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Mixotrophic cultivation of Chlorella pyrenoidosa with diluted primary piggery wastewater to produce lipids
Bioresource Technology 104 (2012) 215–220
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Mixotrophic cultivation of Chlorella pyrenoidosa with diluted primary piggery wastewater to produce lipids Haiying Wang a,⇑, Hairong Xiong a, Zhenglong Hui a, Xiaobo Zeng b,⇑ a b
Key Lab for Microorganisms and Biotransformation, College of Life Science, South-Central University for Nationalities, Wuhan 430074, China Ministry of Education Laboratory of Combinatorial Biosynthesis and Drug Discovery, School of Pharmaceutical Science, Wuhan University, Wuhan 430071, China
a r t i c l e
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Article history: Received 18 July 2011 Received in revised form 2 November 2011 Accepted 5 November 2011 Available online 15 November 2011 Keywords: COD Lipid production Microalgae Primary piggery wastewater
a b s t r a c t Piggery wastewater that is used for microalgae cultivation has usually been treated by secondary treatment processes. The present study investigates mixotrophic cultivation of the green microalgae Chlorella pyrenoidosa with primary piggery wastewater that has merely been diluted before use. The biomass productivity of the microalgae exhibited a positive linear correlation with the initial COD values, which ranged from 250 to 1000 mg/L. Nutrients in the piggery wastewater were removed efficiently; for example, the removal rate of ammonium was over 90% in all diluted samples. Profiles of fatty acids in the algal lipids were different after cultivation in piggery wastewater medium compared to Bristol’s solution. The maximum lipid productivity of 6.3 mg L1 day1, which was achieved when the initial COD was 1000 mg/L, is not superior to other reports, but it suggests a convenient way to reduce the high organic content of piggery waste with the production of algal lipids. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Treatment and disposal of piggery waste are among the most important environmental problems to be solved in many countries. An effective and widely used method of treating animal waste involves anaerobic treatment followed by post-treatment in highrate oxidation ponds (Olguin et al., 2003). In China piggery waste is a vast source of pollution with a total annual discharge of 6.0 billion tons, and most of it is released directly into the environment by the many pig farms that seldom operate their non-profitable sewage treatment facilities or do not even have such facilities. Many treatments of piggery wastewater by microalgae have been investigated, even in pilot-scale operations (de Godos et al., 2009), as a means of providing environmental protection from and recovery of nutrients. Some strains of microalgae yield oil efficiently, which offers the possibility of providing raw material for biodiesel that potentially may be substituted for fossil diesel (Chisti, 2007). Neglecting the cost, processing of piggery wastewater by microalgae with the simultaneous production of oil would seem to be a good choice. However, the high concentrations of urea, ammonium, organic acids, pesticides, and other compounds that are frequently present in the wastewater might inhibit microalgal growth (Hodaifa et al., 2008). ⇑ Corresponding authors. Tel./fax: +86 27 67842689 (H. Wang), tel.: +86 27 68759939; fax: +86 27 68759850 (X. Zeng). E-mail addresses: [email protected] (H. Wang), [email protected] (X. Zeng). 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.11.020
In order to avoid the inhibition caused by high organic strength wastewater, many studies were conducted on secondary wastewater treated by anaerobic digestion (Olguin et al., 2003; Wang et al., 2010) or on effluent from wastewater treatment facilities (Kim et al., 2007; Martinez et al., 2000; Woertz et al., 2009). Few papers have reported the culture of microalgae by directly using diluted original piggery wastewater (Traviseo et al., 2006). Most microalgae used in these studies were mixotrophic species as they are effective at reducing values of chemical oxygen demand (COD) and can eliminate nitrogen and phosphorus. Mixotrophic growth occurs when CO2 and organic carbon are assimilated in cells simultaneously. Culture of microalgae under mixotrophic conditions could overcome the light limitation exhibited in pure photoautotrophic culture (Ceron Garcia et al., 2006; Garci et al., 2000; Sanchez et al., 2001). This offers the possibility of greatly increasing the cell density and productivity by eliminating the requirement of light (Andrade and Costa, 2007; Ip et al., 2004). Direct application of diluted piggery wastewater to microalgae culture in mixotrophic mode might provide a simplified way to produce algal oil and simultaneously treat high organic content wastewater. The use of microalgae in municipal wastewater treatment in ponds is well established (Noüe et al., 1992). Previous studies primarily discussed their capacity to remove inorganic nutrients (An et al., 2003; Aslan and Kapdan, 2006; Converti et al., 2006; Traviseo et al., 2006), and some reported the lipid content of the microalgae that grew in wastewater (Wahlen et al., 2011; Wang et al., 2010; Woertz et al., 2009). However, the studies mentioned above mainly
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focused on algae growth, lipid content, and nutrient recovery efficiency with dairy and piggery wastewater after secondary treatment. Few were concerned with culturing microalgae in original piggery waste with no pretreatment other than dilution. Many studies also revealed that the lipid content, fatty acid profile, and biomass productivity rely on environmental conditions and culturing modes (Chisti, 2007; Illman et al., 2000; Liang et al., 2009). The strain of Chlorella pyrenoidosa used in the present study was found to grow in mixotrophic mode at the expense of glucose. Batchwise experiments were employed to analyze the cultivation properties and lipid productivity of C. pyrenoidosa in diluted piggery wastewater from a local pig farm. The relationships between the initial COD values and properties such as the growth rate, the initial optical density of the medium, and the elimination of organic compounds were individually investigated, as were variations in the algal fatty acid profile.
2.3. Measurement of growth Biomass concentration (DCW, dry-weight of cell powder in culture medium, g/L) was estimated by an equation that corroborated the dry-weight determinations:
DCWðg=LÞ ¼ 0:2538OD625 ; R2 ¼ 0:9860; where OD625 was the absorbance measured at 625 nm by a UV–Vis spectrophotometer (UV-1750, Shimadzu). The specific growth rate was calculated by fitting the cell dry weight for the first 3 days of culture to an exponential function:
lðday1 Þ ¼ ðln X ln X 0 Þ=t; where t (day) was the time between the two measurements, X and X0 (g/L) were the concentrations of biomass at day t (3 days) and t0, respectively.The biomass productivity during the culture period was calculated from the equation:
2. Methods
Pbiomass ðg L1 day Þ ¼ ðX X 0 Þ=t;
2.1. Piggery wastewater used for C. pyrenoidosa cultivation
where X (g/L) was the concentration of biomass at the end of the cultivation, X0 (g/L) was that at the beginning, and t was the duration of cultivation (10 days).
1
Experiments were carried out using piggery wastewater from a local pig farm as a substrate. The characteristics and features of the raw wastewater are summarized in Table 1. The values of ammonium ðNHþ 4 -NÞ, total nitrogen, total phosphorus, COD, total solids, and volatile solids in piggery wastewater were determined following known protocols (Clesceri et al., 1998). The piggery wastewater used as the medium for culturing microalgae was first allowed to settle for several hours. Then the supernatant was separated and diluted with distilled water to COD values of 250, 500, 750, and 1000 mg/L. All experiments were carried out in triplicate and average values or mean values with standard deviation are reported. 2.2. Algal strain, medium, and culture conditions The strain of C. pyrenoidosa was obtained from local fresh water and maintained in a modified Bristol’s solution: 0.25 g/L NaNO3, 0.075 g/L K2HPO43H2O, 0.075 g/L MgSO4, 0.025 g/L CaCl22H2O, 0.175 g/L KH2PO4, 0.025 g/L NaCl, 0.005 g/L FeCl36H2O, 1 mL FeEDTA solution, 40 mL soil extract/L, and 1 mL/L A5 solution, which consisted of 0.22 g/L ZnSO47H2O, 2.86 g/L H3BO3, 1.81 g/L MnCl2 4H2O, 0.079 g/L CuSO45H2O, and 0.039 g/L (NH4)6Mo7O244H2O. After adjusting the pH to 8.0, 500 mL of the diluted piggery water or Bristol’s solution for cultivation were put in 1000-mL conical flasks and autoclaved for 20 min at 121 °C. The inocula in linear growth phase were prepared previously. The culture broth was stirred by bubbling through air that had been sterilized with a 0.2-lm membrane filter at a flow rate of 300 mL/min. Light was continuously supplied (Philips TLD 36W/54 fluorescent lamp) at an intensity of 63 lmol m2 s1 measured at the surface of the flask with an illuminance meter (ZDS-10, Shanghai Cany Precision Instrument Ltd., China). Batchwise cultivation lasted for 10 days at 25–27 °C, and then the broth was harvested for measurement. Table 1 Characteristics of the piggery wastewater used in the experiments.
a b
Parameter
Value
NH3-N (mg N/L)a Total nitrogen (mg/L) Total phosphorus (mg P/L) COD (mg/L)b Total solids (mg/L) Volatile solids (mg/L)
1388 980 158 11,000 7000 4500
NH3-N, ammonium. COD, chemical oxygen demand.
2.4. Measurement of lipid production Total lipids were determined by the method of Bligh and Dyer (Iverson et al., 2001). The cell suspensions were collected and centrifuged at 6000g for 10 min. Then the pellets were freeze-dried and stored at 20 °C before analysis. The lipid contents were calculated from the equation:
C 1 ðg=gÞ ¼ W L =W A where WL (g) was the weight of the extracted lipids and WA (g) was the dry algae biomass. The lipid productivity of the batch cultivation was calculated from the equation: 1
Plipid ðg L1 day Þ ¼ ðC l X C 0 X 0 Þ=t where C0 (g/g) was the content of microalgae lipid at the beginning and C1 (g/g) was that at the end of the cultivation, X0 and X (g/L) were the concentrations of biomass at the corresponding beginning and end, and t was the duration of the experiment (10 days). 2.5. Fatty acid analysis Analysis of the fatty acids in the algae cells by gas chromatography was modified from the procedure of Slover and Lanza (1979). Three milligrams of freeze-dried sample were put into a capped test tube and extracted three times with 2 mL of CHCl3–CH3OH (V/V = 2/1) under N2 with enhancement by ultrasound. To each specimen 0.12 mg of an internal standard (heptadecanoic acid, Sigma, USA) was added. The extracted sample was saponified with 2 mL of KOH saturated methanol at 75 °C for 10 min and then mixed with 1 mL of boron trifluoride methanol solution at 75 °C for another 30 min. After the suspension cooled, 2 mL of hexane and 2 mL of saturated NaCl solution were added to form separated layers in the tube. The upper clear layer of alkane was pipetted out for analysis by a gas chromatograph (Agilent-6890, Agilent Technologies, USA) that was equipped with a flame ionization detector. All operations were conducted under low-light conditions and protection of nitrogen. Separation was achieved on a HP-5 capillary column (30.0 m 320 lm 0.25 lm, 50% phenyl methyl siloxane) with N2 as the carrier gas at a flow speed of 1 mL/min. One microliter of sample solution was injected in split mode (split ratio = 1:30) for each analysis. The injector
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temperature of the machine was set to 250 °C, and the column temperature program was set as follows: the initial temperature was 150 °C; then it rose to 210 °C at a rate of 10 °C/min and sequentially increased to 250 °C at a rate of 2 °C/min; the final column temperature was maintained at 250 °C for 25 min. Identification of the components proceeded by comparing the retention times and fragmentation pattern with a SupelcoÒ 37-Component FAME Mix (Sigma, USA).
3. Results and discussion 3.1. Algal growth in piggery water Algal growth, as indicated by biomass concentrations in diluted piggery wastewater and Bristol’s solution, is shown in Fig. 1. With no obvious lag phase exhibited, this wild-isolated C. pyrenoidosa grew faster in all samples of piggery wastewater than in Bristol’s solution. The biomass at the end of the experiment increased as the initial COD value rose from 250 to 1000 mg/L. Among the serial dilutions, the algae grew faster in 750- and 1000-mg COD/L samples than in the others. Growth of the 250-mg COD/L sample caught up to that of the 500-mg COD/L sample in the latter part of the cultivation period, but the growth speeds of both slowed at the seventh day. Similarly, the growth of algae in the 750- and 1000-mg/L diluted samples also entered a stationary phase at about the seventh day. Considering the high removal rate of ammonium (Fig. 2b), the slowing of the growth speeds might be the result of its rapid consumption. As shown in Fig. 3, the biomass productivities in all of the diluted piggery wastewater samples were much higher than that in Bristol’s solution and reached a maximum at 1000 mg COD/L. During the first 3 days, the average specific growth rates decreased as the initial COD value increased. The relationship between the initial optical density of the culture medium (measured at 680 nm) and the initial COD value is also plotted in Fig. 3. The specific growth rates at the beginning of culture correlated well with the initial COD value (R2 = 0.9709), which in turn provided a good correlation between the specific growth rates and the initial optical density of the wastewater (R2 = 0.9920, data not shown here). Suspended matter or impurities in the piggery waste would decrease the clarity of the water and interfere with light penetration. Therefore, the initial optical density of the wastewater increased as the initial COD rose. At the beginning of culture, the growth rate of the algae was impeded by turbidity in the wastewater, which decreased the light penetration. Since the piggery wastewater was allowed to settle and was diluted before testing, the suspended matter in the diluted samples should be largely attributed to some
Fig. 1. Growth curves of Chlorella pyrenoidosa in Bristol’s solution and diluted piggery wastewater with different initial COD values.
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finely divided inorganic and organic matter, as well as soluble colored organic compounds. This appears to be a reasonable explanation for the decrease in specific growth rates as the initial COD value increased. Fig. 3 shows the biomass productivities at the end of culture were well correlated with the initial COD values (R2 = 0.9639). This indicates that the initial COD is an important parameter that determines the suitability of wastewater for growth of C. pyrenoidosa. Though the initial growth of the algae was suppressed (Fig. 3) due to the limitation of luminous flux caused by high initial COD values, the biomass at the end of the experiment increased as the initial COD increased from 500 to 1000 mg/L (Fig. 1). A probable explanation is that the complex nutrients of piggery wastewater provide some organic components for algae growth in mixotrophic mode by assimilating organic carbon as well as fixing CO2 in light (Wang et al., 2010). It was found that, with no nutrient limitations, the photosynthesis of algae was enhanced by increasing the light intensity until the maximum algal growth rate was achieved at the light saturation point (Torzillo et al., 2003). Then the algal concentration continuously increased until a shading effect of the biomass became prominent. The light intensity was thought to have a small effect on the growth of Chlorella vulgaris under non nutrient-limited conditions as it increased to greater than 31.3 lmol m2 s1, though its saturation point was found to be 47.0 lmol m2 s1 (Bartosh and Banks, 2007). The strain of C. pyrenoidosa investigated in this study is capable of mixotrophic growth that further reduces its dependence on light. Thus, limiting the light intensity does not have a fatal effect on these microalgae when they are cultured in a nutritious medium like diluted piggery wastewater. 3.2. Nutrient removal efficiencies The removal rates of COD were all of double-digit percentages in different samples of diluted piggery wastewater (Fig. 2a). At the tested COD levels, possible toxicity or inhibition of COD was not present for this microalgae strain, which suggests it is a COD tolerant strain. Traviseo et al. (2006) reported a COD removal rate of 88.0% at 190 h for an initial COD concentration of 250 mg/L, which is far higher than the removal rate in this study and Wang’s results (Wang et al., 2010). The reduction in COD could only be attributed to exhaustion of the microalgae in the axenic culture conditions. The good growth and considerable COD reduction in diluted piggery wastewater indicate that C. pyrenoidosa use organic carbon as a source of energy and a substrate for cell growth, like some microalgae reported previously (Liu et al., 2009). Although the results suggest dilution followed by algae cultivation is a convenient way to treat piggery wastewater, it still cannot replace the traditional secondary treatment as its residual COD cannot meet the national emission standards of 125 mg/L (except when the initial COD value is 250 mg/L, as shown in Fig. 2a) in most Western countries. However, in less diluted samples, C. pyrenoidosa could be supported by a higher carbon concentration to obtain more biomass (Fig. 1). More research is required to find the optimum balance between reduction in COD value and biomass production. Ammonium removal of over 90% was achieved for all diluted samples, regardless of its initial concentration (Fig. 2b, from 34.7 to 138.8 mg/L). For all eukaryotic algae, including C. pyrenoidosa, the forms of inorganic nitrogen that can be directly assimilated þ þ are only nitrate ðNOþ 3 Þ, nitrite ðNO2 Þ, and ammonium ðNH4 Þ (Barsanti and Gualtieri, 2006). Remarkable reductions in total nitrogen and total phosphorus were also found for all four diluted wastewater samples (Fig. 2c and d). It is obvious that the removal rate of both total nitrogen and total phosphorus increased along with the rise in COD value. Another species of microalgae, Spirulina, was reported to have removal rates for NHþ 4 -N and phosphate ranging from 84% to 96% depending on the season or pond depth
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Fig. 2. The removal rates of (a) COD, (b) ammonium, (c) total nitrogen, and (d) total phosphorus calculated from their contents at the initial and final stages of the cultivation of Chlorella pyrenoidosa in different diluted piggery wastewaters (COD, chemical oxygen demand; NH3-N, ammonium; Total N, total nitrogen; Total P, total phosphorus).
Fig. 3. The initial specific growth rates (l), biomass productivities (Pbiomass), and initial optical densities of the broth (Ainitial) in the cultures of Chlorella pyrenoidosa with Bristol’s solution and different diluted piggery wastewaters (COD 250–1000 are mg/L of COD in diluted piggery wastewater; the lines and equations are correlations between the three dependent variables and the initial COD values in the medium).
(Olguin et al., 2003). Wang et al. (2010) also found that a wild-type Chlorella sp. cultivated in manure diluted 10–25-fold could provide removal rates of 75.7–82.5% for total nitrogen and 62.5–74.7% for total phosphorus. Data from these studies and the present research suggest that a relatively higher value of COD would enhance the removal rates of nutrients in the culture medium. Some previous studies proposed that high initial concentrations of nutrients would reduce their removal efficiencies. For instance, Aslan and Kapdan (2006) presumed that the uptake of nitrogen and phosphorus by algae does not provide efficient removal of these nutrients from synthetic media at high concentrations because of the light limitation caused by excess chlorophyll a content in the culture broth. And Kumar et al. (2010) reported that the maximum reduction in total ammonia nitrogen by C. vulgaris was 61.8% in digested piggery effluent with 20 mg/L initial total ammonia nitrogen. This rate was much less than the 93.7% removal observed for the 250-mg COD/L sample in this study with a
corresponding ammonia nitrogen concentration of 34.7 mg/L. Analysis of the data in Fig. 1Fig. 2c and d showed that the removal of organic matter increased in accordance with the rise in biomass, which was enhanced by the increase in COD values from 250 to 1000 mg/L. C. pyrenoidosa was found to provide efficient removal of nutrients and to be able to overcome the light limitation exhibited in pure photoautotrophic culture by growing mixotrophicly in media with a high content of nutrients (Wei et al., 2009). Thus, culture of microalgae in diluted piggery wastewater with mixotrophic growth has advantages in reducing the values of total nitrogen, total phosphorus, and ammonia nitrogen. 3.3. Yield of lipids and fatty acids composition Fig. 4 shows the lipid contents produced in Bristol’s solution and diluted piggery wastewater were roughly at equal levels, but the lipid productivities were evidently different. The lower level
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of lipid productivity in Bristol’s solution should be due to its lower biomass, which was almost half of that in diluted piggery wastewater (Fig. 1). The total nitrogen in Bristol’s solution was approximately 41 mg/L (calculated from its composition), which was close to the values of 24.5 mg/L and 49.0 mg/L found for the 250- and 500-mg COD/L samples (in Fig. 2c), respectively. However, the diluted piggery wastewater samples with total nitrogen content of 73.5 and 98.0 mg/L (corresponding to 750- and 1000-mg COD/L samples, respectively) provided lipid contents that were significantly less (p < 0.01) than Bristol’s solution and the 250-mg COD/ L sample (Fig. 4). The phenomenon of nitrogen limitation enhancing the accumulation of lipids (mostly triacylglycerols) in microalgae cells has been reported in many studies (Illman et al., 2000; Shifrin and Chisholm, 1981). (Shifrin and Chisholm, 1981) reported that deprivation of nitrogen increased the lipid content in 30 strains of green algae and diatoms including seven Chlorella. Illman et al. (2000) surveyed five Chlorella cultures in a low nitrogen medium and found an almost twofold rise in lipid content of four strains. The biomass and lipid productivities obtained in the present work were not necessarily superior to those reported elsewhere using different strains of microalgae. For instance, Converti et al. (2009) reported that C. vulgaris growing in Bold’s basal medium had somewhat higher production rates ranging from 8 to 20 mg L1 day1. In Woertz’s report, the maximum lipid productivities achieved for microalgae culture in a semi-continuous mode were 17 mg L1 day1 on dairy wastewater and 24 mg L1 day1 on municipal wastewater (Woertz et al., 2009). This suggests that in continuous culture mode the lipid productivity in piggery wastewater might be improved by continuous supplementation of nutrients such as ammonium, which has a limited concentration in batch cultivation. If this method could be adapted on a commercial scale, transforming piggery wastewater to biodiesel by algae cultivation with lipid production would be a waste-to-biofuel approach with no food competition issues and provide a valuable mode of degrading sewage for environmental protection. The green alga Chlorella yields oil with a fatty acid composition of 14:0, 16:0, 16:1, 16:2, 16:3, 18:0, 18:1, 18:2, and a-18:3 under most kinds of culture conditions such as photoautotrophic and heterotrophic cultivation, nitrogen and phosphorus starvation, and outdoors in a photobioreactor (Petkov and Garcia, 2007). Fig. 5 shows the fatty acid profiles of algae cells cultured with Bristol’s solution and the diluted sample with 500-mg COD/L in this study. There were nine fatty acids identified belonging to the composition mentioned above, among which hexadecanoic acid (C 16:0), linoleic acid (C 18:2), and linolenic acid (C 18:3) were the abundant fatty acids. Compared to Bristol’s solution, there were remarkable reductions in the relative content of hexadecadienoic acid (C 16:2) and oleinic acid (C 18:1) as well as an increase in hexadecanoic acid (C 16:0) for the diluted piggery waste samples. Variations
Fig. 4. Effect of initial COD values on the yield of lipids from Chlorella pyrenoidosa culture in Bristol’s solution and piggery wastewater with different dilutions (COD 250–1000 are mg/L of COD in diluted piggery wastewater).
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Fig. 5. The fatty acid profiles of C. pyrenoidosa culture with Bristol’s solution and piggery wastewater with 500-mg/L COD values (, p < 0.05; SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; Others, undefined peaks in chromatomap).
in contents of these fatty acids also occurred in a strain of Chlorella sp. with increases in oleinic acid (C 18:1) and hexadecanoic acid (C 16:0) and a decrease in hexadecadienoic acid (C 16:2) as the COD values in the medium were reduced by dilution (Wang et al., 2010). The relative content of linolenic acid (C 18:3) among the total fatty acids was about 27% (weight/weight). Although it is difficult to convert this value of a mass ratio into a molar ratio, it seems the value cannot meet the limitation of 12% (mol/mol) in the European Standard EN 14214, which limits the content of linolenic acid methyl ester in biodiesel for vehicle use (Knothe, 2006). This implies that additional treatment is required, such as partial catalytic hydrogenation of the oil (Dijkstra, 2006), which would not be a significant restriction for its application in biodiesel (Chisti, 2007). 4. Conclusions The present study attempts to combine the treatment of primary piggery wastewater with algae oil production using a strain of C. pyrenoidosa. The results indicate that culturing microalgae in mixtrophic mode with diluted primary piggery wastewater provides an effective method for converting high strength livestock wastewater into a profitable byproduct as well as reducing contamination of the environment. Acknowledgements The study was supported by the National Natural Science Foundation of China (50907074). References An, J.Y., Sim, S.J., Lee, J.S., Kim, B.W., 2003. Hydrocarbon production from secondarily treated piggery wastewater by the green alga Botryococcus braunii. J. Appl. Phycol. 15 (2–3), 185–191. Andrade, M.R., Costa, J.A.V., 2007. Mixotrophic cultivation of microalga Spirulina platensis using molasses as organic substrate. Aquaculture 264 (1–4), 130–134. Aslan, S., Kapdan, I.K., 2006. Batch kinetics of nitrogen and phosphorus removal from synthetic wastewater by algae. Ecol. Eng. 28 (1), 64–70. Barsanti, L., Gualtieri, P., 2006. Algae: Anatomy, Biochemistry, and Biotechnology. CRC Press, Boca Raton. Bartosh, Y., Banks, C.J., 2007. Algal growth response and survival in a range of light and temperature conditions: implications for non-steady-state conditions in waste stabilisation ponds. Water Sci. Technol. 55 (11), 211–218. Ceron Garcia, M.C., Camacho, F.G., Miron, A.S., Sevilla, J.M.F., Chisti, Y., Grima, E.M., 2006. Mixotrophic production of marine microalga Phaeodactylum tricornutum on various carbon sources. J. Microbiol. Biotechnol. 16 (5), 689–694. Chisti, Y., 2007. Biodiesel from microalgae. Biotechnol. Adv. 25 (3), 294–306. Clesceri, L.S., Greenberg, A.E., Eaton, A.D., 1998. Standard Methods for the Examination of Water and Wastewater, 20th ed. American Public Health Association, Washington, DC. 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: Process Intensification 48 (6), 1146–1151.
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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 (3), 263–272. Noüe, J., Laliberté, G., Proulx, D., 1992. Algae and waste water. J. Appl. Phycol. 4 (3), 247–254. Olguin, E.J., Galicia, S., Mercado, G., Perez, T., 2003. Annual productivity of Spirulina (Arthrospira) and nutrient removal in a pig wastewater recycling process under tropical conditions. J. Appl. Phycol. 15 (2–3), 249–257. Petkov, G., Garcia, G., 2007. Which are fatty acids of the green alga Chlorella? Biochem. Syst. Ecol. 35 (5), 281–285. Sanchez, S., Martinez, M.E., Espejo, M.T., Pacheco, R., Espinola, F., Hodaifa, G., 2001. Mixotrophic culture of Chlorella pyrenoidosa with olive-mill wastewater as the nutrient medium. J. Appl. Phycol. 13 (5), 443–449. Shifrin, N.S., Chisholm, S.W., 1981. Phytoplankton lipids: interspecific differences and effects on nitrate, silicate and light-dark cycles. J. Phycol. 17 (4), 374–384. Slover, H., Lanza, E., 1979. Quantitative analysis of food fatty acids by capillary gas chromatography. J. Am. Oil Chem. Soc. 56 (12), 933–943. Torzillo, G., Pushparaj, B., Masojidek, J., Vonshak, A., 2003. Biological constraints in algal biotechnology. Biotechnol. Bioprocess Eng. 8 (6), 338–348. Traviseo, L., Benitez, R., Sanchez, E., Borja, R., Martin, A., Colmenarejo, M.F., 2006. Batch mixed culture of Chorella vulgaris using settled and diluted piggery waste. Ecol. Eng. 28 (2), 158–165. Wahlen, B.D., Willis, R.M., Seefeldt, L.C., 2011. Biodiesel production by simultaneous extraction and conversion of total lipids from microalgae, cyanobacteria, and wild mixed-cultures. Bioresour. Technol. 102 (3), 2724–2730. Wang, L., Li, Y.C., Chen, P., Min, M., Chen, Y.F., Zhu, J., Ruan, R.R., 2010. Anaerobic digested dairy manure as a nutrient supplement for cultivation of oil-rich green microalgae Chlorella sp.. Bioresour. Technol. 101 (8), 2623–2628. Wei, A.L., Zhang, X.W., Wei, D., Chen, G., Wu, Q.Y., Yang, S.T., 2009. Effects of cassava starch hydrolysate on cell growth and lipid accumulation of the heterotrophic microalgae Chlorella protothecoides. J. Ind. Microbiol. Biotechnol. 36 (11), 1383– 1389. Woertz, I., Feffer, A., Lundquist, T., Nelson, Y., 2009. Algae grown on dairy and municipal wastewater for simultaneous nutrient removal and lipid production for biofuel feedstock. J. Environ. Eng.-ASCE 135 (11), 1115–1122.
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Mixotrophic cultivation of Chlorella pyrenoidosa with diluted primary piggery wastewater to produce lipids Haiying Wang a,⇑, Hairong Xiong a, Zhenglong Hui a, Xiaobo Zeng b,⇑ a b
Key Lab for Microorganisms and Biotransformation, College of Life Science, South-Central University for Nationalities, Wuhan 430074, China Ministry of Education Laboratory of Combinatorial Biosynthesis and Drug Discovery, School of Pharmaceutical Science, Wuhan University, Wuhan 430071, China
a r t i c l e
i n f o
Article history: Received 18 July 2011 Received in revised form 2 November 2011 Accepted 5 November 2011 Available online 15 November 2011 Keywords: COD Lipid production Microalgae Primary piggery wastewater
a b s t r a c t Piggery wastewater that is used for microalgae cultivation has usually been treated by secondary treatment processes. The present study investigates mixotrophic cultivation of the green microalgae Chlorella pyrenoidosa with primary piggery wastewater that has merely been diluted before use. The biomass productivity of the microalgae exhibited a positive linear correlation with the initial COD values, which ranged from 250 to 1000 mg/L. Nutrients in the piggery wastewater were removed efficiently; for example, the removal rate of ammonium was over 90% in all diluted samples. Profiles of fatty acids in the algal lipids were different after cultivation in piggery wastewater medium compared to Bristol’s solution. The maximum lipid productivity of 6.3 mg L1 day1, which was achieved when the initial COD was 1000 mg/L, is not superior to other reports, but it suggests a convenient way to reduce the high organic content of piggery waste with the production of algal lipids. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Treatment and disposal of piggery waste are among the most important environmental problems to be solved in many countries. An effective and widely used method of treating animal waste involves anaerobic treatment followed by post-treatment in highrate oxidation ponds (Olguin et al., 2003). In China piggery waste is a vast source of pollution with a total annual discharge of 6.0 billion tons, and most of it is released directly into the environment by the many pig farms that seldom operate their non-profitable sewage treatment facilities or do not even have such facilities. Many treatments of piggery wastewater by microalgae have been investigated, even in pilot-scale operations (de Godos et al., 2009), as a means of providing environmental protection from and recovery of nutrients. Some strains of microalgae yield oil efficiently, which offers the possibility of providing raw material for biodiesel that potentially may be substituted for fossil diesel (Chisti, 2007). Neglecting the cost, processing of piggery wastewater by microalgae with the simultaneous production of oil would seem to be a good choice. However, the high concentrations of urea, ammonium, organic acids, pesticides, and other compounds that are frequently present in the wastewater might inhibit microalgal growth (Hodaifa et al., 2008). ⇑ Corresponding authors. Tel./fax: +86 27 67842689 (H. Wang), tel.: +86 27 68759939; fax: +86 27 68759850 (X. Zeng). E-mail addresses: [email protected] (H. Wang), [email protected] (X. Zeng). 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.11.020
In order to avoid the inhibition caused by high organic strength wastewater, many studies were conducted on secondary wastewater treated by anaerobic digestion (Olguin et al., 2003; Wang et al., 2010) or on effluent from wastewater treatment facilities (Kim et al., 2007; Martinez et al., 2000; Woertz et al., 2009). Few papers have reported the culture of microalgae by directly using diluted original piggery wastewater (Traviseo et al., 2006). Most microalgae used in these studies were mixotrophic species as they are effective at reducing values of chemical oxygen demand (COD) and can eliminate nitrogen and phosphorus. Mixotrophic growth occurs when CO2 and organic carbon are assimilated in cells simultaneously. Culture of microalgae under mixotrophic conditions could overcome the light limitation exhibited in pure photoautotrophic culture (Ceron Garcia et al., 2006; Garci et al., 2000; Sanchez et al., 2001). This offers the possibility of greatly increasing the cell density and productivity by eliminating the requirement of light (Andrade and Costa, 2007; Ip et al., 2004). Direct application of diluted piggery wastewater to microalgae culture in mixotrophic mode might provide a simplified way to produce algal oil and simultaneously treat high organic content wastewater. The use of microalgae in municipal wastewater treatment in ponds is well established (Noüe et al., 1992). Previous studies primarily discussed their capacity to remove inorganic nutrients (An et al., 2003; Aslan and Kapdan, 2006; Converti et al., 2006; Traviseo et al., 2006), and some reported the lipid content of the microalgae that grew in wastewater (Wahlen et al., 2011; Wang et al., 2010; Woertz et al., 2009). However, the studies mentioned above mainly
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focused on algae growth, lipid content, and nutrient recovery efficiency with dairy and piggery wastewater after secondary treatment. Few were concerned with culturing microalgae in original piggery waste with no pretreatment other than dilution. Many studies also revealed that the lipid content, fatty acid profile, and biomass productivity rely on environmental conditions and culturing modes (Chisti, 2007; Illman et al., 2000; Liang et al., 2009). The strain of Chlorella pyrenoidosa used in the present study was found to grow in mixotrophic mode at the expense of glucose. Batchwise experiments were employed to analyze the cultivation properties and lipid productivity of C. pyrenoidosa in diluted piggery wastewater from a local pig farm. The relationships between the initial COD values and properties such as the growth rate, the initial optical density of the medium, and the elimination of organic compounds were individually investigated, as were variations in the algal fatty acid profile.
2.3. Measurement of growth Biomass concentration (DCW, dry-weight of cell powder in culture medium, g/L) was estimated by an equation that corroborated the dry-weight determinations:
DCWðg=LÞ ¼ 0:2538OD625 ; R2 ¼ 0:9860; where OD625 was the absorbance measured at 625 nm by a UV–Vis spectrophotometer (UV-1750, Shimadzu). The specific growth rate was calculated by fitting the cell dry weight for the first 3 days of culture to an exponential function:
lðday1 Þ ¼ ðln X ln X 0 Þ=t; where t (day) was the time between the two measurements, X and X0 (g/L) were the concentrations of biomass at day t (3 days) and t0, respectively.The biomass productivity during the culture period was calculated from the equation:
2. Methods
Pbiomass ðg L1 day Þ ¼ ðX X 0 Þ=t;
2.1. Piggery wastewater used for C. pyrenoidosa cultivation
where X (g/L) was the concentration of biomass at the end of the cultivation, X0 (g/L) was that at the beginning, and t was the duration of cultivation (10 days).
1
Experiments were carried out using piggery wastewater from a local pig farm as a substrate. The characteristics and features of the raw wastewater are summarized in Table 1. The values of ammonium ðNHþ 4 -NÞ, total nitrogen, total phosphorus, COD, total solids, and volatile solids in piggery wastewater were determined following known protocols (Clesceri et al., 1998). The piggery wastewater used as the medium for culturing microalgae was first allowed to settle for several hours. Then the supernatant was separated and diluted with distilled water to COD values of 250, 500, 750, and 1000 mg/L. All experiments were carried out in triplicate and average values or mean values with standard deviation are reported. 2.2. Algal strain, medium, and culture conditions The strain of C. pyrenoidosa was obtained from local fresh water and maintained in a modified Bristol’s solution: 0.25 g/L NaNO3, 0.075 g/L K2HPO43H2O, 0.075 g/L MgSO4, 0.025 g/L CaCl22H2O, 0.175 g/L KH2PO4, 0.025 g/L NaCl, 0.005 g/L FeCl36H2O, 1 mL FeEDTA solution, 40 mL soil extract/L, and 1 mL/L A5 solution, which consisted of 0.22 g/L ZnSO47H2O, 2.86 g/L H3BO3, 1.81 g/L MnCl2 4H2O, 0.079 g/L CuSO45H2O, and 0.039 g/L (NH4)6Mo7O244H2O. After adjusting the pH to 8.0, 500 mL of the diluted piggery water or Bristol’s solution for cultivation were put in 1000-mL conical flasks and autoclaved for 20 min at 121 °C. The inocula in linear growth phase were prepared previously. The culture broth was stirred by bubbling through air that had been sterilized with a 0.2-lm membrane filter at a flow rate of 300 mL/min. Light was continuously supplied (Philips TLD 36W/54 fluorescent lamp) at an intensity of 63 lmol m2 s1 measured at the surface of the flask with an illuminance meter (ZDS-10, Shanghai Cany Precision Instrument Ltd., China). Batchwise cultivation lasted for 10 days at 25–27 °C, and then the broth was harvested for measurement. Table 1 Characteristics of the piggery wastewater used in the experiments.
a b
Parameter
Value
NH3-N (mg N/L)a Total nitrogen (mg/L) Total phosphorus (mg P/L) COD (mg/L)b Total solids (mg/L) Volatile solids (mg/L)
1388 980 158 11,000 7000 4500
NH3-N, ammonium. COD, chemical oxygen demand.
2.4. Measurement of lipid production Total lipids were determined by the method of Bligh and Dyer (Iverson et al., 2001). The cell suspensions were collected and centrifuged at 6000g for 10 min. Then the pellets were freeze-dried and stored at 20 °C before analysis. The lipid contents were calculated from the equation:
C 1 ðg=gÞ ¼ W L =W A where WL (g) was the weight of the extracted lipids and WA (g) was the dry algae biomass. The lipid productivity of the batch cultivation was calculated from the equation: 1
Plipid ðg L1 day Þ ¼ ðC l X C 0 X 0 Þ=t where C0 (g/g) was the content of microalgae lipid at the beginning and C1 (g/g) was that at the end of the cultivation, X0 and X (g/L) were the concentrations of biomass at the corresponding beginning and end, and t was the duration of the experiment (10 days). 2.5. Fatty acid analysis Analysis of the fatty acids in the algae cells by gas chromatography was modified from the procedure of Slover and Lanza (1979). Three milligrams of freeze-dried sample were put into a capped test tube and extracted three times with 2 mL of CHCl3–CH3OH (V/V = 2/1) under N2 with enhancement by ultrasound. To each specimen 0.12 mg of an internal standard (heptadecanoic acid, Sigma, USA) was added. The extracted sample was saponified with 2 mL of KOH saturated methanol at 75 °C for 10 min and then mixed with 1 mL of boron trifluoride methanol solution at 75 °C for another 30 min. After the suspension cooled, 2 mL of hexane and 2 mL of saturated NaCl solution were added to form separated layers in the tube. The upper clear layer of alkane was pipetted out for analysis by a gas chromatograph (Agilent-6890, Agilent Technologies, USA) that was equipped with a flame ionization detector. All operations were conducted under low-light conditions and protection of nitrogen. Separation was achieved on a HP-5 capillary column (30.0 m 320 lm 0.25 lm, 50% phenyl methyl siloxane) with N2 as the carrier gas at a flow speed of 1 mL/min. One microliter of sample solution was injected in split mode (split ratio = 1:30) for each analysis. The injector
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temperature of the machine was set to 250 °C, and the column temperature program was set as follows: the initial temperature was 150 °C; then it rose to 210 °C at a rate of 10 °C/min and sequentially increased to 250 °C at a rate of 2 °C/min; the final column temperature was maintained at 250 °C for 25 min. Identification of the components proceeded by comparing the retention times and fragmentation pattern with a SupelcoÒ 37-Component FAME Mix (Sigma, USA).
3. Results and discussion 3.1. Algal growth in piggery water Algal growth, as indicated by biomass concentrations in diluted piggery wastewater and Bristol’s solution, is shown in Fig. 1. With no obvious lag phase exhibited, this wild-isolated C. pyrenoidosa grew faster in all samples of piggery wastewater than in Bristol’s solution. The biomass at the end of the experiment increased as the initial COD value rose from 250 to 1000 mg/L. Among the serial dilutions, the algae grew faster in 750- and 1000-mg COD/L samples than in the others. Growth of the 250-mg COD/L sample caught up to that of the 500-mg COD/L sample in the latter part of the cultivation period, but the growth speeds of both slowed at the seventh day. Similarly, the growth of algae in the 750- and 1000-mg/L diluted samples also entered a stationary phase at about the seventh day. Considering the high removal rate of ammonium (Fig. 2b), the slowing of the growth speeds might be the result of its rapid consumption. As shown in Fig. 3, the biomass productivities in all of the diluted piggery wastewater samples were much higher than that in Bristol’s solution and reached a maximum at 1000 mg COD/L. During the first 3 days, the average specific growth rates decreased as the initial COD value increased. The relationship between the initial optical density of the culture medium (measured at 680 nm) and the initial COD value is also plotted in Fig. 3. The specific growth rates at the beginning of culture correlated well with the initial COD value (R2 = 0.9709), which in turn provided a good correlation between the specific growth rates and the initial optical density of the wastewater (R2 = 0.9920, data not shown here). Suspended matter or impurities in the piggery waste would decrease the clarity of the water and interfere with light penetration. Therefore, the initial optical density of the wastewater increased as the initial COD rose. At the beginning of culture, the growth rate of the algae was impeded by turbidity in the wastewater, which decreased the light penetration. Since the piggery wastewater was allowed to settle and was diluted before testing, the suspended matter in the diluted samples should be largely attributed to some
Fig. 1. Growth curves of Chlorella pyrenoidosa in Bristol’s solution and diluted piggery wastewater with different initial COD values.
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finely divided inorganic and organic matter, as well as soluble colored organic compounds. This appears to be a reasonable explanation for the decrease in specific growth rates as the initial COD value increased. Fig. 3 shows the biomass productivities at the end of culture were well correlated with the initial COD values (R2 = 0.9639). This indicates that the initial COD is an important parameter that determines the suitability of wastewater for growth of C. pyrenoidosa. Though the initial growth of the algae was suppressed (Fig. 3) due to the limitation of luminous flux caused by high initial COD values, the biomass at the end of the experiment increased as the initial COD increased from 500 to 1000 mg/L (Fig. 1). A probable explanation is that the complex nutrients of piggery wastewater provide some organic components for algae growth in mixotrophic mode by assimilating organic carbon as well as fixing CO2 in light (Wang et al., 2010). It was found that, with no nutrient limitations, the photosynthesis of algae was enhanced by increasing the light intensity until the maximum algal growth rate was achieved at the light saturation point (Torzillo et al., 2003). Then the algal concentration continuously increased until a shading effect of the biomass became prominent. The light intensity was thought to have a small effect on the growth of Chlorella vulgaris under non nutrient-limited conditions as it increased to greater than 31.3 lmol m2 s1, though its saturation point was found to be 47.0 lmol m2 s1 (Bartosh and Banks, 2007). The strain of C. pyrenoidosa investigated in this study is capable of mixotrophic growth that further reduces its dependence on light. Thus, limiting the light intensity does not have a fatal effect on these microalgae when they are cultured in a nutritious medium like diluted piggery wastewater. 3.2. Nutrient removal efficiencies The removal rates of COD were all of double-digit percentages in different samples of diluted piggery wastewater (Fig. 2a). At the tested COD levels, possible toxicity or inhibition of COD was not present for this microalgae strain, which suggests it is a COD tolerant strain. Traviseo et al. (2006) reported a COD removal rate of 88.0% at 190 h for an initial COD concentration of 250 mg/L, which is far higher than the removal rate in this study and Wang’s results (Wang et al., 2010). The reduction in COD could only be attributed to exhaustion of the microalgae in the axenic culture conditions. The good growth and considerable COD reduction in diluted piggery wastewater indicate that C. pyrenoidosa use organic carbon as a source of energy and a substrate for cell growth, like some microalgae reported previously (Liu et al., 2009). Although the results suggest dilution followed by algae cultivation is a convenient way to treat piggery wastewater, it still cannot replace the traditional secondary treatment as its residual COD cannot meet the national emission standards of 125 mg/L (except when the initial COD value is 250 mg/L, as shown in Fig. 2a) in most Western countries. However, in less diluted samples, C. pyrenoidosa could be supported by a higher carbon concentration to obtain more biomass (Fig. 1). More research is required to find the optimum balance between reduction in COD value and biomass production. Ammonium removal of over 90% was achieved for all diluted samples, regardless of its initial concentration (Fig. 2b, from 34.7 to 138.8 mg/L). For all eukaryotic algae, including C. pyrenoidosa, the forms of inorganic nitrogen that can be directly assimilated þ þ are only nitrate ðNOþ 3 Þ, nitrite ðNO2 Þ, and ammonium ðNH4 Þ (Barsanti and Gualtieri, 2006). Remarkable reductions in total nitrogen and total phosphorus were also found for all four diluted wastewater samples (Fig. 2c and d). It is obvious that the removal rate of both total nitrogen and total phosphorus increased along with the rise in COD value. Another species of microalgae, Spirulina, was reported to have removal rates for NHþ 4 -N and phosphate ranging from 84% to 96% depending on the season or pond depth
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Fig. 2. The removal rates of (a) COD, (b) ammonium, (c) total nitrogen, and (d) total phosphorus calculated from their contents at the initial and final stages of the cultivation of Chlorella pyrenoidosa in different diluted piggery wastewaters (COD, chemical oxygen demand; NH3-N, ammonium; Total N, total nitrogen; Total P, total phosphorus).
Fig. 3. The initial specific growth rates (l), biomass productivities (Pbiomass), and initial optical densities of the broth (Ainitial) in the cultures of Chlorella pyrenoidosa with Bristol’s solution and different diluted piggery wastewaters (COD 250–1000 are mg/L of COD in diluted piggery wastewater; the lines and equations are correlations between the three dependent variables and the initial COD values in the medium).
(Olguin et al., 2003). Wang et al. (2010) also found that a wild-type Chlorella sp. cultivated in manure diluted 10–25-fold could provide removal rates of 75.7–82.5% for total nitrogen and 62.5–74.7% for total phosphorus. Data from these studies and the present research suggest that a relatively higher value of COD would enhance the removal rates of nutrients in the culture medium. Some previous studies proposed that high initial concentrations of nutrients would reduce their removal efficiencies. For instance, Aslan and Kapdan (2006) presumed that the uptake of nitrogen and phosphorus by algae does not provide efficient removal of these nutrients from synthetic media at high concentrations because of the light limitation caused by excess chlorophyll a content in the culture broth. And Kumar et al. (2010) reported that the maximum reduction in total ammonia nitrogen by C. vulgaris was 61.8% in digested piggery effluent with 20 mg/L initial total ammonia nitrogen. This rate was much less than the 93.7% removal observed for the 250-mg COD/L sample in this study with a
corresponding ammonia nitrogen concentration of 34.7 mg/L. Analysis of the data in Fig. 1Fig. 2c and d showed that the removal of organic matter increased in accordance with the rise in biomass, which was enhanced by the increase in COD values from 250 to 1000 mg/L. C. pyrenoidosa was found to provide efficient removal of nutrients and to be able to overcome the light limitation exhibited in pure photoautotrophic culture by growing mixotrophicly in media with a high content of nutrients (Wei et al., 2009). Thus, culture of microalgae in diluted piggery wastewater with mixotrophic growth has advantages in reducing the values of total nitrogen, total phosphorus, and ammonia nitrogen. 3.3. Yield of lipids and fatty acids composition Fig. 4 shows the lipid contents produced in Bristol’s solution and diluted piggery wastewater were roughly at equal levels, but the lipid productivities were evidently different. The lower level
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of lipid productivity in Bristol’s solution should be due to its lower biomass, which was almost half of that in diluted piggery wastewater (Fig. 1). The total nitrogen in Bristol’s solution was approximately 41 mg/L (calculated from its composition), which was close to the values of 24.5 mg/L and 49.0 mg/L found for the 250- and 500-mg COD/L samples (in Fig. 2c), respectively. However, the diluted piggery wastewater samples with total nitrogen content of 73.5 and 98.0 mg/L (corresponding to 750- and 1000-mg COD/L samples, respectively) provided lipid contents that were significantly less (p < 0.01) than Bristol’s solution and the 250-mg COD/ L sample (Fig. 4). The phenomenon of nitrogen limitation enhancing the accumulation of lipids (mostly triacylglycerols) in microalgae cells has been reported in many studies (Illman et al., 2000; Shifrin and Chisholm, 1981). (Shifrin and Chisholm, 1981) reported that deprivation of nitrogen increased the lipid content in 30 strains of green algae and diatoms including seven Chlorella. Illman et al. (2000) surveyed five Chlorella cultures in a low nitrogen medium and found an almost twofold rise in lipid content of four strains. The biomass and lipid productivities obtained in the present work were not necessarily superior to those reported elsewhere using different strains of microalgae. For instance, Converti et al. (2009) reported that C. vulgaris growing in Bold’s basal medium had somewhat higher production rates ranging from 8 to 20 mg L1 day1. In Woertz’s report, the maximum lipid productivities achieved for microalgae culture in a semi-continuous mode were 17 mg L1 day1 on dairy wastewater and 24 mg L1 day1 on municipal wastewater (Woertz et al., 2009). This suggests that in continuous culture mode the lipid productivity in piggery wastewater might be improved by continuous supplementation of nutrients such as ammonium, which has a limited concentration in batch cultivation. If this method could be adapted on a commercial scale, transforming piggery wastewater to biodiesel by algae cultivation with lipid production would be a waste-to-biofuel approach with no food competition issues and provide a valuable mode of degrading sewage for environmental protection. The green alga Chlorella yields oil with a fatty acid composition of 14:0, 16:0, 16:1, 16:2, 16:3, 18:0, 18:1, 18:2, and a-18:3 under most kinds of culture conditions such as photoautotrophic and heterotrophic cultivation, nitrogen and phosphorus starvation, and outdoors in a photobioreactor (Petkov and Garcia, 2007). Fig. 5 shows the fatty acid profiles of algae cells cultured with Bristol’s solution and the diluted sample with 500-mg COD/L in this study. There were nine fatty acids identified belonging to the composition mentioned above, among which hexadecanoic acid (C 16:0), linoleic acid (C 18:2), and linolenic acid (C 18:3) were the abundant fatty acids. Compared to Bristol’s solution, there were remarkable reductions in the relative content of hexadecadienoic acid (C 16:2) and oleinic acid (C 18:1) as well as an increase in hexadecanoic acid (C 16:0) for the diluted piggery waste samples. Variations
Fig. 4. Effect of initial COD values on the yield of lipids from Chlorella pyrenoidosa culture in Bristol’s solution and piggery wastewater with different dilutions (COD 250–1000 are mg/L of COD in diluted piggery wastewater).
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Fig. 5. The fatty acid profiles of C. pyrenoidosa culture with Bristol’s solution and piggery wastewater with 500-mg/L COD values (, p < 0.05; SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; Others, undefined peaks in chromatomap).
in contents of these fatty acids also occurred in a strain of Chlorella sp. with increases in oleinic acid (C 18:1) and hexadecanoic acid (C 16:0) and a decrease in hexadecadienoic acid (C 16:2) as the COD values in the medium were reduced by dilution (Wang et al., 2010). The relative content of linolenic acid (C 18:3) among the total fatty acids was about 27% (weight/weight). Although it is difficult to convert this value of a mass ratio into a molar ratio, it seems the value cannot meet the limitation of 12% (mol/mol) in the European Standard EN 14214, which limits the content of linolenic acid methyl ester in biodiesel for vehicle use (Knothe, 2006). This implies that additional treatment is required, such as partial catalytic hydrogenation of the oil (Dijkstra, 2006), which would not be a significant restriction for its application in biodiesel (Chisti, 2007). 4. Conclusions The present study attempts to combine the treatment of primary piggery wastewater with algae oil production using a strain of C. pyrenoidosa. The results indicate that culturing microalgae in mixtrophic mode with diluted primary piggery wastewater provides an effective method for converting high strength livestock wastewater into a profitable byproduct as well as reducing contamination of the environment. Acknowledgements The study was supported by the National Natural Science Foundation of China (50907074). References An, J.Y., Sim, S.J., Lee, J.S., Kim, B.W., 2003. Hydrocarbon production from secondarily treated piggery wastewater by the green alga Botryococcus braunii. J. Appl. Phycol. 15 (2–3), 185–191. Andrade, M.R., Costa, J.A.V., 2007. Mixotrophic cultivation of microalga Spirulina platensis using molasses as organic substrate. Aquaculture 264 (1–4), 130–134. Aslan, S., Kapdan, I.K., 2006. Batch kinetics of nitrogen and phosphorus removal from synthetic wastewater by algae. Ecol. Eng. 28 (1), 64–70. Barsanti, L., Gualtieri, P., 2006. Algae: Anatomy, Biochemistry, and Biotechnology. CRC Press, Boca Raton. Bartosh, Y., Banks, C.J., 2007. Algal growth response and survival in a range of light and temperature conditions: implications for non-steady-state conditions in waste stabilisation ponds. Water Sci. Technol. 55 (11), 211–218. Ceron Garcia, M.C., Camacho, F.G., Miron, A.S., Sevilla, J.M.F., Chisti, Y., Grima, E.M., 2006. Mixotrophic production of marine microalga Phaeodactylum tricornutum on various carbon sources. J. Microbiol. Biotechnol. 16 (5), 689–694. Chisti, Y., 2007. Biodiesel from microalgae. Biotechnol. Adv. 25 (3), 294–306. Clesceri, L.S., Greenberg, A.E., Eaton, A.D., 1998. Standard Methods for the Examination of Water and Wastewater, 20th ed. American Public Health Association, Washington, DC. 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: Process Intensification 48 (6), 1146–1151.
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