Bioprocess Biosyst Eng DOI 10.1007/s00449-015-1380-0
ORIGINAL PAPER
Simultaneous treatment of food-waste recycling wastewater and cultivation of Tetraselmis suecica for biodiesel production Sung-Woon Heo • Byung-Gon Ryu • Kibok Nam • Woong Kim • Ji-Won Yang
Received: 17 September 2014 / Accepted: 19 February 2015 Ó Springer-Verlag Berlin Heidelberg 2015
Abstract There is an increasing interest in the use of cultivated microalgae to simultaneously produce biodiesel and remove nutrients from various wastewaters. For this purpose, Tetraselmis suecica was cultivated in flasks and fermenters using diluted food-waste recycling wastewater (FRW). The effect of FRW dilution on T. suecica growth and nutrient removal was initially tested in flasks. The maximal microalgal concentration after 14 days was in medium with a twofold dilution (28.3 9 106 cells/mL) and a fivefold dilution (25.5 9 106 cells/mL). When fivefold diluted medium was used in fermenters, the final dry cell weight of T. suecica was 2.0 g/L. The removal efficiencies of ammonium and phosphate in the fermenters were 99.0 and 52.3 %, respectively. In comparison with the results of previous studies, the growth data of T. suecica in the FRW medium indicate that microalgal cultivation system incorporating removal of nutrients in FRW is feasible at the field level. Keywords Food wastewater Marine microalgae Nutrients Tetraselmis suecica Biodiesel S.-W. Heo K. Nam W. Kim (&) J.-W. Yang (&) Department of Chemical and Biomolecular Engineering, KAIST, 291 Daehakno, Yuseong-Gu, Daejeon 305-701, Republic of Korea e-mail: [email protected] J.-W. Yang e-mail: [email protected] B.-G. Ryu Environmental and Energy Program, KAIST, 291 Daehakno, Yuseong-Gu, Daejeon 305-701, Republic of Korea J.-W. Yang Advanced Biomass R&D Center, KAIST, 291 Daehakno, Yuseong-Gu, Daejeon 305-701, Republic of Korea
Introduction Recent studies of microalgal biotechnology from the EU and the US have estimated that the market is currently valued at €2.4 billion per year and is increasing by 10 % per year [1]. Additionally, the annual dry algal biomass production is about 7000 tons and estimated to be worth around €3.5–5 billion world-wide (http://www.algaemax. eu). These products are sold for diverse applications, such as value-added items, pollution treatments, human or animal nutrition, cosmetics, pharmaceuticals, biofuels, and nitrogen and phosphorus removal [2–5]. Thus, microalgal studies have come to the forefront because of their potential applicability in diverse fields. The removal of nitrogen (N) and phosphorus (P) is essential for wastewater treatment because accumulation of these nutrients leads to eutrophication. However, previous chemical and physical methods for N and P removal have increased costs and were also associated with other pollution risks [6]. Thus, there is currently significant interest in the use of algal-based treatment of N and P in wastewater because it may lower costs and also is relatively easy to control the process [6–8]. In addition, the use of wastewater for algal-based removal of N and P is regarded as an attractive alternative because it can reduce the nutrient load required for microalgal cultivation by as much as 30 % [9]. Among all types of wastewater, food wastewater is the most appropriate for marine microalgal cultivation because of its high salinity. However, only a few studies have investigated the use of food wastewater as a nutrient source because this wastewater has wide variations in pH, seasonal variations in chemical composition, and a high content of organic components [10]. Undiluted raw wastewater is not typically used for cultivation of microalgae because of its high organic content
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Bioprocess Biosyst Eng
and turbidity [11]. However, dilution of food wastewater requires large quantities of fresh water, and fresh water is an important resource whose conservation is an increasingly significant global issue [12]. Thus, cultivation of microalgae in seawater-diluted wastewater can reduce the use of fresh water resources and make the cultivation of microalgae more economically feasible [13]. Tetraselmis suecica, a marine microalga that is one of the most promising biomass- and lipid-producing species (Chlorella sp., Scenedesmus sp., Nannochloropsis sp., and Tetraselmis suecica), was selected from a screen of previous microalgal cultures whose biomass and lipid production were examined [14]. This species can be grown in nitrate concentrations as high as 500 mg/L [15], and can take up more than 99 % of the nitrogen at an initial concentration of 150 mg/L when grown in a bubble column photobioreactor in a high salt medium over 3 days [16]. This study examined the optimal dilution of food-waste recycling wastewater (FRW) for the small-scale and largescale cultivation of T. suecica in synthetic seawater.
Materials and methods Microalgal strain and seed culture conditions Tetraselmis suecica CCAP 66-4 was obtained from the Collection Culture of Algae and Protozoa (CCAP, UK). The seed culture was grown in a modified f/2 medium [17] containing NaCl (30 g/L), NaNO3 (750 mg/L), NaH2PO42H2O (6 mg/L); a trace element solution (1 mL/L) consisting of FeCl36H2O (3.15 mg/L), Na2EDTA (4.36 mg/ L), CuSO45H2O (9.8 mg/L), Na2MoO42H2O (6.3 mg/L), ZnSO47H2O (22.0 mg/L), CoCl26H2O (10.0 mg/L), and MnCl24H2O (180.0 mg/L); and a vitamin solution (0.5 mL) consisting of Vitamin B12 (cyanocobalamin, 1.0 mg/L), biotin (1.0 mg/L), and thiamineHCl (200 mg/ L). This medium contained 10 times more NaNO3 than the original f/2 medium and no silicate. A microalgal seed culture was grown in 250 mL baffled hybrid flasks that contained 200 mL of culture medium aerated with compressed air and supplemented with 2 % CO2. The flasks were cultured at 20 ± 1 °C, under cool-white fluorescent lamps (110 lmol m-2 s-1), and at 150 rpm agitation. Culture conditions FRW was obtained from Food-Waste Recycling Facilities (Yu-seong gu, Daejeon, Republic of Korea) after pressure flotation. This FRW was pretreated by sedimentation and filtration with a 650-lm sieve to remove large and insoluble particles. After adjustment to pH 7.0, the FRW was centrifuged (3500 rpm 9 5 min) and then passed through a 0.2-
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Table 1 Characteristics of filtered food-waste recycling wastewater Parameter
Unit
Concentrationa
NH4?
mg/L
246.7 ± 14.4
NO3-
141.8 ± 11.5
NO2-
ND
3-
PO4
Salinity
438.3 ± 54.4 g/L
30
ND not detected a
Mean ± standard deviation
lm filter. The filtrate was analyzed (Table 1) and stored at 4 °C before use. Synthetic seawater was made from sea salt (Sigma-Aldrich), passed through a 0.2-lm filter, and used for dilution of FRW. Salinity of this synthetic seawater was adjusted to 3 %, equivalent to that of the FRW collected (Table 1), even though the salinity of real seawater was 3.5 % which were slightly different from the adjusted value [18]. The FRW was diluted to twofold, fivefold, or tenfold for cultivation experiments. Seed cells were inoculated into the media with an optical density (OD680) of 0.2. Microalgae were cultured for 14 days under the same conditions used for seed culture. Cultivation in a fermenter (2.5 L working volume) was conducted in fivefold diluted FRW under the same conditions used for seed culture. Approximately 1.5 mL of culture was withdrawn every 24 h, transferred to an Eppendorf tube, centrifuged at 12000 rpm for 10 min, then passed through a 0.2-lm filter and used to measure the nutrient concentrations. Analytical methods Salinity was monitored by the measurement of liquid conductivity in seawater, using seawater refractometer (HI96822 Digital Refractometer, HANNA instruments, USA). The salinity unit was initially designated as psu (practical salinity unit), then converted to usual unit, g/L, for convenience. The salinity unit Cell concentrations were determined by optical microscopy (Leica Microsystems, CMS GmbH, Germany) with a Neubauer hemocytometer (Marienfeld, Germany). The dry cell weight was determined as the difference between the fresh weight after passage through a 0.45 lm nitrocellulose membrane filter (Whatman, USA) and aluminum dish and the weight after drying at 105 °C for 2 h. Ammonium, nitrate, and phosphate concentrations were determined by ion chromatography (IC, 881 Compact IC pro, Metrohm, Swiss) with a conductivity detector, Metrosep C4 150 column with cation analysis, and Metrosep A Supp5 150 column with anion analysis. Fatty acid methyl ester (FAME) content The FAME content was determined by gas chromatography (GC, HP5890, Agilent, USA) with a flame ionization
Bioprocess Biosyst Eng
Fig. 1 Concentrations of a T. suecica cells, b ammonium (NH4?), c nitrate (NO3-), and d phosphate (PO43-) during microalgal cultivation using FRW in flask-scale (200 mL working volume)
experiments (squares, undiluted FRW; circles, twofold diluted FRW; diamonds, fivefold diluted FRW; triangles, tenfold diluted FRW)
detector (FID) and an INNOWAX capillary column (30 m 9 0.32 mm 9 0.5 lm, Agilent, USA). The GC column temperature was increased from 50–200 °C at a rate of 15 °C/min and held for 9 min. When the temperature reached 200 °C, the temperature was gradually increased from 200–250 °C at a rate of 2 °C/min and held for 2 min. The retention times and peak areas of isolated FAMEs were compared with standards.
waste is from production of soy-bean sauce and many kinds of soups [19, 20]. The wastewater used in this study contained higher concentrations of phosphorus than of nitrogen, which is the opposite of what is observed in other food wastewaters [21].
Results and discussion Properties and salinity of filtered FRW Table 1 shows the nitrogen, phosphorus, and salinity of the FRW. This wastewater is somewhat unique in that it has high concentrations of phosphate and salinity because the
Microalgal growth in diluted FRW medium Figure 1 shows the cell concentrations of T. suecica over the course of 14 days when cultured in undiluted FRW medium and in medium that was diluted two-, five-, and tenfold (100 mL working volume). The duration of the lag phase was inversely proportional to dilution, indicating that some component(s) in the FRW inhibited the incipient stage of cell growth. After 14 days, the final concentrations of T. suecica cells were 28.3 9 106/mL (twofold dilution), 25.5 9 106/mL (fivefold dilution), 16.0 9 106/mL (tenfold
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Bioprocess Biosyst Eng Table 2 FAME composition of T. suecica cells that were cultivated in flask-scale reactors (100 mL working volume) with different dilutions of food-waste recycling wastewater FAME
Medium dilution (% Total FAME) Twofold
Fivefold
Tenfold
C6:0
2.1 ± 0.3
0.8 ± 0.1
0.6 ± 0.2
C8:0
2.2 ± 0.4
1.8 ± 0.1
1.7 ± 0.4
C10:0
1.2 ± 0.3
0.9 ± 0.0
ND
C12:0
0.8 ± 0.1
0.3 ± 0.0
ND
C14:0
0.7 ± 0.1
0.7 ± 0.1
0.6 ± 0.1
C14:1
1.3 ± 0.4
0.7 ± 0.0
0.7 ± 0.1
C16:0
27.3 ± 1.8
32.2 ± 1.6
32.9 ± 0.5
C16:1
4.6 ± 0.1
3.9 ± 0.5
2.5 ± 0.2
C18:0
8.5 ± 0.8
5.6 ± 0.4
5.6 ± 0.2
C18:1
20.9 ± 0.2
27.4 ± 0.1
26.5 ± 3.0
C18:2
7.2 ± 0.2
7.4 ± 0.3
7.8 ± 0.3
C18:3
11.6 ± 1.0
9.3 ± 0.3
10.3 ± 1.2
C20:5
2.9 ± 0.7
4.1 ± 0.3
4.0 ± 0.2
Rest
8.7
4.9
6.8
ND not detected
dilution), and 0.8 9 106/mL (no dilution). Undiluted FRW fully suppressed the proliferation of T. suecica, probably because the ammonium concentration was more than 100 mg/L [22]. Although cells grown in twofold diluted medium had the second longest lag phase, these cells had the highest concentration after 14 days. This is likely because sufficient nitrogen sources in the FRW medium were able to compensate for the initial inhibitory effect of ammonium on cell growth [23]. Removal of nutrients and production of FAMEs
Fig. 2 a Dry cell weight of T. suecica, and b concentrations of ammonium, nitrate, and phosphate during microalgal cultivation with fivefold diluted FRW in fermenter-scale (2.5 L working volume) experiments (squares, ammonium; circles, nitrate; triangles, phosphate)
The concentrations of NH4?, NO3-, and PO43- were monitored during cell proliferation to evaluate N and P removal. As expected, there was hardly any removal of NH4? and NO3- in the undiluted medium, but there was complete elimination of nitrogenous compounds in the diluted media (Fig. 1b, c). Microalgae in the twofold diluted medium required 6 days to fully remove the NH4? and NO3-, although cells in the other media removed these nutrients in 2–3 days. The slower removal of these nutrients in the twofold diluted medium is likely related to the need of the microalgae to adapt to the high concentration of ammonium. Interestingly, nitrate reduction was delayed by 1–3 days relative to ammonia (Fig. 1b, c). This implies that T. suecica can take up ammonia, and also utilize nitrate as a nitrogen source for biomass synthesis [24]. Examination of the phosphorus profile indicated no removal in undiluted medium, but complete removal in the five- and tenfold
diluted media after 14 days. In the twofold diluted medium, phosphate was eliminated by 82.0 % after 14 days (Fig. 1d). The FAME compositions of cells grown in the two-, five-, and tenfold diluted media were measured on the 14th day during the cell cultivation (Table 2). There were only slight differences in the FAME profiles in these three groups of cells. Analysis of the three major FAMEs, palmitic (C16:0), oleic (C18:1), stearic (C18:0) acids [25] indicated that cells grown in twofold diluted medium had lower concentrations of palmitic (C16:0) and oleic (C18:1) acids, but a higher concentration of stearic (C18:0) acid. The increase of C16:0 and C18:1 in the more diluted media is likely related to the more rapid consumption of phosphorus. In particular, a previous study reported that rapid depletion of phosphorus stimulated the accumulation of these two FAMEs [26]. In agreement with this
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Bioprocess Biosyst Eng Table 3 Comparison of biomass production and efficiency of nutrient removal by various microalgae in different growth media Medium
Species
Scale (L)
Biomass production (g/L)
Nutrient removal efficiency (%)
References
Anaerobic digested dairy manure
Chlorella sp.
0.25
1.71
NH3-N: 100
[32]
TKN: 78.3 Municipal wastewater F/2 medium Food-waste recycling wastewater
Nannochloropsis sp. T. suecica
1.0
2.23a
–d
[33]
b
d
[34]
16
3.41
0.25
4.48c
–d
[14]
2.5
2.00
NH4?, NO3-: [99
This study
–
PO43-: 52.3 a
50 % municipal wastewater, 150 lmol m-2 s-1 light intensity, 15 % CO2, after 18 days
b
Estimated from biomass of 7.4 g m-2 day-1 after 14 days
c
Estimated from biomass of 0.32 g L-1 day-1 after 14 days, 25 °C, 100 lmol m-2 s-1 light intensity, air/CO2 (95:5, v/v)
d
No data
interpretation, phosphorus removal was relatively slow in cells grown in twofold diluted medium (Fig. 1d). Scaled-up culturing of cells We performed similar experiments with a 2.5 L working volume to confirm the presence of microalgal growth and elimination of N and P in large reactors (Fig. 2). In these experiments, fivefold diluted medium was used based on our previous results regarding total growth rate and nutrient removal. Although the previous experiments showed maximal cell accumulation in twofold diluted medium, fivefold diluted medium was selected because biomass accumulation was nearly the same as in twofold diluted medium, and phosphate removal was more effective in fivefold diluted medium. The results of these experiments (Fig. 2) show that dry cell weight reached 2.0 g/L on the 14th day, 61.5 % of that obtained when the working volume was 100 mL (3.3 g/L, data not shown). The reduced growth rate may be due to differences in the physical properties of the process environment such as light penetration into the vessel, distribution of CO2, and stirring [27]. Nevertheless, the concentration of microalgal production of around 2 g/L in these larger-scale experiments provides insight for the feasible application of cultivation of T. suecica using FRW at the field-scale level. Growth of cells in larger reactors also affected the removal of N and P. Nitrate was almost fully removed by the third day, but the phosphate level stayed at about 20 mg/L, corresponding to 52.3 % removal (Fig. 2b). This might be due to the reduced cell proliferation, because previous research reported that the efficiency of phosphate removal was reduced if the cell growth rate decreased when cultures are scaled up [28]. Phosphate profiles showed the relatively more gradual drop than ammonium or nitrate ones during the algal cultivation, which has been usually reported [29].
Phosphate removal efficiency was also lower than those of nitrate and ammonium, probably owing to the fact that algal phosphorus content is no more than a tenth of nitrogen content [30]. Temporary fluctuation of phosphate concentration on second to fourth points in Fig. 2b is just due to the effect of transitory pH increase from addition of caustic soda for incipient pH adjustment, making the precipitant of phosphate salt. Table 3 compares the results of the present study with those previous studies in which different microalgae were grown in different types of media [32, 33], and in which T. suecica was grown in F/2 medium [14, 34]. Comparison of the results shows that biomass production and nutrient removal efficiencies in the present study were similar to those reported in other studies with wastewater [31, 32]. The final concentration of biomass produced in this study (using FRW as a growth medium) was slightly lower than when F/2 medium was used [14, 34]. Nonetheless, T. suecica cultivation using wastewater seems to be a feasible and perhaps economical means for removal of nitrogen and phosphate from wastewater.
Conclusions Tetraselmis suecica CCAP 66/4 was successfully cultivated in high-saline Korean FRW diluted by seawater. The maximum cell biomass when cells were grown in smallscale experiments (100 mL) and large-scale experiments (2.5 L) were 3.3 g/L and 2.0 g/L, respectively. The cells completely removed nitrogenous compounds from the growth medium under small-scale and large-scale conditions, but phosphate removal was lower under large-scale conditions, possibly due to the reduced cell growth. Comparison with other systems suggests that large-scale growth of marine microalgae using diluted FRW is feasible.
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Bioprocess Biosyst Eng Acknowledgments This work was supported by the Advanced Biomass R&D Center (ABC) of Korea Grant funded by the Ministry of Science, ICT and Future Planning (ABC-2010-0029728).
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18. Culkin F, Smith ND (1980) Determination of the concentration of potassium chloride solution having the same electrical conductivity, at 15 °C and infinite frequency, as standard seawater of salinity 35.0000% (Chlorinity 19.37394%), IEEE-JOE OE-5 (1) 19. Han MJ, Behera SK, Park HS (2012) Anaerobic co-digestion of food waste leachate and piggery wastewater for methane production: statistical optimization of key process parameters. J Chem Technol Biot 87(11):1541–1550 20. Seo JY, Heo JS, Kim TH, Joo WH, Crohn DM (2004) Effect of vermiculite addition on compost produced from Korean food wastes. Waste Manage 24(10):981–987 21. Shin SG, Han G, Lim J, Lee C, Hwang S (2010) A comprehensive microbial insight into two-stage anaerobic digestion of food waste-recycling wastewater. Water Res 44(17):4838–4849 22. Ji F, Liu Y, Hao R, Li G, Zhou Y, Dong R (2014) Biomass production and nutrients removal by a new microalgae strain Desmodesmus sp. in anaerobic digestion wastewater. Bioresour Technol 161:200–207 23. Chen M, Tang HY, Ma HZ, Holland TC, Ng KYS, Salley SO (2011) Effect of nutrients on growth and lipid accumulation in the green algae Dunaliella tertiolecta. Bioresour Technol 102(2):1649–1655 24. Ryu BG, Kim J, Farooq W, Han JI, Yang JW, Kim W (2014) Algal-bacterial process for the simultaneous detoxification of thiocyanate-containing wastewater and maximized lipid production under photoautotrophic/photoheterotrophic conditions. Bioresour Technol 162:70–79 25. Ramos MJ, Fernandez CM, Casas A, Rodriguez L, Perez A (2009) Influence of fatty acid composition of raw materials on biodiesel properties. Bioresour Technol 100(1):261–268 26. Reitan KI, Rainuzzo JR, Olsen Y (1994) Effect of nutrient limitation on fatty-acid and lipid-content of marine microalgae. J Phycol 30(6):972–979 27. Wahidin S, Idris A, Shaleh SRM (2013) The influence of light intensity and photoperiod on the growth and lipid content of microalgae Nannochloropsis sp. Bioresour Technol 129:7–11 28. Wang B, Lan CQ (2011) Biomass production and nitrogen and phosphorus removal by the green alga Neochloris oleoabundans in simulated wastewater and secondary municipal wastewater effluent. Bioresour Technol 102(10):5639–5644 29. Luz EB, Manuel M, Juan-Pablo H, Yoav B (2002) Removal of ammonium and phosphorus ions from synthetic wastewater by the microalgae Chlorella vulgaris coimmobilized in alginate beads with the microalgae growth-promoting bacterium Azospirillum brasilense. Water Res 36(12):2941–2948 30. Yakup N, William JO (1995) Enhanced nutrient removal in highrate ponds. Wat. Sci. Tech 31(12):33–43 31. Levine RB, Costanza-Robinson MS, Spatafora GA (2011) Neochloris oleoabundans grown on anaerobically digested dairy manure for concomitant nutrient removal and biodiesel feedstock production. Biomass Bioenergy 35(1):40–49 32. Wang L, Li YC, Chen P, Min M, Chen YF, Zhu J, Ruan RR (2010) Anaerobic digested dairy manure as a nutrient supplement for cultivation of oil-rich green microalgae Chlorella sp. Bioresour Technol 101(8):2623–2628 33. Jiang LL, Luo SJ, Fan XL, Yang ZM, Guo RB (2011) Biomass and lipid production of marine microalgae using municipal wastewater and high concentration of CO2. Appl Energ 88(10):3336–3341 34. Bondioli P, Della Bella L, Rivolta G, Zittelli GC, Bassi N, Rodolfi L, Casini D, Prussi M, Chiaramonti D, Tredici MR (2012) Oil production by the marine microalgae Nannochloropsis sp F&M-M24 and Tetraselmis suecica F&M-M33. Bioresour Technol 114:567–572
ORIGINAL PAPER
Simultaneous treatment of food-waste recycling wastewater and cultivation of Tetraselmis suecica for biodiesel production Sung-Woon Heo • Byung-Gon Ryu • Kibok Nam • Woong Kim • Ji-Won Yang
Received: 17 September 2014 / Accepted: 19 February 2015 Ó Springer-Verlag Berlin Heidelberg 2015
Abstract There is an increasing interest in the use of cultivated microalgae to simultaneously produce biodiesel and remove nutrients from various wastewaters. For this purpose, Tetraselmis suecica was cultivated in flasks and fermenters using diluted food-waste recycling wastewater (FRW). The effect of FRW dilution on T. suecica growth and nutrient removal was initially tested in flasks. The maximal microalgal concentration after 14 days was in medium with a twofold dilution (28.3 9 106 cells/mL) and a fivefold dilution (25.5 9 106 cells/mL). When fivefold diluted medium was used in fermenters, the final dry cell weight of T. suecica was 2.0 g/L. The removal efficiencies of ammonium and phosphate in the fermenters were 99.0 and 52.3 %, respectively. In comparison with the results of previous studies, the growth data of T. suecica in the FRW medium indicate that microalgal cultivation system incorporating removal of nutrients in FRW is feasible at the field level. Keywords Food wastewater Marine microalgae Nutrients Tetraselmis suecica Biodiesel S.-W. Heo K. Nam W. Kim (&) J.-W. Yang (&) Department of Chemical and Biomolecular Engineering, KAIST, 291 Daehakno, Yuseong-Gu, Daejeon 305-701, Republic of Korea e-mail: [email protected] J.-W. Yang e-mail: [email protected] B.-G. Ryu Environmental and Energy Program, KAIST, 291 Daehakno, Yuseong-Gu, Daejeon 305-701, Republic of Korea J.-W. Yang Advanced Biomass R&D Center, KAIST, 291 Daehakno, Yuseong-Gu, Daejeon 305-701, Republic of Korea
Introduction Recent studies of microalgal biotechnology from the EU and the US have estimated that the market is currently valued at €2.4 billion per year and is increasing by 10 % per year [1]. Additionally, the annual dry algal biomass production is about 7000 tons and estimated to be worth around €3.5–5 billion world-wide (http://www.algaemax. eu). These products are sold for diverse applications, such as value-added items, pollution treatments, human or animal nutrition, cosmetics, pharmaceuticals, biofuels, and nitrogen and phosphorus removal [2–5]. Thus, microalgal studies have come to the forefront because of their potential applicability in diverse fields. The removal of nitrogen (N) and phosphorus (P) is essential for wastewater treatment because accumulation of these nutrients leads to eutrophication. However, previous chemical and physical methods for N and P removal have increased costs and were also associated with other pollution risks [6]. Thus, there is currently significant interest in the use of algal-based treatment of N and P in wastewater because it may lower costs and also is relatively easy to control the process [6–8]. In addition, the use of wastewater for algal-based removal of N and P is regarded as an attractive alternative because it can reduce the nutrient load required for microalgal cultivation by as much as 30 % [9]. Among all types of wastewater, food wastewater is the most appropriate for marine microalgal cultivation because of its high salinity. However, only a few studies have investigated the use of food wastewater as a nutrient source because this wastewater has wide variations in pH, seasonal variations in chemical composition, and a high content of organic components [10]. Undiluted raw wastewater is not typically used for cultivation of microalgae because of its high organic content
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Bioprocess Biosyst Eng
and turbidity [11]. However, dilution of food wastewater requires large quantities of fresh water, and fresh water is an important resource whose conservation is an increasingly significant global issue [12]. Thus, cultivation of microalgae in seawater-diluted wastewater can reduce the use of fresh water resources and make the cultivation of microalgae more economically feasible [13]. Tetraselmis suecica, a marine microalga that is one of the most promising biomass- and lipid-producing species (Chlorella sp., Scenedesmus sp., Nannochloropsis sp., and Tetraselmis suecica), was selected from a screen of previous microalgal cultures whose biomass and lipid production were examined [14]. This species can be grown in nitrate concentrations as high as 500 mg/L [15], and can take up more than 99 % of the nitrogen at an initial concentration of 150 mg/L when grown in a bubble column photobioreactor in a high salt medium over 3 days [16]. This study examined the optimal dilution of food-waste recycling wastewater (FRW) for the small-scale and largescale cultivation of T. suecica in synthetic seawater.
Materials and methods Microalgal strain and seed culture conditions Tetraselmis suecica CCAP 66-4 was obtained from the Collection Culture of Algae and Protozoa (CCAP, UK). The seed culture was grown in a modified f/2 medium [17] containing NaCl (30 g/L), NaNO3 (750 mg/L), NaH2PO42H2O (6 mg/L); a trace element solution (1 mL/L) consisting of FeCl36H2O (3.15 mg/L), Na2EDTA (4.36 mg/ L), CuSO45H2O (9.8 mg/L), Na2MoO42H2O (6.3 mg/L), ZnSO47H2O (22.0 mg/L), CoCl26H2O (10.0 mg/L), and MnCl24H2O (180.0 mg/L); and a vitamin solution (0.5 mL) consisting of Vitamin B12 (cyanocobalamin, 1.0 mg/L), biotin (1.0 mg/L), and thiamineHCl (200 mg/ L). This medium contained 10 times more NaNO3 than the original f/2 medium and no silicate. A microalgal seed culture was grown in 250 mL baffled hybrid flasks that contained 200 mL of culture medium aerated with compressed air and supplemented with 2 % CO2. The flasks were cultured at 20 ± 1 °C, under cool-white fluorescent lamps (110 lmol m-2 s-1), and at 150 rpm agitation. Culture conditions FRW was obtained from Food-Waste Recycling Facilities (Yu-seong gu, Daejeon, Republic of Korea) after pressure flotation. This FRW was pretreated by sedimentation and filtration with a 650-lm sieve to remove large and insoluble particles. After adjustment to pH 7.0, the FRW was centrifuged (3500 rpm 9 5 min) and then passed through a 0.2-
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Table 1 Characteristics of filtered food-waste recycling wastewater Parameter
Unit
Concentrationa
NH4?
mg/L
246.7 ± 14.4
NO3-
141.8 ± 11.5
NO2-
ND
3-
PO4
Salinity
438.3 ± 54.4 g/L
30
ND not detected a
Mean ± standard deviation
lm filter. The filtrate was analyzed (Table 1) and stored at 4 °C before use. Synthetic seawater was made from sea salt (Sigma-Aldrich), passed through a 0.2-lm filter, and used for dilution of FRW. Salinity of this synthetic seawater was adjusted to 3 %, equivalent to that of the FRW collected (Table 1), even though the salinity of real seawater was 3.5 % which were slightly different from the adjusted value [18]. The FRW was diluted to twofold, fivefold, or tenfold for cultivation experiments. Seed cells were inoculated into the media with an optical density (OD680) of 0.2. Microalgae were cultured for 14 days under the same conditions used for seed culture. Cultivation in a fermenter (2.5 L working volume) was conducted in fivefold diluted FRW under the same conditions used for seed culture. Approximately 1.5 mL of culture was withdrawn every 24 h, transferred to an Eppendorf tube, centrifuged at 12000 rpm for 10 min, then passed through a 0.2-lm filter and used to measure the nutrient concentrations. Analytical methods Salinity was monitored by the measurement of liquid conductivity in seawater, using seawater refractometer (HI96822 Digital Refractometer, HANNA instruments, USA). The salinity unit was initially designated as psu (practical salinity unit), then converted to usual unit, g/L, for convenience. The salinity unit Cell concentrations were determined by optical microscopy (Leica Microsystems, CMS GmbH, Germany) with a Neubauer hemocytometer (Marienfeld, Germany). The dry cell weight was determined as the difference between the fresh weight after passage through a 0.45 lm nitrocellulose membrane filter (Whatman, USA) and aluminum dish and the weight after drying at 105 °C for 2 h. Ammonium, nitrate, and phosphate concentrations were determined by ion chromatography (IC, 881 Compact IC pro, Metrohm, Swiss) with a conductivity detector, Metrosep C4 150 column with cation analysis, and Metrosep A Supp5 150 column with anion analysis. Fatty acid methyl ester (FAME) content The FAME content was determined by gas chromatography (GC, HP5890, Agilent, USA) with a flame ionization
Bioprocess Biosyst Eng
Fig. 1 Concentrations of a T. suecica cells, b ammonium (NH4?), c nitrate (NO3-), and d phosphate (PO43-) during microalgal cultivation using FRW in flask-scale (200 mL working volume)
experiments (squares, undiluted FRW; circles, twofold diluted FRW; diamonds, fivefold diluted FRW; triangles, tenfold diluted FRW)
detector (FID) and an INNOWAX capillary column (30 m 9 0.32 mm 9 0.5 lm, Agilent, USA). The GC column temperature was increased from 50–200 °C at a rate of 15 °C/min and held for 9 min. When the temperature reached 200 °C, the temperature was gradually increased from 200–250 °C at a rate of 2 °C/min and held for 2 min. The retention times and peak areas of isolated FAMEs were compared with standards.
waste is from production of soy-bean sauce and many kinds of soups [19, 20]. The wastewater used in this study contained higher concentrations of phosphorus than of nitrogen, which is the opposite of what is observed in other food wastewaters [21].
Results and discussion Properties and salinity of filtered FRW Table 1 shows the nitrogen, phosphorus, and salinity of the FRW. This wastewater is somewhat unique in that it has high concentrations of phosphate and salinity because the
Microalgal growth in diluted FRW medium Figure 1 shows the cell concentrations of T. suecica over the course of 14 days when cultured in undiluted FRW medium and in medium that was diluted two-, five-, and tenfold (100 mL working volume). The duration of the lag phase was inversely proportional to dilution, indicating that some component(s) in the FRW inhibited the incipient stage of cell growth. After 14 days, the final concentrations of T. suecica cells were 28.3 9 106/mL (twofold dilution), 25.5 9 106/mL (fivefold dilution), 16.0 9 106/mL (tenfold
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Bioprocess Biosyst Eng Table 2 FAME composition of T. suecica cells that were cultivated in flask-scale reactors (100 mL working volume) with different dilutions of food-waste recycling wastewater FAME
Medium dilution (% Total FAME) Twofold
Fivefold
Tenfold
C6:0
2.1 ± 0.3
0.8 ± 0.1
0.6 ± 0.2
C8:0
2.2 ± 0.4
1.8 ± 0.1
1.7 ± 0.4
C10:0
1.2 ± 0.3
0.9 ± 0.0
ND
C12:0
0.8 ± 0.1
0.3 ± 0.0
ND
C14:0
0.7 ± 0.1
0.7 ± 0.1
0.6 ± 0.1
C14:1
1.3 ± 0.4
0.7 ± 0.0
0.7 ± 0.1
C16:0
27.3 ± 1.8
32.2 ± 1.6
32.9 ± 0.5
C16:1
4.6 ± 0.1
3.9 ± 0.5
2.5 ± 0.2
C18:0
8.5 ± 0.8
5.6 ± 0.4
5.6 ± 0.2
C18:1
20.9 ± 0.2
27.4 ± 0.1
26.5 ± 3.0
C18:2
7.2 ± 0.2
7.4 ± 0.3
7.8 ± 0.3
C18:3
11.6 ± 1.0
9.3 ± 0.3
10.3 ± 1.2
C20:5
2.9 ± 0.7
4.1 ± 0.3
4.0 ± 0.2
Rest
8.7
4.9
6.8
ND not detected
dilution), and 0.8 9 106/mL (no dilution). Undiluted FRW fully suppressed the proliferation of T. suecica, probably because the ammonium concentration was more than 100 mg/L [22]. Although cells grown in twofold diluted medium had the second longest lag phase, these cells had the highest concentration after 14 days. This is likely because sufficient nitrogen sources in the FRW medium were able to compensate for the initial inhibitory effect of ammonium on cell growth [23]. Removal of nutrients and production of FAMEs
Fig. 2 a Dry cell weight of T. suecica, and b concentrations of ammonium, nitrate, and phosphate during microalgal cultivation with fivefold diluted FRW in fermenter-scale (2.5 L working volume) experiments (squares, ammonium; circles, nitrate; triangles, phosphate)
The concentrations of NH4?, NO3-, and PO43- were monitored during cell proliferation to evaluate N and P removal. As expected, there was hardly any removal of NH4? and NO3- in the undiluted medium, but there was complete elimination of nitrogenous compounds in the diluted media (Fig. 1b, c). Microalgae in the twofold diluted medium required 6 days to fully remove the NH4? and NO3-, although cells in the other media removed these nutrients in 2–3 days. The slower removal of these nutrients in the twofold diluted medium is likely related to the need of the microalgae to adapt to the high concentration of ammonium. Interestingly, nitrate reduction was delayed by 1–3 days relative to ammonia (Fig. 1b, c). This implies that T. suecica can take up ammonia, and also utilize nitrate as a nitrogen source for biomass synthesis [24]. Examination of the phosphorus profile indicated no removal in undiluted medium, but complete removal in the five- and tenfold
diluted media after 14 days. In the twofold diluted medium, phosphate was eliminated by 82.0 % after 14 days (Fig. 1d). The FAME compositions of cells grown in the two-, five-, and tenfold diluted media were measured on the 14th day during the cell cultivation (Table 2). There were only slight differences in the FAME profiles in these three groups of cells. Analysis of the three major FAMEs, palmitic (C16:0), oleic (C18:1), stearic (C18:0) acids [25] indicated that cells grown in twofold diluted medium had lower concentrations of palmitic (C16:0) and oleic (C18:1) acids, but a higher concentration of stearic (C18:0) acid. The increase of C16:0 and C18:1 in the more diluted media is likely related to the more rapid consumption of phosphorus. In particular, a previous study reported that rapid depletion of phosphorus stimulated the accumulation of these two FAMEs [26]. In agreement with this
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Bioprocess Biosyst Eng Table 3 Comparison of biomass production and efficiency of nutrient removal by various microalgae in different growth media Medium
Species
Scale (L)
Biomass production (g/L)
Nutrient removal efficiency (%)
References
Anaerobic digested dairy manure
Chlorella sp.
0.25
1.71
NH3-N: 100
[32]
TKN: 78.3 Municipal wastewater F/2 medium Food-waste recycling wastewater
Nannochloropsis sp. T. suecica
1.0
2.23a
–d
[33]
b
d
[34]
16
3.41
0.25
4.48c
–d
[14]
2.5
2.00
NH4?, NO3-: [99
This study
–
PO43-: 52.3 a
50 % municipal wastewater, 150 lmol m-2 s-1 light intensity, 15 % CO2, after 18 days
b
Estimated from biomass of 7.4 g m-2 day-1 after 14 days
c
Estimated from biomass of 0.32 g L-1 day-1 after 14 days, 25 °C, 100 lmol m-2 s-1 light intensity, air/CO2 (95:5, v/v)
d
No data
interpretation, phosphorus removal was relatively slow in cells grown in twofold diluted medium (Fig. 1d). Scaled-up culturing of cells We performed similar experiments with a 2.5 L working volume to confirm the presence of microalgal growth and elimination of N and P in large reactors (Fig. 2). In these experiments, fivefold diluted medium was used based on our previous results regarding total growth rate and nutrient removal. Although the previous experiments showed maximal cell accumulation in twofold diluted medium, fivefold diluted medium was selected because biomass accumulation was nearly the same as in twofold diluted medium, and phosphate removal was more effective in fivefold diluted medium. The results of these experiments (Fig. 2) show that dry cell weight reached 2.0 g/L on the 14th day, 61.5 % of that obtained when the working volume was 100 mL (3.3 g/L, data not shown). The reduced growth rate may be due to differences in the physical properties of the process environment such as light penetration into the vessel, distribution of CO2, and stirring [27]. Nevertheless, the concentration of microalgal production of around 2 g/L in these larger-scale experiments provides insight for the feasible application of cultivation of T. suecica using FRW at the field-scale level. Growth of cells in larger reactors also affected the removal of N and P. Nitrate was almost fully removed by the third day, but the phosphate level stayed at about 20 mg/L, corresponding to 52.3 % removal (Fig. 2b). This might be due to the reduced cell proliferation, because previous research reported that the efficiency of phosphate removal was reduced if the cell growth rate decreased when cultures are scaled up [28]. Phosphate profiles showed the relatively more gradual drop than ammonium or nitrate ones during the algal cultivation, which has been usually reported [29].
Phosphate removal efficiency was also lower than those of nitrate and ammonium, probably owing to the fact that algal phosphorus content is no more than a tenth of nitrogen content [30]. Temporary fluctuation of phosphate concentration on second to fourth points in Fig. 2b is just due to the effect of transitory pH increase from addition of caustic soda for incipient pH adjustment, making the precipitant of phosphate salt. Table 3 compares the results of the present study with those previous studies in which different microalgae were grown in different types of media [32, 33], and in which T. suecica was grown in F/2 medium [14, 34]. Comparison of the results shows that biomass production and nutrient removal efficiencies in the present study were similar to those reported in other studies with wastewater [31, 32]. The final concentration of biomass produced in this study (using FRW as a growth medium) was slightly lower than when F/2 medium was used [14, 34]. Nonetheless, T. suecica cultivation using wastewater seems to be a feasible and perhaps economical means for removal of nitrogen and phosphate from wastewater.
Conclusions Tetraselmis suecica CCAP 66/4 was successfully cultivated in high-saline Korean FRW diluted by seawater. The maximum cell biomass when cells were grown in smallscale experiments (100 mL) and large-scale experiments (2.5 L) were 3.3 g/L and 2.0 g/L, respectively. The cells completely removed nitrogenous compounds from the growth medium under small-scale and large-scale conditions, but phosphate removal was lower under large-scale conditions, possibly due to the reduced cell growth. Comparison with other systems suggests that large-scale growth of marine microalgae using diluted FRW is feasible.
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Bioprocess Biosyst Eng Acknowledgments This work was supported by the Advanced Biomass R&D Center (ABC) of Korea Grant funded by the Ministry of Science, ICT and Future Planning (ABC-2010-0029728).
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