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Lipid production by Rhodosporidium toruloides Y2 in bioethanol wastewater and evaluation of biomass energetic yield
Bioresource Technology 127 (2013) 435–440
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Lipid production by Rhodosporidium toruloides Y2 in bioethanol wastewater and evaluation of biomass energetic yield Wenwen Zhou, Wenrui Wang, Yonghong Li ⇑, Yongkui Zhang ⇑ Department of Pharmaceutical and Biological Engineering, School of Chemical Engineering, University of Sichuan, Chengdu 610065, China
h i g h l i g h t s " R. toruloides Y2 was cultured in bioethanol wastewater for lipid production. " Biomass energetic yield was evaluated by biomass, lipid content and substrate COD. " A method was chosen to accelerate lipid production and avoid catabolite repression.
a r t i c l e
i n f o
Article history: Received 21 March 2012 Received in revised form 4 August 2012 Accepted 20 September 2012 Available online 28 September 2012 Keywords: Microbial lipid Rhodosporidium toruloides Bioethanol wastewater Energy efficiency Biomass energetic yield
a b s t r a c t The oleaginous yeast Rhodosporidium toruloides Y2 was employed to remove waste nutrients from bioethanol wastewater while simultaneously producing biomass enriched in microbial lipids. Under optimal conditions, the COD degradation ratio, biomass and lipid content reached 72.3%, 3.8 g/l and 34.9%, respectively. For accelerating biomass and lipid accumulation, different feeding strategies of substrate were conducted. The biomass and lipid production increased by 39.5% and 53.8%, respectively, when glucose at 1.2 g/(l d) was added during the last three days of the cultivation. An equation was established to estimate biomass energetic yield. Under optimal conditions, the biomass energetic yield was 50.9% and an increase of 26.0% was obtained by feeding glucose at 1.2 g/(l d) during the last three days. The fatty acid composition of the lipids was similar to that from plant oils and other microbial lipids, and could thus be used as raw material for feed additives and biodiesel production. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction The bottle neck for the industrialization of biodiesel is the high cost of its raw material which accounts for 70–85% of total cost (Yousuf et al., 2010). Microbial lipids are being explored as raw materials of the production of biodiesel and functional oils (Minkevich et al., 2010; Papanikolaou and Aggelis, 2011). An important advantage offered by the application of the oleaginous microorganisms is their ability to produce lipids from waste organic matters. Consequently, to optimize the cost of the process, as well as to increase its environmental benefit, some waste materials had been studied as feedstock for single-cell oil production by fermentation with oleaginous microorganisms (Chi et al., 2011; Chinnasamy et al., 2010; Pittman et al., 2011; Xue et al., 2010), but more waste streams remain to be investigated for cost-effective microbial lipid production. Wastewater from bioethanol manufacturing is difficult to treat (Li et al., 2009). Although upflow anaerobic sludge blanket (UASB) ⇑ Corresponding authors. Tel./fax: +86 028 85405221 (Y. Li); +86 028 85408255 (Y. Zhang). E-mail addresses: [email protected] (Y. Li), [email protected] (Y. Zhang). 0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2012.09.067
and aerobic activated sludge process can remove 99% of chemical oxygen demand (COD), five-day biochemical oxygen demand (BOD5) and suspended solids (SS) and produce biogas (Liu et al., 2011b), they require start-up periods of up to 3 months and produce sludge (Liu et al., 2011a). Therefore, it is desirable to develop an efficient and economical treatment approach for bioethanol wastewater. Therefore, the present study aimed to: (1) recycle the energy contained in bioethanol wastewater and transfer it into lipidenriched cells by an oleaginous yeast Rhodosporidium toruloides Y2, (2) develop a method to evaluate the energy yield of the fermentation, (3) investigate the effects of different glucose feeding strategies on the treatment of the wastewater, (4) analyze the fatty acid composition of the lipids to estimate their potential application for the production of biodiesel and feed additives. 2. Methods 2.1. Organism, media and chemicals Yeast extract (containing 3.0% ammonium-N and 9.0% total nitrogen) and peptone (containing 3.0% ammonium-N and 14.5%
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1986). cx represents the number of available electrons per carbon atom in the biomass
total nitrogen) were obtained from Aoboxing Bio-tech. Co., Ltd. (Beijing, China). All other chemicals and reagents were in analytical reagent (AR) grade. The oleaginous yeast, R. toruloides Y2, originally from the China General Microbiological Culture Collection Center was maintained at 4 °C on YPD agar slants with glucose, 20 g/l; peptone, 10 g/l; yeast extract, 10 g/l; agar, 20 g/l. The yeast was grown in YPD liquid medium containing glucose, 20 g/l; peptone, 10 g/l; yeast extract, 10 g/l; pH 6.0. The bioethanol wastewater was provided by Sichuan Guanghe Energy technology Co., Ltd. (Chengdu, China) which produces bioethanol from sweet potato hydrolysate. The bioethanol wastewater contains COD, 34,000 ± 1000 mg/l; total nitrogen (TN), 179 ± 5 mg/l; total phosphorous (TP), 105 ± 10 mg/l; reducing sugar, 1.6 ± 0.1 g/l; pH 2.5–3.0. Wastewater with different COD was prepared by diluting the original wastewater with distilled water. A solution of 600 g/l glucose was used in fed-batch processes. All media were sterilized by autoclaving at 115 °C for 20 min.
where YX/S is the biomass yield (g/g substrate); rx and rs are the weight fractions of carbon in biomass and substrate, respectively; and cx and cs are the reductance degrees of biomass and substrate, respectively. On the basis of the definition of YL/X, Eq. (3) was obtained
2.2. Culture conditions
g¼
The yeast was grown for 28 h at 30 °C with rotation speed 180 r/min, and then inoculated 5–15% (v/v) into 50 ml of bioethanol wastewater in 250-ml flasks and cultured for 5 days. To determine an appropriate substrate feeding strategy, three different modes were examined. The first feeding mode added 0.1 ml glucose solution into the culture each day on the first three days. The second and third feeding modes added 0.1 and 0.2 ml glucose solution into the culture on each of the last three days of the fermentation, respectively. Aliquots were withdrawn every 24 h for analysis of residual reducing sugar, nitrogen, pH, cell biomass, lipid content and COD.
where S (g/l) and X (g/l) are the substrate consumed by the microorganism and the biomass obtained per liter of broth, respectively. The numerator of Eq. (3) is the amount of available electrons in the biomass, while the denominator is the amount of available electrons in the substrate. A new parameter, rxmol, was defined as the molal concentration of carbon contained in 1 g of biomass. Thus, rxmol could be determined by Eq. (4)
2.3. Analytic methods
cx rx ¼ 0:0255Y L=X þ 1:6653
Reducing sugar was determined using the 3,5-dinitrosalycylic acid (DNS) method (Miller, 1959). The total nitrogen of the medium was determined by the Kjeldahl method. COD was measured by K2Cr2O7 method (Xue et al., 2006). A pH meter was used to determine the pH of the culture. Cells were harvested by centrifugation (4600g, 5 min), washed twice with distilled water, and dried at 105 °C to constant weight to obtain biomass (g/l). Full-wave scanning of the wastewater was carried out with a UV-1800 spectrophotometer (Mapada, Shanghai, China).
By combining Eqs. (3)–(5), the molal concentration of available electrons in the biomass (mx, mol/l) was determined by Eq. (6)
cx ¼ 4 þ p 2n 3q:
ð1Þ
Biomass energetic yield (g) was the ratio of the heat produced by oxidation of the biomass to that of the substrate utilized in the fermentation process, where the oxidation resulted in the production of CO2, H2O and NH3. According to Eroshin and Krylova (1983), microbial growth energetic yield was calculated with Eq. (2)
g¼
cx rx Y : cs rs X=S
ð2Þ
cx rx X : cs rs S
ð3Þ
rxmol ¼ rx =12:
ð4Þ
According to Zhou et al. (2012), there is a liner relationship between cxrx and lipid content as stated in Eq. (5):
ð5Þ
mx ¼ ð0:0255Y L=X þ 1:6653Þ X=12:
ð6Þ
According to the definition of COD, the molal concentration of available electrons in the substrate (ms, mol/l) was determined by Eq. (7)
ms ¼ COD=8000:
ð7Þ
When Eqs. (6) and (7) were combined, Eq. (8) was established 2.4. Analysis of lipid and fatty acid composition
gx ¼ ð0:0255Y L=X þ 1:6653Þ Total lipid was extracted according to Li et al. (2001). To determine fatty acid composition, 0.1 ml of lipid extracted from the cells was transmethylated in 0.6 ml 0.1% KOH in methanol at 65 °C for 90 min followed by the addition of 1 ml hexane. The organic layer was washed with distilled water. The fatty acid methyl esters were analyzed using a QP2010 gas chromatographer-mass spectrometer (GC–MS) (Shimadzu; Japan) equipped with an Rtx-5 Sil MS column (30 m 0.25 mm 0.25 lm; Restekcorp; America) (Lu et al., 2009) with helium as carrier gas at a flow rate of 40 ml/min, interface temperature of 250 °C, ion source temperature of 200 °C and a segregation ratio of 1:44. The profile of the column temperature was as follows: initial temperature 100 °C, raised from 100 to 180 °C at 10 °C/min, maintained for 20 min; raised to 220 °C at 5 °C/min, maintained for 2 min; finally, raised to 250 °C at 30 °C/ min, maintained for 5 min.
X 12
ðCOD=8000Þ:
ð8Þ
Considering the glucose added to the culture and its oxidation to CO2 and H2O (Eq. (9)), the COD generated by glucose (CODg, mg/l) was determined by Eq. (10). Thus, Eq. (8) could be also written as Eq. (11)
C6 H12 O6 þ 6O2 ¼ 6CO2 þ 6H2 O
ð9Þ
CODg ¼ C g 1000 192=180
ð10Þ
gx ¼ ð0:0255Y L=X þ 1:6653Þ
X 12
½ðCOD þ C g 1000 192=180Þ=8000:
ð11Þ
where Cg is the glucose added into the broth (g/l). 3. Results and discussion
2.5. Calculation of biomass energetic yield
3.1. Effects of initial COD, pH and inoculum concentration
The reductance degree (c) of organic substance with a chemical formula CHpOnNq was calculated based on Eq. (1) (Pan and Rhee,
Bioethanol wastewater is rich in metabolites of Saccharomyces cerevisiae and the pH is as low as 2.5. The data of Table 1 showed
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3.2. Effects of substrate feeding strategies According to previous studies, high C:N ratio accelerated lipid accumulation of oleaginous microorganisms (Chi et al., 2011; Li
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9 COD degradation ratio Biomass Lipid content
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that the initial COD concentration had a significant effect on biomass. As the COD of the wastewater was diluted from 34,000 mg/l to 4250 mg/l, the COD degradation ratio dropped by 37.6% and biomass decreased by 74.6%, but the lipid content reached a maximum of 27.7% when the initial COD of the wastewater was 8500 mg/l. At an initial COD 4250 mg/l, the lipid content decreased to 17.9%. Overall, bioethanol wastewater with a high COD had no negative influence on growth of the yeast. In order to obtain a high COD degradation ratio and a low outlet COD concentration, an initial COD of 17,000 mg/l was adopted. R. toruloides Y2 was able to grow at pH 3.0–9.0 and a relatively stable COD degradation ratio, lipid content and biomass was observed in this pH range (Fig. 1). The COD degradation ratio was almost the same, but biomass and lipid content were different when inoculums of 5–15% (v/v) were used (Fig. 2). Although the biomass increased to 4.9 g/l, the lipid content of the cultures inoculated at 15% was as low as 15%, much lower than the 34.7% achieved with 5% inoculums. The lipid productions inoculated at inoculums 5%, 10% and 15% were 1.4, 1.2 and 0.7 g/l, respectively. Therefore, an inoculation level of 5% was chosen. Growth of R. toruloides Y2 in wastewater displayed a lag phase from 0 to 24 h, an exponential phase from 24 to 72 h and a relatively stationary phase after 72 h (Fig. 3). The lipid content reached a maximum at 72 h and COD degradation ratio reached a maximum at 96 h (Fig. 4a). Different from the study of Xue et al. (2008) who used monosodium glutamate wastewater to culture Rhodotorula glutinis, release of intracellular products into the broth was not observed in this study because COD degradation ratio was stable at 70% after 72 h. The pH of the medium increased from 4.0 to 8.3, perhaps due to the utilization of acidic compounds by R. toruloides Y2. From full-wavelength spectrogram of treated and original wastewater (Fig. 5), it can be seen that compounds absorbing at 200–400 nm were degraded by the yeast. Bioethanol fermentation with carbohydrate can generate organic acids, such as pyruvic, a-ketoglutaric, succinic and lactic acids, which maximally absorb light at 200–215 nm (Yuan et al., 2009). The reducing sugar content of the wastewater decreased in the first 24 h and then remained at about 0.5 g/l after 72 h, suggesting that the yeast could not use a fraction of the sugar (Fig. 4a–d). The cultures were nitrogen-sufficient as indicated by a residual nitrogen concentration of 107 mg/l in the 120-h wastewater sample. Under optimal culture conditions of initial COD, 17,000 mg/l; pH 4.0; inoculation level, 5% (v/v), lipid production reached 1.3 g/l which was 30.0% higher than the maximum obtained before condition optimization (Table 1). A few studies on oleaginous yeasts employed in wastewater treatment without added nutrients have previously been carried out, but the lipid content obtained in the present study was 2–3 and four times that of Lipomyces starkeyi and R. glutinis, respectively, when cultured in monosodium glutamate wastewater (Liu et al., 2012; Xue et al., 2006).
COD degradation ratio (%), Lipid content (%)
W. Zhou et al. / Bioresource Technology 127 (2013) 435–440
5%
10%
15%
0
Inoculum concentration (v/v) Fig. 2. Effect of inoculum concentration on COD degradation ratio, biomass and lipid content of R. toruloides Y2 grown in bioethanol wastewater.
et al., 2007; Xue et al., 2008). The bioethanol wastewater was nitrogen-sufficient, but carbon-deficient, which goes against lipid accumulation. There are some studies which added glucose in industrial wastewater to culture oleaginous yeasts (Liu et al., 2012; Xue et al., 2008). However, the COD degradation ratios were low because of the catabolite repression. It is necessary to supply the carbon resource in such a way as to prevent catabolite repression. In the present study, experiments were carried out to investigate effects of timing and glucose concentration on the treatment of the bioethanol wastewater and the growth of the yeast (Table 2). When 1.2 g/(l d) glucose was supplemented during last three days, biomass and lipid production increased by 39.5% and 53.8%, respectively. Higher biomass levels, lipid contents and COD degradation ratios were obtained by feeding glucose during the last three days (entry 3) than when the glucose was added during
Table 1 Effect of initial COD on final COD, COD degradation ratio, lipid content and biomass of R. toruloides Y2.a Initial COD (mg/l)
Final COD (mg/l)
COD degradation ratio (%)
Lipid content (%)
Biomass (g/l)
Lipid production (g/l)
34,000 17,000 8500 4250
9636 ± 582 5578 ± 237 3555 ± 147 2349 ± 80
71.7 ± 1.7 67.2 ± 1.4 58.2 ± 1.7 44.7 ± 1.9
15.5 ± 0.7 25.4 ± 1.0 27.7 ± 0.5 17.9 ± 0.2
6.7 ± 0.2 3.9 ± 0.1 2.2 ± 0.2 1.7 ± 0.2
1.0 ± 0.04 1.0 ± 0.02 0.6 ± 0.02 0.3 ± 0.01
Values represent averages of data from three parallel samples. a The culture conditions were: temperature, 30 °C; rotational speed, 180 r/min; inoculum concentration, 10% (v/v); pH 4.0; culture time, 5 d.
W. Zhou et al. / Bioresource Technology 127 (2013) 435–440
5.0
50 Lipid content Biomass
4.0
35
3.5
30
3.0
25
2.5
20
2.0
15
1.5
10
1.0
5
0.5 80
100
0.0 200
120
300
Culture time (h) Fig. 3. Biomass and lipid content during growth of R. toruloides Y2 in bioethanol wastewater.
the first three days (entry 2), presumably because most organic substances were consumed by the yeast during the first two days of fermentation. Although adding higher amounts of glucose increased the lipid content by 12.9% (Table 2), there was little effect on biomass and a negative effect on the COD degradation ratio as a decrease by 18.2% was observed. The nitrogen consumption ratio was higher when glucose was added (Fig. 4b–d). 3.3. Energy efficiency evaluation of the fermentation Previous studies indicated that R. toruloides was able to accumulate lipid as high as 70–80% of dry cell weight and the biomass
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energetic yield and the combustion heat of the cells could reach more than 80% and 33 kJ/g when using glucose as carbon source (Zhou et al., 2012). R. toruloides Y2 converted more than 50% of the energy contained in the wastewater into biomass energy (Fig. 6). The biomass energetic yields of different cultures are compared in Fig. 6. The highest yield of 64.3% was achieved when adding glucose at 1.2 g/(l d) to a culture growing in bioethanol wastewater during the last three days (group 3). Saccharomyces cerevisiae-1 and R. toruloides Y2 had a stronger ability to recover the energy contained in wastewater than other yeasts. This type of energy evaluation can supply important information for strain selection and optimization of the fermentation process.
10
160
0
500
Fig. 5. UV Spectrum of original and treated bioethanol wastewater.
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(a)
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Nitrogen COD degradation ratio
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45
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438
10 0
Fig. 4. Reducing sugar concentration, pH, nitrogen concentration and COD degradation ratio during growth of R. toruloides Y2 in bioethanol wastewater.
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W. Zhou et al. / Bioresource Technology 127 (2013) 435–440 Table 2 COD degradation ratio, biomass and lipid content of R. toruloides Y2 grown in bioethanol wastewater under different glucose feeding strategies.a Entry b
1 2c 3d 4e
COD degradation ratio (%)
Lipid content (%)
Biomass (g/l)
Lipid production (g/l)
72.3 ± 3.4 62.5 ± 2.6 66.0 ± 2.3 54.0 ± 3.0
34.9 ± 1.0 31.3 ± 0.2 37.2 ± 0.7 42.0 ± 1.5
3.8 ± 0.1 4.8 ± 0.3 5.3 ± 0.2 5.8 ± 0.1
1.3 ± 0.03 1.5 ± 0.03 2.0 ± 0.01 2.4 ± 0.02
Values represent averages of data from three parallel samples. a The culture conditions were: temperature, 30 °C; rotational speed, 180 r/min; inoculum concentration, 5% (v/v); initial COD, 17,000 mg/l; pH 4.0; culture time, 5 d. b No glucose was added. c Glucose at 1.2 g/(l d) was added during the first three days. d Glucose at 1.2 g/(l d) was added during the last three days. e Glucose at 2.4 g/(l d) was added during the last three days.
L. oil and lower than what has been determined for rapeseed oil (Li et al., 2006; Lu et al., 2009). The combustion heat of the cells was 24.7 kJ/g, which makes them an ideal material for high-energy feed (Zhou et al., 2012). The lipid content of the biomass (37.2%) obtained from the wastewater reached the level of those from other common oil materials such as rapeseed and J. curcas L. (Li et al., 2006; Lu et al., 2009).
70
Biomass energy yield (%)
60 50 40 30
4. Conclusions 20 10 0
1
2
3
4
5
6
7
8
9
Group Fig. 6. Comparison of the biomass energetic yield of several oleaginous yeasts using wastewater as culture medium.
3.4. Composition of the fatty acids in R. toruloides Y2 grown in bioethanol wastewater The average fatty acid composition of the lipids is listed in Table 3. Oleic acid (18:1) was the most abundant acid followed by stearic (18:0), linoleic (18:2) and palmitoleic acid (16:1). These four fatty acids accounted for over 90% of the total fatty acids. This profile is similar to that of Jatropha curcas L., Cryptococcus curvatus and R. toruloides Y4 oli (Table 3). Unsaturated fatty acids (UFAs) accounted for 75.8%, which is comparable to what is found in J. curcas Table 3 Fatty acid composition of Rhodosporidium toruloides Y2, Jatropha curcas L., rapeseed, Cryptococcus curvatus, and Rhodosporidium toruloides Y4 oils. Fatty acid
Jatropha curcas L. oil (Lu et al., 2009)
Rapeseed oil (Li et al., 2006)
Cryptococcus curvatus (Liang et al., 2010)
Rhodosporidium toruloides Y4 (Zhao et al., 2011)
This work
C12:0 C14:0 C16:0 C16:1 C18:3 C18:2 C18:1 C18:0 C20:1 C22:1 C22:0 C26:0 UFAs (%) Lipid content (%)
– 0.1 19.0 1.2 – 38.4 35.3 5.6 – – – 0.4 74.9 39.8
– – 3.6 0.1 13.6 15.5 14.5 1.1 – 48.0 – – 91.7 33.4
– – 23.0 0.9 0.7 15.2 39.6 16.7 – – – – 56.4 52.9
– 2.6 35.1 2.1 0.3 2.9 49.6 6.6 – – – – 54.9 58.6
0.1 1.4 0.9 11.2 – 13.6 49.9 16.9 1.0 0.1 1.5 3.6 75.8 37.2
‘‘–’’ Standing for trace or none.
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Xue, F.Y., Gao, B., Zhu, Y.Q., Zhang, X., Feng, W., Tan, T.W., 2010. Pilot-scale production of microbial lipid using starch wastewater as raw material. Bioresour. Technol. 101, 6092–6095. Yousuf, A., Sannino, F., Addorisio, V., Pirozzi, D., 2010. Microbial conversion of olive oil mill wastewaters into lipids suitable for biodiesel production. J. Agric. Food Chem. 58, 8630–8635. Yuan, W.J., Kong, L., Zi, L.H., Bai, F.W., 2009. Simultaneous determination of ethanol, sugar and organic acids in Kluveromyces marxious broth by high performance liquid chromatography–ultra violet/refractive index detector. Chin. J. Anal. Chem. 37, 850–854. Zhao, X., Hu, C.M., Wu, S.G., Shen, H.W., Zhao, Z.B.K., 2011. Lipid production by Rhodosporidium toruloides Y4 using different substrate feeding strategies. J. Ind. Microbiol. Biotechnol. 38, 627–632. Zhou, W.W., Li, Y.H., Zhang, Y.K., Zhao, Z.B., 2012. Energy efficiency evaluation of lipid production by oleaginous yeast Rhodosporidium toruloides. J. Therm. Anal. Calorim. 108, 119–126.
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Lipid production by Rhodosporidium toruloides Y2 in bioethanol wastewater and evaluation of biomass energetic yield Wenwen Zhou, Wenrui Wang, Yonghong Li ⇑, Yongkui Zhang ⇑ Department of Pharmaceutical and Biological Engineering, School of Chemical Engineering, University of Sichuan, Chengdu 610065, China
h i g h l i g h t s " R. toruloides Y2 was cultured in bioethanol wastewater for lipid production. " Biomass energetic yield was evaluated by biomass, lipid content and substrate COD. " A method was chosen to accelerate lipid production and avoid catabolite repression.
a r t i c l e
i n f o
Article history: Received 21 March 2012 Received in revised form 4 August 2012 Accepted 20 September 2012 Available online 28 September 2012 Keywords: Microbial lipid Rhodosporidium toruloides Bioethanol wastewater Energy efficiency Biomass energetic yield
a b s t r a c t The oleaginous yeast Rhodosporidium toruloides Y2 was employed to remove waste nutrients from bioethanol wastewater while simultaneously producing biomass enriched in microbial lipids. Under optimal conditions, the COD degradation ratio, biomass and lipid content reached 72.3%, 3.8 g/l and 34.9%, respectively. For accelerating biomass and lipid accumulation, different feeding strategies of substrate were conducted. The biomass and lipid production increased by 39.5% and 53.8%, respectively, when glucose at 1.2 g/(l d) was added during the last three days of the cultivation. An equation was established to estimate biomass energetic yield. Under optimal conditions, the biomass energetic yield was 50.9% and an increase of 26.0% was obtained by feeding glucose at 1.2 g/(l d) during the last three days. The fatty acid composition of the lipids was similar to that from plant oils and other microbial lipids, and could thus be used as raw material for feed additives and biodiesel production. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction The bottle neck for the industrialization of biodiesel is the high cost of its raw material which accounts for 70–85% of total cost (Yousuf et al., 2010). Microbial lipids are being explored as raw materials of the production of biodiesel and functional oils (Minkevich et al., 2010; Papanikolaou and Aggelis, 2011). An important advantage offered by the application of the oleaginous microorganisms is their ability to produce lipids from waste organic matters. Consequently, to optimize the cost of the process, as well as to increase its environmental benefit, some waste materials had been studied as feedstock for single-cell oil production by fermentation with oleaginous microorganisms (Chi et al., 2011; Chinnasamy et al., 2010; Pittman et al., 2011; Xue et al., 2010), but more waste streams remain to be investigated for cost-effective microbial lipid production. Wastewater from bioethanol manufacturing is difficult to treat (Li et al., 2009). Although upflow anaerobic sludge blanket (UASB) ⇑ Corresponding authors. Tel./fax: +86 028 85405221 (Y. Li); +86 028 85408255 (Y. Zhang). E-mail addresses: [email protected] (Y. Li), [email protected] (Y. Zhang). 0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2012.09.067
and aerobic activated sludge process can remove 99% of chemical oxygen demand (COD), five-day biochemical oxygen demand (BOD5) and suspended solids (SS) and produce biogas (Liu et al., 2011b), they require start-up periods of up to 3 months and produce sludge (Liu et al., 2011a). Therefore, it is desirable to develop an efficient and economical treatment approach for bioethanol wastewater. Therefore, the present study aimed to: (1) recycle the energy contained in bioethanol wastewater and transfer it into lipidenriched cells by an oleaginous yeast Rhodosporidium toruloides Y2, (2) develop a method to evaluate the energy yield of the fermentation, (3) investigate the effects of different glucose feeding strategies on the treatment of the wastewater, (4) analyze the fatty acid composition of the lipids to estimate their potential application for the production of biodiesel and feed additives. 2. Methods 2.1. Organism, media and chemicals Yeast extract (containing 3.0% ammonium-N and 9.0% total nitrogen) and peptone (containing 3.0% ammonium-N and 14.5%
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1986). cx represents the number of available electrons per carbon atom in the biomass
total nitrogen) were obtained from Aoboxing Bio-tech. Co., Ltd. (Beijing, China). All other chemicals and reagents were in analytical reagent (AR) grade. The oleaginous yeast, R. toruloides Y2, originally from the China General Microbiological Culture Collection Center was maintained at 4 °C on YPD agar slants with glucose, 20 g/l; peptone, 10 g/l; yeast extract, 10 g/l; agar, 20 g/l. The yeast was grown in YPD liquid medium containing glucose, 20 g/l; peptone, 10 g/l; yeast extract, 10 g/l; pH 6.0. The bioethanol wastewater was provided by Sichuan Guanghe Energy technology Co., Ltd. (Chengdu, China) which produces bioethanol from sweet potato hydrolysate. The bioethanol wastewater contains COD, 34,000 ± 1000 mg/l; total nitrogen (TN), 179 ± 5 mg/l; total phosphorous (TP), 105 ± 10 mg/l; reducing sugar, 1.6 ± 0.1 g/l; pH 2.5–3.0. Wastewater with different COD was prepared by diluting the original wastewater with distilled water. A solution of 600 g/l glucose was used in fed-batch processes. All media were sterilized by autoclaving at 115 °C for 20 min.
where YX/S is the biomass yield (g/g substrate); rx and rs are the weight fractions of carbon in biomass and substrate, respectively; and cx and cs are the reductance degrees of biomass and substrate, respectively. On the basis of the definition of YL/X, Eq. (3) was obtained
2.2. Culture conditions
g¼
The yeast was grown for 28 h at 30 °C with rotation speed 180 r/min, and then inoculated 5–15% (v/v) into 50 ml of bioethanol wastewater in 250-ml flasks and cultured for 5 days. To determine an appropriate substrate feeding strategy, three different modes were examined. The first feeding mode added 0.1 ml glucose solution into the culture each day on the first three days. The second and third feeding modes added 0.1 and 0.2 ml glucose solution into the culture on each of the last three days of the fermentation, respectively. Aliquots were withdrawn every 24 h for analysis of residual reducing sugar, nitrogen, pH, cell biomass, lipid content and COD.
where S (g/l) and X (g/l) are the substrate consumed by the microorganism and the biomass obtained per liter of broth, respectively. The numerator of Eq. (3) is the amount of available electrons in the biomass, while the denominator is the amount of available electrons in the substrate. A new parameter, rxmol, was defined as the molal concentration of carbon contained in 1 g of biomass. Thus, rxmol could be determined by Eq. (4)
2.3. Analytic methods
cx rx ¼ 0:0255Y L=X þ 1:6653
Reducing sugar was determined using the 3,5-dinitrosalycylic acid (DNS) method (Miller, 1959). The total nitrogen of the medium was determined by the Kjeldahl method. COD was measured by K2Cr2O7 method (Xue et al., 2006). A pH meter was used to determine the pH of the culture. Cells were harvested by centrifugation (4600g, 5 min), washed twice with distilled water, and dried at 105 °C to constant weight to obtain biomass (g/l). Full-wave scanning of the wastewater was carried out with a UV-1800 spectrophotometer (Mapada, Shanghai, China).
By combining Eqs. (3)–(5), the molal concentration of available electrons in the biomass (mx, mol/l) was determined by Eq. (6)
cx ¼ 4 þ p 2n 3q:
ð1Þ
Biomass energetic yield (g) was the ratio of the heat produced by oxidation of the biomass to that of the substrate utilized in the fermentation process, where the oxidation resulted in the production of CO2, H2O and NH3. According to Eroshin and Krylova (1983), microbial growth energetic yield was calculated with Eq. (2)
g¼
cx rx Y : cs rs X=S
ð2Þ
cx rx X : cs rs S
ð3Þ
rxmol ¼ rx =12:
ð4Þ
According to Zhou et al. (2012), there is a liner relationship between cxrx and lipid content as stated in Eq. (5):
ð5Þ
mx ¼ ð0:0255Y L=X þ 1:6653Þ X=12:
ð6Þ
According to the definition of COD, the molal concentration of available electrons in the substrate (ms, mol/l) was determined by Eq. (7)
ms ¼ COD=8000:
ð7Þ
When Eqs. (6) and (7) were combined, Eq. (8) was established 2.4. Analysis of lipid and fatty acid composition
gx ¼ ð0:0255Y L=X þ 1:6653Þ Total lipid was extracted according to Li et al. (2001). To determine fatty acid composition, 0.1 ml of lipid extracted from the cells was transmethylated in 0.6 ml 0.1% KOH in methanol at 65 °C for 90 min followed by the addition of 1 ml hexane. The organic layer was washed with distilled water. The fatty acid methyl esters were analyzed using a QP2010 gas chromatographer-mass spectrometer (GC–MS) (Shimadzu; Japan) equipped with an Rtx-5 Sil MS column (30 m 0.25 mm 0.25 lm; Restekcorp; America) (Lu et al., 2009) with helium as carrier gas at a flow rate of 40 ml/min, interface temperature of 250 °C, ion source temperature of 200 °C and a segregation ratio of 1:44. The profile of the column temperature was as follows: initial temperature 100 °C, raised from 100 to 180 °C at 10 °C/min, maintained for 20 min; raised to 220 °C at 5 °C/min, maintained for 2 min; finally, raised to 250 °C at 30 °C/ min, maintained for 5 min.
X 12
ðCOD=8000Þ:
ð8Þ
Considering the glucose added to the culture and its oxidation to CO2 and H2O (Eq. (9)), the COD generated by glucose (CODg, mg/l) was determined by Eq. (10). Thus, Eq. (8) could be also written as Eq. (11)
C6 H12 O6 þ 6O2 ¼ 6CO2 þ 6H2 O
ð9Þ
CODg ¼ C g 1000 192=180
ð10Þ
gx ¼ ð0:0255Y L=X þ 1:6653Þ
X 12
½ðCOD þ C g 1000 192=180Þ=8000:
ð11Þ
where Cg is the glucose added into the broth (g/l). 3. Results and discussion
2.5. Calculation of biomass energetic yield
3.1. Effects of initial COD, pH and inoculum concentration
The reductance degree (c) of organic substance with a chemical formula CHpOnNq was calculated based on Eq. (1) (Pan and Rhee,
Bioethanol wastewater is rich in metabolites of Saccharomyces cerevisiae and the pH is as low as 2.5. The data of Table 1 showed
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3.2. Effects of substrate feeding strategies According to previous studies, high C:N ratio accelerated lipid accumulation of oleaginous microorganisms (Chi et al., 2011; Li
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that the initial COD concentration had a significant effect on biomass. As the COD of the wastewater was diluted from 34,000 mg/l to 4250 mg/l, the COD degradation ratio dropped by 37.6% and biomass decreased by 74.6%, but the lipid content reached a maximum of 27.7% when the initial COD of the wastewater was 8500 mg/l. At an initial COD 4250 mg/l, the lipid content decreased to 17.9%. Overall, bioethanol wastewater with a high COD had no negative influence on growth of the yeast. In order to obtain a high COD degradation ratio and a low outlet COD concentration, an initial COD of 17,000 mg/l was adopted. R. toruloides Y2 was able to grow at pH 3.0–9.0 and a relatively stable COD degradation ratio, lipid content and biomass was observed in this pH range (Fig. 1). The COD degradation ratio was almost the same, but biomass and lipid content were different when inoculums of 5–15% (v/v) were used (Fig. 2). Although the biomass increased to 4.9 g/l, the lipid content of the cultures inoculated at 15% was as low as 15%, much lower than the 34.7% achieved with 5% inoculums. The lipid productions inoculated at inoculums 5%, 10% and 15% were 1.4, 1.2 and 0.7 g/l, respectively. Therefore, an inoculation level of 5% was chosen. Growth of R. toruloides Y2 in wastewater displayed a lag phase from 0 to 24 h, an exponential phase from 24 to 72 h and a relatively stationary phase after 72 h (Fig. 3). The lipid content reached a maximum at 72 h and COD degradation ratio reached a maximum at 96 h (Fig. 4a). Different from the study of Xue et al. (2008) who used monosodium glutamate wastewater to culture Rhodotorula glutinis, release of intracellular products into the broth was not observed in this study because COD degradation ratio was stable at 70% after 72 h. The pH of the medium increased from 4.0 to 8.3, perhaps due to the utilization of acidic compounds by R. toruloides Y2. From full-wavelength spectrogram of treated and original wastewater (Fig. 5), it can be seen that compounds absorbing at 200–400 nm were degraded by the yeast. Bioethanol fermentation with carbohydrate can generate organic acids, such as pyruvic, a-ketoglutaric, succinic and lactic acids, which maximally absorb light at 200–215 nm (Yuan et al., 2009). The reducing sugar content of the wastewater decreased in the first 24 h and then remained at about 0.5 g/l after 72 h, suggesting that the yeast could not use a fraction of the sugar (Fig. 4a–d). The cultures were nitrogen-sufficient as indicated by a residual nitrogen concentration of 107 mg/l in the 120-h wastewater sample. Under optimal culture conditions of initial COD, 17,000 mg/l; pH 4.0; inoculation level, 5% (v/v), lipid production reached 1.3 g/l which was 30.0% higher than the maximum obtained before condition optimization (Table 1). A few studies on oleaginous yeasts employed in wastewater treatment without added nutrients have previously been carried out, but the lipid content obtained in the present study was 2–3 and four times that of Lipomyces starkeyi and R. glutinis, respectively, when cultured in monosodium glutamate wastewater (Liu et al., 2012; Xue et al., 2006).
COD degradation ratio (%), Lipid content (%)
W. Zhou et al. / Bioresource Technology 127 (2013) 435–440
5%
10%
15%
0
Inoculum concentration (v/v) Fig. 2. Effect of inoculum concentration on COD degradation ratio, biomass and lipid content of R. toruloides Y2 grown in bioethanol wastewater.
et al., 2007; Xue et al., 2008). The bioethanol wastewater was nitrogen-sufficient, but carbon-deficient, which goes against lipid accumulation. There are some studies which added glucose in industrial wastewater to culture oleaginous yeasts (Liu et al., 2012; Xue et al., 2008). However, the COD degradation ratios were low because of the catabolite repression. It is necessary to supply the carbon resource in such a way as to prevent catabolite repression. In the present study, experiments were carried out to investigate effects of timing and glucose concentration on the treatment of the bioethanol wastewater and the growth of the yeast (Table 2). When 1.2 g/(l d) glucose was supplemented during last three days, biomass and lipid production increased by 39.5% and 53.8%, respectively. Higher biomass levels, lipid contents and COD degradation ratios were obtained by feeding glucose during the last three days (entry 3) than when the glucose was added during
Table 1 Effect of initial COD on final COD, COD degradation ratio, lipid content and biomass of R. toruloides Y2.a Initial COD (mg/l)
Final COD (mg/l)
COD degradation ratio (%)
Lipid content (%)
Biomass (g/l)
Lipid production (g/l)
34,000 17,000 8500 4250
9636 ± 582 5578 ± 237 3555 ± 147 2349 ± 80
71.7 ± 1.7 67.2 ± 1.4 58.2 ± 1.7 44.7 ± 1.9
15.5 ± 0.7 25.4 ± 1.0 27.7 ± 0.5 17.9 ± 0.2
6.7 ± 0.2 3.9 ± 0.1 2.2 ± 0.2 1.7 ± 0.2
1.0 ± 0.04 1.0 ± 0.02 0.6 ± 0.02 0.3 ± 0.01
Values represent averages of data from three parallel samples. a The culture conditions were: temperature, 30 °C; rotational speed, 180 r/min; inoculum concentration, 10% (v/v); pH 4.0; culture time, 5 d.
W. Zhou et al. / Bioresource Technology 127 (2013) 435–440
5.0
50 Lipid content Biomass
4.0
35
3.5
30
3.0
25
2.5
20
2.0
15
1.5
10
1.0
5
0.5 80
100
0.0 200
120
300
Culture time (h) Fig. 3. Biomass and lipid content during growth of R. toruloides Y2 in bioethanol wastewater.
the first three days (entry 2), presumably because most organic substances were consumed by the yeast during the first two days of fermentation. Although adding higher amounts of glucose increased the lipid content by 12.9% (Table 2), there was little effect on biomass and a negative effect on the COD degradation ratio as a decrease by 18.2% was observed. The nitrogen consumption ratio was higher when glucose was added (Fig. 4b–d). 3.3. Energy efficiency evaluation of the fermentation Previous studies indicated that R. toruloides was able to accumulate lipid as high as 70–80% of dry cell weight and the biomass
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energetic yield and the combustion heat of the cells could reach more than 80% and 33 kJ/g when using glucose as carbon source (Zhou et al., 2012). R. toruloides Y2 converted more than 50% of the energy contained in the wastewater into biomass energy (Fig. 6). The biomass energetic yields of different cultures are compared in Fig. 6. The highest yield of 64.3% was achieved when adding glucose at 1.2 g/(l d) to a culture growing in bioethanol wastewater during the last three days (group 3). Saccharomyces cerevisiae-1 and R. toruloides Y2 had a stronger ability to recover the energy contained in wastewater than other yeasts. This type of energy evaluation can supply important information for strain selection and optimization of the fermentation process.
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Fig. 5. UV Spectrum of original and treated bioethanol wastewater.
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(a)
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W. Zhou et al. / Bioresource Technology 127 (2013) 435–440 Table 2 COD degradation ratio, biomass and lipid content of R. toruloides Y2 grown in bioethanol wastewater under different glucose feeding strategies.a Entry b
1 2c 3d 4e
COD degradation ratio (%)
Lipid content (%)
Biomass (g/l)
Lipid production (g/l)
72.3 ± 3.4 62.5 ± 2.6 66.0 ± 2.3 54.0 ± 3.0
34.9 ± 1.0 31.3 ± 0.2 37.2 ± 0.7 42.0 ± 1.5
3.8 ± 0.1 4.8 ± 0.3 5.3 ± 0.2 5.8 ± 0.1
1.3 ± 0.03 1.5 ± 0.03 2.0 ± 0.01 2.4 ± 0.02
Values represent averages of data from three parallel samples. a The culture conditions were: temperature, 30 °C; rotational speed, 180 r/min; inoculum concentration, 5% (v/v); initial COD, 17,000 mg/l; pH 4.0; culture time, 5 d. b No glucose was added. c Glucose at 1.2 g/(l d) was added during the first three days. d Glucose at 1.2 g/(l d) was added during the last three days. e Glucose at 2.4 g/(l d) was added during the last three days.
L. oil and lower than what has been determined for rapeseed oil (Li et al., 2006; Lu et al., 2009). The combustion heat of the cells was 24.7 kJ/g, which makes them an ideal material for high-energy feed (Zhou et al., 2012). The lipid content of the biomass (37.2%) obtained from the wastewater reached the level of those from other common oil materials such as rapeseed and J. curcas L. (Li et al., 2006; Lu et al., 2009).
70
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4. Conclusions 20 10 0
1
2
3
4
5
6
7
8
9
Group Fig. 6. Comparison of the biomass energetic yield of several oleaginous yeasts using wastewater as culture medium.
3.4. Composition of the fatty acids in R. toruloides Y2 grown in bioethanol wastewater The average fatty acid composition of the lipids is listed in Table 3. Oleic acid (18:1) was the most abundant acid followed by stearic (18:0), linoleic (18:2) and palmitoleic acid (16:1). These four fatty acids accounted for over 90% of the total fatty acids. This profile is similar to that of Jatropha curcas L., Cryptococcus curvatus and R. toruloides Y4 oli (Table 3). Unsaturated fatty acids (UFAs) accounted for 75.8%, which is comparable to what is found in J. curcas Table 3 Fatty acid composition of Rhodosporidium toruloides Y2, Jatropha curcas L., rapeseed, Cryptococcus curvatus, and Rhodosporidium toruloides Y4 oils. Fatty acid
Jatropha curcas L. oil (Lu et al., 2009)
Rapeseed oil (Li et al., 2006)
Cryptococcus curvatus (Liang et al., 2010)
Rhodosporidium toruloides Y4 (Zhao et al., 2011)
This work
C12:0 C14:0 C16:0 C16:1 C18:3 C18:2 C18:1 C18:0 C20:1 C22:1 C22:0 C26:0 UFAs (%) Lipid content (%)
– 0.1 19.0 1.2 – 38.4 35.3 5.6 – – – 0.4 74.9 39.8
– – 3.6 0.1 13.6 15.5 14.5 1.1 – 48.0 – – 91.7 33.4
– – 23.0 0.9 0.7 15.2 39.6 16.7 – – – – 56.4 52.9
– 2.6 35.1 2.1 0.3 2.9 49.6 6.6 – – – – 54.9 58.6
0.1 1.4 0.9 11.2 – 13.6 49.9 16.9 1.0 0.1 1.5 3.6 75.8 37.2
‘‘–’’ Standing for trace or none.
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