- Email: [email protected]
Cultivation of Chlorella pyrenoidosa in soybean processing wastewater
Bioresource Technology 102 (2011) 9884–9890
Contents lists available at SciVerse ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Cultivation of Chlorella pyrenoidosa in soybean processing wastewater Su Hongyang a,b, Zhang Yalei a,⇑, Zhang Chunmin a, Zhou Xuefei a,⇑, Li Jinpeng a a b
State Key Laboratory of Pollution Control and Resources Reuse, College of Environmental Science and Engineering, Tongji University, Shanghai 200092, China School of Resource and Environment, Fujian Agriculture and Forestry University, Fuzhou 350002, China
a r t i c l e
i n f o
Article history: Received 11 April 2011 Received in revised form 28 July 2011 Accepted 3 August 2011 Available online 16 August 2011 Keywords: Chlorella pyrenoidosa Soybean processing wastewater Fed-batch culture Anaerobic hydrolysis
a b s t r a c t Chlorella pyrenoidosa was cultivated in soybean processing wastewater (SPW) in batch and fed-batch cultures without a supply of additional nutrients. The alga was able to remove 77.8 ± 5.7%, 88.8 ± 1.0%, 89.1 ± 0.6% and 70.3 ± 11.4% of soluble chemical oxygen demand (SCODCr), total nitrogen (TN), NH4+-N and total phosphate (TP), respectively, after 120 h in fed-batch culture. C. pyrenoidosa attained an average biomass productivity of 0.64 g L1 d1, an average lipid content of 37.00 ± 9.34%, and a high lipid productivity of 0.40 g L1 d1. Therefore, cultivation of C. pyrenoidosa in SPW could yield cleaner water and useful biomass. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Processing soybeans into bean curd generates about 7–10 tons of wastewater with a chemical oxygen demand (COD) of 10– 20 g L1 per ton of processed soybeans (Yu et al., 2002). Studies on soybean processing wastewater (SPW) treatment have focused on anaerobic filters (AF) (Yu et al., 2002), upflow anaerobic sludge beds (UASB), and sequencing batch reactors (SBR) (Su and Yu, 2005). Although anaerobic processing is appropriate for treatment of high-strength wastewaters with energy recovery (Maroun and El Fadel, 2007; Metcalf and Eddy, 2003), the method has several disadvantages. Anaerobic treatments are unable to remove biological nitrogen and phosphorus, and require frequent adjustments for alkalinity (Metcalf and Eddy, 2003). Aerobic treatments suffer from high investment and energy costs to meet COD biotransformation requirements, nitrogen and phosphorus removal goals, and sludge thickening and other needs. The aerobic process not only wastes organic carbon and nutrient resources, but also generates CO2 and sludge. An optimal treatment process should be able to utilize the production of useful organisms and, at the same time, remove organic matter, nitrogen, and phosphorus pollutants. SPW contains monosaccharide, oligosaccharides, vitamins, organic acids, amino acids, lipids, whey protein, isoflavone, saponin, P, Ca, Fe, and other nutrients (Lopes Barbosa et al., 2006; Tang and Ma, 2009). Some studies have explored the recovery of oligosaccharides, proteins or isoflavones with membrane technologies (Cassini et al., 2010; Lopes Barbosa et al., 2006; Tang and Ma,
2009), but this approach only recovers some of the components of the wastewater and leaves effluents that might still require further treatment. Some researchers have explored the cultivation of microalgae in conjunction with wastewater treatment (Table 1). Some of these microalgae are not only capable of removing pollutants, but also produce lipids that can be converted into biodiesel (Perez-Garcia et al., 2011; Bhatnagar et al., 2010; Li et al., 2008). Since SPW contains significant usable nutrients generally without toxic and hazardous substances that could inhibit growth of microalgae, cultivation of oleaginous microalgae in such a medium may reduce the cost of algae-based biodiesel. C. pyrenoidosa is a green algae that is used as human and animal nutrition supplement, and for the extraction of high-value compounds such as fatty acids, pigments, carotenoids and xanthophylls (Spolaore et al., 2006), but also for wastewater treatment and biofuel production (Perez-Garcia et al., 2011). C. pyrenoidosa can grow autotrophically, heterotrophically or mixotrophically, and we hypothesized that under the latter growth condition, the algae could grow and remove nutrients from SPW. Thus, the aim of the present study was to investigate growth, nutrient removal, and lipid production by C. pyrenoidosa in batch and fed-batch cultures in SPW.
2. Methods 2.1. Composition and pretreatment of SPW
⇑ Corresponding authors. Tel.: +86 21 65982503; fax: +86 21 65988885. E-mail addresses: [email protected] (Z. Yalei), [email protected] (Z. Xuefei). 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.08.016
SPW was obtained from a local soybean processing plant, and its overall composition was shown in Table 2 together with that
9885
S. Hongyang et al. / Bioresource Technology 102 (2011) 9884–9890 Table 1 Wastewaters treatment by various microalgae reported in the literature. Wastewater
Microalgae
Reactor
Culture time (h)
Removal (%) TP
NH4+-
COD
Productivity (g L1 d1)
Biomass (g L1)
References
0.03
<0.5
Martínez et al. (2000) Bhatnagar et al. (2010) Kumar et al. (2010)
N Urban wastewater Municipal Wastewaters Digested piggery effluent Urine Industrial wastewater
Scenedesmus obliquus Chlorella minutissima Chlorella vulgaris
Jacketed cylindrical bioreactors Oxidation pond
188
98
100
360
30
>41
Plastic bags
240
100
78
Spirulina platensis Chlorella vulgaris
Photobioreactor
192
99
99
Acrylic bioreactor
120
28
72
>75
>0.5
1.05 61
Yang et al. (2008) Valderrama et al. (2002)
Table 2 Composition of soybean processing wastewater, SE and Basal medium.
SWa SW1 SW2 SE Basal a b
COD mg L1
TN
NH4+-N
TP
Na
Cu
Zn
Mg
Mn
Co
Fe
Ca
SS
13215 10160 8087 / 10000b
267.1 198.2 189.9 41.2 152
52.1 78.4 169.8 0 0
56.3 47.4 45.6 53.2 285
1387 / / 77.5 /
0.55 / / 0.02 4.02
6.91 / / 0.05 19.98
173.5 / / 7.3 97.6
0.21 / / 0.35 3.94
/ / / / 0.99
5.16 / / / 10.03
51.47 / / 6.80 30.20
1125 / / 0 0
Soybean processing wastewater. Glucose concentration.
of SE (Song and Liu, 1999) and Basal medium (Sorokin and Krauss, 1958). To obtain media for the cultivation of C. pyrenoidosa, SPW from the equalization basin (SW) and the anaerobic hydrolysis reactor were collected separately and centrifuged at 10,355g, 10 min at 4 °C in a CT15RT Benchtop High Speed Refrigerated Centrifuge, TECHCOMP LTD, Shanghai, China. The centrifuged supernatant from the equalization basin (SW1) and the anaerobic hydrolysis reactor (SW2) contained 10,160 and 8087 mg L1 of COD, respectively. The pH of the supernatants was adjusted to 6.5, autoclaved at 121 °C for 20 min, and stored at 1 °C.
were fed with SW2 medium at the same time, and the dosing quantity of the SW2 were: 75, 100, 0 and 50 ml, respectively. The above experiments were performed in a light incubator. The cultures were incubated for 5 days at an initial algal biomass concentration of 0.3 g L1 for all treatments. The cultivation conditions were as follows: light intensity = 40.5 lmole photons m2 s1, light/dark ratio = 14:10, temperature = 27 ± 1 °C. All the experiments were done in triplicate.
2.4. Analytical methods 2.2. Microalgal strain and pre-culture conditions C. pyrenoidosa, FACHB-9 was purchased from the Institute of Hydrobiology (the Chinese Academy of Sciences, Wuhan, China). Inoculation was performed under sterile conditions, and C. pyrenoidosa was cultivated with 100 mL of autoclaved SE medium in 250 mL conical flasks, and then placed in a light incubator (GZX300BS-III, CIMO Medical Instrument, Shanghai, China). The cultivation conditions were as follows: light intensity = 27 lmolephotonsm2 s1, light/dark ratio = 14:10, temperature = 25 ± 1 °C and artificial intermittent shaking (three times in a day) for 6–7 days. 2.3. Experimental design (a) Batch culture experiments. Batch experiments were conducted first to compare the effects of the SW1, SW2 and SE growth medium on the growth of C. pyrenoidosa as well as the removal efficiencies of nutrients and organic compounds from soybean wastewater. Three 500 ml conical flasks were used as photobioreactors (P1, P2 and P3), the solutions were mixed using a magnetic stirrer. In P1 and P2, a total of 100 mL of the inoculum was combined with 100 ml of wastewater medium and 70 mL of distilled water. In P3, 170 mL of the SE culture medium was combined with 100 mL of the inoculum, which was used as a contrast. (b) Fed-batch culture experiments. A 500-ml conical flask was used as photobioreactor (P4). Mixing was accomplished using a magnetic stirrer. In P4, a total of 100 mL of the inoculum was combined with 50 ml of SW2 medium. In the next four days, the algae
C. pyrenoidosa biomass was determined in triplicate by collecting 1 ml of algae suspension, centrifugation at 5074g, 4 °C for 10 min, drying the pellet to constant weight at 65 °C. Triplicate blanks for each sample were also treated in the same way. Samples without algae were used as controls. For water quality measurements, the algal culture was centrifuged (10,355g, 10 min at 4 °C), and the supernatant was filtered through a 0.45-lm membrane. The filtrate was analyzed for the determination of soluble chemical oxygen demand (SCODCr), total nitrogen (TN) and total phosphate (TP). All of the measurements were conducted according to standard methods (State Environmental Protection Administration of China, 2002). C. pyrenoidosa was analyzed for carbon (C), hydrogen (H), oxygen (O), and nitrogen (N) content, using an elemental analyzer (Vario EL III, Elementar Analysen Systeme, USA). Carbohydrate was measured by the phenol–sulfuric method with glucose as standard (Herbert et al., 1971). Total lipids were analyzed according to Bligh and Dyer (1959) with modifications as described by Yoo et al. (2010). To analyze volatile fatty acids (VFAs), the filtrate was collected in a 1.5-mL gas chromatography (GC) vial, and 3% H3PO4 was added to adjust the pH to approximately 4.0. A gas chromatograph (HP6890II, USA) equipped with a flame ionization detector (FID) and analytical column CPWAX52CB (30 m 0.53 mm 1 lm) was utilized to determine the concentrations of VFAs (C2–C5). The sample injection volume was 1.0 lL. The temperature of the injector and FID were 200 and 220 °C, respectively. Nitrogen was used as the carrier gas at a flow rate of 50 mL min1. The GC oven was programmed to begin
9886
S. Hongyang et al. / Bioresource Technology 102 (2011) 9884–9890
at 110 °C and remain there 2 min, then increased to 220 °C at a rate of 10 °C min1, and hold at 220 °C for 2 min. The calculation of the organic producing algae coefficient was shown as Eq. (1).
2.0
ð1Þ
Biomass (g L-1)
where YB/COD (g (g COD)1) is the yield coefficient, Vt (L) is the algae liquid volume at time t, t (d) is the time, SBH (mg L1) is the heterotrophic algae concentration, SBMt (mg L1) is the total algae concentration at time t, SBAt (mg L1) is the autotrophic algae concentration at time t, SS (mg L1) is the organic matter concentration, and SSt (mg L1) is the total organic concentration in liquid algae at time t. The calculation of the organic producing activated sludge coefficient was shown as Eq. (2).
Yobs ¼
1.5
1.0
0.5
0.0 0
20
40
60
80
100
120
Time (h)
Y 1 þ kd hc
ð2Þ 1
1
where Yobs (g (g COD) ) is the net yield coefficient, Y (g (g COD) ) is the yield coefficient, hc (d) is the sludge age, Kd (d1) is the coefficient of endogenous respiration (attenuation).
Fig. 1. Growth of C. pyrenoidosa in soybean processing wastewater in batch and fedbatch culture.
6000
Batch-fed SW2 COD Batch SW2 COD Batch SW1 COD
5500
3. Results
-1
SCODCr (mg L )
3.1. Batch and fed-batch cultivation of C. pyrenoidosa with SPW
5000
As shown in Fig. 1, the biomass concentration of C. pyrenoidosa with SW1 medium and SW2 medium after 120 h were 2.09 ± 0.13 and 2.15 ± 0.21 g L1, respectively. In contrast, the biomass concentration of C. pyrenoidosa using SE medium achieved only 0.66 ± 0.03 g L1. The average biomass productivity of C. pyrenoidosa by using SW1 medium and SW2 medium in batch reactors were 0.37 (with a maximum value of 0.64 ± 0.09) and 0.38 g L1 d1 (with a maximum value of 0.76 ± 0.05), respectively. 3.2. Removal of pollutants removal from SPW by C. pyrenoidosa 3.2.1. COD removal from SPW by C. pyrenoidosa As can be seen from Fig. 2, the SCODCr concentration in fedbatch culture process increased to a high value after the addition of SPW, but it was rapidly reduced to a lower value due to the absorption and consumption of organic compounds by C. pyrenoids. The SCODCr removal efficiencies gradually increased for the batch and fed-batch cultures. The SCODCr removal efficiency in the batch culture with SW1 and SW2 medium after 120 h were 80 and 84%, respectively, while, 77.8 ± 5.7% of SCODCr removal efficiency was achieved in the fed-batch culture with SW2 medium. However, SCODCr removal rates with SW1 and SW2 medium in batch were only 0.60 and 0.51 g L1 d1, respectively whereas the average SCODCr removal rate in the fed-batch mode with SW2 medium was 1.40 g L1 d1. 3.2.2. Removal of N, P from SPW by C. pyrenoidosa As shown in Fig. 3, the concentrations of TN, NH4+-N and TP increased to high values after addition of SPW, but rapidly decreased due to absorption and consumption of nutrients by Chlorella. At the end of the mixotrophic period, the concentrations of TN, NH4+-N and TP decreased to 19.1 ± 0.7, 16.7 ± 0.8 and 11.7 ± 4.7 mg L1, respectively. Correspondingly, the removal efficiencies of TN, NH4+-N and TP were 88.8 ± 1.0%, 89.1 ± 0.6%, and 70.3 ± 11.4%, respectively. 3.2.3. Removal of organic matter from SPW with C. pyrenoidosa 3.2.3.1. Removal of carbohydrate from SPW after anaerobic hydrolysis and acidification pretreatment by C. pyrenoidosa. Fig. 4 shows the
R1 R2 R3
110 100 90
4500
80
4000
70
3500
60
3000
50
2500
40
2000
Removal (%)
Y B=COD
Pn DSBH DðSBM SBA Þ ðSBMt SBAT Þ P ¼ ¼ ¼ nt¼1 DS S DS s ðS t¼1 sðt1Þ Sst Þ
Fed-batch SW2 Batch SW2 Batch SW1 Batch SE
2.5
30
1500
20
1000
10
500 0
24
48
72
96
120
0
Time (h) Fig. 2. Removal of SCODCr in soybean processing wastewater medium by C. pyrenoidosa in batch and fed-batch culture. SW1 represent the supernatant of the centrifuged soybean processing wastewater from the equalization basin. SW2 represent the supernatant of the centrifuged soybean processing wastewater from the anaerobic hydrolysis reactor. R1, R2 and R3 represent the SCODCr removal rates in fed-batch culture with SW2 medium, batch culture with SW2 medium and batch culture with SW1 medium, respectively.
fate of the carbohydrates in pretreated SPW medium during fedbatch culture. After 120 h of cultivation, the carbohydrate concentration in SPW was 59.8 ± 12.1 mg L1 and the corresponding removal efficiency was 41.1 ± 1.9%. The main constituents of carbohydrates in SPW were oligosaccharides, including stachyose, raffinose, sucrose and small amounts of glucose, fructose, D-inositol ether, galactose, inositnol ether (Zhao, 2006). After anaerobic hydrolysis and acidification pretreatment, the majority of carbohydrates were converted to volatile fatty acids (VFAs) and other small molecules (Rittmann and McCarty, 2001). A small part of the carbohydrates were not utilized C. pyrenoidosa. 3.2.3.2. Removal of VFAs from SPW after anaerobic hydrolysis and acidification pretreatment by C. pyrenoidosa. Fig. 4 shows the fate of VFAs in pretreated SPW during fed-batch culture. After 120 h of cultivation, the VFAs concentration in the SPW decreased to 162.4 ± 68.6 mg L1 and the corresponding removal efficiency was 96.7 ± 1.4%. Fig. 5 shows the changes in individual VFAs concentration in pretreated SPW medium during fed-batch culture. C. pyrenoidosa prefered to utilize acetic acid, propionic acid, butyric acid,
9887
S. Hongyang et al. / Bioresource Technology 102 (2011) 9884–9890
100 TN concentration
100
rTN
50 40
40
30
VFAs-COD (mg L-1)
60
Removal (%)
Concentration (mg L-1)
70
60
100
2500
80 80
rVFAs-COD rSC-COD
VFAs-COD SC
3000
90
80 2000 60 1500 40
1000 500
20
20
20
0
10
0
0
0 0
24
48
72
96
0
120
20
40
+
NH4-N concentration
100
rNH4-N
80
100
120
Fig. 4. Removal of total VFAs and soluble carbohydrates from soybean processing wastewater medium by C. pyrenoidosa in a fed-batch culture. SC represent soluble carbohydrates.
100 +
60
time (h)
Time(h)
b
Removal(%)
a
90
70
60
50 40
40
30 20
20
10 0
0 0
24
48
72
96
120
c
600 500 400 300 200
rTP
-100
90
80
50 40
40
Removal (%)
60
24
24
48
48
72
72
96
96
120
Fig. 5. Changes in individual VFA concentrations in soybean processing wastewater medium by C. pyrenoidosa during fed-batch culture.
70
60
0
Time (h)
80
Concentration (mg L-1)
700
0 100
TP concentration
acetic propionic n-butyric iso-butyric iso-valeric n-valeric
800
100
Time(h)
100
900
concentration (mg/L)
60
Removal (%)
Concentration (mg L-1)
80 80
30
As shown in Fig. 6, the V/SCODCr was increased to a high value after the addition of SPW, but it was rapidly decrease to a lower value due to the absorption and consumption of VFAs by Chlorella.
20
20
10 0
0 0
24
48
72
96
120
Time(h) Fig. 3. Removal of total nitrogen (TN), NH4+-N and total phosphate (TP) from soybean processing wastewater by C. pyrenoidosa in fed-batch culture. rTN, rNH4+-N and rTP represent the relative removal rates of TN, NH4+-N and TP in soybean wastewater medium, respectively.
3.3. Lipid productivity of C. pyrenoidosa in mixotrophic culture C. pyrenoidosa achieved an average biomass productivity of 0.64 g L1 d1, a final lipid content of 37.00 ± 9.34% and an average lipid productivity of 0.24 g L1 d1. It is worthy of note that the highest biomass productivity was 1.07 ± 0.09 g L1 d1 with a lipid productivity of 0.40 g L1 d1 using fed-batch culture.
4. Discussion isovaleric acid, and n-valeric acid, resulting in average removal rates of 442.5, 257.6, 174.3, 2.5, and 49.5 mg L1 d1, respectively. The main VFAs showed good performance on removal efficiency during fed-batch culture. The removal efficiency of acetic acid, propionic acid, butyric acid, isovaleric acid, and n-valeric were 99.3 ± 0.5, 92.8 ± 4.0, 99.5 ± 0.8, 88.0 ± 10.4, and 100.0 ± 0.0%, respectively. This indicated that C. pyrenoidosa was able to make full use of acetate, propionate, butyrate, and iso-valerate in SPW. After the anaerobic hydrolysis and acidification process, the proportion of VFAs to COD increased from 16.1% to 69.2%, indicating VFAs were the main component of COD in pretreated SPW.
As shown in Table 2, SPW contained a high concentration of nutrients (COD > 10,000 mg L1). In addition, the concentration of inorganic nutrients in SPW was also higher than that in the SE medium, but lower than that in the Basal medium, which implying that the SPW may be able to provide sufficient nutrients for meeting the growth needs of C. pyrenoidosa under photoautotrophic or heterotrophic culture conditions. The results indicated that SPW can be used directly as a culture medium for the mixotrophic growth of C. pyrenoidosa. This can be explained by the fact that the SPW does not contain compounds that inhibit the growth of C. pyrenoidosa. In addition, the organic matter, nitrogen and phos-
S. Hongyang et al. / Bioresource Technology 102 (2011) 9884–9890
-1
Soluble organic materials (mg COD L )
4000
100
SO/SCODCr
SO SC V
4500
SC/SCODCr
90
V/SCODCr
80
3500
70
3000
60
2500
50
2000
40
1500
30
1000
20
500
10
0
Soluble organic materials/SCOD Cr (%)
9888
0 0
24
48
72
96
120
Time(h) Fig. 6. Changes in total VFAs (V), soluble carbohydrates (SC), undetermined soluble organic materials (SO) concentration and percentage of each in SCODCr in soybean processing wastewater medium during fed-batch culture of C. pyrenoidosa.
phorus in the SPW meet the requirement of the growth of C. pyrenoidosa. Batch culture, fed-batch culture and continuous culture methods can eliminate substrate inhibition in industrial fermentation processes, which have been widely used to produce high cell densities in yeast production systems. Fed-batch culture and continuous culture techniques were more controlled. Especially when substrate concentrations were high, fed-batch culture or continuous culture systems could be better targeted to control substrate concentrations in the medium. As shown in Fig. 1, the biomass concentration was diminished during fed-batch culture due to the dilution of SPW. However, C. pyrenoidas can adapt and utilize high concentrations of organic substrates with a large growth rate; it was found that the resultant biomass concentration further increased the next day. Although no significant difference was observed for the biomass concentration between fed-batch culture process and batch culture process, the average biomass productivity of C. pyrenoidosaon in fed-batch culture process achieved 0.64 g L1 d1 and with a maximum value of 1.01 ± 0.11 g L1 d1, which was higher than that by batch culture process. To investigate the removal of organic pollutants by C. pyrenoidosa, the SCODCr reduction in SPW treated by C. pyrenoidosa was compared for batch and fed-batch culture. The results indicated that SCODCr removal efficiency showed small difference between batch and fed-batch culture. This can be explained by the fact that C. pyrenoidosa can tolerate high concentrations of organic compounds. It was also found that both cultures showed good performance for the utilization and removal of organic pollutants in SPW. However, SCODCr removal rates in fed-batch culture were different from that of batch culture. The concentration of organic compounds was decreased continuously, which correspondingly decreased the reaction driving force and the SCODCr removal rate. The results showed that fed-batch culture was more effective for the removal of organic compounds in SPW by C. pyrenoidosa than that by batch culture. This promising result was the reason that fed-batch culture for C. pyrenoidosa production was higher. Mixotrophy of microalgae assimilates CO2 and organic carbon simultaneously (Marquez et al., 1993). Mixotrophic algal growth processes may consist of autotrophic and heterotrophic processes, which can proceed noncompetitively for some microalgae (Endo et al., 1977). It was reported that the presence of organic matter did not significantly prevent the absorption and utilization of inorganic carbon in the mixotrophic cultures (Bhatnagar et al., 2010). The growth rate of some microalgae in the mixotrophic cultures was approximately equal to the sum of the growth rates for photo-
autotrophic and heterotrophic cultures (Endo et al., 1977; Marquez et al., 1993). Yang et al. (2008) used cassava ethanol fermentation under continuous light to grow C. pyrenoidosa, the results showed that the maximal CODCr removal rate reached 71.2%, algae biomass concentration reached 3.6 ± 0.1 g L1 and the YB/COD for increase of algae biomass to the decrease of organic matters was 0.5 g (g COD)1. But this calculation did not take into account autotrophic algae. In the present study, the YB/COD in batch culture process by using SW2 and SW1 medium were calculated to be 0.58 and 0.50 g (g COD)1, respectively. The YB/COD in fed-batch culture by using SW2 medium was 0.39 g (g COD)1. By elemental analysis it was found that the composition of C. pyrenoidosa cells was CH1.76O0.60N0.15. The COD equivalent of pyrenoidosa cell was about 1.53 g COD (g C. pyrenoidosa)1. Therefore, the Yobs for the batch culture with SW2 and SW1 and the fed-batch culture with SW2 were calculated to be 0.89, 0.76 and 0.60 g algal COD (g substrate COD)1, respectively. The results also showed that the fed-batch culture with SW2 medium removed most of the COD but with a low net yield coefficient. This can be explained by that algae consumed more organic matter by endogenous respiration, thus the concentration of algae formed in the fed-batch culture with SW2 medium was less than that of the batch culture. Activated sludge was used to treat organic wastewater containing ammonia nitrogen, with the yield coefficient (Y) being 0.67 (0.40–0.72)g bacteria COD (g substrate COD)1, the sludge age (hc) being 15 (10–20) days, and the coefficient of endogenous respiration (attenuation) (kd) being 0.06 (0.04–0.075) d1 (Henze, 2000). According to Eq. (2), Y value could be converted into net yield coefficient (Yobs), the Yobs was lower than 0.4. Therefore, higher biomass and lower emissions of CO2 could be achieved during the treatment of SPW using C. pyrenoidosa under a heterotrophic condition. Microalgae could efficiently remove different nitrogen and phosphorus substances from sewage (Martínez et al., 2000) and livestock waste (Kumar et al., 2010). This was because the cell required a high amount of nitrogen and phosphorous for protein, nucleic acid and phospholipid synthesis. Multiple factors including light, pH, nitrogen and phosphorus ratio, temperature, carbon source and bacteria concentration were crucial to nitrogen and phosphorus removal efficiency by algae. Nitrogen was removed mainly in the ammonia form, but C. pyrenoidosa also used organic nitrogen. According to the molecular formula for algae by Stumm (C106H263O110N16P) (Stumm and Morgan, 1981), 0.063 g nitrogen and 0.009 g phosphorus were required to generate 1 g of algae. Thus, with an algae production rate of 0.64 g L1 d1 in the SW2 medium, the corresponding average removal rate of TN and TP should be 40.32 and 5.76 mg L1 d1, respectively. In fact, the average removal rates of TN, NH4+-N and TP in SW2 medium were 42.84, 36.73 and 6.95 mg L1 d1, respectively, which were very close to the calculated values. It was found that the removal of nitrogen was a faster process than that of phosphate, and the removed nitrogen was mainly used for the synthesis of C. pyrenoidosa. However, most of the removed phosphorus was assimilated by microalgae in this experiment, while, about 17% of the removed TP was possibly caused by the precipitation because of the increase of pH in the algal reactor. The pH value was slowly increased from 6.5 to 8.5 due to the photosynthesis of algae and the removal of the organic pollutant. The increase of pH in medium was also reported (Munoz and Guieysse, 2006), which could lead to the precipitation of phosphorus and increasing phosphate adsorption on alginate gels (Tam and Wong, 2000). The removed nitrogen and phosphorus in SPW was mainly used for the synthesis of Chlorella, while the organic carbon was more suitable for the cultivation of Chlorella than inorganic carbon. Therefore, the presence of high concentrations of organic carbon that could be utilized by C. pyrenoidosa contributed significantly to the nitrogen and phosphorus removal rate.
S. Hongyang et al. / Bioresource Technology 102 (2011) 9884–9890
Acetate was an appropriate organic carbon source for heterotrophic or mixotrophic cultivation of microalgae (Bhatnagar et al., 2010). Particulate organic matter and organic macromolecules could be converted into acetate and other small organic molecules via anaerobic hydrolysis, which may promote algae growth. There had been reported that Na-acetate were the best carbon sources for heterotrophic growth (Perez-Garcia et al., 2011), and the similar conclusion was also found in this paper. From Fig. 2, it can be also observed that the pretreatment of SPW by anaerobic hydrolysis and acidification improved growth of C. pyrenoidosa. During the entire culture process, the decrease of VFAs accounted for the decrease of the total organic matter. While the proportions of sugar and other organic compounds were increased, this indicated that VFAs were the main group of organic compounds in SPW utilized by C. pyrenoidosa. In addition, the number of organic compounds that can be treated by C. pyrenoidosa was limited. This was probably because some complex organic compounds, especially with large molecular weight, could not be directly utilized by C. pyrenoidosa. Pretreatment by anaerobic hydrolysis and acidogenesis can convert complex organic components in SPW such as proteins, fats and polysaccharides to simple organic compounds such as lactate, propionate and acetate by heterogeneous groups of facultative and anaerobic bacteria. Therefore, the pretreated SPW was more suitable for the cultivation of C. pyrenoidosa. The lipid productivity of C. pyrenoidosa is generally about 0.040 g L1 d1, the growth of C. pyrenoidosa is a photoautotrophic process with low biomass productivity but high lipid content of C. pyrenoidosa cell (Chisti, 2007; Lv et al., 2010). Rodolfi et al. cultivated Chlorella (Chlorella sorokiniana, Chlorella sp., Chlorella vulgaris) in 0.6 L glass tubes while bubbling a sterile air/CO2 mixture (97/3, v/v) using continuous artificial illumination. A biomass productivity of 0.17–0.23 g L1 d1 and a lipid content of 18.7–19.3% were achieved in their study (Rodolfi et al., 2009). The biomass productivity of C. pyrenoidosa was only 0.1 g L1 d1 in photoautotrophic cultures without the bubbling of a sterile air/CO2 mixture, and the reported lipid content of Chlorella was only 28–32% (Chisti, 2007). In order to enhance the economic feasibility of algal oil production, the algal growth rate and oil content of the biomass are the key parameters which should be improved (Chisti, 2007; Subramaniam et al., 2010). Some studies have changed the concentration of nutrients such as nitrogen, phosphorous and organic carbon sources (Li et al., 2008; Rodolfi et al., 2009; Subramaniam et al., 2010; Xiong et al., 2010) to achieve high growth rates for algae cells and to enhance the lipid accumulation. Previous study also found that some bacterium favored the lipid accumulation of the microalgae Chlorella species (Valderrama et al., 2002). Xiong et al. adopted a photosynthesis-fermentation model for Chlorella protothecoide cultivation, 69% higher lipid yield was achieved by using glucose at the fermentation stage (Xiong et al., 2010). However, most of these studies required the addition of glucose and other chemicals which increased the production costs. In addition, some studies also have shown that the conditions favored the growth of algal cells but resulted in a small lipid fraction in the cells and vice versa (Lv et al., 2010; Yoo et al., 2010). In the present study, C. pyrenoidosa achieved higher biomass productivity and lipid productivity. The results also showed that fed-batch culture presented better performance on biomass productivity and lipid productivity. The treatment of SPW by mixotrophic cultivation of C. pyrenoidosa could effectively utilize nutrients and organic pollutants in the wastewater to efficiently promote microalgae growth. This process can greatly minimize the use of both freshwater and the required chemicals for the culture of microalgae, therefore, by which could reduce the production cost of microalgae for biodiesel production. Yet, there are still a number of technical aspects that could be developed, such as the relationship between bacteria and C. pyre-
9889
noidosa in SPW, optimizing cultivation of C. pyrenoidosa in SPW, and the important changes in metabolism induced by other factors. Solving these shortcomings will promote C. pyrenoidosa cultivated in large volumes of SPW for commercial use. 5. Conclusions Pollutants in soybean product wastewater were efficiently utilized by C. pyrenoidosa and no additional nutrients were required to achieve an average biomass productivity of 0.64 g L1 d1 in fed-batch culture. Treatment of soybean product wastewater could be coupled with cultivation of C. pyrenoidosa to reduce the production cost of microalgae and the treatment cost of wastewater simultaneously. Acknowledgement This study was supported by China National Science Fund (20976139), the National Key Technology R&D Program (2009BAC62B02) and the Research Foundation of Shanghai (09JC1413900, 09160707900 and 10230712400). The authors want to acknowledge Prof. Nigel W.T. Quinn from Lawrence Berkeley National Laboratory for his constructive suggestions in the paper writing process. References Bhatnagar, A., Bhatnagar, M., Chinnasamy, S., Das, K., 2010. Chlorella minutissima— a promising fuel alga for cultivation in municipal wastewaters. Appl. Biochem. Biotechnol. 161, 523–536. Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911–917. Cassini, A.S., Tessaro, I.C., Marczak, L.D.F., Pertile, C., 2010. Ultrafiltration of wastewater from isolated soy protein production: A comparison of three UF membranes. J. Clean. Prod. 18, 260–265. Chisti, Y., 2007. Biodiesel from microalgae. Biotechnol. Adv. 25, 294–306. Endo, H., Sansawa, H., Nakajima, K., 1977. Studies on Chlorella regularis, heterotrophic fastgrowing strain II. Mixotrophic growth in relation to light intensity and acetate concentration. Plant Cell Physiol. 18, 199–205. Henze, M., 2000. Activated sludge models ASM1, ASM2, ASM2d and ASM3. Intl Water Assn. Herbert, D., Philipps, P.J., Strange, R.E., 1971. Methods Enzymol. 5B, 265–277. Kumar, M.S., Miao, Z.H.H., Wyatt, S.K., 2010. Influence of nutrient loads, feeding frequency and inoculum source on growth of Chlorella vulgaris in digested piggery effluent culture medium. Bioresour. Technol. 101, 6012–6018. Li, Q., Du, W., Liu, D., 2008a. Perspectives of microbial oils for biodiesel production. Appl. Microbiol. Biotechnol. 80, 749–756. Li, Y., Horsman, M., Wu, N., Lan, C.Q., Dubois-Calero, N., 2008b. Biofuels from microalgae. Biotechnol. Progr. 24, 815–820. Lopes Barbosa, A.C., Lajolo, F.M., Genovese, M.I., 2006. Influence of temperature, pH and ionic strength on the production of isoflavone-rich soy protein isolates. Food Chem. 98, 757–766. Lv, J.M., Cheng, L.H., Xu, X.H., Zhang, L., Chen, H.L., 2010. Enhanced lipid production of Chlorella vulgaris by adjustment of cultivation conditions. Bioresour. Technol. 101, 6797–6804. Maroun, R., El Fadel, M., 2007. Start-up of Anaerobic Digestion of Source-Sorted Organic Municipal Solid Waste in the Absence of Classical Inocula. Environ. Sci. Technol. 41, 6808–6814. Marquez, F.J., Sasaki, K., Kakizono, T., Nishio, N., Nagai, S., 1993. Growth characteristics of Spirulina platensis in mixotrophic and heterotrophic conditions. J. Ferment. Bioeng. 76, 408–410. Martínez, M.E., Sánchez, S., Jiménez, J.M., El Yousfi, F., Mu noz, L., 2000. Nitrogen and phosphorus removal from urban wastewater by the microalga Scenedesmus obliquus. Bioresour. Technol. 73, 263–272. Metcalf, E., Eddy, H., 2003. Wastewater Engineering: Treatment and Reuse. MeGraw Hill, New York. Munoz, R., Guieysse, B., 2006. Algal–bacterial processes for the treatment of hazardous contaminants: A review. Water Res. 40, 2799–2815. Perez-Garcia, O., Escalante, F.M.E., de-Bashan, L.E., Bashan, Y., 2011. Heterotrophic cultures of microalgae: metabolism and potential products. Water Res. 45, 11– 36. Rittmann, B.E., McCarty, P.L., 2001. Environmental Biotechnology: Principles and Applications. McGraw, Hill New York. Rodolfi, L., Chini Zittelli, G., Bassi, N., Padovani, G., Biondi, N., Bonini, G., Tredici, M., 2009. Microalgae for oil: Strain selection, induction of lipid synthesis and outdoor mass cultivation in a low-cost photobioreactor. Biotechnol. Bioeng. 102, 100–112.
9890
S. Hongyang et al. / Bioresource Technology 102 (2011) 9884–9890
Song, Liu, Y., 1999. FACHB-Collection: List of Strains. Institute of Hydrobiology, the Chinese Academy of Sciences. Wuhan, China. Sorokin, C., Krauss, R., 1958. The Effects of Light Intensity on the Growth Rates of Green Algae. Plant Physiol. 33, 109–113. Spolaore, P., Joannis-Cassan, C., Duran, E., Isambert, A., 2006. Commercial applications of microalgae. J. Biosci. Bioeng. 101, 87–96. State Environmental Protection Administration of China, 2002. Water and Wastewater Analyzing Methods, fourth ed. China Environmental Science. Stumm, W., Morgan, J., 1981. Aquatic Chemistry. Wiley New York. Su, K., Yu, H., 2005. Formation and characterization of aerobic granules in a sequencing batch reactor treating soybean-processing wastewater. Environ. Sci. Technol. 39, 2818–2827. Subramaniam, R., Dufreche, S., Zappi, M., Bajpai, R., 2010. Microbial lipids from renewable resources: production and characterization. J. Ind. Microbiol. Biotechnol. 37, 1271–1287. Tam, N., Wong, Y., 2000. Effect of immobilized microalgal bead concentrations on wastewater nutrient removal. Environ. Pollut. 107, 145–151. Tang, C.-H., Ma, C.-Y., 2009. Effect of high pressure treatment on aggregation and structural properties of soy protein isolate. LWT – Food Sci. Technol. 42, 606– 611.
Valderrama, L.T., Del Campo, C.M., Rodriguez, C.M., de-Bashan, L.E., Bashan, Y., 2002. Treatment of recalcitrant wastewater from ethanol and citric acid production using the microalga Chlorella vulgaris and the macrophyte Lemna minuscula. Water Res. 36, 4185–4192. Xiong, W., Gao, C., Yan, D., Wu, C., Wu, Q., 2010. Double CO2 fixation in photosynthesis-fermentation model enhances algal lipid synthesis for biodiesel production. Bioresour. Technol. 101, 2287–2293. Yang, C.F., Ding, Z.Y., Zhang, K.C., 2008. Growth of Chlorella pyrenoidosa in wastewater from cassava ethanol fermentation. World J. Microbiol. Biotechnol. 24, 2919–2925. Yoo, C., Jun, S., Lee, J., Ahn, C., Oh, H., 2010. Selection of microalgae for lipid production under high levels carbon dioxide. Bioresour. Technol. 101, S71–S74. Yu, H.Q., Hu, Z.H., Hong, T.Q., Gu, G.W., 2002. Performance of an anaerobic filter treating soybean processing wastewater with and without effluent recycle. Process Biochem. 38, 507–513. Zhao D.M, 2006. The study of intensified technology processes the high concentrated bean product processing wastewater. Beijing University of Chemical Technology (in Chinese with English Abstract).
Contents lists available at SciVerse ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Cultivation of Chlorella pyrenoidosa in soybean processing wastewater Su Hongyang a,b, Zhang Yalei a,⇑, Zhang Chunmin a, Zhou Xuefei a,⇑, Li Jinpeng a a b
State Key Laboratory of Pollution Control and Resources Reuse, College of Environmental Science and Engineering, Tongji University, Shanghai 200092, China School of Resource and Environment, Fujian Agriculture and Forestry University, Fuzhou 350002, China
a r t i c l e
i n f o
Article history: Received 11 April 2011 Received in revised form 28 July 2011 Accepted 3 August 2011 Available online 16 August 2011 Keywords: Chlorella pyrenoidosa Soybean processing wastewater Fed-batch culture Anaerobic hydrolysis
a b s t r a c t Chlorella pyrenoidosa was cultivated in soybean processing wastewater (SPW) in batch and fed-batch cultures without a supply of additional nutrients. The alga was able to remove 77.8 ± 5.7%, 88.8 ± 1.0%, 89.1 ± 0.6% and 70.3 ± 11.4% of soluble chemical oxygen demand (SCODCr), total nitrogen (TN), NH4+-N and total phosphate (TP), respectively, after 120 h in fed-batch culture. C. pyrenoidosa attained an average biomass productivity of 0.64 g L1 d1, an average lipid content of 37.00 ± 9.34%, and a high lipid productivity of 0.40 g L1 d1. Therefore, cultivation of C. pyrenoidosa in SPW could yield cleaner water and useful biomass. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Processing soybeans into bean curd generates about 7–10 tons of wastewater with a chemical oxygen demand (COD) of 10– 20 g L1 per ton of processed soybeans (Yu et al., 2002). Studies on soybean processing wastewater (SPW) treatment have focused on anaerobic filters (AF) (Yu et al., 2002), upflow anaerobic sludge beds (UASB), and sequencing batch reactors (SBR) (Su and Yu, 2005). Although anaerobic processing is appropriate for treatment of high-strength wastewaters with energy recovery (Maroun and El Fadel, 2007; Metcalf and Eddy, 2003), the method has several disadvantages. Anaerobic treatments are unable to remove biological nitrogen and phosphorus, and require frequent adjustments for alkalinity (Metcalf and Eddy, 2003). Aerobic treatments suffer from high investment and energy costs to meet COD biotransformation requirements, nitrogen and phosphorus removal goals, and sludge thickening and other needs. The aerobic process not only wastes organic carbon and nutrient resources, but also generates CO2 and sludge. An optimal treatment process should be able to utilize the production of useful organisms and, at the same time, remove organic matter, nitrogen, and phosphorus pollutants. SPW contains monosaccharide, oligosaccharides, vitamins, organic acids, amino acids, lipids, whey protein, isoflavone, saponin, P, Ca, Fe, and other nutrients (Lopes Barbosa et al., 2006; Tang and Ma, 2009). Some studies have explored the recovery of oligosaccharides, proteins or isoflavones with membrane technologies (Cassini et al., 2010; Lopes Barbosa et al., 2006; Tang and Ma,
2009), but this approach only recovers some of the components of the wastewater and leaves effluents that might still require further treatment. Some researchers have explored the cultivation of microalgae in conjunction with wastewater treatment (Table 1). Some of these microalgae are not only capable of removing pollutants, but also produce lipids that can be converted into biodiesel (Perez-Garcia et al., 2011; Bhatnagar et al., 2010; Li et al., 2008). Since SPW contains significant usable nutrients generally without toxic and hazardous substances that could inhibit growth of microalgae, cultivation of oleaginous microalgae in such a medium may reduce the cost of algae-based biodiesel. C. pyrenoidosa is a green algae that is used as human and animal nutrition supplement, and for the extraction of high-value compounds such as fatty acids, pigments, carotenoids and xanthophylls (Spolaore et al., 2006), but also for wastewater treatment and biofuel production (Perez-Garcia et al., 2011). C. pyrenoidosa can grow autotrophically, heterotrophically or mixotrophically, and we hypothesized that under the latter growth condition, the algae could grow and remove nutrients from SPW. Thus, the aim of the present study was to investigate growth, nutrient removal, and lipid production by C. pyrenoidosa in batch and fed-batch cultures in SPW.
2. Methods 2.1. Composition and pretreatment of SPW
⇑ Corresponding authors. Tel.: +86 21 65982503; fax: +86 21 65988885. E-mail addresses: [email protected] (Z. Yalei), [email protected] (Z. Xuefei). 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.08.016
SPW was obtained from a local soybean processing plant, and its overall composition was shown in Table 2 together with that
9885
S. Hongyang et al. / Bioresource Technology 102 (2011) 9884–9890 Table 1 Wastewaters treatment by various microalgae reported in the literature. Wastewater
Microalgae
Reactor
Culture time (h)
Removal (%) TP
NH4+-
COD
Productivity (g L1 d1)
Biomass (g L1)
References
0.03
<0.5
Martínez et al. (2000) Bhatnagar et al. (2010) Kumar et al. (2010)
N Urban wastewater Municipal Wastewaters Digested piggery effluent Urine Industrial wastewater
Scenedesmus obliquus Chlorella minutissima Chlorella vulgaris
Jacketed cylindrical bioreactors Oxidation pond
188
98
100
360
30
>41
Plastic bags
240
100
78
Spirulina platensis Chlorella vulgaris
Photobioreactor
192
99
99
Acrylic bioreactor
120
28
72
>75
>0.5
1.05 61
Yang et al. (2008) Valderrama et al. (2002)
Table 2 Composition of soybean processing wastewater, SE and Basal medium.
SWa SW1 SW2 SE Basal a b
COD mg L1
TN
NH4+-N
TP
Na
Cu
Zn
Mg
Mn
Co
Fe
Ca
SS
13215 10160 8087 / 10000b
267.1 198.2 189.9 41.2 152
52.1 78.4 169.8 0 0
56.3 47.4 45.6 53.2 285
1387 / / 77.5 /
0.55 / / 0.02 4.02
6.91 / / 0.05 19.98
173.5 / / 7.3 97.6
0.21 / / 0.35 3.94
/ / / / 0.99
5.16 / / / 10.03
51.47 / / 6.80 30.20
1125 / / 0 0
Soybean processing wastewater. Glucose concentration.
of SE (Song and Liu, 1999) and Basal medium (Sorokin and Krauss, 1958). To obtain media for the cultivation of C. pyrenoidosa, SPW from the equalization basin (SW) and the anaerobic hydrolysis reactor were collected separately and centrifuged at 10,355g, 10 min at 4 °C in a CT15RT Benchtop High Speed Refrigerated Centrifuge, TECHCOMP LTD, Shanghai, China. The centrifuged supernatant from the equalization basin (SW1) and the anaerobic hydrolysis reactor (SW2) contained 10,160 and 8087 mg L1 of COD, respectively. The pH of the supernatants was adjusted to 6.5, autoclaved at 121 °C for 20 min, and stored at 1 °C.
were fed with SW2 medium at the same time, and the dosing quantity of the SW2 were: 75, 100, 0 and 50 ml, respectively. The above experiments were performed in a light incubator. The cultures were incubated for 5 days at an initial algal biomass concentration of 0.3 g L1 for all treatments. The cultivation conditions were as follows: light intensity = 40.5 lmole photons m2 s1, light/dark ratio = 14:10, temperature = 27 ± 1 °C. All the experiments were done in triplicate.
2.4. Analytical methods 2.2. Microalgal strain and pre-culture conditions C. pyrenoidosa, FACHB-9 was purchased from the Institute of Hydrobiology (the Chinese Academy of Sciences, Wuhan, China). Inoculation was performed under sterile conditions, and C. pyrenoidosa was cultivated with 100 mL of autoclaved SE medium in 250 mL conical flasks, and then placed in a light incubator (GZX300BS-III, CIMO Medical Instrument, Shanghai, China). The cultivation conditions were as follows: light intensity = 27 lmolephotonsm2 s1, light/dark ratio = 14:10, temperature = 25 ± 1 °C and artificial intermittent shaking (three times in a day) for 6–7 days. 2.3. Experimental design (a) Batch culture experiments. Batch experiments were conducted first to compare the effects of the SW1, SW2 and SE growth medium on the growth of C. pyrenoidosa as well as the removal efficiencies of nutrients and organic compounds from soybean wastewater. Three 500 ml conical flasks were used as photobioreactors (P1, P2 and P3), the solutions were mixed using a magnetic stirrer. In P1 and P2, a total of 100 mL of the inoculum was combined with 100 ml of wastewater medium and 70 mL of distilled water. In P3, 170 mL of the SE culture medium was combined with 100 mL of the inoculum, which was used as a contrast. (b) Fed-batch culture experiments. A 500-ml conical flask was used as photobioreactor (P4). Mixing was accomplished using a magnetic stirrer. In P4, a total of 100 mL of the inoculum was combined with 50 ml of SW2 medium. In the next four days, the algae
C. pyrenoidosa biomass was determined in triplicate by collecting 1 ml of algae suspension, centrifugation at 5074g, 4 °C for 10 min, drying the pellet to constant weight at 65 °C. Triplicate blanks for each sample were also treated in the same way. Samples without algae were used as controls. For water quality measurements, the algal culture was centrifuged (10,355g, 10 min at 4 °C), and the supernatant was filtered through a 0.45-lm membrane. The filtrate was analyzed for the determination of soluble chemical oxygen demand (SCODCr), total nitrogen (TN) and total phosphate (TP). All of the measurements were conducted according to standard methods (State Environmental Protection Administration of China, 2002). C. pyrenoidosa was analyzed for carbon (C), hydrogen (H), oxygen (O), and nitrogen (N) content, using an elemental analyzer (Vario EL III, Elementar Analysen Systeme, USA). Carbohydrate was measured by the phenol–sulfuric method with glucose as standard (Herbert et al., 1971). Total lipids were analyzed according to Bligh and Dyer (1959) with modifications as described by Yoo et al. (2010). To analyze volatile fatty acids (VFAs), the filtrate was collected in a 1.5-mL gas chromatography (GC) vial, and 3% H3PO4 was added to adjust the pH to approximately 4.0. A gas chromatograph (HP6890II, USA) equipped with a flame ionization detector (FID) and analytical column CPWAX52CB (30 m 0.53 mm 1 lm) was utilized to determine the concentrations of VFAs (C2–C5). The sample injection volume was 1.0 lL. The temperature of the injector and FID were 200 and 220 °C, respectively. Nitrogen was used as the carrier gas at a flow rate of 50 mL min1. The GC oven was programmed to begin
9886
S. Hongyang et al. / Bioresource Technology 102 (2011) 9884–9890
at 110 °C and remain there 2 min, then increased to 220 °C at a rate of 10 °C min1, and hold at 220 °C for 2 min. The calculation of the organic producing algae coefficient was shown as Eq. (1).
2.0
ð1Þ
Biomass (g L-1)
where YB/COD (g (g COD)1) is the yield coefficient, Vt (L) is the algae liquid volume at time t, t (d) is the time, SBH (mg L1) is the heterotrophic algae concentration, SBMt (mg L1) is the total algae concentration at time t, SBAt (mg L1) is the autotrophic algae concentration at time t, SS (mg L1) is the organic matter concentration, and SSt (mg L1) is the total organic concentration in liquid algae at time t. The calculation of the organic producing activated sludge coefficient was shown as Eq. (2).
Yobs ¼
1.5
1.0
0.5
0.0 0
20
40
60
80
100
120
Time (h)
Y 1 þ kd hc
ð2Þ 1
1
where Yobs (g (g COD) ) is the net yield coefficient, Y (g (g COD) ) is the yield coefficient, hc (d) is the sludge age, Kd (d1) is the coefficient of endogenous respiration (attenuation).
Fig. 1. Growth of C. pyrenoidosa in soybean processing wastewater in batch and fedbatch culture.
6000
Batch-fed SW2 COD Batch SW2 COD Batch SW1 COD
5500
3. Results
-1
SCODCr (mg L )
3.1. Batch and fed-batch cultivation of C. pyrenoidosa with SPW
5000
As shown in Fig. 1, the biomass concentration of C. pyrenoidosa with SW1 medium and SW2 medium after 120 h were 2.09 ± 0.13 and 2.15 ± 0.21 g L1, respectively. In contrast, the biomass concentration of C. pyrenoidosa using SE medium achieved only 0.66 ± 0.03 g L1. The average biomass productivity of C. pyrenoidosa by using SW1 medium and SW2 medium in batch reactors were 0.37 (with a maximum value of 0.64 ± 0.09) and 0.38 g L1 d1 (with a maximum value of 0.76 ± 0.05), respectively. 3.2. Removal of pollutants removal from SPW by C. pyrenoidosa 3.2.1. COD removal from SPW by C. pyrenoidosa As can be seen from Fig. 2, the SCODCr concentration in fedbatch culture process increased to a high value after the addition of SPW, but it was rapidly reduced to a lower value due to the absorption and consumption of organic compounds by C. pyrenoids. The SCODCr removal efficiencies gradually increased for the batch and fed-batch cultures. The SCODCr removal efficiency in the batch culture with SW1 and SW2 medium after 120 h were 80 and 84%, respectively, while, 77.8 ± 5.7% of SCODCr removal efficiency was achieved in the fed-batch culture with SW2 medium. However, SCODCr removal rates with SW1 and SW2 medium in batch were only 0.60 and 0.51 g L1 d1, respectively whereas the average SCODCr removal rate in the fed-batch mode with SW2 medium was 1.40 g L1 d1. 3.2.2. Removal of N, P from SPW by C. pyrenoidosa As shown in Fig. 3, the concentrations of TN, NH4+-N and TP increased to high values after addition of SPW, but rapidly decreased due to absorption and consumption of nutrients by Chlorella. At the end of the mixotrophic period, the concentrations of TN, NH4+-N and TP decreased to 19.1 ± 0.7, 16.7 ± 0.8 and 11.7 ± 4.7 mg L1, respectively. Correspondingly, the removal efficiencies of TN, NH4+-N and TP were 88.8 ± 1.0%, 89.1 ± 0.6%, and 70.3 ± 11.4%, respectively. 3.2.3. Removal of organic matter from SPW with C. pyrenoidosa 3.2.3.1. Removal of carbohydrate from SPW after anaerobic hydrolysis and acidification pretreatment by C. pyrenoidosa. Fig. 4 shows the
R1 R2 R3
110 100 90
4500
80
4000
70
3500
60
3000
50
2500
40
2000
Removal (%)
Y B=COD
Pn DSBH DðSBM SBA Þ ðSBMt SBAT Þ P ¼ ¼ ¼ nt¼1 DS S DS s ðS t¼1 sðt1Þ Sst Þ
Fed-batch SW2 Batch SW2 Batch SW1 Batch SE
2.5
30
1500
20
1000
10
500 0
24
48
72
96
120
0
Time (h) Fig. 2. Removal of SCODCr in soybean processing wastewater medium by C. pyrenoidosa in batch and fed-batch culture. SW1 represent the supernatant of the centrifuged soybean processing wastewater from the equalization basin. SW2 represent the supernatant of the centrifuged soybean processing wastewater from the anaerobic hydrolysis reactor. R1, R2 and R3 represent the SCODCr removal rates in fed-batch culture with SW2 medium, batch culture with SW2 medium and batch culture with SW1 medium, respectively.
fate of the carbohydrates in pretreated SPW medium during fedbatch culture. After 120 h of cultivation, the carbohydrate concentration in SPW was 59.8 ± 12.1 mg L1 and the corresponding removal efficiency was 41.1 ± 1.9%. The main constituents of carbohydrates in SPW were oligosaccharides, including stachyose, raffinose, sucrose and small amounts of glucose, fructose, D-inositol ether, galactose, inositnol ether (Zhao, 2006). After anaerobic hydrolysis and acidification pretreatment, the majority of carbohydrates were converted to volatile fatty acids (VFAs) and other small molecules (Rittmann and McCarty, 2001). A small part of the carbohydrates were not utilized C. pyrenoidosa. 3.2.3.2. Removal of VFAs from SPW after anaerobic hydrolysis and acidification pretreatment by C. pyrenoidosa. Fig. 4 shows the fate of VFAs in pretreated SPW during fed-batch culture. After 120 h of cultivation, the VFAs concentration in the SPW decreased to 162.4 ± 68.6 mg L1 and the corresponding removal efficiency was 96.7 ± 1.4%. Fig. 5 shows the changes in individual VFAs concentration in pretreated SPW medium during fed-batch culture. C. pyrenoidosa prefered to utilize acetic acid, propionic acid, butyric acid,
9887
S. Hongyang et al. / Bioresource Technology 102 (2011) 9884–9890
100 TN concentration
100
rTN
50 40
40
30
VFAs-COD (mg L-1)
60
Removal (%)
Concentration (mg L-1)
70
60
100
2500
80 80
rVFAs-COD rSC-COD
VFAs-COD SC
3000
90
80 2000 60 1500 40
1000 500
20
20
20
0
10
0
0
0 0
24
48
72
96
0
120
20
40
+
NH4-N concentration
100
rNH4-N
80
100
120
Fig. 4. Removal of total VFAs and soluble carbohydrates from soybean processing wastewater medium by C. pyrenoidosa in a fed-batch culture. SC represent soluble carbohydrates.
100 +
60
time (h)
Time(h)
b
Removal(%)
a
90
70
60
50 40
40
30 20
20
10 0
0 0
24
48
72
96
120
c
600 500 400 300 200
rTP
-100
90
80
50 40
40
Removal (%)
60
24
24
48
48
72
72
96
96
120
Fig. 5. Changes in individual VFA concentrations in soybean processing wastewater medium by C. pyrenoidosa during fed-batch culture.
70
60
0
Time (h)
80
Concentration (mg L-1)
700
0 100
TP concentration
acetic propionic n-butyric iso-butyric iso-valeric n-valeric
800
100
Time(h)
100
900
concentration (mg/L)
60
Removal (%)
Concentration (mg L-1)
80 80
30
As shown in Fig. 6, the V/SCODCr was increased to a high value after the addition of SPW, but it was rapidly decrease to a lower value due to the absorption and consumption of VFAs by Chlorella.
20
20
10 0
0 0
24
48
72
96
120
Time(h) Fig. 3. Removal of total nitrogen (TN), NH4+-N and total phosphate (TP) from soybean processing wastewater by C. pyrenoidosa in fed-batch culture. rTN, rNH4+-N and rTP represent the relative removal rates of TN, NH4+-N and TP in soybean wastewater medium, respectively.
3.3. Lipid productivity of C. pyrenoidosa in mixotrophic culture C. pyrenoidosa achieved an average biomass productivity of 0.64 g L1 d1, a final lipid content of 37.00 ± 9.34% and an average lipid productivity of 0.24 g L1 d1. It is worthy of note that the highest biomass productivity was 1.07 ± 0.09 g L1 d1 with a lipid productivity of 0.40 g L1 d1 using fed-batch culture.
4. Discussion isovaleric acid, and n-valeric acid, resulting in average removal rates of 442.5, 257.6, 174.3, 2.5, and 49.5 mg L1 d1, respectively. The main VFAs showed good performance on removal efficiency during fed-batch culture. The removal efficiency of acetic acid, propionic acid, butyric acid, isovaleric acid, and n-valeric were 99.3 ± 0.5, 92.8 ± 4.0, 99.5 ± 0.8, 88.0 ± 10.4, and 100.0 ± 0.0%, respectively. This indicated that C. pyrenoidosa was able to make full use of acetate, propionate, butyrate, and iso-valerate in SPW. After the anaerobic hydrolysis and acidification process, the proportion of VFAs to COD increased from 16.1% to 69.2%, indicating VFAs were the main component of COD in pretreated SPW.
As shown in Table 2, SPW contained a high concentration of nutrients (COD > 10,000 mg L1). In addition, the concentration of inorganic nutrients in SPW was also higher than that in the SE medium, but lower than that in the Basal medium, which implying that the SPW may be able to provide sufficient nutrients for meeting the growth needs of C. pyrenoidosa under photoautotrophic or heterotrophic culture conditions. The results indicated that SPW can be used directly as a culture medium for the mixotrophic growth of C. pyrenoidosa. This can be explained by the fact that the SPW does not contain compounds that inhibit the growth of C. pyrenoidosa. In addition, the organic matter, nitrogen and phos-
S. Hongyang et al. / Bioresource Technology 102 (2011) 9884–9890
-1
Soluble organic materials (mg COD L )
4000
100
SO/SCODCr
SO SC V
4500
SC/SCODCr
90
V/SCODCr
80
3500
70
3000
60
2500
50
2000
40
1500
30
1000
20
500
10
0
Soluble organic materials/SCOD Cr (%)
9888
0 0
24
48
72
96
120
Time(h) Fig. 6. Changes in total VFAs (V), soluble carbohydrates (SC), undetermined soluble organic materials (SO) concentration and percentage of each in SCODCr in soybean processing wastewater medium during fed-batch culture of C. pyrenoidosa.
phorus in the SPW meet the requirement of the growth of C. pyrenoidosa. Batch culture, fed-batch culture and continuous culture methods can eliminate substrate inhibition in industrial fermentation processes, which have been widely used to produce high cell densities in yeast production systems. Fed-batch culture and continuous culture techniques were more controlled. Especially when substrate concentrations were high, fed-batch culture or continuous culture systems could be better targeted to control substrate concentrations in the medium. As shown in Fig. 1, the biomass concentration was diminished during fed-batch culture due to the dilution of SPW. However, C. pyrenoidas can adapt and utilize high concentrations of organic substrates with a large growth rate; it was found that the resultant biomass concentration further increased the next day. Although no significant difference was observed for the biomass concentration between fed-batch culture process and batch culture process, the average biomass productivity of C. pyrenoidosaon in fed-batch culture process achieved 0.64 g L1 d1 and with a maximum value of 1.01 ± 0.11 g L1 d1, which was higher than that by batch culture process. To investigate the removal of organic pollutants by C. pyrenoidosa, the SCODCr reduction in SPW treated by C. pyrenoidosa was compared for batch and fed-batch culture. The results indicated that SCODCr removal efficiency showed small difference between batch and fed-batch culture. This can be explained by the fact that C. pyrenoidosa can tolerate high concentrations of organic compounds. It was also found that both cultures showed good performance for the utilization and removal of organic pollutants in SPW. However, SCODCr removal rates in fed-batch culture were different from that of batch culture. The concentration of organic compounds was decreased continuously, which correspondingly decreased the reaction driving force and the SCODCr removal rate. The results showed that fed-batch culture was more effective for the removal of organic compounds in SPW by C. pyrenoidosa than that by batch culture. This promising result was the reason that fed-batch culture for C. pyrenoidosa production was higher. Mixotrophy of microalgae assimilates CO2 and organic carbon simultaneously (Marquez et al., 1993). Mixotrophic algal growth processes may consist of autotrophic and heterotrophic processes, which can proceed noncompetitively for some microalgae (Endo et al., 1977). It was reported that the presence of organic matter did not significantly prevent the absorption and utilization of inorganic carbon in the mixotrophic cultures (Bhatnagar et al., 2010). The growth rate of some microalgae in the mixotrophic cultures was approximately equal to the sum of the growth rates for photo-
autotrophic and heterotrophic cultures (Endo et al., 1977; Marquez et al., 1993). Yang et al. (2008) used cassava ethanol fermentation under continuous light to grow C. pyrenoidosa, the results showed that the maximal CODCr removal rate reached 71.2%, algae biomass concentration reached 3.6 ± 0.1 g L1 and the YB/COD for increase of algae biomass to the decrease of organic matters was 0.5 g (g COD)1. But this calculation did not take into account autotrophic algae. In the present study, the YB/COD in batch culture process by using SW2 and SW1 medium were calculated to be 0.58 and 0.50 g (g COD)1, respectively. The YB/COD in fed-batch culture by using SW2 medium was 0.39 g (g COD)1. By elemental analysis it was found that the composition of C. pyrenoidosa cells was CH1.76O0.60N0.15. The COD equivalent of pyrenoidosa cell was about 1.53 g COD (g C. pyrenoidosa)1. Therefore, the Yobs for the batch culture with SW2 and SW1 and the fed-batch culture with SW2 were calculated to be 0.89, 0.76 and 0.60 g algal COD (g substrate COD)1, respectively. The results also showed that the fed-batch culture with SW2 medium removed most of the COD but with a low net yield coefficient. This can be explained by that algae consumed more organic matter by endogenous respiration, thus the concentration of algae formed in the fed-batch culture with SW2 medium was less than that of the batch culture. Activated sludge was used to treat organic wastewater containing ammonia nitrogen, with the yield coefficient (Y) being 0.67 (0.40–0.72)g bacteria COD (g substrate COD)1, the sludge age (hc) being 15 (10–20) days, and the coefficient of endogenous respiration (attenuation) (kd) being 0.06 (0.04–0.075) d1 (Henze, 2000). According to Eq. (2), Y value could be converted into net yield coefficient (Yobs), the Yobs was lower than 0.4. Therefore, higher biomass and lower emissions of CO2 could be achieved during the treatment of SPW using C. pyrenoidosa under a heterotrophic condition. Microalgae could efficiently remove different nitrogen and phosphorus substances from sewage (Martínez et al., 2000) and livestock waste (Kumar et al., 2010). This was because the cell required a high amount of nitrogen and phosphorous for protein, nucleic acid and phospholipid synthesis. Multiple factors including light, pH, nitrogen and phosphorus ratio, temperature, carbon source and bacteria concentration were crucial to nitrogen and phosphorus removal efficiency by algae. Nitrogen was removed mainly in the ammonia form, but C. pyrenoidosa also used organic nitrogen. According to the molecular formula for algae by Stumm (C106H263O110N16P) (Stumm and Morgan, 1981), 0.063 g nitrogen and 0.009 g phosphorus were required to generate 1 g of algae. Thus, with an algae production rate of 0.64 g L1 d1 in the SW2 medium, the corresponding average removal rate of TN and TP should be 40.32 and 5.76 mg L1 d1, respectively. In fact, the average removal rates of TN, NH4+-N and TP in SW2 medium were 42.84, 36.73 and 6.95 mg L1 d1, respectively, which were very close to the calculated values. It was found that the removal of nitrogen was a faster process than that of phosphate, and the removed nitrogen was mainly used for the synthesis of C. pyrenoidosa. However, most of the removed phosphorus was assimilated by microalgae in this experiment, while, about 17% of the removed TP was possibly caused by the precipitation because of the increase of pH in the algal reactor. The pH value was slowly increased from 6.5 to 8.5 due to the photosynthesis of algae and the removal of the organic pollutant. The increase of pH in medium was also reported (Munoz and Guieysse, 2006), which could lead to the precipitation of phosphorus and increasing phosphate adsorption on alginate gels (Tam and Wong, 2000). The removed nitrogen and phosphorus in SPW was mainly used for the synthesis of Chlorella, while the organic carbon was more suitable for the cultivation of Chlorella than inorganic carbon. Therefore, the presence of high concentrations of organic carbon that could be utilized by C. pyrenoidosa contributed significantly to the nitrogen and phosphorus removal rate.
S. Hongyang et al. / Bioresource Technology 102 (2011) 9884–9890
Acetate was an appropriate organic carbon source for heterotrophic or mixotrophic cultivation of microalgae (Bhatnagar et al., 2010). Particulate organic matter and organic macromolecules could be converted into acetate and other small organic molecules via anaerobic hydrolysis, which may promote algae growth. There had been reported that Na-acetate were the best carbon sources for heterotrophic growth (Perez-Garcia et al., 2011), and the similar conclusion was also found in this paper. From Fig. 2, it can be also observed that the pretreatment of SPW by anaerobic hydrolysis and acidification improved growth of C. pyrenoidosa. During the entire culture process, the decrease of VFAs accounted for the decrease of the total organic matter. While the proportions of sugar and other organic compounds were increased, this indicated that VFAs were the main group of organic compounds in SPW utilized by C. pyrenoidosa. In addition, the number of organic compounds that can be treated by C. pyrenoidosa was limited. This was probably because some complex organic compounds, especially with large molecular weight, could not be directly utilized by C. pyrenoidosa. Pretreatment by anaerobic hydrolysis and acidogenesis can convert complex organic components in SPW such as proteins, fats and polysaccharides to simple organic compounds such as lactate, propionate and acetate by heterogeneous groups of facultative and anaerobic bacteria. Therefore, the pretreated SPW was more suitable for the cultivation of C. pyrenoidosa. The lipid productivity of C. pyrenoidosa is generally about 0.040 g L1 d1, the growth of C. pyrenoidosa is a photoautotrophic process with low biomass productivity but high lipid content of C. pyrenoidosa cell (Chisti, 2007; Lv et al., 2010). Rodolfi et al. cultivated Chlorella (Chlorella sorokiniana, Chlorella sp., Chlorella vulgaris) in 0.6 L glass tubes while bubbling a sterile air/CO2 mixture (97/3, v/v) using continuous artificial illumination. A biomass productivity of 0.17–0.23 g L1 d1 and a lipid content of 18.7–19.3% were achieved in their study (Rodolfi et al., 2009). The biomass productivity of C. pyrenoidosa was only 0.1 g L1 d1 in photoautotrophic cultures without the bubbling of a sterile air/CO2 mixture, and the reported lipid content of Chlorella was only 28–32% (Chisti, 2007). In order to enhance the economic feasibility of algal oil production, the algal growth rate and oil content of the biomass are the key parameters which should be improved (Chisti, 2007; Subramaniam et al., 2010). Some studies have changed the concentration of nutrients such as nitrogen, phosphorous and organic carbon sources (Li et al., 2008; Rodolfi et al., 2009; Subramaniam et al., 2010; Xiong et al., 2010) to achieve high growth rates for algae cells and to enhance the lipid accumulation. Previous study also found that some bacterium favored the lipid accumulation of the microalgae Chlorella species (Valderrama et al., 2002). Xiong et al. adopted a photosynthesis-fermentation model for Chlorella protothecoide cultivation, 69% higher lipid yield was achieved by using glucose at the fermentation stage (Xiong et al., 2010). However, most of these studies required the addition of glucose and other chemicals which increased the production costs. In addition, some studies also have shown that the conditions favored the growth of algal cells but resulted in a small lipid fraction in the cells and vice versa (Lv et al., 2010; Yoo et al., 2010). In the present study, C. pyrenoidosa achieved higher biomass productivity and lipid productivity. The results also showed that fed-batch culture presented better performance on biomass productivity and lipid productivity. The treatment of SPW by mixotrophic cultivation of C. pyrenoidosa could effectively utilize nutrients and organic pollutants in the wastewater to efficiently promote microalgae growth. This process can greatly minimize the use of both freshwater and the required chemicals for the culture of microalgae, therefore, by which could reduce the production cost of microalgae for biodiesel production. Yet, there are still a number of technical aspects that could be developed, such as the relationship between bacteria and C. pyre-
9889
noidosa in SPW, optimizing cultivation of C. pyrenoidosa in SPW, and the important changes in metabolism induced by other factors. Solving these shortcomings will promote C. pyrenoidosa cultivated in large volumes of SPW for commercial use. 5. Conclusions Pollutants in soybean product wastewater were efficiently utilized by C. pyrenoidosa and no additional nutrients were required to achieve an average biomass productivity of 0.64 g L1 d1 in fed-batch culture. Treatment of soybean product wastewater could be coupled with cultivation of C. pyrenoidosa to reduce the production cost of microalgae and the treatment cost of wastewater simultaneously. Acknowledgement This study was supported by China National Science Fund (20976139), the National Key Technology R&D Program (2009BAC62B02) and the Research Foundation of Shanghai (09JC1413900, 09160707900 and 10230712400). The authors want to acknowledge Prof. Nigel W.T. Quinn from Lawrence Berkeley National Laboratory for his constructive suggestions in the paper writing process. References Bhatnagar, A., Bhatnagar, M., Chinnasamy, S., Das, K., 2010. Chlorella minutissima— a promising fuel alga for cultivation in municipal wastewaters. Appl. Biochem. Biotechnol. 161, 523–536. Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911–917. Cassini, A.S., Tessaro, I.C., Marczak, L.D.F., Pertile, C., 2010. Ultrafiltration of wastewater from isolated soy protein production: A comparison of three UF membranes. J. Clean. Prod. 18, 260–265. Chisti, Y., 2007. Biodiesel from microalgae. Biotechnol. Adv. 25, 294–306. Endo, H., Sansawa, H., Nakajima, K., 1977. Studies on Chlorella regularis, heterotrophic fastgrowing strain II. Mixotrophic growth in relation to light intensity and acetate concentration. Plant Cell Physiol. 18, 199–205. Henze, M., 2000. Activated sludge models ASM1, ASM2, ASM2d and ASM3. Intl Water Assn. Herbert, D., Philipps, P.J., Strange, R.E., 1971. Methods Enzymol. 5B, 265–277. Kumar, M.S., Miao, Z.H.H., Wyatt, S.K., 2010. Influence of nutrient loads, feeding frequency and inoculum source on growth of Chlorella vulgaris in digested piggery effluent culture medium. Bioresour. Technol. 101, 6012–6018. Li, Q., Du, W., Liu, D., 2008a. Perspectives of microbial oils for biodiesel production. Appl. Microbiol. Biotechnol. 80, 749–756. Li, Y., Horsman, M., Wu, N., Lan, C.Q., Dubois-Calero, N., 2008b. Biofuels from microalgae. Biotechnol. Progr. 24, 815–820. Lopes Barbosa, A.C., Lajolo, F.M., Genovese, M.I., 2006. Influence of temperature, pH and ionic strength on the production of isoflavone-rich soy protein isolates. Food Chem. 98, 757–766. Lv, J.M., Cheng, L.H., Xu, X.H., Zhang, L., Chen, H.L., 2010. Enhanced lipid production of Chlorella vulgaris by adjustment of cultivation conditions. Bioresour. Technol. 101, 6797–6804. Maroun, R., El Fadel, M., 2007. Start-up of Anaerobic Digestion of Source-Sorted Organic Municipal Solid Waste in the Absence of Classical Inocula. Environ. Sci. Technol. 41, 6808–6814. Marquez, F.J., Sasaki, K., Kakizono, T., Nishio, N., Nagai, S., 1993. Growth characteristics of Spirulina platensis in mixotrophic and heterotrophic conditions. J. Ferment. Bioeng. 76, 408–410. Martínez, M.E., Sánchez, S., Jiménez, J.M., El Yousfi, F., Mu noz, L., 2000. Nitrogen and phosphorus removal from urban wastewater by the microalga Scenedesmus obliquus. Bioresour. Technol. 73, 263–272. Metcalf, E., Eddy, H., 2003. Wastewater Engineering: Treatment and Reuse. MeGraw Hill, New York. Munoz, R., Guieysse, B., 2006. Algal–bacterial processes for the treatment of hazardous contaminants: A review. Water Res. 40, 2799–2815. Perez-Garcia, O., Escalante, F.M.E., de-Bashan, L.E., Bashan, Y., 2011. Heterotrophic cultures of microalgae: metabolism and potential products. Water Res. 45, 11– 36. Rittmann, B.E., McCarty, P.L., 2001. Environmental Biotechnology: Principles and Applications. McGraw, Hill New York. Rodolfi, L., Chini Zittelli, G., Bassi, N., Padovani, G., Biondi, N., Bonini, G., Tredici, M., 2009. Microalgae for oil: Strain selection, induction of lipid synthesis and outdoor mass cultivation in a low-cost photobioreactor. Biotechnol. Bioeng. 102, 100–112.
9890
S. Hongyang et al. / Bioresource Technology 102 (2011) 9884–9890
Song, Liu, Y., 1999. FACHB-Collection: List of Strains. Institute of Hydrobiology, the Chinese Academy of Sciences. Wuhan, China. Sorokin, C., Krauss, R., 1958. The Effects of Light Intensity on the Growth Rates of Green Algae. Plant Physiol. 33, 109–113. Spolaore, P., Joannis-Cassan, C., Duran, E., Isambert, A., 2006. Commercial applications of microalgae. J. Biosci. Bioeng. 101, 87–96. State Environmental Protection Administration of China, 2002. Water and Wastewater Analyzing Methods, fourth ed. China Environmental Science. Stumm, W., Morgan, J., 1981. Aquatic Chemistry. Wiley New York. Su, K., Yu, H., 2005. Formation and characterization of aerobic granules in a sequencing batch reactor treating soybean-processing wastewater. Environ. Sci. Technol. 39, 2818–2827. Subramaniam, R., Dufreche, S., Zappi, M., Bajpai, R., 2010. Microbial lipids from renewable resources: production and characterization. J. Ind. Microbiol. Biotechnol. 37, 1271–1287. Tam, N., Wong, Y., 2000. Effect of immobilized microalgal bead concentrations on wastewater nutrient removal. Environ. Pollut. 107, 145–151. Tang, C.-H., Ma, C.-Y., 2009. Effect of high pressure treatment on aggregation and structural properties of soy protein isolate. LWT – Food Sci. Technol. 42, 606– 611.
Valderrama, L.T., Del Campo, C.M., Rodriguez, C.M., de-Bashan, L.E., Bashan, Y., 2002. Treatment of recalcitrant wastewater from ethanol and citric acid production using the microalga Chlorella vulgaris and the macrophyte Lemna minuscula. Water Res. 36, 4185–4192. Xiong, W., Gao, C., Yan, D., Wu, C., Wu, Q., 2010. Double CO2 fixation in photosynthesis-fermentation model enhances algal lipid synthesis for biodiesel production. Bioresour. Technol. 101, 2287–2293. Yang, C.F., Ding, Z.Y., Zhang, K.C., 2008. Growth of Chlorella pyrenoidosa in wastewater from cassava ethanol fermentation. World J. Microbiol. Biotechnol. 24, 2919–2925. Yoo, C., Jun, S., Lee, J., Ahn, C., Oh, H., 2010. Selection of microalgae for lipid production under high levels carbon dioxide. Bioresour. Technol. 101, S71–S74. Yu, H.Q., Hu, Z.H., Hong, T.Q., Gu, G.W., 2002. Performance of an anaerobic filter treating soybean processing wastewater with and without effluent recycle. Process Biochem. 38, 507–513. Zhao D.M, 2006. The study of intensified technology processes the high concentrated bean product processing wastewater. Beijing University of Chemical Technology (in Chinese with English Abstract).