemical precipitation (Crannell et al., 2000).

3.2. Microalgal growth and productivity

The effects of nutrient concentrations and pH controls on bio-mass productivities with the effluent from the SSFV were investi-gated. Cell growths (DCW) varied from 0.67 g/L for 15% effluentto 4.23 g/L for 100% effluent with the pH control (pH 100%)(Fig. 1). C. vulgaris could grow under all conditions showing slightlag phases in 100% and 50% effluents. Biomass productivities in thegrowth phase of C. vulgaris except stationary phase indicated thatthe highest biomass productivity was achieved in pH 100% culture(433 ± 11.9 mg/L/d), followed by pH 50% (393 ± 8.5 mg/L/d), 50%(358 ± 6.5 mg/L/d), 100% (296 ± 6.7 mg/L/d), 15% (177 ± 11.0 mg/L/d), and pH 15% (172 ± 2.2 mg/L/d) cultures (Table 2). The resultsshowed that there were significant differences (P < 0.05) betweenthe initial effluent concentrations and the biomass productivities.The biomass productivities obtained from this study were similarto or higher than those reported previously by Cabanelas et al.(2013) who attained values ranging from 39 to 195 mg/L/d withC. vulgaris cultivated in sludge centrates. Maximum biomass con-centration increased as the initial nutrient concentration wasincreased; this result implies that the organic matter and nutrientsin the effluent from the SSFV did not include compounds that inhi-bit the growth of C. vulgaris. The pH values of 100%, 50%, and 15%effluents were elevated during the cell growth due to photosynthe-sis of microalgae and reduction of organic acids in the effluents(Fig. 2). The relatively low cell growths in 100% and 50% effluentscompared to pH 100% and pH 50% effluents could be attributedto the fast increase in pH during the cultivation, resulting ininhibiting the cell growth. This result showed that pH wouldbe an important factor that can trigger increased biomass

s of effluent from the SSFV.

50% pH 50% 15% pH 15%

5 5 3 3358 ± 6.5 393 ± 8.5 177 ± 11.0 172 ± 2.2

centration on microbial oil production from Chlorella vulgaris cultivated inlatile fatty acids. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/

Fig. 2. pH variations during the cultivation of C. vulgaris in different concentrationsof effluent from the SSFV.

4 H.U. Cho et al. / Bioresource Technology xxx (2014) xxx–xxx

concentrations. However, biomass concentration did not differsignificantly between 15% and pH 15% cultures, which might bedue to the limit of soluble metabolites available. These resultsconform to other reports that microalgal growth is directlyinfluenced by nutrient concentrations and the stability of pH(Aslan and Kapdan, 2006; Akerstrom et al., 2014).

Fig. 3. Profiles of (a) SCOD removal, (b) NH4+-N removal, and (c) PO4

�3 r

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3.3. The removal of nutrients and VFAs in the effluent from the SSFV

SCOD concentrations were significantly decreased in all condi-tions as a result of absorption of organic compounds by C. vulgaris(Fig. 3a). At the end of the cultivation, the SCOD removalefficiencies were 76.13%, 89.61%, 87.33%, 90.06%, 85.65%, and87.89% in the 100%, pH 100%, 50%, pH 50%, 15%, and pH 15% efflu-ents, respectively, indicating that the increased utilization of car-bon sources by C. vulgaris was observed under pH controlconditions. The concentrations of VFAs in the effluents accountedfor over 75% of the SCOD concentrations (Fig. 4a); this observationsuggests that the VFAs were used as the major carbon sources forthe growth of C. vulgaris. The VFAs were almost completely assim-ilated in pH 100%, 50%, pH 50%, 15%, and pH 15% effluents, while asmall portion of the VFAs was left in 100% effluent on day 11resulting from the high initial concentration of those and elevatedpH during the cultivation. These observations confirm that the highconcentration of VFAs produced biologically from the sewagesludge could be absorbed rapidly and efficiently at neutral pHalthough the microalgae could consume VFAs at alkaline pH to acertain degree; this observation is consistent with previous find-ings that organic carbon sources were assimilated thoroughly byChlorella sp. at adjacent neutral pH (Shi et al., 2006). The individualVFA reductions on day 5 of 100% and pH 100% effluent showed thepreference for individual VFAs consumed by C. vulgaris (Fig. 4b).The most rapidly absorbed component was acetate, followed byiso-butyrate, valerate, propionate, butyrate, and iso-valerate in100% effluent, whereas the rapid consumption of acetate and

emoval in the effluents from the SSFV during a cultivation period.

centration on microbial oil production from Chlorella vulgaris cultivated inolatile fatty acids. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/

Fig. 4. Profiles of (a) VFA concentrations and (b) individual VFA removal rates onday 5.

Fig. 5. Lipid productivities and contents of C. vulgaris grown in different concen-trations of effluent from the SSFV.

Table 3Fatty acid profiles of C. vulgaris grown in different concentrations of effluent from theSSFV (% of total FAME).

Fatty acid 100% pH 100% 50% pH 50% 15% pH 15%

C14:0 0 0 0 0 0 0C16:0 15.19 16.36 20.71 18.16 25.66 22.08C16:1 2.09 0.96 0 0 0 0C18:0 1.34 1.48 0 2.33 1.32 0C18:1 20.78 24.72 37.73 15.04 7.90 12.94C18:2 56.44 54.17 41.56 54.86 25.80 32.28C18:3 4.17 2.31 0 9.62 39.32 32.70C20:0 0 0 0 0 0 0C22:0 0 0 0 0 0 0C22:1 0 0 0 0 0 0C24:0 0 0 0 0 0 0C16–C18 100 100 100 100 100 100Unsaturated 83.48 82.16 79.29 79.52 73.02 77.92Saturated 16.53 17.84 20.71 20.49 26.98 22.08

Note: ‘‘pH’’ means the pH was controlled at 7.0 periodically.

H.U. Cho et al. / Bioresource Technology xxx (2014) xxx–xxx 5

iso-butyrate occurred simultaneously, followed by consumption ofthe others in the same order as in pH 100% effluent. It is knownthat acetate is one of the best organic carbon sources for cultivat-ing microalgae mixotrophically or heterotrophically and its lowmolecular weight and structural simplicity compared to the otherVFAs might enable microalgal cells to absorb it rapidly (Bhatnagaret al., 2010; Boyle and Morgan, 2009).

Microalgae have been believed to have an ability to removenitrogen and phosphorus from various wastewaters (Boelee et al.,2012; Li et al., 2011; Chinnasamy et al., 2010). The main form ofnitrogen present in the effluent from the anaerobic fermentationof organic wastes is ammonium, which is the form of nitrogen thatmicroalgae prefer. The ammonium removal rates were in the rangeof 19.77–31.83% in each condition, which were relatively lowerthan those in other studies resulting from the high ammoniumconcentrations in the effluents used in this study (Fig. 3b). Accord-ing to the literature (Wang et al., 2010), the removal rates ofammonium in municipal wastewaters by Chlorella sp. were from74.7% to 82.4% but they used the wastewaters with low initial con-centrations of ammonium (32.2 mg/L to 71.8 mg/L). Phosphateremoval rates increased from 41.42% to 90.85% with the decreasein the initial concentrations of phosphate (Fig. 3c) and the results

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were comparable to previous studies that about 55% removal ofphosphorus was achieved by C. vulgaris cultivated in industrialwastewaters containing phosphorus concentration of 112 mg/L(Gonzalez et al., 1997). The results observed in this study showedthat C. vulgaris was very efficient in reducing high concentrationsof ammonium and phosphate, which might be due to the presenceof high concentrations of organic carbon sources such as VFAsassimilated readily by C. vulgaris together with the nutrients. Inaddition, appropriate pH control would be required to enhancenot only the removal rate of organic carbon, nitrogen, and phos-phorus but also the growth of microalgae when VFAs are used asthe main carbon sources.

3.4. Lipid and fatty acid profile

The lipid contents of C. vulgaris ranged between 12.87% and20.01% under six different conditions (Fig. 5). The highest lipidaccumulation of C. vulgaris occurred in pH 15% effluent and thelowest lipid accumulation of it was observed in pH 100% effluent,indicating that the lipid contents increased with the decrease inthe initial concentrations of the effluent. In general, accumulationof microbial lipids can be promoted by environmental stressessuch as nitrogen or phosphorus deficiencies; the relatively lowconcentrations of ammonium and phosphate in 15% effluents com-pared to the 50% and 100% effluents resulted in increased accumu-lation of lipids in the cells (Wang et al., 2012; Hu et al., 2008;Illman et al., 2000). pH stresses also seemed to affect lipid contentssince those were slightly higher in 100% and 50% cultures than in

centration on microbial oil production from Chlorella vulgaris cultivated inlatile fatty acids. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/

6 H.U. Cho et al. / Bioresource Technology xxx (2014) xxx–xxx

pH 100% and pH 50% cultures, which was in agreement with pre-vious findings that the lipid accumulation can be induced whenthe Chlorella sp. is exposed to elevated pH (Guckert and Cooksey,1990). However, no significant difference for the lipid contentsbetween 15% and pH 15% cultures might be due to the fact thatphosphate limitation outweighed the pH stresses. Although lipidcontents were high in 15% and pH 15% cultures, their lipid produc-tivities were lower than in the other cultures because biomass pro-ductivity differed more than did the lipid content. Therefore, thehighest lipid productivity (66.98 ± 6.48 mg/L/d) was achieved inthe pH 50% culture (Fig. 5). The results also indicated that therewere significant differences (P < 0.05) between the initial nutrientconcentrations and the lipid contents.

Fatty acid compositions vary with cultivation conditions andspecies (Petkov and Garcia, 2007). It can be seen that the main fattyacid methyl esters (FAME) produced from C. vulgaris in this studywere C16:0 (palmitic acid), C18:1 (oleic acid), C18:2 (linoleic acid),and C18:3 (linolenic acid) accounting for 96.58%, 97.56%, 100%,97.68%, 98.68%, and 100% in the 100%, pH 100%, 50%, pH 50%,15%, and pH 15% cultures, respectively (Table 3). In terms of thequality of biodiesel, containing 16 or 18 carbons are favorable forupgrading it and the results obtained from this study compliedwith the requirement of high-quality biodiesel (Huang et al., 2010).

4. Conclusions

VFAs produced from the SSFV were demonstrated to be favor-able carbon sources for cultivating C. vulgaris, and combining theSSFV with microalgae cultivation is a promising process that canreduce the cost of microalgae production by utilizing the low-costorganic waste as the feedstock. However, the lipid contents of theproduced biomass were not high enough to satisfy the condition ofoleaginous species. Therefore, further researches on finding outrobust strains accumulating more lipids in their cells or adding astarvation phase, which includes low concentrations of nutrients,to the above system might be necessary to increase the lipidproductivity.

Acknowledgements

This research was supported by the Advanced Biomass R&DCenter (ABC) of Korea Grant funded by the Ministry of Education,Science and Technology (ABC-2013059453). This research was alsopartially supported by a Grant from Korea and the ManpowerDevelopment Program for Marine Energy funded by Ministry ofLand, Transportation and Maritime Affairs (MLTM) of Korean gov-ernment, a part of the project entitled ‘Technology Development ofMarine Industrial Biomaterials’ funded by the Ministry of Oceansand Fisheries, Korea, POSCO and the Korea Institute of EnergyTechnology Evaluation and Planning (KETEP) Grant funded by theKorea Government Ministry of Knowledge Economy (Nos.2012K130, SUBJID_ 0000000014497), and BK21+ program throughthe National Research Foundation of Korea funded by the Ministryof Education, Science and Technology.

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