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Inhibitory effects of soluble algae products (SAP) released by Scenedesmus sp. LX1 on its growth and lipid production
Bioresource Technology 146 (2013) 643–648
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Inhibitory effects of soluble algae products (SAP) released by Scenedesmus sp. LX1 on its growth and lipid production Zhang Tian-Yuan a, Yu Yin b, Wu Yin-Hu a, Hu Hong-Ying a,c,⇑ a
Environmental Simulation and Pollution Control State Key Joint Laboratory, School of Environment, Tsinghua University, Beijing 100084, PR China State Key Laboratory of Environmental Criteria and Risk Assessment, Research Center of Water Pollution Control Technology, Chinese Research Academy of Environmental Sciences, Beijing 100012, PR China c State Environmental Protection Key Laboratory of Microorganism Application and Risk Control (MARC), Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, PR China b
h i g h l i g h t s Soluble algae products could significantly inhibit the growth of Scenedesmus sp. LX1. All of the fractions of SAP could inhibit the growth of Scenedesmus sp. LX1. Organic bases and HIA expressed the strongest inhibition on growth. HIA could significantly inhibit the lipid accumulation of Scenedesmus sp. LX1. Molecular weight and fluorescence spectroscopy of HIA were investigated.
a r t i c l e
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Article history: Received 14 June 2013 Received in revised form 25 July 2013 Accepted 29 July 2013 Available online 6 August 2013 Keywords: Soluble algal products (SAP) Scenedesmus sp. Organic fractions Microalgal biodiesel Lipid production
a b s t r a c t Soluble algal products (SAP) accumulated in culture medium via water reuse may affect the growth of microalga during the cultivation. Scenedesmus sp. LX1, a freshwater microalga, was used in this study to investigate the effect of SAP on growth and lipid production of microalga. Under the SAP concentrations of 6.4–25.8 mg L 1, maximum algal density (K) and maximum growth rate (Rmax) of Scenedesmus sp. LX1 were decreased by 50–80% and 35–70% compared with the control group, respectively. The effect of SAP on lipid accumulation of Scenedesmus sp. LX1 was non-significant. According to hydrophilic– hydrophobic and acid–base properties, SAP was fractionized into six fractions. All of the fractions could inhibit the growth of Scenedesmus sp. LX1. Organic bases (HIB, HOB) and hydrophilic acids (HIA) showed the strongest inhibition. HIA could also decrease the lipid content of Scenedesmus sp. LX1 by 59.2%. As the inhibitory effect, SAP should be seriously treated before water reuse. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction The massive and excessive consumption of fossil biodiesel has aroused the risk of energy independence (Gouveia and Oliveira, 2009) and global warming (Brennan and Owende, 2010). Derived from oil crops, biodiesel provides a sustainable alternative to fossil fuels as it is renewable and carbon neutral so that attracts considerable attention in recent years (Xu et al., 2006). The federal government of US has passed the energy independence and security act (EISA) in 2007 which aims to increase the production of renewable fuels to 36 billion gallons per year by 2022 (Congress, ⇑ Corresponding author at: Environmental Simulation and Pollution Control State Key Joint Laboratory, School of Environment, Tsinghua University, Beijing 100084, PR China. Tel.: +86 10 62794005; fax: +86 10 62797265. E-mail addresses: [email protected], [email protected] (H.-Y. Hu). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.07.142
2007). As microalga is much more productive than conventional oil bearing crops (Chisti, 2007) and has no adverse effect on food supply (Chisti, 2008), it has become one of the most promising sources to meet the huge demand of renewable transport fuels by large-scale production (Behzadi and Farid, 2007; Schenk et al., 2008). One of the bottlenecks in the production of microalgae is the huge consumption of water. Without the recycling of culture supernatant, to achieve the EISA goal of biodiesel production in 2022, the water consumption would account for 85% of the total national usage of US (Yang et al., 2011), which makes the largescale cultivation of microalgae looks impossible. On the other hand, if the harvest water could be recycled, the water and nutrients usage would be sharply reduced by 84% and 55% (Yang et al., 2011). Therefore, the recycling of the water is essential to the large-scale cultivation of microalgae.
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However, during the cultivation process, microalgae continuously releases extracellular organic matter into the medium (Yu et al., 2012), with various constituents including carbohydrates, amino acids, proteins, lipids and organic acids (Biddanda and Benner, 1997). These soluble algal products (SAP) could not be removed effectively by usual harvesting processes for algal cells and thus will accumulate in the medium with the reuse of the old culture supernatant. Some studies have found that the residual SAP could have a negative impact on the growth of the microalga. Pratt and Fong (1940) reported that Chlorella cells could produce and liberate a growth-inhibiting substance into the external solution. This inhibitor is probably a thermal-instable organic base with a molecular diameter less than 15 Å (Pratt, 1942). Water recycling during the cultivation of Scenedesmus obliquus reduced the consumption of water by 63%, but a depression of algal productivity caused by SAP residue in medium was also observed (Lívansky´ et al., 1996). The SAP could be extracted by some organic solvent, such as ethyl aceta, ethyl alcohol, ether and petroleum ether. The inhibitory effect on algae growth would become more serious with the increase of exposure time and the addition amount of SAP extract in the medium (Liu et al., 2002; Pratt, 1942). Although the inhibitory effect of SAP on the growth of microalgae has been realized, the specific inhibitory fractions in SAP have not been identified. Also the effect of SAP on lipid accumulation of microalgae, especially under the nutrient level of secondary effluent has not been reported. Scenedesmus sp. LX1, isolated from freshwater by Li et al. (2010b), was used in this study to investigate the effect of SAP on growth and lipid production of microalga. This species grew well in typical secondary effluent of domestic wastewater treatment plant with high lipid content (Li et al., 2010a) and high nutrient removal efficiency (Li et al., 2010b). The kinetics of SAP accumulation of Scenedesmus sp. LX1 during batch cultivation process at the nutrient level similar to secondary effluent has been studied by Yu et al. (2012). On the basis of previous researches, this study focused on the effect of SAP and its organic fractions on the growth and lipid accumulation of Scenedesmus sp. LX1. It gave special attention to the identifying of the specific inhibitory fraction in SAP. 2. Methods 2.1. Microalga and medium Scenedesmus sp. LX1 (Patent No. CGMCC 3036 in China General Microbiological Culture Collection Center) used in this study was originally isolated from tap water (Li et al., 2010a). The strain was kept in modified BG11 medium (mBG11) to simulate the nutrient content of secondary effluent of domestic wastewater treatment plants. Nitrate and phosphate were used as the nitrogen and phosphorus sources, respectively. The growth medium mBG11 contained the following: 91.1 mg L 1 NaNO3, 11.0 mg L 1 K2HPO43H2O, 37.5 mg L 1 MgSO47H2O, 18 mg L 1 CaCl22H2O, 3 mg L 1 citric acid, 3 mg L 1 ferric ammonium citrate, 0.5 mg L 1 Na2EDTA, 10 mg L 1 Na2CO3, and 1.0 mg L 1 A5 + Co solution. The A5 + Co solution contained 2.86 g L 1 H3BO3, 1.81 g L 1 MnCl2H2O, 222 mg L 1 ZnSO47H2O, 79 mg L 1 CuSO45H2O, 390 mg L 1 Na2MoO42H2O, and 49 mg L 1 Co(NO3)26H2O. 2.2. Acquirement of SAP Scenedesmus sp. LX1 were cultivated in 200 mL autoclaved growth medium in 500 mL flasks and maintained in an artificial climate chamber (HPG-280H). The cultivation conditions were as follows: light intensity 2300 lx, light/dark ratio = 14:10, initial pH = 7
and temperature = 25 °C. After 15 days of cultivation, the algae liquid was filtered by 0.45 lm membrane (PALL Corporation, USA) to separate the algae cells from the media and acquired the SAP. At this time, Scenedesmus sp. LX1 had entered into stationary phase with the density of 3.4 106 cells per liter. The dissolved organic carbon (DOC) content of the SAP acquired was 25.8 mg L 1. 2.3. Isolation and fractionation of SAP The method developed by Leenheer (1981) and modified by Zhang et al. (2009) was used to separate the SAP into six fractions: hydrophobic acids (HOA), hydrophobic bases (HOB), hydrophobic neutrals (HON), hydrophilic acids (HIA), hydrophilic bases (HIB), and hydrophilic neutrals (HIN). Dialysis bag (MWCO, 100–500) was used to remove the inorganic salt introduced into the SAP in fraction process. Finally, the volume of each fraction acquired was controlled as the same with the original volume of the SAP, so that the fractions were not concentrated or diluted during the isolation. The test result of ion exchange chromatography (IEC) indicated that after dialysis the residual salt in SAP sample was totally NaCl and the concentration of the salt was less than 100 mg L 1. Preliminary experiments showed that the addition of 100 mg L 1 NaCl in mBG11 medium showed no effect on the growth characteristics of Scenedesmus sp. LX1. 2.4. Effect of SAP on the growth of Scenedesmus sp. LX1 The cultivation experiment was operated under a bacteria-controlled condition. Fifty milliliter flasks were used to cultivate Scenedesmus sp. LX1 with 20 mL of mixed culture medium composed by autoclaved mBG11 medium and the SAP. Dissolved organic carbon (DOC) of the original SAP acquired in this work was 25.8 mg L 1. For the experiments on the effects of different SAP concentrations, the addition amount of SAP in each set of medium was prepared as 25.8/19.3/12.9/6.4/0 mg L 1 DOC, respectively. For the experiments on the effects of different SAP fractions, six fractions were added into the sets respectively with no dilution. Except the content of SAP, the nutrient conditions of all experiment sets were same with mBG11. Scenedesmus sp. LX1 cells were inoculated by 2.5% (v/v) into medium. The condition of the cultivation was same with the description in 2.2. 2.5. Analytical methods The amount of SAP was determined as the dissolved organic carbon (DOC). Under sterile condition, 20 mL of the alga culture was sampled and then filtered by 0.45 lm membrane (PALL Corporation, USA). After that the DOC were measured by a TOC analyzer (TOC-VCPH, SHIMADZU). The algal density was measured by cell counting using a blood cell counting chamber. The dry weight of algal biomass was determined using the method of suspended solid (SS) measurement according to Chinese state standard testing methods (Administration, 2002). The total lipids were extracted with a chloroform/methanol solution (1/1, v/v) and were quantified gravimetrically (Bligh and Dyer, 1959). Triacylglycerols (TAG) is the main raw material in biodiesel production (Schenk et al., 2008) and an important index to indicate the lipid content in algae cells. After the measurement of total lipid, the dried lipid was dissolved in 0.4 mL of isopropyl alcohol. Then the TAG was estimated by enzymatic colorimetric method using commercial kit from Beijing BHKT Clinical Reagent Co., Ltd., No. 2400076. The logistic model, a classic model describing the relationship between a microorganism’s growth and density in limited environ-
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Algal density (cells·mL-1)
108
SAP (mg DOC·L-1) 0 6.4 12.9 19.3 25.8
107
a
106
105
0
5
10
15
20
25
Cultivation time (d)
8
6
b
6 4 4 2 2
0
0
5
10
15
20
25
Rmax (105 cells·mL-1·d-1)
K Rmax
K (106 cells·mL-1)
mental conditions (Li et al., 2010a), was applied in this study. Maximal algal density (K) and maximal population growth rate (Rmax) were measured to describe the growth condition of Scenedesmus sp. LX1. K (cells mL 1) is the carrying capacity, which is the maximal microalgal density reached in the culture. According to the logistic model, when the microalgal density reaches half the value of K, the population growth rate reaches its maximal value (Rmax). All tests were carried out in triplicate (n = 3) and an Independent-Samples t-test was used for significant difference analysis by using SPSS (version 20) statistical software. The fluorescence spectrum of the fraction in SAP was determined using a fluorescence spectrophotometer (Model: F-7000, Hitachi, Japan). Three-dimensional spectra were obtained as the method described by Chen et al. (2003). Size exclusion chromatography (SEC) was used to determine the molecular weight (MW) distribution of the fraction in SAP on a Shimadzu LC-20 high-performance liquid chromatography system. The detection was carried out at 40 °C with a Shimadzu SPD-M20A UV detector and two connected columns (a TSK-GEL G3000PWXL column followed by a TSK-GEL G2500PWXL column). The mobile phase was composed of MILLI-Q ultrapure water buffered with phosphate (0.0024 M NaH2PO4 and 0.0016 M Na2HPO4) and 0.025 M sodium sulfate. The column was calibrated with the standard polyethylene glycol with molecular weights of 330, 700, 1050, 5250, 10,225, 30,000 Da (American Polymer Standards Corp.) and acetone. The retention time in the SEC chromatogram of SAP was converted into the MW according to the calibration.
0 30
SAP (mg·L-1)
3.1. Effects of SAP on the growth of Scenedesmus sp. LX1 The changes of algal densities of Scenedesmus sp. LX1 with different initial concentrations of SAP are shown in Fig. 1a. At the beginning of the cultivation, no obvious difference was found among the growth curves of microalga. After 12 days of cultivation, SAP (6.4–25.8 mg L 1) expressed a significant inhibition on algal density compared with the control group. Under the SAP concentration of 6.4, 12.9 and 19.3 mg L 1, the decline of algal density appeared earlier with the increase of SAP concentration. Under the SAP concentration of 19.3 and 25.8 mg L 1, no significant difference in algal density was observed. Through linear regression analysis (Logistic Model), the K and Rmax of Scenedesmus sp. LX1 under different SAP concentrations were obtained and are shown in Fig. 1b. In the culture medium with no SAP addition (control group), the K and Rmax of Scenedesmus sp. LX1 were over 7.2 106 cells mL 1 and 5.0 105 cells(mL d) 1, respectively. With the addition of SAP, K and Rmax of Scenedesmus sp. LX1 dropped to only 1.6 106–3.6 106 cells mL 1 and 1.5 105–3.2 105 cells(mL d) 1, decreasing by 50–80% and 35–70% compared with control group, respectively. When the concentration of SAP was in the range of 6.4– 19.3 mg L 1, the K and Rmax of Scenedesmus sp. LX1 decreased with the increase of SAP concentration. Liu et al. (2002) reported that the more crude ethyl acetate extract of SAP was added into the algal culture, the more serious the growth of Parietochloris incisa was inhibited. When it came to Scenedesmus sp. LX1, it could not be simply concluded that the inhibition on growth of microalgae increased correspondingly with the SAP concentration, since once the SAP concentration was up to 19.3 mg L 1, the inhibitory effect did not continue to increase. More studies are needed to reveal whether the inhibitory effects would aggravate with the higher addition amount of SAP and how much amount of SAP is allowed on a specific cultivation condition.
Fig. 1. (a) Algal density curves of Scenedesmus sp. LX1 with the different concentrations of SAP released by Scenedesmus sp. LX1. Fig. 1(b) K and Rmax of Scenedesmus sp. LX1 with the different concentrations of SAP released by Scenedesmus sp. LX1.
3.2. Effects of SAP on lipid accumulation of Scenedesmus sp. LX1 As the SAP of Scenedesmus sp. LX1 in the stationary phase expressed a significant inhibitory effect on the growth of this microalga, more researches had been done to investigate the effect of SAP on the lipid accumulation of Scenedesmus sp. LX1, including lipid content in dry weight and TAG content in lipid.Fig. 2. presents the lipid accumulation of Scenedesmus sp. LX1 after 14-day cultivation with the SAP concentration of 25.8 mg DOC L 1 and with no SAP addition. The lipid content in dry weight and the TAGs content in lipid in all groups were around 15% and 20%, respectively. Statistic analysis showed that the addition of SAP has no significant (P > 0.1) impact on the lipid accumulation of Scenedesmus sp. LX1.
35
Lipid or TAGs content (%)
3. Results and discussion
Lipid content in dry weight TAGs content in lipid
30 25 20 15 10 5 0
Without SAP
With SAP
Fig. 2. Lipid accumulation of Scenedesmus sp. LX1 after 14-day cultivation with the SAP concentration of 25.8 mg DOC L 1 and with no SAP addition.
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-1
Some researches revealed that environmental stress was an effective trigger to enhance the lipid accumulation in microalgal cells (Hu et al., 2008; Khozin-Goldberg and Cohen, 2006; Rodolfi et al., 2009). Although the existence of SAP is also an adverse environmental condition to the growth of microalga, this condition seems cannot stimulate the lipid accumulation of Scenedesmus sp. LX1 in cells. As the growth of Scenedesmus sp. LX1 was significantly inhibited by SAP and the lipid or TAG content in cells were not enhanced, thus in general the addition of SAP significantly decreased the total lipid and TAG production of Scenedesmus sp. LX1. The results demonstrated that the SAP should be removed before the reuse of the water to minimize the adverse effect on biodiesel production based on Scenedesmus sp. LX1.
Algal density (cells·mL )
646
10
7
10
6
10
5
10
4
10
3
After 15 days of cultivation, the lipid accumulation of Scenedesmus sp. LX1 under different fractions of SAP were determined and shown in Fig. 4. The effects of different fractions of SAP on lipid
Table 1 The DOC content and the percentage in total SAP of each fraction in SAP released by Scenedesmus sp. LX1. Parameter
HOA
HOB
HON
HIA
HIB
HIN
DOC (mg L 1) Percentage in total SAP (%)
2.75 10.23
1.96 7.29
10.77 40.03
5.17 19.22
1.45 5.4
4.79 17.82
2
4
HIA HIB HIN
6
10
8
12
0.5
b
Rmax(106 cells·mL-1·d-1)
Ctrl 0.4
0.3
HIN HOA
0.2
HON 0.1
HIA HOB
0.0 0.0
HIB 0.5
1.0
1.5
2.0
2.5
3.0
3.5
K (106 cells·mL-1) Fig. 3. (a) Algal density curves of Scenedesmus sp. LX1 with different fractions of SAP released by Scenedesmus sp. LX1. Fig. 3(b) K and Rmax of Scenedesmus sp. LX1 with the different fractions of SAP released by Scenedesmus sp. LX1.
accumulation were various. Setting the lipid accumulation data of control group as the original point, the data points of lipid and TAGs contents in Scenedesmus sp. LX1 with the addition of different fractions of SAP were divided into different quadrants. Several fractions inhibited the lipid accumulation of Scenedesmus sp. LX1, and others showed positive or non-significant effects. The organic bases (HOB, HIB) and HON, which were located in Quadrant IV, could increase the lipid content in dry weight by 36.2%, 26.2% and 61.5%, respectively. Also the HOA, which was located in Quadrant II, showed an enhancing effect on the TAGs contents in lipid. However, the positive impacts of these fractions on
70 TAGs content in lipid (%)
3.4. Effect of different fractions of SAP on lipid accumulation of Scenedesmus sp. LX1
0
Without SAP HOA HOB HON Cultivation time (d)
3.3. Effects of different fractions of SAP on growth of Scenedesmus sp. LX1 In order to identify the main inhibitory factions, the SAP of Scenedesmus sp. LX1 was isolated into six fractions: hydrophobic acids (HOA), hydrophobic bases (HOB), hydrophobic neutrals (HON), hydrophilic acids (HIA), hydrophilic bases (HIB), and hydrophilic neutrals (HIN). The DOC content and the percentage in total SAP of each fraction was shown in Table 1. The recovery rate of DOC though the fraction process was 104%, which was acceptable in this study. Results showed that HON was the main fraction in SAP, which accounted for 40% of DOC content, and the organic bases (HIB, HOB) only accounted for less than 15%. After 15-day cultivation with the addition of different SAP fractions, the impact of each fraction on the growth of Scenedesmus sp. LX1 was shown in Fig. 3a. The initial inoculation density was about 5 103 cells mL 1. At the beginning of the cultivation, no obvious difference of the growth of microalgae among groups was observed. However, from the fourth day of the cultivation, all of the fractions of SAP showed significant inhibitory effect on the growth of Scenedesmus sp. LX1, inducing the algal densities in stationary phase were much less than the group without SAP. Through linear regression analysis, the growth parameters of Scenedesmus sp. LX1 under different fractions of SAP were obtained and shown in Fig. 3b. With the existence of the fractions of SAP, K and Rmax decreased by 40–90% compared with the control group. HIB and HIA expressed the most significant inhibitory effect, which decreased the maximal algae density by 90.0% and 83.2%, respectively. According to the growth parameters, the sequence of the inhibition effects were HIB > HIA > HOB > HON > HOA > HIN. Therefore, although the DOC of the organic bases (HIB and HOB) only accounted for less than 15% in the SAP, these two fractions were among the three strongest inhibitory fractions within SAP. This result was similar with the conclusion drawn by Pratt (1942) which found the growth-inhibiting substance in SAP of Chlorella probably belonging to organic base.
a
60
HOA
50
Ctrl
40
HIB HON
HOB
30 HIA
20
HIN
10 0
0
5
10
15
20
25
30
35
Lipid content in dry weight (%) Fig. 4. Lipid accumulation of Scenedesmus sp. LX1 after 15-day cultivation with different fractions of SAP released by Scenedesmus sp. LX1.
T.-Y. Zhang et al. / Bioresource Technology 146 (2013) 643–648
lipid accumulations of Scenedesmus sp. LX1 were non-significant via T-test (P > 0.1). In Quadrant III, HIA showed the significant inhibition on the lipid accumulation of Scenedesmus sp. LX1 (P < 0.01). With the addition of HIA, the lipid content in dry weight and the TAGs content in lipid were decreased by 59.2% and 51.2% compared with the control group, respectively. HIN could also inhibit the lipid and TAGs accumulation, yet the inhibition was not significant via T-test. No fraction was located in Quadrant I meaning that all of the fractions could not increase both the lipid content in dry weight and the TAG content in lipid of Scenedesmus sp. LX1. In general, as the fractions of SAP expressed various effects on the lipid accumulation of Scenedesmus sp. LX1, the overall effect of SAP was the integrated result of these six fractions. The inhibitory fractions should be removed before the reuse of the culture solution in order to avoid the adverse effect on the biodiesel production based on Scenedesmus sp. LX1. 3.5. Further analysis of the inhibitory substance in HIA of SAP As the HIA fraction expressed the most significant inhibition on growth and lipid accumulation of Scenedesmus sp. LX1, further analysis has been done to identify the composition of HIA. The three-dimensional fluorescence spectroscopy, which was widely used to analyze the composition of dissolved organic materials (DOM), was applied in this study to characterize the composition of HIA in SAP. The three-dimensional spectra of HIA was showed in Fig. 5 and defined into five regions to represent different fractions according to the reference (Chen et al., 2003). The strongest fluorescent peak of HIA located in the range between excitation wavelengths (EX) of 270–310 nm and emission wavelength (EM) of 370–380 nm. Based on literatures, Chen et al. (2003) summarized that peaks at intermediate excitation wavelengths (250–350 nm) and shorter emission wavelength (250–380 nm) were related to soluble microbial byproduct-like material (Region IV). The fluorescent response was also observed in Region II, III and V. These regions were representative for simple aromatic proteins (Region II), humic acid-like organics (Region V) and fulvic acid-like materials (Region III). The SEC system with UV detector was used to determine the molecular weight (MW) distribution of HIA in SAP. The result of SEC at UV254 showed that the molecular weight (MW) of the organic materials in SAP mainly distributed in the range of 0.15–7 kDa. The response peaks of MW appeared at 1.90, 2.83 and 4.32 kDa. Ni et al. (2010) reported that the biomass-associated products in soluble microbial products (SMP) of activated sludge were mainly macromolecules with an MW in a range of 290–
Fig. 5. Fluorescence spectroscopy of HIA in SAP released by Scenedesmus sp. LX1.
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5000 kDa, which was much higher than the MW of HIA in SAP. Hadj-Romdhane et al. (2013) researched the MW distribution of SAP released by Chlorella vulgaris and identified some small organic molecules richer in nitrogen with a molecular size ranging from 1 to 3 kDa in SAP, which was similar with the MW distribution of HIA in SAP released by Scenedesmus sp. LX1. In order to identify the specific molecular structure of inhibitory matters within HIA, some advanced analysis methods should be utilized in further research, such as LC-MS–MS. 4. Conclusions SAP and all of its organic fractions could inhibit the growth of Scenedesmus sp. LX1. Under the SAP concentrations of 6.4– 25.8 mg L 1, K and Rmax were decreased by 50–80% and 35–70%, respectively. HIA and organic bases expressed the strongest inhibition. SAP showed non-significant effect on lipid accumulation of Scenedesmus sp. LX1. HIA could significantly decrease the lipid content of Scenedesmus sp. LX1 by 59.2%. The strongest fluorescent peaks of HIA in SAP located in range between EX of 270–300 nm and EM of 370–380 nm. MW of HIA mainly distributed in the range of 0.15–7 kDa. Acknowledgements This study was supported by China National Science Fund Key Program (No. 51138006). References Administration, S.E.P. 2002. Monitoring Method of Water and Wastewater, (fourth ed.). China Environmental Science Press, Beijing, pp. 105, 246–248, 255–257. Behzadi, S., Farid, M., 2007. Review: examining the use of different feedstock for the production of biodiesel. Asia-Pacific Journal of Chemical Engineering 2 (5), 480– 486. Biddanda, B., Benner, R., 1997. Carbon, nitrogen, and carbohydrate fluxes during the production of particulate and dissolved organic matter by marine phytoplankton. Limnology and Oceanography 42 (3), 506–518. Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Canadian Journal of Biochemistry and Physiology 37 (8), 911–917. Brennan, L., Owende, P., 2010. Biofuels from microalgae–a review of technologies for production, processing, and extractions of biofuels and co-products. Renewable & Sustainable Energy Reviews 14 (2), 557–577. Chen, W., Westerhoff, P., Leenheer, J.A., Booksh, K., 2003. Fluorescence excitation– emission matrix regional integration to quantify spectra for dissolved organic matter. Environmental Science & Technology 37 (24), 5701–5710. Chisti, Y., 2007. Biodiesel from microalgae. Biotechnology Advances 25 (3), 294– 306. Chisti, Y., 2008. Biodiesel from microalgae beats bioethanol. Trends in Biotechnology 26 (3), 126–131. Congress, U., 2007. Energy independence and security act of 2007. Public Law (110– 140). Gouveia, L., Oliveira, A.C., 2009. Microalgae as a raw material for biofuels production. Journal of Industrial Microbiology & Biotechnology 36 (2), 269–274. Hadj-Romdhane, F., Zheng, X., Jaouen, P., Pruvost, J., Grizeau, D., Croué, J., Bourseau, P., 2013. The culture of Chlorella vulgaris in a recycled supernatant: effects on biomass production and medium quality. Bioresource Technology 132, 285– 292. 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. The Plant Journal 54 (4), 621–639. Khozin-Goldberg, I., Cohen, Z., 2006. The effect of phosphate starvation on the lipid and fatty acid composition of the fresh water eustigmatophyte Monodus subterraneus. Phytochemistry 67 (7), 696–701. Leenheer, J.A., 1981. Comprehensive approach to preparative isolation and fractionation of dissolved organic-carbon from natural-waters and wastewaters. Environmental Science & Technology 15 (5), 578–587. Li, X., Hu, H.Y., Gan, K., Sun, Y.X., 2010a. Effects of different nitrogen and phosphorus concentrations on the growth, nutrient uptake, and lipid accumulation of a freshwater microalga Scenedesmus sp.. Bioresource Technology 101 (14), 5494– 5500. 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 Biotechnology 27 (1), 59–63.
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Inhibitory effects of soluble algae products (SAP) released by Scenedesmus sp. LX1 on its growth and lipid production Zhang Tian-Yuan a, Yu Yin b, Wu Yin-Hu a, Hu Hong-Ying a,c,⇑ a
Environmental Simulation and Pollution Control State Key Joint Laboratory, School of Environment, Tsinghua University, Beijing 100084, PR China State Key Laboratory of Environmental Criteria and Risk Assessment, Research Center of Water Pollution Control Technology, Chinese Research Academy of Environmental Sciences, Beijing 100012, PR China c State Environmental Protection Key Laboratory of Microorganism Application and Risk Control (MARC), Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, PR China b
h i g h l i g h t s Soluble algae products could significantly inhibit the growth of Scenedesmus sp. LX1. All of the fractions of SAP could inhibit the growth of Scenedesmus sp. LX1. Organic bases and HIA expressed the strongest inhibition on growth. HIA could significantly inhibit the lipid accumulation of Scenedesmus sp. LX1. Molecular weight and fluorescence spectroscopy of HIA were investigated.
a r t i c l e
i n f o
Article history: Received 14 June 2013 Received in revised form 25 July 2013 Accepted 29 July 2013 Available online 6 August 2013 Keywords: Soluble algal products (SAP) Scenedesmus sp. Organic fractions Microalgal biodiesel Lipid production
a b s t r a c t Soluble algal products (SAP) accumulated in culture medium via water reuse may affect the growth of microalga during the cultivation. Scenedesmus sp. LX1, a freshwater microalga, was used in this study to investigate the effect of SAP on growth and lipid production of microalga. Under the SAP concentrations of 6.4–25.8 mg L 1, maximum algal density (K) and maximum growth rate (Rmax) of Scenedesmus sp. LX1 were decreased by 50–80% and 35–70% compared with the control group, respectively. The effect of SAP on lipid accumulation of Scenedesmus sp. LX1 was non-significant. According to hydrophilic– hydrophobic and acid–base properties, SAP was fractionized into six fractions. All of the fractions could inhibit the growth of Scenedesmus sp. LX1. Organic bases (HIB, HOB) and hydrophilic acids (HIA) showed the strongest inhibition. HIA could also decrease the lipid content of Scenedesmus sp. LX1 by 59.2%. As the inhibitory effect, SAP should be seriously treated before water reuse. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction The massive and excessive consumption of fossil biodiesel has aroused the risk of energy independence (Gouveia and Oliveira, 2009) and global warming (Brennan and Owende, 2010). Derived from oil crops, biodiesel provides a sustainable alternative to fossil fuels as it is renewable and carbon neutral so that attracts considerable attention in recent years (Xu et al., 2006). The federal government of US has passed the energy independence and security act (EISA) in 2007 which aims to increase the production of renewable fuels to 36 billion gallons per year by 2022 (Congress, ⇑ Corresponding author at: Environmental Simulation and Pollution Control State Key Joint Laboratory, School of Environment, Tsinghua University, Beijing 100084, PR China. Tel.: +86 10 62794005; fax: +86 10 62797265. E-mail addresses: [email protected], [email protected] (H.-Y. Hu). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.07.142
2007). As microalga is much more productive than conventional oil bearing crops (Chisti, 2007) and has no adverse effect on food supply (Chisti, 2008), it has become one of the most promising sources to meet the huge demand of renewable transport fuels by large-scale production (Behzadi and Farid, 2007; Schenk et al., 2008). One of the bottlenecks in the production of microalgae is the huge consumption of water. Without the recycling of culture supernatant, to achieve the EISA goal of biodiesel production in 2022, the water consumption would account for 85% of the total national usage of US (Yang et al., 2011), which makes the largescale cultivation of microalgae looks impossible. On the other hand, if the harvest water could be recycled, the water and nutrients usage would be sharply reduced by 84% and 55% (Yang et al., 2011). Therefore, the recycling of the water is essential to the large-scale cultivation of microalgae.
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However, during the cultivation process, microalgae continuously releases extracellular organic matter into the medium (Yu et al., 2012), with various constituents including carbohydrates, amino acids, proteins, lipids and organic acids (Biddanda and Benner, 1997). These soluble algal products (SAP) could not be removed effectively by usual harvesting processes for algal cells and thus will accumulate in the medium with the reuse of the old culture supernatant. Some studies have found that the residual SAP could have a negative impact on the growth of the microalga. Pratt and Fong (1940) reported that Chlorella cells could produce and liberate a growth-inhibiting substance into the external solution. This inhibitor is probably a thermal-instable organic base with a molecular diameter less than 15 Å (Pratt, 1942). Water recycling during the cultivation of Scenedesmus obliquus reduced the consumption of water by 63%, but a depression of algal productivity caused by SAP residue in medium was also observed (Lívansky´ et al., 1996). The SAP could be extracted by some organic solvent, such as ethyl aceta, ethyl alcohol, ether and petroleum ether. The inhibitory effect on algae growth would become more serious with the increase of exposure time and the addition amount of SAP extract in the medium (Liu et al., 2002; Pratt, 1942). Although the inhibitory effect of SAP on the growth of microalgae has been realized, the specific inhibitory fractions in SAP have not been identified. Also the effect of SAP on lipid accumulation of microalgae, especially under the nutrient level of secondary effluent has not been reported. Scenedesmus sp. LX1, isolated from freshwater by Li et al. (2010b), was used in this study to investigate the effect of SAP on growth and lipid production of microalga. This species grew well in typical secondary effluent of domestic wastewater treatment plant with high lipid content (Li et al., 2010a) and high nutrient removal efficiency (Li et al., 2010b). The kinetics of SAP accumulation of Scenedesmus sp. LX1 during batch cultivation process at the nutrient level similar to secondary effluent has been studied by Yu et al. (2012). On the basis of previous researches, this study focused on the effect of SAP and its organic fractions on the growth and lipid accumulation of Scenedesmus sp. LX1. It gave special attention to the identifying of the specific inhibitory fraction in SAP. 2. Methods 2.1. Microalga and medium Scenedesmus sp. LX1 (Patent No. CGMCC 3036 in China General Microbiological Culture Collection Center) used in this study was originally isolated from tap water (Li et al., 2010a). The strain was kept in modified BG11 medium (mBG11) to simulate the nutrient content of secondary effluent of domestic wastewater treatment plants. Nitrate and phosphate were used as the nitrogen and phosphorus sources, respectively. The growth medium mBG11 contained the following: 91.1 mg L 1 NaNO3, 11.0 mg L 1 K2HPO43H2O, 37.5 mg L 1 MgSO47H2O, 18 mg L 1 CaCl22H2O, 3 mg L 1 citric acid, 3 mg L 1 ferric ammonium citrate, 0.5 mg L 1 Na2EDTA, 10 mg L 1 Na2CO3, and 1.0 mg L 1 A5 + Co solution. The A5 + Co solution contained 2.86 g L 1 H3BO3, 1.81 g L 1 MnCl2H2O, 222 mg L 1 ZnSO47H2O, 79 mg L 1 CuSO45H2O, 390 mg L 1 Na2MoO42H2O, and 49 mg L 1 Co(NO3)26H2O. 2.2. Acquirement of SAP Scenedesmus sp. LX1 were cultivated in 200 mL autoclaved growth medium in 500 mL flasks and maintained in an artificial climate chamber (HPG-280H). The cultivation conditions were as follows: light intensity 2300 lx, light/dark ratio = 14:10, initial pH = 7
and temperature = 25 °C. After 15 days of cultivation, the algae liquid was filtered by 0.45 lm membrane (PALL Corporation, USA) to separate the algae cells from the media and acquired the SAP. At this time, Scenedesmus sp. LX1 had entered into stationary phase with the density of 3.4 106 cells per liter. The dissolved organic carbon (DOC) content of the SAP acquired was 25.8 mg L 1. 2.3. Isolation and fractionation of SAP The method developed by Leenheer (1981) and modified by Zhang et al. (2009) was used to separate the SAP into six fractions: hydrophobic acids (HOA), hydrophobic bases (HOB), hydrophobic neutrals (HON), hydrophilic acids (HIA), hydrophilic bases (HIB), and hydrophilic neutrals (HIN). Dialysis bag (MWCO, 100–500) was used to remove the inorganic salt introduced into the SAP in fraction process. Finally, the volume of each fraction acquired was controlled as the same with the original volume of the SAP, so that the fractions were not concentrated or diluted during the isolation. The test result of ion exchange chromatography (IEC) indicated that after dialysis the residual salt in SAP sample was totally NaCl and the concentration of the salt was less than 100 mg L 1. Preliminary experiments showed that the addition of 100 mg L 1 NaCl in mBG11 medium showed no effect on the growth characteristics of Scenedesmus sp. LX1. 2.4. Effect of SAP on the growth of Scenedesmus sp. LX1 The cultivation experiment was operated under a bacteria-controlled condition. Fifty milliliter flasks were used to cultivate Scenedesmus sp. LX1 with 20 mL of mixed culture medium composed by autoclaved mBG11 medium and the SAP. Dissolved organic carbon (DOC) of the original SAP acquired in this work was 25.8 mg L 1. For the experiments on the effects of different SAP concentrations, the addition amount of SAP in each set of medium was prepared as 25.8/19.3/12.9/6.4/0 mg L 1 DOC, respectively. For the experiments on the effects of different SAP fractions, six fractions were added into the sets respectively with no dilution. Except the content of SAP, the nutrient conditions of all experiment sets were same with mBG11. Scenedesmus sp. LX1 cells were inoculated by 2.5% (v/v) into medium. The condition of the cultivation was same with the description in 2.2. 2.5. Analytical methods The amount of SAP was determined as the dissolved organic carbon (DOC). Under sterile condition, 20 mL of the alga culture was sampled and then filtered by 0.45 lm membrane (PALL Corporation, USA). After that the DOC were measured by a TOC analyzer (TOC-VCPH, SHIMADZU). The algal density was measured by cell counting using a blood cell counting chamber. The dry weight of algal biomass was determined using the method of suspended solid (SS) measurement according to Chinese state standard testing methods (Administration, 2002). The total lipids were extracted with a chloroform/methanol solution (1/1, v/v) and were quantified gravimetrically (Bligh and Dyer, 1959). Triacylglycerols (TAG) is the main raw material in biodiesel production (Schenk et al., 2008) and an important index to indicate the lipid content in algae cells. After the measurement of total lipid, the dried lipid was dissolved in 0.4 mL of isopropyl alcohol. Then the TAG was estimated by enzymatic colorimetric method using commercial kit from Beijing BHKT Clinical Reagent Co., Ltd., No. 2400076. The logistic model, a classic model describing the relationship between a microorganism’s growth and density in limited environ-
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Algal density (cells·mL-1)
108
SAP (mg DOC·L-1) 0 6.4 12.9 19.3 25.8
107
a
106
105
0
5
10
15
20
25
Cultivation time (d)
8
6
b
6 4 4 2 2
0
0
5
10
15
20
25
Rmax (105 cells·mL-1·d-1)
K Rmax
K (106 cells·mL-1)
mental conditions (Li et al., 2010a), was applied in this study. Maximal algal density (K) and maximal population growth rate (Rmax) were measured to describe the growth condition of Scenedesmus sp. LX1. K (cells mL 1) is the carrying capacity, which is the maximal microalgal density reached in the culture. According to the logistic model, when the microalgal density reaches half the value of K, the population growth rate reaches its maximal value (Rmax). All tests were carried out in triplicate (n = 3) and an Independent-Samples t-test was used for significant difference analysis by using SPSS (version 20) statistical software. The fluorescence spectrum of the fraction in SAP was determined using a fluorescence spectrophotometer (Model: F-7000, Hitachi, Japan). Three-dimensional spectra were obtained as the method described by Chen et al. (2003). Size exclusion chromatography (SEC) was used to determine the molecular weight (MW) distribution of the fraction in SAP on a Shimadzu LC-20 high-performance liquid chromatography system. The detection was carried out at 40 °C with a Shimadzu SPD-M20A UV detector and two connected columns (a TSK-GEL G3000PWXL column followed by a TSK-GEL G2500PWXL column). The mobile phase was composed of MILLI-Q ultrapure water buffered with phosphate (0.0024 M NaH2PO4 and 0.0016 M Na2HPO4) and 0.025 M sodium sulfate. The column was calibrated with the standard polyethylene glycol with molecular weights of 330, 700, 1050, 5250, 10,225, 30,000 Da (American Polymer Standards Corp.) and acetone. The retention time in the SEC chromatogram of SAP was converted into the MW according to the calibration.
0 30
SAP (mg·L-1)
3.1. Effects of SAP on the growth of Scenedesmus sp. LX1 The changes of algal densities of Scenedesmus sp. LX1 with different initial concentrations of SAP are shown in Fig. 1a. At the beginning of the cultivation, no obvious difference was found among the growth curves of microalga. After 12 days of cultivation, SAP (6.4–25.8 mg L 1) expressed a significant inhibition on algal density compared with the control group. Under the SAP concentration of 6.4, 12.9 and 19.3 mg L 1, the decline of algal density appeared earlier with the increase of SAP concentration. Under the SAP concentration of 19.3 and 25.8 mg L 1, no significant difference in algal density was observed. Through linear regression analysis (Logistic Model), the K and Rmax of Scenedesmus sp. LX1 under different SAP concentrations were obtained and are shown in Fig. 1b. In the culture medium with no SAP addition (control group), the K and Rmax of Scenedesmus sp. LX1 were over 7.2 106 cells mL 1 and 5.0 105 cells(mL d) 1, respectively. With the addition of SAP, K and Rmax of Scenedesmus sp. LX1 dropped to only 1.6 106–3.6 106 cells mL 1 and 1.5 105–3.2 105 cells(mL d) 1, decreasing by 50–80% and 35–70% compared with control group, respectively. When the concentration of SAP was in the range of 6.4– 19.3 mg L 1, the K and Rmax of Scenedesmus sp. LX1 decreased with the increase of SAP concentration. Liu et al. (2002) reported that the more crude ethyl acetate extract of SAP was added into the algal culture, the more serious the growth of Parietochloris incisa was inhibited. When it came to Scenedesmus sp. LX1, it could not be simply concluded that the inhibition on growth of microalgae increased correspondingly with the SAP concentration, since once the SAP concentration was up to 19.3 mg L 1, the inhibitory effect did not continue to increase. More studies are needed to reveal whether the inhibitory effects would aggravate with the higher addition amount of SAP and how much amount of SAP is allowed on a specific cultivation condition.
Fig. 1. (a) Algal density curves of Scenedesmus sp. LX1 with the different concentrations of SAP released by Scenedesmus sp. LX1. Fig. 1(b) K and Rmax of Scenedesmus sp. LX1 with the different concentrations of SAP released by Scenedesmus sp. LX1.
3.2. Effects of SAP on lipid accumulation of Scenedesmus sp. LX1 As the SAP of Scenedesmus sp. LX1 in the stationary phase expressed a significant inhibitory effect on the growth of this microalga, more researches had been done to investigate the effect of SAP on the lipid accumulation of Scenedesmus sp. LX1, including lipid content in dry weight and TAG content in lipid.Fig. 2. presents the lipid accumulation of Scenedesmus sp. LX1 after 14-day cultivation with the SAP concentration of 25.8 mg DOC L 1 and with no SAP addition. The lipid content in dry weight and the TAGs content in lipid in all groups were around 15% and 20%, respectively. Statistic analysis showed that the addition of SAP has no significant (P > 0.1) impact on the lipid accumulation of Scenedesmus sp. LX1.
35
Lipid or TAGs content (%)
3. Results and discussion
Lipid content in dry weight TAGs content in lipid
30 25 20 15 10 5 0
Without SAP
With SAP
Fig. 2. Lipid accumulation of Scenedesmus sp. LX1 after 14-day cultivation with the SAP concentration of 25.8 mg DOC L 1 and with no SAP addition.
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-1
Some researches revealed that environmental stress was an effective trigger to enhance the lipid accumulation in microalgal cells (Hu et al., 2008; Khozin-Goldberg and Cohen, 2006; Rodolfi et al., 2009). Although the existence of SAP is also an adverse environmental condition to the growth of microalga, this condition seems cannot stimulate the lipid accumulation of Scenedesmus sp. LX1 in cells. As the growth of Scenedesmus sp. LX1 was significantly inhibited by SAP and the lipid or TAG content in cells were not enhanced, thus in general the addition of SAP significantly decreased the total lipid and TAG production of Scenedesmus sp. LX1. The results demonstrated that the SAP should be removed before the reuse of the water to minimize the adverse effect on biodiesel production based on Scenedesmus sp. LX1.
Algal density (cells·mL )
646
10
7
10
6
10
5
10
4
10
3
After 15 days of cultivation, the lipid accumulation of Scenedesmus sp. LX1 under different fractions of SAP were determined and shown in Fig. 4. The effects of different fractions of SAP on lipid
Table 1 The DOC content and the percentage in total SAP of each fraction in SAP released by Scenedesmus sp. LX1. Parameter
HOA
HOB
HON
HIA
HIB
HIN
DOC (mg L 1) Percentage in total SAP (%)
2.75 10.23
1.96 7.29
10.77 40.03
5.17 19.22
1.45 5.4
4.79 17.82
2
4
HIA HIB HIN
6
10
8
12
0.5
b
Rmax(106 cells·mL-1·d-1)
Ctrl 0.4
0.3
HIN HOA
0.2
HON 0.1
HIA HOB
0.0 0.0
HIB 0.5
1.0
1.5
2.0
2.5
3.0
3.5
K (106 cells·mL-1) Fig. 3. (a) Algal density curves of Scenedesmus sp. LX1 with different fractions of SAP released by Scenedesmus sp. LX1. Fig. 3(b) K and Rmax of Scenedesmus sp. LX1 with the different fractions of SAP released by Scenedesmus sp. LX1.
accumulation were various. Setting the lipid accumulation data of control group as the original point, the data points of lipid and TAGs contents in Scenedesmus sp. LX1 with the addition of different fractions of SAP were divided into different quadrants. Several fractions inhibited the lipid accumulation of Scenedesmus sp. LX1, and others showed positive or non-significant effects. The organic bases (HOB, HIB) and HON, which were located in Quadrant IV, could increase the lipid content in dry weight by 36.2%, 26.2% and 61.5%, respectively. Also the HOA, which was located in Quadrant II, showed an enhancing effect on the TAGs contents in lipid. However, the positive impacts of these fractions on
70 TAGs content in lipid (%)
3.4. Effect of different fractions of SAP on lipid accumulation of Scenedesmus sp. LX1
0
Without SAP HOA HOB HON Cultivation time (d)
3.3. Effects of different fractions of SAP on growth of Scenedesmus sp. LX1 In order to identify the main inhibitory factions, the SAP of Scenedesmus sp. LX1 was isolated into six fractions: hydrophobic acids (HOA), hydrophobic bases (HOB), hydrophobic neutrals (HON), hydrophilic acids (HIA), hydrophilic bases (HIB), and hydrophilic neutrals (HIN). The DOC content and the percentage in total SAP of each fraction was shown in Table 1. The recovery rate of DOC though the fraction process was 104%, which was acceptable in this study. Results showed that HON was the main fraction in SAP, which accounted for 40% of DOC content, and the organic bases (HIB, HOB) only accounted for less than 15%. After 15-day cultivation with the addition of different SAP fractions, the impact of each fraction on the growth of Scenedesmus sp. LX1 was shown in Fig. 3a. The initial inoculation density was about 5 103 cells mL 1. At the beginning of the cultivation, no obvious difference of the growth of microalgae among groups was observed. However, from the fourth day of the cultivation, all of the fractions of SAP showed significant inhibitory effect on the growth of Scenedesmus sp. LX1, inducing the algal densities in stationary phase were much less than the group without SAP. Through linear regression analysis, the growth parameters of Scenedesmus sp. LX1 under different fractions of SAP were obtained and shown in Fig. 3b. With the existence of the fractions of SAP, K and Rmax decreased by 40–90% compared with the control group. HIB and HIA expressed the most significant inhibitory effect, which decreased the maximal algae density by 90.0% and 83.2%, respectively. According to the growth parameters, the sequence of the inhibition effects were HIB > HIA > HOB > HON > HOA > HIN. Therefore, although the DOC of the organic bases (HIB and HOB) only accounted for less than 15% in the SAP, these two fractions were among the three strongest inhibitory fractions within SAP. This result was similar with the conclusion drawn by Pratt (1942) which found the growth-inhibiting substance in SAP of Chlorella probably belonging to organic base.
a
60
HOA
50
Ctrl
40
HIB HON
HOB
30 HIA
20
HIN
10 0
0
5
10
15
20
25
30
35
Lipid content in dry weight (%) Fig. 4. Lipid accumulation of Scenedesmus sp. LX1 after 15-day cultivation with different fractions of SAP released by Scenedesmus sp. LX1.
T.-Y. Zhang et al. / Bioresource Technology 146 (2013) 643–648
lipid accumulations of Scenedesmus sp. LX1 were non-significant via T-test (P > 0.1). In Quadrant III, HIA showed the significant inhibition on the lipid accumulation of Scenedesmus sp. LX1 (P < 0.01). With the addition of HIA, the lipid content in dry weight and the TAGs content in lipid were decreased by 59.2% and 51.2% compared with the control group, respectively. HIN could also inhibit the lipid and TAGs accumulation, yet the inhibition was not significant via T-test. No fraction was located in Quadrant I meaning that all of the fractions could not increase both the lipid content in dry weight and the TAG content in lipid of Scenedesmus sp. LX1. In general, as the fractions of SAP expressed various effects on the lipid accumulation of Scenedesmus sp. LX1, the overall effect of SAP was the integrated result of these six fractions. The inhibitory fractions should be removed before the reuse of the culture solution in order to avoid the adverse effect on the biodiesel production based on Scenedesmus sp. LX1. 3.5. Further analysis of the inhibitory substance in HIA of SAP As the HIA fraction expressed the most significant inhibition on growth and lipid accumulation of Scenedesmus sp. LX1, further analysis has been done to identify the composition of HIA. The three-dimensional fluorescence spectroscopy, which was widely used to analyze the composition of dissolved organic materials (DOM), was applied in this study to characterize the composition of HIA in SAP. The three-dimensional spectra of HIA was showed in Fig. 5 and defined into five regions to represent different fractions according to the reference (Chen et al., 2003). The strongest fluorescent peak of HIA located in the range between excitation wavelengths (EX) of 270–310 nm and emission wavelength (EM) of 370–380 nm. Based on literatures, Chen et al. (2003) summarized that peaks at intermediate excitation wavelengths (250–350 nm) and shorter emission wavelength (250–380 nm) were related to soluble microbial byproduct-like material (Region IV). The fluorescent response was also observed in Region II, III and V. These regions were representative for simple aromatic proteins (Region II), humic acid-like organics (Region V) and fulvic acid-like materials (Region III). The SEC system with UV detector was used to determine the molecular weight (MW) distribution of HIA in SAP. The result of SEC at UV254 showed that the molecular weight (MW) of the organic materials in SAP mainly distributed in the range of 0.15–7 kDa. The response peaks of MW appeared at 1.90, 2.83 and 4.32 kDa. Ni et al. (2010) reported that the biomass-associated products in soluble microbial products (SMP) of activated sludge were mainly macromolecules with an MW in a range of 290–
Fig. 5. Fluorescence spectroscopy of HIA in SAP released by Scenedesmus sp. LX1.
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5000 kDa, which was much higher than the MW of HIA in SAP. Hadj-Romdhane et al. (2013) researched the MW distribution of SAP released by Chlorella vulgaris and identified some small organic molecules richer in nitrogen with a molecular size ranging from 1 to 3 kDa in SAP, which was similar with the MW distribution of HIA in SAP released by Scenedesmus sp. LX1. In order to identify the specific molecular structure of inhibitory matters within HIA, some advanced analysis methods should be utilized in further research, such as LC-MS–MS. 4. Conclusions SAP and all of its organic fractions could inhibit the growth of Scenedesmus sp. LX1. Under the SAP concentrations of 6.4– 25.8 mg L 1, K and Rmax were decreased by 50–80% and 35–70%, respectively. HIA and organic bases expressed the strongest inhibition. SAP showed non-significant effect on lipid accumulation of Scenedesmus sp. LX1. HIA could significantly decrease the lipid content of Scenedesmus sp. LX1 by 59.2%. The strongest fluorescent peaks of HIA in SAP located in range between EX of 270–300 nm and EM of 370–380 nm. MW of HIA mainly distributed in the range of 0.15–7 kDa. Acknowledgements This study was supported by China National Science Fund Key Program (No. 51138006). References Administration, S.E.P. 2002. Monitoring Method of Water and Wastewater, (fourth ed.). China Environmental Science Press, Beijing, pp. 105, 246–248, 255–257. Behzadi, S., Farid, M., 2007. Review: examining the use of different feedstock for the production of biodiesel. Asia-Pacific Journal of Chemical Engineering 2 (5), 480– 486. Biddanda, B., Benner, R., 1997. Carbon, nitrogen, and carbohydrate fluxes during the production of particulate and dissolved organic matter by marine phytoplankton. Limnology and Oceanography 42 (3), 506–518. Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Canadian Journal of Biochemistry and Physiology 37 (8), 911–917. Brennan, L., Owende, P., 2010. Biofuels from microalgae–a review of technologies for production, processing, and extractions of biofuels and co-products. Renewable & Sustainable Energy Reviews 14 (2), 557–577. Chen, W., Westerhoff, P., Leenheer, J.A., Booksh, K., 2003. Fluorescence excitation– emission matrix regional integration to quantify spectra for dissolved organic matter. Environmental Science & Technology 37 (24), 5701–5710. Chisti, Y., 2007. Biodiesel from microalgae. Biotechnology Advances 25 (3), 294– 306. Chisti, Y., 2008. Biodiesel from microalgae beats bioethanol. Trends in Biotechnology 26 (3), 126–131. Congress, U., 2007. Energy independence and security act of 2007. Public Law (110– 140). Gouveia, L., Oliveira, A.C., 2009. Microalgae as a raw material for biofuels production. Journal of Industrial Microbiology & Biotechnology 36 (2), 269–274. Hadj-Romdhane, F., Zheng, X., Jaouen, P., Pruvost, J., Grizeau, D., Croué, J., Bourseau, P., 2013. The culture of Chlorella vulgaris in a recycled supernatant: effects on biomass production and medium quality. Bioresource Technology 132, 285– 292. 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. The Plant Journal 54 (4), 621–639. Khozin-Goldberg, I., Cohen, Z., 2006. The effect of phosphate starvation on the lipid and fatty acid composition of the fresh water eustigmatophyte Monodus subterraneus. Phytochemistry 67 (7), 696–701. Leenheer, J.A., 1981. Comprehensive approach to preparative isolation and fractionation of dissolved organic-carbon from natural-waters and wastewaters. Environmental Science & Technology 15 (5), 578–587. Li, X., Hu, H.Y., Gan, K., Sun, Y.X., 2010a. Effects of different nitrogen and phosphorus concentrations on the growth, nutrient uptake, and lipid accumulation of a freshwater microalga Scenedesmus sp.. Bioresource Technology 101 (14), 5494– 5500. 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 Biotechnology 27 (1), 59–63.
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