Identification and characterization of a freshwater microalga Scenedesmus SDEC-8 for nutrient removal and biodiesel production

Bioresource Technology 162 (2014) 129–135

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Identification and characterization of a freshwater microalga Scenedesmus SDEC-8 for nutrient removal and biodiesel production Mingming Song a, Haiyan Pei a,b,⇑, Wenrong Hu a,b, Shuo Zhang a, Guixia Ma a, Lin Han a, Yan Ji a a b

School of Environmental Science and Engineering, Shandong University, 27 Shanda Nan Road, Jinan 250100, China Shandong Provincial Engineering Centre on Environmental Science and Technology, 17923 Jingshi Road, Jinan 250061, China

h i g h l i g h t s  Six newly isolated algae species were investigated for biodiesel production.  Lipid accumulation, fatty acid profiles and biodiesel properties were examined.  Ultrastructure and molecular analysis of the most potential strain were examined.  Scenedesmus SDEC-8 is rich in fatty acid profiles of C16:0 and C18:1.  Scenedesmus SDEC-8 is promising for algae-biofuel production and nutrient removal.

a r t i c l e

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Article history: Received 9 February 2014 Received in revised form 21 March 2014 Accepted 24 March 2014 Available online 3 April 2014 Keywords: Microalgae Identification Lipid production FAME production N, P removal

a b s t r a c t The selection of the right strains is of fundamental important to the success of the algae-based oil industry. From the six newly isolated microalgae strains tested for growth, fatty acid methyl ester (FAME) profiles and biodiesel properties, Scenedesmus SDEC-8, with favorable C16:0 fatty acids (73.43%), showed the best combined results. Then, morphological and molecular identification were examined. From the three wastewaters samples, Scenedesmus SDEC-8 showed good ability to yield oil and remove nutrients, which were comparable with other reports. In b artificial wastewater (TN 40 mg L1, TP 8 mg L1), Scenedesmus SDEC-8 achieved the highest value of lipid productivity (53.84 mg L1 d1), MUFA content (35.35%) and total FAME content (59.57 ± 0.02 mg g1 DW), besides higher removal efficiencies of TN (99.18%) and TP (98.86%) helped effluent directly discharge and smaller dilution factor of N, P (3.3 and 9) which was good for lessening water utilization. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction As increasing use of fossil fuels aggravates the energy crisis and global climate change, a new renewable energy source is urgently needed. Recently, microalgae-based biodiesel production has attracted significantly increased interest (Hoekman et al., 2012; Mata et al., 2010). Besides their inherent advantageous qualities, microalgae have proven to be highly effective lipid producers (Sivakumar et al., 2012; Hoekman et al., 2012), yielding about 15–300 times more oil than conventional crops (Li et al., 2010b). However, inadequate numbers of microalgal species and relatively little information on their detailed fatty acid compositional profiles have limited the development of the algal bio-resource (Hoekman et al., 2012), ⇑ Corresponding author at: School of Environmental Science and Engineering, Shandong University, 27 Shanda Nan Road, Jinan 250100, China. Tel./fax: +86 531 88392983. E-mail address: [email protected] (H. Pei). http://dx.doi.org/10.1016/j.biortech.2014.03.135 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.

so we urgently need to identify more microalgal species that can be exploited as a bio-resource (Grobbelaar, 2000). Selection of highly productive, oil-rich algal strains is fundamentally important to enrich the algae collection available for biodiesel production (Griffiths et al., 2012). Several high oil algal strains have been commonly reported, such as Botryococcus braunii, Nannochloropsis sp., Schizochytrium sp., which can have a total lipid content of 30–60% of their dry weight (Chisti, 2007). However, current studies are mainly focused on several locally isolated microalgal strains such as Chlorella (Li et al., 2011a; Lv et al., 2010), Scenedesmus (Li et al., 2011b), and Nannochloropsis sp. (Rodolfi et al., 2008), which showed faster growth and easier cultivation besides their high lipid content. Recent studies have paid more attention to the application of algae in wastewater treatment for nutrient removal (Zhou et al., 2011; Yang et al., 2011). Inorganic nutrients are necessary to develop algae as biodiesel feedstock; however, their high cost is the main block in microalgal application (Behzadi and Farid, 2007). The combination of wastewater

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treatment and algal biomass cultivation for biodiesel has proven to be an economic and environmentally sustainable way to solve two problems at once (Zhou et al., 2011). Hence, the selection of algal strains with high nutrient removal rate and high lipid productivity from a local wastewater environment is one of the major challenges for algae-biofuel development. Although Scenedesmus (Li et al., 2011b) and Chlorella (Yang et al., 2011) have been the most widely studied for nitrogen and phosphorus removal and lipid accumulation, the reported quantitative data is insufficient on their fatty acid (FA) compositional profiles, which will determine the properties of the final biodiesel (Griffiths et al., 2012; Knothe, 2011; Tan and Lin, 2011). Thus, the specific objectives of this study were (1) to isolate and identify robust microalgae strains from freshwater habitats in China; (2) to characterize the selected strains for their biomass and lipid production and determine the most promising strains with high oil productivity and suitable fatty acid (FA) compositional profiles for biofuel production; and (3) to study the potential of this promising microalgae for growth, lipid accumulation, FA compositional profiles and nutrient removal from wastewater. 2. Methods 2.1. Algal strains and cultivation The microalgae strains were obtained from the local freshwater lake (Quancheng Lake in Jinan) that had been slightly polluted by wastewater. And they were isolated by the throat spray-plate method combined with 96 orifice plate method (Zhou et al., 2011). Quancheng Lake water quality was as follows: COD 143.61 ± 1.1 mg L1, NH4-N 17.41 ± 0.8 mg L1, TN 20.23 ± 1.0 mg L1, TP 1.129 ± 0.7 mg L1, Turbidity 4.5 ± 0.01, Dissolved Oxygen 5.68 ± 0.1, pH 7.6 ± 0.2. The isolated six strains were preserved in BG11 medium: NaNO3 1.5 g L1, K2HPO4 40 mg L1, MgSO47H2O 75 mg L1, CaCl22H2O 36 mg L1, citric acid 6 mg L1, ferric ammonium citrate 6 mg L1, EDTANa2 1 mg L1, Na2CO3 20 mg L1, A5 solution 1 mg L1. A5 solution: MnCl24H2O 1.86 g L1, ZnSO47H2O 0.22 g L1, Na2MoO42H2O 0.39 g L1, H3BO3 2.86 g L1, CuSO45H2O 0.08 g L1, Co(NO3)26H2O 0.05 g L1. The microalgae strains were enriched from a volume of 20 mL of stock culture in a 250 mL Erlenmeyer at 25 ± 1 °C, under continuous illumination (40 lmol photons m2 s1) provided by daylight fluorescent tubes. Every week, three times the volume of culture media was added to the culture flask, which was manually shaken twice per day to prevent biomass sedimentation. Then each strain was cultivated in a single batch in 500 mL flasks for 18 days. 2.2. Experimental set up As ammonium is the main nitrogen source in wastewater (Li et al., 2010a; Zhang et al., 2004), (NH4)2CO3 was selected as the main nitrogen source in the three artificial wastewaters in this study. K2HPO4 was selected as the main phosphorus source. Besides nitrogen and phosphorus, the composition of other elements was the same as BG11 medium. Based on typical domestic sewage (Zhang et al., 2004), three artificial wastewaters (a, b, c) with different nitrogen and phosphorus concentrations: a (TN 20 mg L1, TP 4 mg L1), b (TN 40 mg L1, TP 8 mg L1), c (TN 85 mg L1, TP 15 mg L1), were created to investigate the effect of different nitrogen and phosphorus concentrations on the growth and lipid compositions of the algae during the 18 day study, each trial with three replicates. BG11 (TN 246.96 mg L1, TP 7.12 mg L1) was designed as the control medium. Scenedesmus SDEC-8 was cultivated in 800 mL autoclaved growth medium in 1 L flasks. After reaching

the late exponential phase, the microalgae cells were recovered by centrifugation and suspended in 15 mg L1 NaHCO3 solution before inoculation. All cultures were continuously bubbled with filtered compressed air at the rate of 0.2 L min1. 2.3. Algal growth and lipid determination Microalgae growth was measured every 24 h based on enumeration as described in our previous work (Song et al., 2013). Specific growth rate (d1) was calculated according to the following equation:

k ¼ ðln N1  ln N2 Þ=ðt 1  t2 Þ

ð1Þ

1

where k (d ) is the specific growth rate in exponential growth phase, N1 and N2 are the biomass concentration at day t1 and t2. Biomass productivity was obtained according to the following calculation (Song et al., 2013).

PDMDW ¼ DM  k

ð2Þ 1

1

where PDMDW is the biomass productivity (mg L d ) in the exponential growth phase and DM (mg L1) is the biomass concentration at the end of the exponential phase. The total lipid contents were extracted with a chloroform/methanol (2:1, v/v) mixture and were quantified gravimetrically (Song et al., 2013). The lipid productivity (PL) was calculated according to the following equation:

PL ¼ PDMDW  LW

ð3Þ 1

1

where PL is the lipid productivity (mg L d ) in the exponential growth phase. LW stands by lipid content based on dry weight. Fatty acid content and composition analysis were determined by two steps including preparation of fatty acids methyl ester (FAME) and Gas Chromatography–Mass Spectrometry analysis. FAME was prepared by acid-catalyzed esterification in our previous study. Dried algae samples (about 300 mg) were reacted directly with 1.5 ml mixture of methanol and solid acid (30 mg) (Amberlyst 15, Sigma–Aldrich, USA). Transesterification was firstly carried out in a 60 °C water bath for 40 min and then 1.5 ml KOH– methanol solution (4% of m:v) was added the solution for 60 °C water bath for 60 min. Upon completion of the reaction, hexane (1 mL) was added into the solution, and the whole solution was homogenized (Ultrasonic Homogenizer SCIENTZ-IID, China) for 10 min, and then separated into two layers. Heptadecanoic acid methyl ester (50 lL, 2 mg mL1) was added to upper layer solution (200 lL) for methyl ester analysis on GC–MS (Trace GC-DSQII, Thermo Fisher, USA). Solution (1 lL) was injected under splitless mode and the injector temperature was 210 °C. The oven temperature was set at 140 °C, and held for 2 min, then raised to 200 °C at a rate of 3 °C min1, and held at 200 °C for 10 min. Mass spectra was recorded under electron ionization (70 eV) at a frequency of 5 scans for 1 s and the ion source was 250 °C and full scan was 150–650 amus. In order to calculate the FAME yield of samples, a mixture of FAME standards (Sigma Aldrich 47,885, 10 mg mL1) was analyzed at the same GC–MS condition as the depicted above. FAME yield were calculated by Eq. (4), where fi is the correction factor of section i and this data is obtained from the analysis of the mixture of FAME standards, Ai is the peak area of section i, As is the peak area of internal standard, Cs is the concentration of internal standard and v is the volume of upper layer.

FAME yields ¼

X

fi

Ai v As =C s



ð4Þ

2.4. Nitrogen and phosphorus analysis For the measurement of water quality, the microalgae culture was centrifuged (4000 rpm at 3 °C for 10 min) and the

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supernatant was filtered through a 0.45 mm membrane for use in the determination of TN and TP. All the measurements were conducted according to Chinese state standard testing methods (Monitoring Methods for Water and Wastewater, 2002). 2.5. Microscopy and molecular identification The cell morphology of the isolated strain was observed by microscope (CX31, OLYMPUS, Japan), scanning electron microscopy (SEM) (Hitachi S-3400N, Japan) and transmission electron morphology (TEM) (Hitachi JEOL-1200EX, Japan) (Ren et al., 2013; Nemcova et al., 2011). Genomic DNA was extracted with a DNeasy Plant Mini kit according to the manufacturer’s instructions (Zhou et al., 2011). 18S rDNA PCR was amplified as described previously (Ren et al., 2013). For PCR amplification, a dilute part of extracted DNA was used to amplify the 18S rRNA gene with two primers, 18 S-F (CCTGGTTGATCCTGCCAG) and 18 S-R (TTG ATCCTTCTGCAGGTTCA) (Wan, 2012). The PCR products were purified by SK1131 kit (Sangon Biotech Co. Ltd., Shanghai, China) and then sent to Sangon Biotech Co. Ltd., (Shanghai, China) for DNA sequencing. Alignment of consequent 18S rRNA gene sequences and analysis of the similarity of the18S rRNA were completed with Basic Local Alignment Search Tool (BLAST) in GenBank database of the National Center for Biotechnology Information. 3. Results and discussion 3.1. Selection and identification of the most promising strain among the candidate strains To determine the most promising algal strain for biodiesel production, the specific growth rate, biomass concentration and lipid productivity of the six newly isolated algal strains were investigated (Table 1). Five of them produced higher lipid contents than that average value of 20% reported for algal species (Rodolfi et al., 2008; Li et al., 2011b) under the same cultivation conditions. Chlorella SDEC-6 attained the highest lipid and FAME content of 37.6% and 11.7 mg g1, respectively, but it had lower lipid productivity than Scenedesmus SDEC-8 because of non-competitive biomass concentration. The local strain Scenedesmus SDEC-8 achieved the highest biomass productivity of 253.45 mg L1 and the highest lipid productivity of 13.52 mg L1 d1, and it was comparable with those green algal strains with lipid productivities of 0.8–9 mg L1 d1 (Song et al., 2013). The GC–MS analysis showed that the main fatty acid compositions of the six microalgae strains were C16–C18 fatty acids, accounting for over 95% of total fatty acids (Table 2). It is well known that C16–C18 fatty acids are the most common feedstock suitable for biodiesel production (Knothe, 2009). It is interesting to note that the six locally green algal strains could produce higher C16:0 contents (70–80% of total fatty acids) than those in the previous report (Song et al., 2013). Scenedesmus SDEC-8 achieved C16:0 content of 73.43%, which was comparable with the highest one. In addition, the C18:1 content of the six strains ranged from 20% to 30%. Oleic acid (18:1) and palmitoleic acid (16:1) were

considered as the optimum biodiesel compositions, as they can give the finest compromise between oxidative stability and cold flow properties (Knothe, 2009; Hoekman et al., 2012). To further evaluate the most important biodiesel properties derived from the six algal oil, kinematic viscosity, cloud point, specific gravity, cetane number, iodine number and higher heating value were estimated based on the predictive models (Song et al., 2013). Table 2 indicated that the six biodiesel properties of the six candidates almost satisfied the quality standards of ASTM D6751 and EN 14214 and the ranges of qualities occurring in common biodiesel feedstock (Song et al., 2013; Hoekman et al., 2012; Knothe, 2011). Higher cetane number (CN) and lower iodine number (IN) than those standards may be due to high content of saturated fatty acid (SFA) (70–80%), which ultimately decided the oxidation stability (Francisco et al., 2010; Hoekman et al., 2012). Scenedesmus SDEC-8 reached the CN of 61.38, which was almost equal to the highest CN (61.61) achieved by Chlorella SDEC-4. In addition, the IN of Scenedesmus SDEC-8 was about 29.38, which was close to the lowest IN (26.88) got by Chlorella SDEC-4. So combined with highest lipid productivity and favorable biodiesel properties, Scenedesmus SDEC-8 was considered as the most promising species in this study. 3.2. Morphological and molecular identification of Scenedesmus SDEC-8 The microalgae strain was preliminary identified as genus Scenedesmus by microscopic and SEM analysis. The green microalgae colonies were composed of 2–4 cells in oblong or ovate shapes. And it bore large spines with the length of about 6 lm at the four poles of colonies. The length and width of colonies ranged from 9 to 26 lm and 8 to 14 lm, respectively, depending on different growth phase. The ultrastructure showed that each cell contained a single chloroplast that occupied half of the cell besides nucleus and mitochondrion. The chloroplast contained a noticeable pyrenoid surrounded by a starch envelope of two halves. A different type of vacuoles with electron-transparent content and oil-like content in some vacuoles were visible. To further identify the taxonomic position of Scenedesmus SDEC-8, molecular phylogenetic analysis was used in Fig. 1. The partial 18S rRNA sequence from Scenedesmus SDEC-8 consisting of 1054 bases was determined and submitted to the GenBank (Accession No.: KF999643). In the phylogram (Fig. 1), the 18S rDNA sequence of Scenedesmus SDEC8 confirmed its identification as Scenedesmus sp.. The partial 18S rRNA sequence from Scenedesmus SDEC-8 was 98% identical to other Scenedesmus species tested, for example, Scenedesmus sp. KMMCC 872 (1672 bases) (Accession No.: JQ315579.1), and the Scenedesmus sp. Lake Las Vegas (1697 bases) (Accession No.: JX910112.1). 3.3. Growth and lipid accumulation properties of Scenedesmus SDEC-8 in different culture media To further enhance the biomass production and avoid biomass sedimentation, Scenedesmus SDEC-8 was cultured in BG11 medium with air aeration of gas liquid ratio of 0.2 vvm and the culture with

Table 1 Specific growth rate, lipid productivity and FAME content of six newly isolated microalgal strains cultivated in 500 mL flasks for 18 days. Algal species

Specific growth rate k (d1)

Biomass concentration DM (mg L1)

Lipid content (%)

Lipid productivity PL (mg L1 d1)

FAME (mg g1 DW)

Chlorella SDEC-4 Chlorella SDEC-5 Chlorella SDEC-6 Scenedesmus SDEC-7 Scenedesmus SDEC-8 Scenedesmus SDEC-9

0.18 0.17 0.16 0.14 0.17 0.12

157.30 148.44 159.33 116.80 253.45 84.72

19.40 20.13 37.60 31.60 31.80 30.70

5.49 5.08 9.58 5.17 13.52 3.12

2.59 5.28 11.7 5.54 9.55 5.47

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Table 2 Fatty acid compositional profiles (% of total FAME) and six biodiesel properties of screened microalgal strains. Fatty acid profiles

C14:0 C15:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 SFA MUFA PUFA Kinematic viscosity 40 °C (mm2 s1) Specific gravity (kg L1) Cloud point (°C) Cetane number Iodine number (gI2/100 g) HHV(MJ/kg)

Strains Chlorella SDEC-4

Chlorella SDEC-5

Chlorella DEC-6

Scenedesmus SDEC-7

Scenedesmus SDEC-8

Scenedesmus SDEC-9

– – 80.95 – – 19.05 – – 80.95 19.05 0.00 5.09 0.87 17.45 61.61 26.88 38.87

– – 76.23 – – 23.77 – – 76.23 23.77 0.00 5.06 0.87 16.82 61.29 30.39 38.95

– – 63.44 – – 25.65 6.28 4.63 63.44 25.65 10.92 4.91 0.88 13.65 59.71 48.01 39.37

– – 68.80 – – 31.20 – – 68.80 31.20 0.00 5.01 0.87 15.83 60.80 35.91 39.08

2.49 1.11 73.43 3.54 1.92 16.18 1.35 – 78.94 19.71 1.35 5.06 0.87 17.00 61.38 29.38 38.93

– – 71.85 – – 28.15 – – 71.85 28.15 0.005 5.03 0.87 16.23 61.00 33.65 39.03

SFA = saturated fatty acids (14:0, 16:0, 18:0, 24:0); MUFA = monounsaturated fatty acids (16:1, 18:1); PUFA = polyunsaturated fatty acids (16:2, 16:3, 18:2, 18:3, 20:4, 20:5, 22:6).

Fig. 1. Phylogenetic tree of Scenedesmus SDEC-8 and closely related strains, and their related informations. This tree was analyzed by the neighbor-joining method.

no air aeration was used as a control. Fig. 2a and Table 3 indicated that Scenedesmus SDEC-8 performed well in air-aerated culture. In air aerated culture system, the biomass productivity (41.06 mg L1 d1) and the lipid productivity (13.52 mg L1 d1) showed significant advantage over those in the culture system without aeration. The higher biomass productivity under aeration may be attributed to radial mixing, which provoke mass transfer and high frequency of light exposition, increasing the photosynthetic efficiency of algal cells. So in the three artificial wastewaters (a, b, c), Scenedesmus SDEC-8 was cultured under aeration with gas liquid ratio of 0.2 vvm. As ammonium was the main nitrogen source in wastewater (Li et al., 2010a; Zhang et al., 2004), ammonium was selected as the main nitrogen source in a, b, c artificial wastewaters in this study. Scenedesmus SDEC-8 reached higher growth rate of 0.28– 0.42 d1 in a, b, c artificial wastewaters than that (0.27 d1) in the BG11 control medium (Fig. 2b and Table 3). This result was consistent with the report that Ellipsoidion achieved a higher growth rate in ammonium-N treatment than that in nitrate-N and urea-N treatment (Xu et al., 2001; Li et al., 2010a). Some reports agree that nitrate-N must be transformed into ammoniumN before being utilized by microalgae, so more energy would be needed for microalgae to assimilate nitrate-N than ammonium-N (Xu et al., 2001; Flores et al., 1980). This may be the possible

explanation of why Scenedesmus SDEC-8 grew vigorously in the ammonium medium. In addition, Scenedesmus SDEC-8 produced higher lipid content (16–19%) and FAME content (23–60 mg g1) in a, b, c artificial wastewaters than those of 11.90 ± 2.29% and 10.61 ± 0.08 mg g1 in BG11, respectively. This result was consistent with the report that S. rubescens like alga achieved the highest FAME productivity in ammonium treatment (Lin and Lin, 2011). Although Scenedesmus SDEC-8 achieved the highest biomass of 1.29 ± 0.10 mg L1 in BG11 for the highest TN concentration, it had lower lipid productivity and FAME productivity than a, b, c artificial wastewaters because of non-competitive lipid content and FAME content. So these results showed that Scenedesmus SDEC-8 showed good ability to grow and yield oil in the wastewaters. In a, b, c artificial wastewaters, the higher the concentrations of TN and TP, the higher the biomass concentration. As the concentration of TN and TP increased, FAME content and FAME productivity also increased, but those in c (TN 85 mg L1, TP 15 mg L1) and b (TN 40 mg L1, TP 8 mg L1) wastewaters showed no significant differences. The highest lipid and FAME productivity were achieved in b wastewater, while the lipid productivities in c and a wastewaters were closer, with the values of 45.72 mg L1 d1 and 45.86 mg L1 d1, respectively, which indicated that the high concentrations of TN, TP in c wastewater inhibited algal growth.

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Fatty acid profiles (% of total FAME)

M. Song et al. / Bioresource Technology 162 (2014) 129–135

100

Others C18:3

80

C18:2 C18:1

60

C18:0 C16:2

40

C16:1 C16:0

20

0

C15:0 C14:0 a

b

c

BG11

Culture medium Fig. 3. Fatty acid compositional profiles of Scenedesmus SDEC-8 in wastewaters with different nitrogen and phosphorus concentrations based on typical domestic sewage (% of total FAME). The fatty acids content were listed from bottom to top by the number of carbon in fatty acid. a (TN 20 mg L1, TP 4 mg L1), b (TN 40 mg L1, TP 8 mg L1), c (TN 85 mg L1, TP 15 mg L1).

Fig. 2. (a) Growth curves of Scenedesmus SDEC-8 cultured with no aeration and air aeration of gas liquid ratio of 0.2 vvm in BG11. (b) Growth curves of Scenedesmus SDEC-8 in artificial wastewaters with different nitrogen and phosphorus concentrations based on typical domestic sewage. a (TN 20 mg L1, TP 4 mg L1), b (TN 40 mg L1, TP 8 mg L1), c (TN 85 mg L1, TP 15 mg L1). Values are expressed as means ± s.d. (n = 3).

It was consistent with the report that ammonia nitrogen exceeding 50 mg L1 would inhibit algal growth (Ip et al., 1982). 3.4. Fatty acid properties of Scenedesmus SDEC-8 in different culture media The FAME compositional profiles in a, b, c artificial wastewaters and in BG11 were significantly different (Fig. 3). For example, the contents of C18 series in b and c ammonium-N treatments were 42.34% and 38.01%, respectively, which were higher than that in BG11 nitrate-N treatment (24.35%). Lin and Lin (2011) also reported that the C18 content of Scenedesmus rubescens fed with ammonium-N was higher than that with nitrate-N and urea-N. In addition, the contents of C16–C18 (around 99%) in a, b, c artificial wastewaters were higher than that in BG11 (around 96%) (Table 4). In a word, artificial wastewater was more suitable for biodiesel production than BG11. Xu et al. (2001) found that the C18:1 content of Ellipsoidion sp. cultured in nitrate-N medium was higher than that in ammonium-N medium. However, in the present study, contents of C18:1 in b and c ammonium-N treatments (33.18% and

27.67%) were higher than that in BG11 nitrate-N treatment (19.44%), and the values of C18:1 of Scenedesmus SDEC-8 were much higher than that in many other algae reported, such as S. rubescens like alga (Lin and Lin, 2011), Haematococcus pluvialis (Damiani et al., 2010) and Chlorella vulgaris (Converti et al., 2009). Many reports have evaluated the fatty acids composition of microalgae. The highest C16:0 content (as % of total FAME) of Ellipsoidion sp. at post-logarithmic growth phase achieved 40% under the different ammonia concentrations (Xu et al., 2001). Both C. vulgaris and N. oculata got high values of C16:0 content (as % of total FAME) at around 60% (Converti et al., 2009). Damiani et al. (2010) reported that the C16:0 content (as % of total FAME) of H. pluvialis was 22.49% in control condition. In this study, the contents of C16:0 of Scenedesmus SDEC-8 were siginificantly higher with the values ranged 50–80% under different culture media. Therefore, in the artificial wastewaters Scenedesmus SDEC-8 showed significant advantage for biodiesel production over other microalgae species reported before.

Table 4 Fatty acid compositional profiles of Scenedesmus SDEC-8 in wastewaters with different nitrogen and phosphorus concentrations (% of total FAME). Fatty acid profile

BG11

a

b

c

C16–C18 SFA MUFA PUFA

96.24 72.04 23.05 4.91

98.91 80.87 19.13 0.00

98.69 57.76 35.85 6.39

99.30 62.17 28.21 9.62

SFA = saturated fatty acids (14:0, 16:0, 18:0, 24:0); MUFA = monounsaturated fatty acids (16:1, 18:1); PUFA = polyunsaturated fatty acids (16:2, 16:3, 18:2, 18:3, 20:4, 20:5, 22:6). a (TN 20 mg L1, TP 4 mg L1), b (TN 40 mg L1, TP 8 mg L1), c (TN 85 mg L1, TP 15 mg L1).

Table 3 Specific growth rate, lipid productivity and FAME productivity of Scenedesmus SDEC-8 in BG11 and artificial wastewaters with different nitrogen and phosphorus concentrations based on typical domestic sewage. a (TN 20 mg L1, TP 4 mg L1), b (TN 40 mg L1, TP 8 mg L1), c (TN 85 mg L1, TP 15 mg L1).

BG11 no areation BG11 areation a b c

Specific growth rate k (d1)

Biomass concentration DM (g L1)

Total lipid content (%)

Lipid productivity PL (mg L1 d1)

FAME content (mg g1 DW)

FAME productivity (mg L1 d1)

0.17 0.27 0.42 0.37 0.28

0.25 ± 0.08 1.29 ± 0.10 0.66 ± 0.02 0.80 ± 0.12 0.96 ± 0.02

31.80 ± 0.56 11.90 ± 2.29 16.36 ± 0.29 18.29 ± 0.64 17.19 ± 0.67

13.52 41.06 45.86 53.84 45.72

9.55 ± 0.26 10.61 ± 0.08 22.82 ± 0.10 59.57 ± 0.02 57.74 ± 0.25

0.41 3.66 6.40 17.53 15.36

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The fatty acids composition in some microalgae could be easily changed through altering the cultivation conditions (Damiani et al., 2010). The fatty acids composition also fluctuated significantly in a, b, c artificial wastewaters. Lin and Lin (2011) reported that the content (%) of C18:3 decreased during the nitrogen starvation phase. In the present study, as the concentration of TN and TP increased, the content (%) of C18:2 and C18:3 also increased, but the content of C16:0 decreased dramatically. In addition, with higher concentration of TN and TP, the content of PUFA was higher, but the content of SFA was lower. In b artificial wastewater, Scenedesmus SDEC-8 got the highest MUFA content of 35.35%, highest total FAME content of 59.57 ± 0.02 mg g1 DW and highest FAME productivity of 17.53 mg L1 d1. Therefore, the excessive utilization of TN and TP decreased the FAME content in the algae. So of the three artificial wastewaters, b (TN 40 mg L1, TP 8 mg L1) was most advantageous for Scenedesmus SDEC-8 to grow and produce biodiesel.

3.5. Nutrient removal properties of Scenedesmus SDEC-8 in different culture media The nitrogen (N) and phosphorus (P) removal efficiency, dilution factors and lipid productivity obtained in previous studies were summarized in Table 5. The removal efficiencies achieved by Scenedesmus SDEC-8 in this study were comparable with other dominant species under the same nutrient concentration and the lipid productivity obtained from in present study ranked among the top ones of those from previous studies. In particular, the N, P removal efficiencies of Scenedesmus SDEC-8 in b (TN 40 mg L1, TP 8 mg L1) and c (TN 85 mg L1, TP 15 mg L1) were far more than those efficiencies of C. vulgaris when initial NH4-N concentration ranged of 41.8–92.8 mg L1 with the concentration of PO4-P above 7 mg L1 at the 10th day (Aslan and Kapdan, 2006). In addition, it is notable that, whereas the treated effluents used as a, b, c culture media, a (TN 20 mg L1, TP 4 mg L1), b (TN 40 mg L1, TP 8 mg L1), c (TN 85 mg L1, TP 15 mg L1) would require dilution factors of 400, 800, 1500 to make the N and P concentrations of the discharge smaller than the risk limits for the eutrophication of the water. The treatments represented by the Scenedesmus SDEC-8 significantly reduce these values, as shown in Table 5. It also showed that dilution factors of the effluents in this study were smaller than others under the same nutrient concentration, so less water was needed to dilute the effluent before it was discharged into a freshwater.

Fig. 4. Changes in TN and TP concentration of Scenedesmus SDEC-8 in wastewaters with different nitrogen and phosphorus concentrations based on typical domestic sewage. a (TN 20 mg L1, TP 4 mg L1), b (TN 40 mg L1, TP 8 mg L1), c (TN 85 mg L1, TP 15 mg L1).

In a, b, c artificial wastewaters, the highest N and P removal efficiencies showed no significant differences but were reached in less time at lower N and P concentration. The residual P concentration showed a prominent decrease during the first 3 day of the culture (In Fig. 4). Over 50% of this P removal was not mainly caused by the dissolved nature of the substance but depended primarily on the concentration and the surface area available for adsorption (Martinez et al., 2000). Combining these results and microscopic examination, it can be speculated that the surface of the Scenedesmus SDEC-8 cells was large for adsorption. After 3 days, the

Table 5 Summary of nitrogen (N) and phosphorus (P) removal efficiency, dilution factors and lipid productivity in previous studies.

a

Dilution factors fN Dilution factors fP Lipid Incubation References time (day) productivity PL (mg L1 d1)

Nitrogen and phosphorus type

Microalgae species

Initial nutrient concentration (mg L1) N

P

N

P

NH4-N, PO4-Pa NH4-N, PO4-Pa NH4-N, PO4-Pa TN, TPa (NO3-N, PO4-Pa) TN, TPb TN, TPb TN, TPa (NH4-N, PO4-P)

Scenedesmus obliquus Chlorella vulgaris Chlorella vulgaris Chlorella vulgaris

27.4 13.2–21.2 41.8–92.8 60.0

11.8 7.7 >7.7 2.28

>99 >99 50 95

97 78 <30 –

7.8 <1.0–1.7 163–362 30

3.9 66 >210

Scenedesmus sp. LX1 Chlorella ellipsoidea YJ1 Scenedesmus SDEC-8 Scenedesmus SDEC-8 Scenedesmus SDEC-8

15.5 7.0 20 40 85

0.50 0.46 4 8 15

98.5 99.3 >99 >99 98.2

98 95.2 >99 98.9 96.8

2.3 0.49 0.5 3.3 15.3

1 2.21 0.1 9 48

Removal efficiency (%)

– – – 35

8 10 5

Martinez et al. (2000) Aslan and Kapdan (2006) Aslan and Kapdan (2006) Lv et al. (2010)

8 12.7 45.86 53.84 45.72

15 22 18 18 18

Li et al. (2010b) Yang et al. (2011) Current study Current study Current study

Nutrient from artificial medium. Nutrient from secondary effluent of wastewater treatment plant. The dilution factors are expressed so that after removal of the biomass, the N and P concentrations of the effluent into a freshwater current are within the risk limits for the eutrophication of the water (Schelef et al., 1980). N = 0.1 mg L1, P = 0.01 mg L1; fN = N/0.1, fP = P/0.01. b

M. Song et al. / Bioresource Technology 162 (2014) 129–135

eliminations of P decreased and the concentrations of P kept stable, which can be explained by suggesting that ruptured cells of microalgae spilled their P content into the culture medium (Martinez et al., 2000). 4. Conclusions The isolated Scenedesmus SDEC-8 achieved the highest value of lipid productivity (53.84 mg L1 d1), total FAME content (59.57 ± 0.02 mg g1 DW) and FAME productivity (17.53 mg L1 d1) in b artificial wastewater (TN: 40 mg L1, TP: 8 mg L1), besides the better nitrogen and phosphorus removal (99.18% and 98.86%, respectively) and less dilution factor of fN (3.3) and fP (9) for reducing risk limits for the water eutrophication. These results indicated that the algae appeared to have great potential for use in biodiesel production and nitrogen and phosphorus removal for wastewater treatment. Acknowledgements Funding for this research was provided by Natural Science Foundation of China (51078221), Science and Technology Development Planning of Shandong Province (2012GGE27027), The Program for New Century Excellent Talents in University of the Ministry of Education of China (Grant No. NCET-12-0341). The authors thank Findlay A Nicol of Shandong University of Finance and Economics for revising the English in the manuscript. References Aslan, S., Kapdan, I.K., 2006. Batch kinetics of nitrogen and phosphorus removal from synthetic wastewater by algae. Ecol. Eng. 28 (1), 64–70. Behzadi, S., Farid, M.M., 2007. Review: examining the use of different feedstock for the production of biodiesel. Asia Pac. J. Chem. Eng. 2, 480–486. Chisti, Y., 2007. Biodiesel from microalgae. Biotechnol. Adv. 25, 294–306. Converti, A., Casazza, A.A., Ortiz, E.Y., Perego, P., Del Borghi, M., 2009. Effect of temperature and nitrogen concentration on the growth and lipid content of Nannochloropsis oculata and Chlorella vulgaris for biodiesel production. Chem. Eng. Process. 48, 1146–1151. Damiani, M.C., Popovich, C.A., Constenla, D., Leonardi, P.I., 2010. Lipid analysis in Haematococcus pluvialis to assess its potential use as a biodiesel feedstock. Bioresour. Technol. 101, 3801–3807. Flores, E., Guerrero, M.G., Losadh, M., 1980. Short term ammonium inhibition of nitrate utilization in Anacystis nidulans and other cyanobateria. Arch. Microbiol. 128, 137–140. Francisco, E.C., Neves, D.B., Jacob-Lopes, E., Franco, T.T., 2010. Microalgaes feedstock for biodiesel production: carbon dioxide sequestration, lipid production and biofuel quality. J. Chem. Technol. Biotechnol. 85, 395–403. Griffiths, M.J., van Hille, R.P., Harrison, S.T.L., 2012. Lipid productivity, settling potential and fatty acid profile of 11 microalgal species grown under nitrogen replete and limited conditions. J. Appl. Phycol. 24, 989–1001. Grobbelaar, J.U., 2000. Physiological and technological considerations for optimising mass algal cultures. J. Appl. Phycol. 12, 201–206. Hoekman, S.K., Broch, A., Robbins, C., Ceniceros, E., Natarajan, M., 2012. Review of biodiesel composition, properties, and specifications. Renewable Sustainable Energy Rev. 16, 143–169. Ip, S.Y., Bridger, J.S., Chin, C.T., Martin, W.R.B., Raper, W.G.C., 1982. Algal growth in primary settled sewage: the effects of five key variables. Water Res. 16, 621– 632.

135

Knothe, G., 2009. Improving biodiesel fuel properties by modifying fatty ester composition. Energy Environ. Sci. 2, 759–766. Knothe, G., 2011. A technical evaluation of biodiesel from vegetable oils vs. algae. Will algae-derived biodiesel perform? Green Chem. 13, 3048–3065. Li, Y., Chen, Y.F., Chen, P., Min, M., Zhou, W., Martinez, B., Zhu, J., Ruan, R., 2011a. Characterization of a microalga Chlorella sp. well adapted to highly concentrated municipal wastewater for nutrient removal and biodiesel production. Bioresour. Technol. 102, 5138–5144. Li, X., Hu, H.Y., Zhang, Y.P., 2011b. Growth and lipid accumulation properties of a freshwater microalga Scenedesmus sp. under different cultivation temperature. Bioresour. Technol. 102, 3098–3102. Li, X., Hu, H.Y., Gan, K., Yang, J., 2010a. Growth and nutrient removal properties of a freshwater microalga Scenedesmus sp. LX1 under different kinds of nitrogen sources. Ecol. Eng. 36 (4), 379–381. Li, X., Hu, H.Y., Yang, J., 2010b. Lipid accumulation and nutrient removal properties of a newly isolated freshwater microalga, Scenedesmus sp. LX1, growing in secondary effluent. New Biotechnol. 27 (1), 59–63. Lin, Q., Lin, J., 2011. Effects of nitrogen source and concentration on biomass and oil production of a Scenedesmus rubescens like microalga. Bioresour. Technol. 102 (2), 1615–1621. Lv, J.M., Cheng, L.H., Xu, X.H., Zhang, L., Chen, H.L., 2010. Enhanced lipid production of Chlorella vulgaris by adjustment of cultivation conditions. Bioresour. Technol. 101, 6797–6804. Mata, T.M., Martins, A.A., Caetano, N., 2010. Microalgae for biodiesel production and other applications: a review. Renewable Sustainable Energy Rev. 14 (1), 217– 232. Martinez, M.E., Sanchez, S., Jimenez, J.M., El Yousfi, F., Munoz, L., 2000. Nitrogen and phosphorus removal from urban wastewater by the microalga Scenedesmus obliquus. Bioresour. Technol. 73, 263–272. Nemcova, Y., Elias, M., Skaloud, P., Hodac, L., Neustupa, J., 2011. Jenufa gen. nov.: a new genus of coccoid green algae (Chlorophyceae, Incertae Sedis) previously recorded by environmental sequencing. J. Phycol. 47, 928–938. Ren, H.Y., Liu, B.F., Ma, C., Zhao, L., Ren, N.Q., 2013. A new lipid-rich microalga Scenedesmus sp. strain R-16 isolated using Nile red staining: effects of carbon and nitrogen sources and initial pH on the biomass and lipid production. Biotechnol. Biofuels 6, 143. Rodolfi, L., Zittelli, G.C., Bassi, N., Padovani, G., Biondi, N., Bonini, G., Tredici, M.R., 2008. Microalgae for oil: strain selection, induction of lipid synthesis and outdoor mass cultivation in a low-cost photobioreactor. Biotechnol. Bioeng. 102, 100–112. Schelef, G., Azov, Y., Moraine, R., Oron, G., 1980. Algal mass production as an integral part of a wastewater treatment and reclamation system. In: Shelef, G., Soedeer, C.J. (Eds.), Algae Biomass. Elsevier/ North-Holland Biomedical Press, Amsterdam, pp. 163±189. Sivakumar, G., Xu, J., Thompson, R.W., Yang, Y., Randol-Smith, P., Weathers, P.J., 2012. Integrated green algal technology for bioremediation and biofuel. Bioresour. Technol. 107, 1–9. State Environmental Protection Administration, 2002, . Monitoring Method of Water and Wastewater, fourth ed., vol. 105. China Environmental Science Press, Beijing, pp. 246–248, 255–257. Song, M., Pei, H., Hu, W., Ma, G., 2013. Evaluation of the potential of 10 microalgal strains for biodiesel production. Bioresour. Technol. 141, 245. Xu, N., Zhang, X., Fan, X., Han, L., Zeng, C., 2001. Effects of nitrogen source and concentration on growth rate and fatty acid composition of Ellipsoidion sp. (Eustigmatophyta). J. Appl. Phycol. 13, 463–469. Tan, Y.X., Lin, J., 2011. Biomass production and fatty acid profile of a Scenedesmus rubescens-like microalga. Bioresour. Technol. 102, 10131–10135. Wan, M.X., 2012. Isolation and the Basic Study on Genetic Engineering of Microalgae for Biofuels. Doctor Degree. Central South University, China. Yang, J., Li, X., Hu, H.Y., Zhang, X., Yu, Y., Chen, Y., 2011. Growth and lipid accumulation properties of a freshwater microalga, Chlorella ellipsoidea YJ1, in domestic secondary effluents. Appl. Energy 88, 3295–3299. Zhang, Z.H., Hang, S.J., Li, Y., 2004. The Fifth Water Supply and Drainage Design Manual: Town Drainage, second ed. China Building Industry Press, Beijing. Zhou, W., Li, Y., Min, M., Hu, B., Chen, P., Ruan, R., 2011. Local bioprospecting for high-lipid producing microalgal strains to be grown on concentrated municipal wastewater for biofuel production. Bioresour. Technol. 102, 6909–6919.