Bioresource Technology 164 (2014) 93–99
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Synergistic effects of oleaginous yeast Rhodotorula glutinis and microalga Chlorella vulgaris for enhancement of biomass and lipid yields Zhiping Zhang, Hairui Ji, Guiping Gong, Xu Zhang ⇑, Tianwei Tan National Energy R&D Center for Biorefinery, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, PR China
h i g h l i g h t s Biomass and lipid yields achieved a theoretical model of 1+1>2 by mixed culture. A double system bubble column photo-bioreactor was designed for mixing cultivation. Growth curves of yeast and alga were confirmed in mixing cultivation system. Real-time online detection of off-gas showed synergistic effects on O2/CO2 balance. Analysis of metabolite variations proved synergistic effects on substance exchange.
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Article history: Received 22 February 2014 Received in revised form 9 April 2014 Accepted 10 April 2014 Available online 21 April 2014 Keywords: Rhodotorula glutinis Chlorella vulgaris Mixed culture Synergistic effects Lipid
a b s t r a c t The optimal mixed culture model of oleaginous yeast Rhodotorula glutinis and microalga Chlorella vulgaris was confirmed to enhance lipid production. A double system bubble column photo-bioreactor was designed and used for demonstrating the relationship of yeast and alga in mixed culture. The results showed that using the log-phase cultures of yeast and alga as seeds for mixed culture, the improvements of biomass and lipid yields reached 17.3% and 70.9%, respectively, compared with those of monocultures. Growth curves of two species were confirmed in the double system bubble column photo-bioreactor, and the second growth of yeast was observed during 36–48 h of mixed culture. Synergistic effects of two species for cell growth and lipid accumulation were demonstrated on O2/CO2 balance, substance exchange, dissolved oxygen and pH adjustment in mixed culture. This study provided a theoretical basis and culture model for producing lipids by mixed culture in place of monoculture. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Oleaginous microorganisms, involving bacterium, yeasts, moulds and algae, have been extensively studied because the microbial lipids were very similar to soybean lipids to promise potential feedstock for biodiesel production (Cheirsilp et al., 2011). Among oleaginous microorganisms, the yeast Rhodotorula glutinis, as a high-yielding lipid strain, could utilize some wastewater for lipid production (Xue et al., 2006, 2010a). In particular, it has been reported that light irradiation could stimulate the synthesis of pigment which resists light damage, and affect cell growth rate and lipid content (Zhang et al., 2014; Yen and Zhang, 2011a; Yen and Yang, 2012). Also, R. glutinis has a great growth rate in aerobic condition wherein large amount of O2 must be required and CO2 be emitted (Li et al., 2007). Yen and Zhang (2011b) reported ⇑ Corresponding author. Address: No. 15 Beisanhuan East Road, Chaoyang District, Beijing 100029, PR China. Tel./fax: +86 10 64448962. E-mail address: [email protected] (X. Zhang). http://dx.doi.org/10.1016/j.biortech.2014.04.039 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
that low dissolved oxygen (DO) could retard cell growth and enhance lipid accumulation. However, under enough nutrients and low DO conditions, partial yeast cells would be anaerobic respiration to produce a large number of volatile organic acids (Chen and Gutmanis, 1976), which decreased the pH value of cultivation system. The optimum initial pH for the growth rate of R. glutinis is 6.0 (Bhosale and Gadre, 2001). Undissociated organic acid could lower intracellular pH following translocation across the yeast plasma membrane so that yeast growth is inhibited (Nguyen et al., 2001). Microalgae are considered as another attractive source for biodiesel production due to their high lipid content, photosynthesis efficiency and CO2 reduction efficiency (Eugenia, 2012; Xiong et al., 2010). Some algal cells can grow not only in photosynthesis system, but also in heterotrophic system or mixotrophy when the culture contains both inorganic and organic substrates (Zhao et al., 2012). Chlorella vulgaris, as a kind of mixotrophy algal species, could utilize the high concentration N, P and other ions of waste-
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water in photosynthesis for lipid production meanwhile release O2 (Li et al., 2010; Liu et al., 2008). Moreover, C. vulgaris grows better at pH 6.5–7.0 and accumulates lipids at pH 7.0–8.5 (Wang et al., 2010). However, when CO2 is consumed by microalgae, the pH becomes more alkaline (Eugenia, 2012). Therefore, the algal growth will be inhibited after a period of culture due to the increasing pH value. In theory, there are synergistic effects on gas, substance exchange and pH adjustment in the mixed culture system of oleaginous yeast R. glutinis and microalga C. vulgaris based on the above mentioned researches. However, the reports of lipid production by mixed culture mainly focused on the different alga species (Su et al., 2012; Fradinho et al., 2013; Olgui9 n et al., 2013; Chen et al., 2014) and the mixed culture of yeast and alga based on the mutually beneficial relationship of gas exchange (Shu et al., 2013; Santos et al., 2013). In this study, one of the aims was to confirm that mixed culture of both microorganisms could significantly enhance biomass and lipid production. In order to demonstrate the synergistic effects of the mixed culture, the authors designed a double-system bubble column photo-bioreactor consisting of an association of two cultivation systems, in which one was used for R. glutinis cultivation and the other was for C. vulgaris. A polyester fabric filter (2.6 lm) was fixed between the two systems, which could achieve the exchange of substances and gases and keep microorganisms grow independently. 2. Methods
surface of flasks. Mixed culture was taken in 500 mL flask or double system bubble column photo-bioreactor using logarithmic phase cultures as seed. The monocultures of yeast and alga at the log-phase with 10% (v/v) were transferred into 260 mL double system bubble column photo-bioreactor containing 100 mL mixed medium for measuring the change of gases and substances (Fig. 1). During the mixed culture, the aeration rate was 0.8 L air/min. Aseptic air filter (0.22 lm) was accessed to the bottom of bioreactor through a pipeline and was dispersed by an air distributor. Temperature was controlled by the insulation layer circulating water from electric-heated thermostatic water bath (MPE-40C, China). The bioreactor was illuminated with white fluorescent light tubes assembled outside surface of bioreactor, which supplied a light intensity of 100 lmol/m2 s. 2.3. Measurement of glucose, biomass and lipid content Glucose concentration was measured using a glucose biosensor (SBA-40C, Biological Institute of Shandong Academy of Sciences). The biomass concentrations of yeast and alga were determined by optical density reading at 600 nm (OD600) and 680 nm (OD680), respectively. A calibration curve of yeast dry cell weight corresponded to OD600 value as follows: Biomass (g/L) = 1.052 OD600 + 0.041, R2 = 0.9982, and that curve of alga at OD680 as follows: Biomass (g/L) = 0.324 OD680 + 0.0099, R2 = 0.9991. Biomass concentration of mixed culture was carried out following the method of Zhao et al. (2012) by cell dry weight. The lipid content was determined by sulfo-phospho-vanillin method (Izard and Limber, 2003).
2.1. Strains 2.4. Measurement of off-gas, dissolved oxygen and pH value The yeast R. glutinis (CGMCC No. 2258) was supplied by the China National Research Institute of Food and Fermentation Industries and kept at Beijing University of Chemical Technology. The strain was maintained on yeast extract, peptone and dextrose (YPD) agar slant at 4 °C. The microalga C. vulgaris was stored in National Energy R&D Center for Biorefinery at Beijing University of Chemical Technology after being provided by Institute of Hydrobiology, Chinese Academy of Sciences. The strain could conduct mixotrophy in the culture containing both inorganic and organic substrates and was maintained on alga medium BG-11 (described below) agar slant with glucose (2 g/L) at 4 °C. 2.2. Culture media and conditions Seed media of R. glutinis and C. vulgaris were described as follow. Yeast medium composition (Xue et al., 2008): glucose 40 g/L, yeast extract 1.5 g/L, (NH4)2SO4 2 g/L, KH2PO4 7 g/L, NaSO4 2 g/L, MgSO47H2O 1.5 g/L. The initial pH was adjusted to 5.5. Alga medium BG-11 composition (Zhao et al., 2012): citric acid 6.0 mg/L, ferric ammonium citrate 6.0 mg/L, EDTA 1.0 mg/L, NaNO3 1.5 g/L, K2HPO42H2O 0.051 g/L, MgSO47H2O 0.075 g/L, CaCl2 0.024 g/L, Na2CO3 0.02 g/L, A5 trace mineral solution 1.0 mL/L. The composition of A5 was: H3BO4 2.86 g/L, MnCl24H2O 1.81 g/L, ZnSO47H2O 0.222 g/L, Na2MoO42H2O 0.391 g/L, CuSO45H2O 0.079 g/L, Co(NO3)26H2O 0.049 g/L. The initial pH was adjusted to 6.5–7.5. The mixotrophic culture medium was the alga medium containing 5 g/L of glucose. Mixed medium were adducts of yeast and alga media adjusting the concentration of glucose to 20 g/L (Xue et al., 2010b). Yeast and alga were respectively incubated from agar slant culture to 500 mL Erlenmeyer flasks containing 100 mL seed media. The flasks were placed in a rotary shaker at 180 rpm, 30 °C and around 80 lmol/m2 s. Light intensity was measured by a light-meter (TES-1339, Taiwan Taishi) on the outside
Real-time online detection of the different gas concentrations (O2, CO2 and N2) in off-gas was performed by using the Industrial Gas Analyzer (MAX300-LG, Extrel, USA). The online dissolved oxygen (DO) and pH value were determined by using DO meter (OXYFERM FDA 225, Hamilton) and pH meter (M-10, AMER), respectively. 2.5. Analysis of extracellular metabolites Extracellular metabolites were analyzed by the method of gas chromatography–mass spectrometry (GC–MS) (GC–MS-QP2010, SHIMADZU, Japan) according to the modified procedure of Coutinho et al. (2013) and Cai et al. (2007). Samples (1 mL) taken from mixed culture and controls were centrifuged at 12,000 rpm for 10 min, then dried the supernatant to get the metabolite powder by centrifugal concentrator (HR/T16MM, China) at 30 °C. A volume (100 lL) of methoxyamine pyridine solution (2 g/L) was added into the metabolite powder for suspension and oximation reaction at 30 °C for 2 h, and then 100 lL of N-MethylN-(trimethylsilyl)trifluoroacetamide (MSTFA) agent was added into the reaction system for derivatization at constant temperature of 30 °C for 6 h. After filtered through filter paper (0.22 lm), sample of 1 lL was injected into GC–MS with a column of 30 mm 0.25 mm 0.25 lm. 3. Results and discussion 3.1. The mixed culture of R. glutinis and C. vulgaris 3.1.1. Directly mixed culture Because microalga grew slower than yeast, the increasing initial amount of algal cells were attempted to mixed culture with constant amount of yeast cells. Even so, the biomass in the mixing
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pH probe
Gas outlet DO probe
Insulation layer
Fluorescent light
Fluorescent light
Thermostatic water Insulation layer Filter plate Sampling port
Sampling port Thermostatic water
Air distributor Gas inlet
Air Filter
Fig. 1. Apparatus of the double system bubble column photo-bioreactor for mixed culture of R. glutinis and C. vulgaris with fluorescent light.
3.1.2. Using the log-phase cultures of yeast and alga for mixed culture As the alga grew slower than the yeast, the directly mixed culture in the initial fermentation was not conducive to photoautotrophic growth of alga and play the maximum capacity for enhancing lipid production. In addition, the light may have hardly penetrated and its intensity attenuated drastically because of the high concentration of yeast cells (Cheirsilp et al., 2011). Therefore, a vibrant growth alga culture at the log-phase using mixed medium was
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cultivation system was mainly from the growth of yeast, which could be inferred to the color of fermentation broth. Thus, the directly mixed culture of R. glutinis and C. vulgaris was compared with monoculture of yeast. According to the different initial inoculums, three groups were taken. The initial inoculums of group I, group II and control group were 3% yeast and 3% alga (v/v), 3% yeast and 6% alga (v/v) and 3% yeast (v/v), respectively. Yeast and alga seed cultures were at the logarithmic phase with OD600 0.4–0.5 of yeast (after 50 times dilution) and OD680 0.5–0.6 of alga (after 10 times dilution). The results were shown in Fig. 2. According to Fig. 2A, it was could be divided to three growth phases. During the first phase (0–65 h), the specific growth rate of mixed culture group I was very similar to that of control group. Although the lower growth of mixed culture group II was observed, the glucose utilization rate was faster. It was possible that the pH was increased up to 7.0, which was not suitable for yeast growth, but pH 7.0 was suitable for lipid synthesis (Bhosale and Gadre, 2001). During the second phase (65–90 h), the growth of control group was into the stable phase, but the biomasses of both mixed culture groups were still increasing in a small amount. A new faster increase of biomass in both mixed culture groups was observed in the third phase. Finally, the maximum biomass concentrations of mixed group I, mixed group II and control were 10.3, 8.7 and 8.1 g/L, respectively. It was obvious that mixed culture could enhance the biomass compared with monoculture of yeast. However, when a higher concentration of biomass was reached, the lipid content and lipid production decreased (Fig. 2B). The maximum lipid content of control was higher than that of two mixed culture groups, measuring 20.8%, 15.1% and 11.8%, respectively. Due to the maximum of biomass obtained in group I, the total amounts of lipid production of group I reached 1.56 g/L, which was similar to control (1.68 g/L). This result was different from previously observed that higher oil production was obtained with a higher seed ratio of microalga to yeast (Shu et al., 2013).
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Time (hour) Fig. 2. Comparison of the growth curve (A), lipid content and total lipid (B) of directly mixed culture of yeast R. glutinis and alga C. vulgaris with monoculture of yeast.
added into the culture of yeast cultured at same conditions. Mixing ratio of volume was 1:1 (100 mL alga and 100 mL yeast into 500 mL flask). The results indicated that the biomass concentration in the initial mixed culture decreased compared with the monoculture of yeast (Fig. 3). This was because of the low biomass concentration in monoculture of alga. Finally, the maximum of biomass
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Fig. 3. Results of the growth curve on equivalent volume by directly mixed culture using the log phase monocultures.
group was the mixed culture group (11.5 g/L) at 120 h, while those of the monocultures of yeast and alga were 9.9 and 2.4 g/L (not shown), respectively. This phenomenon obviously demonstrated that the biomass yield (2.3 g of 200 mL) of mixed culture was higher than those (1.3 g of 200 mL) of adding up to pure yeast (0.99 g of 100 mL) and pure alga (0.24 g of 100 mL). Thus, biomass in the mixed culture system have achieved a theoretical model of 1+1>2 compared with that of monocultures. Furthermore, comparisons of lipid content and lipid production at two different of fermentation times (24 and 48 h) after mixed culture were shown in Table 1. The lipid content of mixed culture group was higher than that of pure alga but lower than that of pure yeast at 24 and 48 h after mixed culture. Even so, the lipid production of mixed culture was also the highest in three groups because of the highest biomass obtained in mixed culture group. Consequently, the lipid production in the mixed cultivation system have also achieved a theoretical model of 1+1>2 compared with that of monocultures.
fermentation broth (organic acids, amino acids, polysaccharides, etc) could be exchanged and detected; and (iii) water-soluble gas can be exchanged and utilized. The results of time course data of individual growth curves of species and lipid formation in the mixing cultivation of R. glutinis and C. vulgaris were shown in Fig. 4 compared with control cultures (a bulkhead replaced filter in the middle of the bioreactor). The yeast was the dominant species during the initial 24 h of cultivation (Fig. 4A). The growth ratio of R. glutinis over that of C. vulgaris reached its maximum at 18.9 g/L after 48 h of cultivation, and it appeared the second growth during 36–48 h. However, it decreased gradually to 17.2 g/L at the end of the cultivation, which revealed that the growth of yeast stopped. On the contrary, the growth ratio of alga remained high and the biomass concentration reached 2.4 g/L at 60 h (Fig. 4C). It was presumed that microalga adapted the culture environment gradually, and the autotrophic metabolism was the major mode at the end of the cultivation (Shu et al., 2013). Considering the yeast was the dominant species in mixing cultivation system and the growth of yeast reached stationary growth at 48 h, the mixed culture was terminated at 60 h. Finally, the maximum biomass concentrations of yeast and alga were 22.3% and 25.2% higher than those of the monocultures, respectively. About lipid content, no obvious difference was observed between mixed culture and pure culture of yeast in the initial 24 h of cultivation (Fig. 4B). Likewise, the alga lipid content of mixed culture compared with that of monoculture was remarkably similar in the whole cultivation (Fig. 4D). Obviously, the increase of alga biomass led to the increase of yeast lipid content in the mixing cultivation at the following time. This might be due to that photoautotrophic culture of alga provided extra substrates for lipid synthesis (Cheirsilp et al., 2011). Consequently, combined with higher biomass concentrations in mixed culture, the lipid productions of two species in the mixing cultivation system were also higher than those of monocultures. Therefore, mixed culture of two species would have symbiotic relationship and synergistic effects, which resulted in more biomass and lipid accumulation as compared with those of the monocultures. 3.3. Synergistic effects on gas, DO and pH value in the mixed culture
3.2. The individual growth curves of two species under mixed culture condition As discussed above, although the fact that biomass concentration and lipid production enhanced by mixed culture of yeast and alga in the same medium was confirmed, little information was available on the individual growth curves of two species and the relationship of alga and yeast in mixed culture (Shu et al., 2013). In this study, a double-system bubble column photo-bioreactor was used for mixing cultivation (Fig. 1). In order to assure that the medium kept the same and uniform on both side of the filter, one gas outlet was used on the top of one cultivation system, while the another one was closed. So, there was a pressure difference between the two cultivation systems. Meanwhile, the gas outlets on two systems were opened and closed on turns every few hours to achieve trans-membrane exchange. The equipment has particular significance in three aspects: (i) the growth of separated microorganisms is easy to be investigated; (ii) the nutrients in Table 1 Results of lipid contents and lipid yields of mixed culture after 24 and 48 h compared with those of monocultures of yeast and alga. X1, X2 and X3 are lipid contents of mixed culture, yeast and alga, respectively. Y1 and Y2 are lipid yields of mixed culture and those of adding up to monocultures of yeast and alga, respectively. Time (h)
X1 (%)
X2 (%)
X3 (%)
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Y2 (g/L)
24 48
18.8 22.4
21.6 26.3
14.7 20.2
2.09 2.58
1.24 1.56
The time course data of different gas concentrations (v/v) in offgas, DO and pH value in mixed culture of R. glutinis and C. vulgaris were shown in Fig. 5. The concentration of O2 in off-gas decreased gradually from 20.4% to 20.1% (Fig. 5A) and that of CO2 increased from 0.04% to 0.15% (Fig. 5B) during the initial 24 h of cultivation, which were resulted from the growth of yeast. However, the concentration of O2 increased gradually from 20.1% to 20.6% during 24–36 h due to the slow growth of yeast (Fig. 4A) and the substantial growth of microalga (Fig. 4C). With the rapid algal growth, the concentrations of both O2 and CO2 in off-gas declined rapidly from 20.6% to 20.0% and 0.27% to 0.15%, respectively, during 36–48 h. It indicated that yeast grew again following with the growth of alga. Moreover, increasing rapidly of concentration of N2 in off-gas pointed that both O2 and CO2 were utilized in the mixing cultivation (Fig. 5C). In particular, the concentrations of three gases reached a dynamic equilibrium during 48–54 h, which demonstrated that there was an O2/CO2 balance for enhancing both of yeast and alga growths. At the end of cultivation, the concentration of O2 increased rapidly due to the stop growth of yeast, while the concentration of CO2 decreased rapidly since the rapid growth of alga with phototrophy. The results are very similar to those reported by Cai et al. (2007) that although the initial growth of the alga is slightly inhibited by the yeast in the mixed culture, the alga can rapidly grow by overcoming the inhibitory effect, and that the two species can adopt well to the coexisting circumstance and build up the beneficial balance of mutualism.
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Control 18
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Time (hour) Fig. 4. Results of the biomass (A), lipid content and total lipid (B) of R. glutinis and the biomass (C), lipid content and total lipid (D) of C. vulgaris in a mixed culture system aerated by 0.8 L air/min in a 260 mL double system bubble column photobioreactor illuminated at 100 lmol/m2 s.
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Time (hour) Fig. 5. Time-course data of O2 (A), CO2 (B) and N2 (C) concentrations (v/v) in off-gas, DO and pH value (D) in a mixed culture system aerated by 0.5 vvm of air in a 260 mL double system bubble column photo-bioreactor illuminated at 100 lmol/ m2 s.
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R. glutinis has a great growth rate in aerobic condition wherein large amount of O2 must be required and CO2 be emitted (Li et al., 2007). In this study, yeast was the dominant species in the beginning of the mixed culture that caused a decline of DO from 100% to 53% (Fig. 5D). However, 0.042–0.130 M of dissolved CO2 was produced in the yeast fermentation, which would lead partial yeast to anaerobic respiration to produce a large number of volatile organic acids (Chen and Gutmanis, 1976). Organic acids synthesized not only wasted nutrients for lipid production, but also acidified the media of pH value from 6.0 to 3.2 during the initial 36 h of mixing cultivation (Fig. 5D). Due to the depletion of nutrients, yeast cells stopped to multiply and alga began to grow rapidly, which resulted in increasing the dissolved oxygen and pH value from 53% to 78% and 3.2 to 4.9, respectively. It was inferred that under the low concentrations of dissolved O2 and the high concentration of dissolved CO2, C. vulgaris would be into autotrophic metabolic that could be improved the level of DO in the mixing cultivation system (Pope, 1975). In addition, bicarbonate (HCO3 ) is formed when CO2 dissolves in water at neutral pH. During microalgae photosynthesis metabolic, HCO3 is converted to CO2 and hydroxide ion (OH ). Hence, when CO2 is consumed by microalgae, the pH becomes more alkaline (Eugenia, 2012). These results indicated that there were synergistic effects on gas exchange, DO and pH adjustment by mixed culture of oleaginous yeast and microalga. It was partially explained to the fact that the mixed culture could eventually lead to a higher productivity. However, the cause of the second growth of yeast is not only synergistic effects on gas exchange, DO and pH adjustment in mixed culture. The alga growth would have some secondary metabolites for stimulating the growth of yeast (Cheirsilp et al., 2011; Xue et al., 2010b). 3.4. Synergistic effects on substance exchange in the mixed culture To characterize the extracellular metabolite variations between monocultures and mixed culture, the mixed culture medium without yeast extract was used to cultivation of R. glutinis and C. vulgaris in 500 mL flask trials for GC–MS analysis. Since the aim of this study was to find the extracellular metabolic changes between monocultures and mixed culture, the samples were collected from monocultures of yeast and alga at the end of cultures, the initial mixed culture and the mixing cultivation for 48 h. Library searches of the mass spectrum were used to identify individual compounds from the chromatograms of samples (threshold P 90%). The results indicated that eleven metabolites were detected in the sample of yeast, while seven metabolites were identified in the sample of alga (not shown). Comparison of the metabolite variations between monocultures of yeast and alga, four different metabolites of yeast including glycerol, acetic acid, glycine, and proline were not detected in the sample of alga, while some other metabolites including glycinamide, acetamide and some derivatives of sugar were observed in the sample of alga but not in the yeast sample. In addition, the content of glycerol ether in the yeast sample was significantly higher than that of alga sample, while the content of palmitic acid in the alga sample was higher than that of yeast sample. As the above results clearly showed that the volatile compounds of monocultures of yeast and alga were different. The GC–MS results of the initial mixed culture sample was compared with those of after mixed culture 48 h. Obviously, the contents of some organic acids (propionic acid, pyruvic and acetic acids) glycidyl ether and palmitic acid were decreased, and the contents of some amino acids (glycine and proline) were increased. These results explained that a new faster increase of biomass in mixed culture groups was observed after mixing cultivation (Fig. 2A). On the one hand, the mixed culture of yeast and alga may alleviate and/or eliminate the stresses caused by CO2 on the yeast and O2 on
the alga to maintain an O2/CO2 balance that enhances the growth of two species. This result was similar to the reports by Cheirsilp et al. (2012) and Shu et al. (2013). On the other hand, the inhibition of yeast growth by organic acids could be alleviated through utilized by alga. Xue et al. (2010b) reported that some of yeast metabolites such as pyruvic and acetic acids might be utilized by microalgae. Moreover, higher palmitic acid content in alga cultivation significantly affects the yeast cell membrane permeability and rigidity (Hama et al., 2004), and that higher cell permeability is conducive to nutrient absorption and utilization. Therefore, based on the synergistic effects on substance exchange in the mixed culture, the biomass and lipid accumulation of two species were enhanced, which were performed on the increasing amino acids contents after mixing cultivation. 4. Conclusion The mixed culture strategy resulted in significant improvements on biomass concentration and lipid productivity, especially using the log-phase cultures of oleaginous yeast and microalga as seeds. The individual growth curves of two species in mixing cultivation system were identified by cultivation in the double system bubble column photo-bioreactor. The second growth of yeast was observed following with the beginning into the log-phase growth of alga. Symbiotic relationships and synergistic effects of two species for cell growth and lipid accumulation were demonstrated on O2/CO2 balance, substance exchange, DO and pH adjustment in the mixed culture system. Acknowledgement Authors acknowledge the financial support from the National High Technology Research and Development 863 Program of China (Grant Nos. 2013AA050702, 2012AA022304). References Bhosale, P., Gadre, R.V., 2001. b-Carotene production in sugarcane molasses by a Rhodotorula glutinis mutant. J. Ind. Microbiol. Biotechnol. 26 (6), 327–332. Cai, S.Q., Hu, C.Q., Du, S.B., 2007. Comparisons of growth and biochemical composition between mixed culture of alga and yeast and monocultures. J. Biosci. Bioeng. 104 (5), 391–397. Cheirsilp, B., Kitcha, S., Torpee, S., 2012. Co-culture of an oleaginous yeast Rhodotorula glutinis and a microalga Chlorella vulgaris for biomass and lipid production using pure and crude glycerol as a sole carbon source. Ann. Microbiol. 62 (3), 987–993. Cheirsilp, B., Suwannarat, W., Niyomdecha, R., 2011. Mixed culture of oleaginous yeast Rhodotorula glutinis and microalga Chlorella vulgaris for lipid production from industrial wastes and its use as biodiesel feedstock. New Biotechnol. 28 (4), 362–368. Chen, S.L., Gutmanis, F., 1976. Carbon dioxide inhibition of yeast growth in biomass production. Biotechnol. Bioeng. 18 (10), 1455–1462. Chen, W.T., Zhang, Y.H., Zhang, J.X., Yu, G., Schideman, L.C., Zhang, P., Minarick, M., 2014. Hydrothermal liquefaction of mixed-culture algal biomass from wastewater treatment system into bio-crude oil. Bioresour. Technol. 152, 130–139. Coutinho, J.O.P.A., Silva, M.P.S., Moraes, P.M., Monteiro, A.S., Barcelos, J.C.C., Siqueira, E.P., Santos, V.L., 2013. Demulsifying properties of extracellular products and cells of Pseudomonas aeruginosa MSJ isolated from petroleum-contaminated soil. Bioresour. Technol. 128, 646–654. Eugenia, J.O., 2012. Dual purpose microalgae-bacterial-based systems that treat wastewater and produce biodiesel and chemical products within a biorefinery. Biotechnol. Adv. 30, 1031–1046. Fradinho, J.C., Oehmen, A., Reis, M.A.M., 2013. Effect of dark/light periods on the polyhydroxyalkanoate production of a photosynthetic mixed culture. Bioresour. Technol. 148, 474–479. Hama, S., Yamaji, H., Kaieda, M., Oda, M., Kondo, A., Fukuda, H., 2004. Effect of fatty acid membrane composition on whole-cell biocatalysts for biodiesel-fuel production. Biochem. Eng. J. 21, 155–160. Izard, J., Limber, R., 2003. Rapid screening method for quantitation of bacterial cell lipids from whole cells. J. Microbiol. Methods 55, 411–418. Li, X., Hu, H.Y., Gan, K., Sun, Y.X., 2010. Effects of different nitrogen and phosphorus concentrations on the growth nutrient uptake, and lipid accumulation of a freshwater microalga Scenedesmus sp.. Bioresour. Technol. 101, 5494–5500.
Z. Zhang et al. / Bioresource Technology 164 (2014) 93–99 Li, Y.H., Zhao, Z.B., Bai, F.W., 2007. High-density cultivation of oleaginous yeast Rhodosporidium toruloides Y4 in fed-batch culture. Enzyme Microb. Technol. 41 (3), 312–317. Liu, Z.Y., Wang, G.C., Zhou, B.C., 2008. Effect of iron on growth and lipid accumulation in Chlorella vulgaris. Bioresour. Technol. 99, 4717–4722. Nguyen, V.A.T., Senoo, K., Mishina, T., Hisamatsu, M., 2001. Multiple tolerance of Rhodotorula glutinis R-1 to acid, aluminum ion and manganese ion, and its unusual ability of neutralizing acidic medium. J. Biosci. Bioeng. 92 (4), 366–371. Olgui9 n, E.J., Mendoza, A., Gonzlez-Portela, R.E., Novelo, E., 2013. Population dynamics in mixed cultures of Neochloris oleoabundans and native microalgae from water of a polluted river and isolation of a diatom consortium for the production of lipid rich biomass. New Biotechnol. 30 (6), 705–715. Pope, D.H., 1975. Effects of light intensity, oxygen concentration, and carbon dioxide concentration on photosynthesis in alga. Microb. Ecol. 2, 1–16. Santos, C.A., Caldeira, M.L., Sliva, T.L., Novais, J.M., Reis, A., 2013. Enhanced lipidic algae biomass production using gas transfer from a fermentative Rhodosporidium toruloides culture to an autotrophic Chlorella protothecoides culture. Bioresour. Technol. 138, 48–54. Shu, C.H., Tsai, C.C., Chen, K.Y., Liao, W.H., Huang, H.C., 2013. Enhancing high quality oil accumulation and carbon dioxide fixation by a mixed culture of Chlorella sp. and Saccharomyces cerevisiae. J. Taiwan Inst. Chem. Eng. 44, 936–942. Su, Y.Y., Mennerich, A., Urban, B., 2012. Coupled nutrient removal and biomass production with mixed algal culture: impact of biotic and abiotic factors. Bioresour. Technol. 118, 469–476. Wang, C., Li, H., Wang, Q.Q., Wei, P., 2010. Effect of pH on growth and lipid content of Chlorella vulgaris cultured in biogas slurry. Chin. J. Biotechnol. 26 (8), 1074–1079.
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Xiong, W., Gao, C.F., Yan, D., Wu, Q.Y., 2010. Double CO2 fixation in photosynthesisfermentation model enhances algal lipid synthesis for biodiesel production. Bioresour. Technol. 101, 2287–2293. Xue, F.Y., Gao, B., Zhu, Y.Q., Zhang, X., Feng, W., Tan, T.W., 2010a. Pilot-scale production of microbial lipid using starch wastewater as raw material. Bioresour. Technol. 101, 6092–6095. Xue, F.Y., Miao, J.X., Zhang, X., Luo, H., Tan, T.W., 2008. Studies on lipid production by Rhodotorula glutinis fermentation using monosodium glutamate wastewater as culture medium. Bioresour. Technol. 99, 5923–5927. Xue, F.Y., Miao, J.X., Zhang, X., Tan, T.W., 2010b. A new strategy for lipid production by mix cultivation of Spirulina platensis and Rhodotorula glutinis. Appl. Biochem. Biotechnol. 160, 498–503. Xue, F.Y., Zhang, X., Luo, H., Tan, T.W., 2006. A new method for preparing raw material for biodiesel production. Process Biochem. 41, 1699–1702. Yen, H.W., Yang, Y.C., 2012. The effects of irradiation and microfiltration on the cells growing and total lipids production in the cultivation of Rhodotorula glutinis. Bioresour. Technol. 107, 539–541. Yen, H.W., Zhang, Z.Y., 2011a. Enhancement of cell growth rate by light irradiation in the cultivation of Rhodotorula glutinis. Bioresour. Technol. 102, 9279–9281. Yen, H.W., Zhang, Z.Y., 2011b. Effects of dissolved oxygen level on cell growth and total lipid accumulation in the cultivation of Rhodotorula glutinis. J. Biosci. Bioeng. 112, 71–74. Zhang, Z.P., Zhang, X., Tan, T.W., 2014. Lipid and carotenoid production by Rhodotorula glutinis under irradiation/high-temperature and dark/lowtemperature cultivation. Bioresour. Technol. 157, 149–153. Zhao, G.L., Yu, J.Y., Jiang, F.F., Zhang, X., Tan, T.W., 2012. The effect of different trophic modes on lipid accumulation of Scenedesmus quadricauda. Bioresour. Technol. 114, 466–471.
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Synergistic effects of oleaginous yeast Rhodotorula glutinis and microalga Chlorella vulgaris for enhancement of biomass and lipid yields Zhiping Zhang, Hairui Ji, Guiping Gong, Xu Zhang ⇑, Tianwei Tan National Energy R&D Center for Biorefinery, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, PR China
h i g h l i g h t s Biomass and lipid yields achieved a theoretical model of 1+1>2 by mixed culture. A double system bubble column photo-bioreactor was designed for mixing cultivation. Growth curves of yeast and alga were confirmed in mixing cultivation system. Real-time online detection of off-gas showed synergistic effects on O2/CO2 balance. Analysis of metabolite variations proved synergistic effects on substance exchange.
a r t i c l e
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Article history: Received 22 February 2014 Received in revised form 9 April 2014 Accepted 10 April 2014 Available online 21 April 2014 Keywords: Rhodotorula glutinis Chlorella vulgaris Mixed culture Synergistic effects Lipid
a b s t r a c t The optimal mixed culture model of oleaginous yeast Rhodotorula glutinis and microalga Chlorella vulgaris was confirmed to enhance lipid production. A double system bubble column photo-bioreactor was designed and used for demonstrating the relationship of yeast and alga in mixed culture. The results showed that using the log-phase cultures of yeast and alga as seeds for mixed culture, the improvements of biomass and lipid yields reached 17.3% and 70.9%, respectively, compared with those of monocultures. Growth curves of two species were confirmed in the double system bubble column photo-bioreactor, and the second growth of yeast was observed during 36–48 h of mixed culture. Synergistic effects of two species for cell growth and lipid accumulation were demonstrated on O2/CO2 balance, substance exchange, dissolved oxygen and pH adjustment in mixed culture. This study provided a theoretical basis and culture model for producing lipids by mixed culture in place of monoculture. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Oleaginous microorganisms, involving bacterium, yeasts, moulds and algae, have been extensively studied because the microbial lipids were very similar to soybean lipids to promise potential feedstock for biodiesel production (Cheirsilp et al., 2011). Among oleaginous microorganisms, the yeast Rhodotorula glutinis, as a high-yielding lipid strain, could utilize some wastewater for lipid production (Xue et al., 2006, 2010a). In particular, it has been reported that light irradiation could stimulate the synthesis of pigment which resists light damage, and affect cell growth rate and lipid content (Zhang et al., 2014; Yen and Zhang, 2011a; Yen and Yang, 2012). Also, R. glutinis has a great growth rate in aerobic condition wherein large amount of O2 must be required and CO2 be emitted (Li et al., 2007). Yen and Zhang (2011b) reported ⇑ Corresponding author. Address: No. 15 Beisanhuan East Road, Chaoyang District, Beijing 100029, PR China. Tel./fax: +86 10 64448962. E-mail address: [email protected] (X. Zhang). http://dx.doi.org/10.1016/j.biortech.2014.04.039 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
that low dissolved oxygen (DO) could retard cell growth and enhance lipid accumulation. However, under enough nutrients and low DO conditions, partial yeast cells would be anaerobic respiration to produce a large number of volatile organic acids (Chen and Gutmanis, 1976), which decreased the pH value of cultivation system. The optimum initial pH for the growth rate of R. glutinis is 6.0 (Bhosale and Gadre, 2001). Undissociated organic acid could lower intracellular pH following translocation across the yeast plasma membrane so that yeast growth is inhibited (Nguyen et al., 2001). Microalgae are considered as another attractive source for biodiesel production due to their high lipid content, photosynthesis efficiency and CO2 reduction efficiency (Eugenia, 2012; Xiong et al., 2010). Some algal cells can grow not only in photosynthesis system, but also in heterotrophic system or mixotrophy when the culture contains both inorganic and organic substrates (Zhao et al., 2012). Chlorella vulgaris, as a kind of mixotrophy algal species, could utilize the high concentration N, P and other ions of waste-
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water in photosynthesis for lipid production meanwhile release O2 (Li et al., 2010; Liu et al., 2008). Moreover, C. vulgaris grows better at pH 6.5–7.0 and accumulates lipids at pH 7.0–8.5 (Wang et al., 2010). However, when CO2 is consumed by microalgae, the pH becomes more alkaline (Eugenia, 2012). Therefore, the algal growth will be inhibited after a period of culture due to the increasing pH value. In theory, there are synergistic effects on gas, substance exchange and pH adjustment in the mixed culture system of oleaginous yeast R. glutinis and microalga C. vulgaris based on the above mentioned researches. However, the reports of lipid production by mixed culture mainly focused on the different alga species (Su et al., 2012; Fradinho et al., 2013; Olgui9 n et al., 2013; Chen et al., 2014) and the mixed culture of yeast and alga based on the mutually beneficial relationship of gas exchange (Shu et al., 2013; Santos et al., 2013). In this study, one of the aims was to confirm that mixed culture of both microorganisms could significantly enhance biomass and lipid production. In order to demonstrate the synergistic effects of the mixed culture, the authors designed a double-system bubble column photo-bioreactor consisting of an association of two cultivation systems, in which one was used for R. glutinis cultivation and the other was for C. vulgaris. A polyester fabric filter (2.6 lm) was fixed between the two systems, which could achieve the exchange of substances and gases and keep microorganisms grow independently. 2. Methods
surface of flasks. Mixed culture was taken in 500 mL flask or double system bubble column photo-bioreactor using logarithmic phase cultures as seed. The monocultures of yeast and alga at the log-phase with 10% (v/v) were transferred into 260 mL double system bubble column photo-bioreactor containing 100 mL mixed medium for measuring the change of gases and substances (Fig. 1). During the mixed culture, the aeration rate was 0.8 L air/min. Aseptic air filter (0.22 lm) was accessed to the bottom of bioreactor through a pipeline and was dispersed by an air distributor. Temperature was controlled by the insulation layer circulating water from electric-heated thermostatic water bath (MPE-40C, China). The bioreactor was illuminated with white fluorescent light tubes assembled outside surface of bioreactor, which supplied a light intensity of 100 lmol/m2 s. 2.3. Measurement of glucose, biomass and lipid content Glucose concentration was measured using a glucose biosensor (SBA-40C, Biological Institute of Shandong Academy of Sciences). The biomass concentrations of yeast and alga were determined by optical density reading at 600 nm (OD600) and 680 nm (OD680), respectively. A calibration curve of yeast dry cell weight corresponded to OD600 value as follows: Biomass (g/L) = 1.052 OD600 + 0.041, R2 = 0.9982, and that curve of alga at OD680 as follows: Biomass (g/L) = 0.324 OD680 + 0.0099, R2 = 0.9991. Biomass concentration of mixed culture was carried out following the method of Zhao et al. (2012) by cell dry weight. The lipid content was determined by sulfo-phospho-vanillin method (Izard and Limber, 2003).
2.1. Strains 2.4. Measurement of off-gas, dissolved oxygen and pH value The yeast R. glutinis (CGMCC No. 2258) was supplied by the China National Research Institute of Food and Fermentation Industries and kept at Beijing University of Chemical Technology. The strain was maintained on yeast extract, peptone and dextrose (YPD) agar slant at 4 °C. The microalga C. vulgaris was stored in National Energy R&D Center for Biorefinery at Beijing University of Chemical Technology after being provided by Institute of Hydrobiology, Chinese Academy of Sciences. The strain could conduct mixotrophy in the culture containing both inorganic and organic substrates and was maintained on alga medium BG-11 (described below) agar slant with glucose (2 g/L) at 4 °C. 2.2. Culture media and conditions Seed media of R. glutinis and C. vulgaris were described as follow. Yeast medium composition (Xue et al., 2008): glucose 40 g/L, yeast extract 1.5 g/L, (NH4)2SO4 2 g/L, KH2PO4 7 g/L, NaSO4 2 g/L, MgSO47H2O 1.5 g/L. The initial pH was adjusted to 5.5. Alga medium BG-11 composition (Zhao et al., 2012): citric acid 6.0 mg/L, ferric ammonium citrate 6.0 mg/L, EDTA 1.0 mg/L, NaNO3 1.5 g/L, K2HPO42H2O 0.051 g/L, MgSO47H2O 0.075 g/L, CaCl2 0.024 g/L, Na2CO3 0.02 g/L, A5 trace mineral solution 1.0 mL/L. The composition of A5 was: H3BO4 2.86 g/L, MnCl24H2O 1.81 g/L, ZnSO47H2O 0.222 g/L, Na2MoO42H2O 0.391 g/L, CuSO45H2O 0.079 g/L, Co(NO3)26H2O 0.049 g/L. The initial pH was adjusted to 6.5–7.5. The mixotrophic culture medium was the alga medium containing 5 g/L of glucose. Mixed medium were adducts of yeast and alga media adjusting the concentration of glucose to 20 g/L (Xue et al., 2010b). Yeast and alga were respectively incubated from agar slant culture to 500 mL Erlenmeyer flasks containing 100 mL seed media. The flasks were placed in a rotary shaker at 180 rpm, 30 °C and around 80 lmol/m2 s. Light intensity was measured by a light-meter (TES-1339, Taiwan Taishi) on the outside
Real-time online detection of the different gas concentrations (O2, CO2 and N2) in off-gas was performed by using the Industrial Gas Analyzer (MAX300-LG, Extrel, USA). The online dissolved oxygen (DO) and pH value were determined by using DO meter (OXYFERM FDA 225, Hamilton) and pH meter (M-10, AMER), respectively. 2.5. Analysis of extracellular metabolites Extracellular metabolites were analyzed by the method of gas chromatography–mass spectrometry (GC–MS) (GC–MS-QP2010, SHIMADZU, Japan) according to the modified procedure of Coutinho et al. (2013) and Cai et al. (2007). Samples (1 mL) taken from mixed culture and controls were centrifuged at 12,000 rpm for 10 min, then dried the supernatant to get the metabolite powder by centrifugal concentrator (HR/T16MM, China) at 30 °C. A volume (100 lL) of methoxyamine pyridine solution (2 g/L) was added into the metabolite powder for suspension and oximation reaction at 30 °C for 2 h, and then 100 lL of N-MethylN-(trimethylsilyl)trifluoroacetamide (MSTFA) agent was added into the reaction system for derivatization at constant temperature of 30 °C for 6 h. After filtered through filter paper (0.22 lm), sample of 1 lL was injected into GC–MS with a column of 30 mm 0.25 mm 0.25 lm. 3. Results and discussion 3.1. The mixed culture of R. glutinis and C. vulgaris 3.1.1. Directly mixed culture Because microalga grew slower than yeast, the increasing initial amount of algal cells were attempted to mixed culture with constant amount of yeast cells. Even so, the biomass in the mixing
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pH probe
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Fig. 1. Apparatus of the double system bubble column photo-bioreactor for mixed culture of R. glutinis and C. vulgaris with fluorescent light.
3.1.2. Using the log-phase cultures of yeast and alga for mixed culture As the alga grew slower than the yeast, the directly mixed culture in the initial fermentation was not conducive to photoautotrophic growth of alga and play the maximum capacity for enhancing lipid production. In addition, the light may have hardly penetrated and its intensity attenuated drastically because of the high concentration of yeast cells (Cheirsilp et al., 2011). Therefore, a vibrant growth alga culture at the log-phase using mixed medium was
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cultivation system was mainly from the growth of yeast, which could be inferred to the color of fermentation broth. Thus, the directly mixed culture of R. glutinis and C. vulgaris was compared with monoculture of yeast. According to the different initial inoculums, three groups were taken. The initial inoculums of group I, group II and control group were 3% yeast and 3% alga (v/v), 3% yeast and 6% alga (v/v) and 3% yeast (v/v), respectively. Yeast and alga seed cultures were at the logarithmic phase with OD600 0.4–0.5 of yeast (after 50 times dilution) and OD680 0.5–0.6 of alga (after 10 times dilution). The results were shown in Fig. 2. According to Fig. 2A, it was could be divided to three growth phases. During the first phase (0–65 h), the specific growth rate of mixed culture group I was very similar to that of control group. Although the lower growth of mixed culture group II was observed, the glucose utilization rate was faster. It was possible that the pH was increased up to 7.0, which was not suitable for yeast growth, but pH 7.0 was suitable for lipid synthesis (Bhosale and Gadre, 2001). During the second phase (65–90 h), the growth of control group was into the stable phase, but the biomasses of both mixed culture groups were still increasing in a small amount. A new faster increase of biomass in both mixed culture groups was observed in the third phase. Finally, the maximum biomass concentrations of mixed group I, mixed group II and control were 10.3, 8.7 and 8.1 g/L, respectively. It was obvious that mixed culture could enhance the biomass compared with monoculture of yeast. However, when a higher concentration of biomass was reached, the lipid content and lipid production decreased (Fig. 2B). The maximum lipid content of control was higher than that of two mixed culture groups, measuring 20.8%, 15.1% and 11.8%, respectively. Due to the maximum of biomass obtained in group I, the total amounts of lipid production of group I reached 1.56 g/L, which was similar to control (1.68 g/L). This result was different from previously observed that higher oil production was obtained with a higher seed ratio of microalga to yeast (Shu et al., 2013).
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added into the culture of yeast cultured at same conditions. Mixing ratio of volume was 1:1 (100 mL alga and 100 mL yeast into 500 mL flask). The results indicated that the biomass concentration in the initial mixed culture decreased compared with the monoculture of yeast (Fig. 3). This was because of the low biomass concentration in monoculture of alga. Finally, the maximum of biomass
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group was the mixed culture group (11.5 g/L) at 120 h, while those of the monocultures of yeast and alga were 9.9 and 2.4 g/L (not shown), respectively. This phenomenon obviously demonstrated that the biomass yield (2.3 g of 200 mL) of mixed culture was higher than those (1.3 g of 200 mL) of adding up to pure yeast (0.99 g of 100 mL) and pure alga (0.24 g of 100 mL). Thus, biomass in the mixed culture system have achieved a theoretical model of 1+1>2 compared with that of monocultures. Furthermore, comparisons of lipid content and lipid production at two different of fermentation times (24 and 48 h) after mixed culture were shown in Table 1. The lipid content of mixed culture group was higher than that of pure alga but lower than that of pure yeast at 24 and 48 h after mixed culture. Even so, the lipid production of mixed culture was also the highest in three groups because of the highest biomass obtained in mixed culture group. Consequently, the lipid production in the mixed cultivation system have also achieved a theoretical model of 1+1>2 compared with that of monocultures.
fermentation broth (organic acids, amino acids, polysaccharides, etc) could be exchanged and detected; and (iii) water-soluble gas can be exchanged and utilized. The results of time course data of individual growth curves of species and lipid formation in the mixing cultivation of R. glutinis and C. vulgaris were shown in Fig. 4 compared with control cultures (a bulkhead replaced filter in the middle of the bioreactor). The yeast was the dominant species during the initial 24 h of cultivation (Fig. 4A). The growth ratio of R. glutinis over that of C. vulgaris reached its maximum at 18.9 g/L after 48 h of cultivation, and it appeared the second growth during 36–48 h. However, it decreased gradually to 17.2 g/L at the end of the cultivation, which revealed that the growth of yeast stopped. On the contrary, the growth ratio of alga remained high and the biomass concentration reached 2.4 g/L at 60 h (Fig. 4C). It was presumed that microalga adapted the culture environment gradually, and the autotrophic metabolism was the major mode at the end of the cultivation (Shu et al., 2013). Considering the yeast was the dominant species in mixing cultivation system and the growth of yeast reached stationary growth at 48 h, the mixed culture was terminated at 60 h. Finally, the maximum biomass concentrations of yeast and alga were 22.3% and 25.2% higher than those of the monocultures, respectively. About lipid content, no obvious difference was observed between mixed culture and pure culture of yeast in the initial 24 h of cultivation (Fig. 4B). Likewise, the alga lipid content of mixed culture compared with that of monoculture was remarkably similar in the whole cultivation (Fig. 4D). Obviously, the increase of alga biomass led to the increase of yeast lipid content in the mixing cultivation at the following time. This might be due to that photoautotrophic culture of alga provided extra substrates for lipid synthesis (Cheirsilp et al., 2011). Consequently, combined with higher biomass concentrations in mixed culture, the lipid productions of two species in the mixing cultivation system were also higher than those of monocultures. Therefore, mixed culture of two species would have symbiotic relationship and synergistic effects, which resulted in more biomass and lipid accumulation as compared with those of the monocultures. 3.3. Synergistic effects on gas, DO and pH value in the mixed culture
3.2. The individual growth curves of two species under mixed culture condition As discussed above, although the fact that biomass concentration and lipid production enhanced by mixed culture of yeast and alga in the same medium was confirmed, little information was available on the individual growth curves of two species and the relationship of alga and yeast in mixed culture (Shu et al., 2013). In this study, a double-system bubble column photo-bioreactor was used for mixing cultivation (Fig. 1). In order to assure that the medium kept the same and uniform on both side of the filter, one gas outlet was used on the top of one cultivation system, while the another one was closed. So, there was a pressure difference between the two cultivation systems. Meanwhile, the gas outlets on two systems were opened and closed on turns every few hours to achieve trans-membrane exchange. The equipment has particular significance in three aspects: (i) the growth of separated microorganisms is easy to be investigated; (ii) the nutrients in Table 1 Results of lipid contents and lipid yields of mixed culture after 24 and 48 h compared with those of monocultures of yeast and alga. X1, X2 and X3 are lipid contents of mixed culture, yeast and alga, respectively. Y1 and Y2 are lipid yields of mixed culture and those of adding up to monocultures of yeast and alga, respectively. Time (h)
X1 (%)
X2 (%)
X3 (%)
Y1 (g/L)
Y2 (g/L)
24 48
18.8 22.4
21.6 26.3
14.7 20.2
2.09 2.58
1.24 1.56
The time course data of different gas concentrations (v/v) in offgas, DO and pH value in mixed culture of R. glutinis and C. vulgaris were shown in Fig. 5. The concentration of O2 in off-gas decreased gradually from 20.4% to 20.1% (Fig. 5A) and that of CO2 increased from 0.04% to 0.15% (Fig. 5B) during the initial 24 h of cultivation, which were resulted from the growth of yeast. However, the concentration of O2 increased gradually from 20.1% to 20.6% during 24–36 h due to the slow growth of yeast (Fig. 4A) and the substantial growth of microalga (Fig. 4C). With the rapid algal growth, the concentrations of both O2 and CO2 in off-gas declined rapidly from 20.6% to 20.0% and 0.27% to 0.15%, respectively, during 36–48 h. It indicated that yeast grew again following with the growth of alga. Moreover, increasing rapidly of concentration of N2 in off-gas pointed that both O2 and CO2 were utilized in the mixing cultivation (Fig. 5C). In particular, the concentrations of three gases reached a dynamic equilibrium during 48–54 h, which demonstrated that there was an O2/CO2 balance for enhancing both of yeast and alga growths. At the end of cultivation, the concentration of O2 increased rapidly due to the stop growth of yeast, while the concentration of CO2 decreased rapidly since the rapid growth of alga with phototrophy. The results are very similar to those reported by Cai et al. (2007) that although the initial growth of the alga is slightly inhibited by the yeast in the mixed culture, the alga can rapidly grow by overcoming the inhibitory effect, and that the two species can adopt well to the coexisting circumstance and build up the beneficial balance of mutualism.
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Control 18
20.7 20.6
Mixed culture Oxygen concentration (%)
Biomass of yeast (g/L)
A
20
16 14 12 10 8 6
20.5 20.4 20.3 20.2 20.1 20.0
0
12
24
36
48
60
0
12
Time (hour)
15
B
8
Lipid content Total lipid Control Mixed cuture
6 14 4
13 12
2 11 10 12
24
36
48
C
0.05
12
1.8 1.6 1.4 1.2 1.0 0.8
36
79.1
78.9 78.8 78.7 78.6 78.5 78.4 78.3
0.4 0
12
24
36
48
0
60
12
D
0.8
36
14
0.6
12 0.4 10 0.2
120
8 DO pH value
100
DO (%)
Control Mixed culture
Total lipid of alga (g/L)
1.0
Lipid content Total lipid
16
24
Time (hour)
Time (hour)
Lipid content of alga (%)
24
79.0
2.0
18
60
0.10
Time (hour)
0.6
D
48
0.15
0
Nitrogen concentration (%)
Biomass of alga (g/L)
2.2
60
0.20
60
Control Mixed culture
2.4
48
0.25
Time (hour)
C 2.6
60
0.00
0 0
48
0.30
Carbon dioxide concentration (%)
16
36
Time (hour)
Total lipid of yeast (g/L)
Lipid content of yeast (%)
B
24
7
80
6
60
5
40
4
20
3
pH value
A
8 0.0 0
12
24
36
48
60
Time (hour) Fig. 4. Results of the biomass (A), lipid content and total lipid (B) of R. glutinis and the biomass (C), lipid content and total lipid (D) of C. vulgaris in a mixed culture system aerated by 0.8 L air/min in a 260 mL double system bubble column photobioreactor illuminated at 100 lmol/m2 s.
0
2 0
12
24
36
48
60
Time (hour) Fig. 5. Time-course data of O2 (A), CO2 (B) and N2 (C) concentrations (v/v) in off-gas, DO and pH value (D) in a mixed culture system aerated by 0.5 vvm of air in a 260 mL double system bubble column photo-bioreactor illuminated at 100 lmol/ m2 s.
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R. glutinis has a great growth rate in aerobic condition wherein large amount of O2 must be required and CO2 be emitted (Li et al., 2007). In this study, yeast was the dominant species in the beginning of the mixed culture that caused a decline of DO from 100% to 53% (Fig. 5D). However, 0.042–0.130 M of dissolved CO2 was produced in the yeast fermentation, which would lead partial yeast to anaerobic respiration to produce a large number of volatile organic acids (Chen and Gutmanis, 1976). Organic acids synthesized not only wasted nutrients for lipid production, but also acidified the media of pH value from 6.0 to 3.2 during the initial 36 h of mixing cultivation (Fig. 5D). Due to the depletion of nutrients, yeast cells stopped to multiply and alga began to grow rapidly, which resulted in increasing the dissolved oxygen and pH value from 53% to 78% and 3.2 to 4.9, respectively. It was inferred that under the low concentrations of dissolved O2 and the high concentration of dissolved CO2, C. vulgaris would be into autotrophic metabolic that could be improved the level of DO in the mixing cultivation system (Pope, 1975). In addition, bicarbonate (HCO3 ) is formed when CO2 dissolves in water at neutral pH. During microalgae photosynthesis metabolic, HCO3 is converted to CO2 and hydroxide ion (OH ). Hence, when CO2 is consumed by microalgae, the pH becomes more alkaline (Eugenia, 2012). These results indicated that there were synergistic effects on gas exchange, DO and pH adjustment by mixed culture of oleaginous yeast and microalga. It was partially explained to the fact that the mixed culture could eventually lead to a higher productivity. However, the cause of the second growth of yeast is not only synergistic effects on gas exchange, DO and pH adjustment in mixed culture. The alga growth would have some secondary metabolites for stimulating the growth of yeast (Cheirsilp et al., 2011; Xue et al., 2010b). 3.4. Synergistic effects on substance exchange in the mixed culture To characterize the extracellular metabolite variations between monocultures and mixed culture, the mixed culture medium without yeast extract was used to cultivation of R. glutinis and C. vulgaris in 500 mL flask trials for GC–MS analysis. Since the aim of this study was to find the extracellular metabolic changes between monocultures and mixed culture, the samples were collected from monocultures of yeast and alga at the end of cultures, the initial mixed culture and the mixing cultivation for 48 h. Library searches of the mass spectrum were used to identify individual compounds from the chromatograms of samples (threshold P 90%). The results indicated that eleven metabolites were detected in the sample of yeast, while seven metabolites were identified in the sample of alga (not shown). Comparison of the metabolite variations between monocultures of yeast and alga, four different metabolites of yeast including glycerol, acetic acid, glycine, and proline were not detected in the sample of alga, while some other metabolites including glycinamide, acetamide and some derivatives of sugar were observed in the sample of alga but not in the yeast sample. In addition, the content of glycerol ether in the yeast sample was significantly higher than that of alga sample, while the content of palmitic acid in the alga sample was higher than that of yeast sample. As the above results clearly showed that the volatile compounds of monocultures of yeast and alga were different. The GC–MS results of the initial mixed culture sample was compared with those of after mixed culture 48 h. Obviously, the contents of some organic acids (propionic acid, pyruvic and acetic acids) glycidyl ether and palmitic acid were decreased, and the contents of some amino acids (glycine and proline) were increased. These results explained that a new faster increase of biomass in mixed culture groups was observed after mixing cultivation (Fig. 2A). On the one hand, the mixed culture of yeast and alga may alleviate and/or eliminate the stresses caused by CO2 on the yeast and O2 on
the alga to maintain an O2/CO2 balance that enhances the growth of two species. This result was similar to the reports by Cheirsilp et al. (2012) and Shu et al. (2013). On the other hand, the inhibition of yeast growth by organic acids could be alleviated through utilized by alga. Xue et al. (2010b) reported that some of yeast metabolites such as pyruvic and acetic acids might be utilized by microalgae. Moreover, higher palmitic acid content in alga cultivation significantly affects the yeast cell membrane permeability and rigidity (Hama et al., 2004), and that higher cell permeability is conducive to nutrient absorption and utilization. Therefore, based on the synergistic effects on substance exchange in the mixed culture, the biomass and lipid accumulation of two species were enhanced, which were performed on the increasing amino acids contents after mixing cultivation. 4. Conclusion The mixed culture strategy resulted in significant improvements on biomass concentration and lipid productivity, especially using the log-phase cultures of oleaginous yeast and microalga as seeds. The individual growth curves of two species in mixing cultivation system were identified by cultivation in the double system bubble column photo-bioreactor. The second growth of yeast was observed following with the beginning into the log-phase growth of alga. Symbiotic relationships and synergistic effects of two species for cell growth and lipid accumulation were demonstrated on O2/CO2 balance, substance exchange, DO and pH adjustment in the mixed culture system. Acknowledgement Authors acknowledge the financial support from the National High Technology Research and Development 863 Program of China (Grant Nos. 2013AA050702, 2012AA022304). References Bhosale, P., Gadre, R.V., 2001. b-Carotene production in sugarcane molasses by a Rhodotorula glutinis mutant. J. Ind. Microbiol. Biotechnol. 26 (6), 327–332. Cai, S.Q., Hu, C.Q., Du, S.B., 2007. Comparisons of growth and biochemical composition between mixed culture of alga and yeast and monocultures. J. Biosci. Bioeng. 104 (5), 391–397. Cheirsilp, B., Kitcha, S., Torpee, S., 2012. Co-culture of an oleaginous yeast Rhodotorula glutinis and a microalga Chlorella vulgaris for biomass and lipid production using pure and crude glycerol as a sole carbon source. Ann. Microbiol. 62 (3), 987–993. Cheirsilp, B., Suwannarat, W., Niyomdecha, R., 2011. Mixed culture of oleaginous yeast Rhodotorula glutinis and microalga Chlorella vulgaris for lipid production from industrial wastes and its use as biodiesel feedstock. New Biotechnol. 28 (4), 362–368. Chen, S.L., Gutmanis, F., 1976. Carbon dioxide inhibition of yeast growth in biomass production. Biotechnol. Bioeng. 18 (10), 1455–1462. Chen, W.T., Zhang, Y.H., Zhang, J.X., Yu, G., Schideman, L.C., Zhang, P., Minarick, M., 2014. Hydrothermal liquefaction of mixed-culture algal biomass from wastewater treatment system into bio-crude oil. Bioresour. Technol. 152, 130–139. Coutinho, J.O.P.A., Silva, M.P.S., Moraes, P.M., Monteiro, A.S., Barcelos, J.C.C., Siqueira, E.P., Santos, V.L., 2013. Demulsifying properties of extracellular products and cells of Pseudomonas aeruginosa MSJ isolated from petroleum-contaminated soil. Bioresour. Technol. 128, 646–654. Eugenia, J.O., 2012. Dual purpose microalgae-bacterial-based systems that treat wastewater and produce biodiesel and chemical products within a biorefinery. Biotechnol. Adv. 30, 1031–1046. Fradinho, J.C., Oehmen, A., Reis, M.A.M., 2013. Effect of dark/light periods on the polyhydroxyalkanoate production of a photosynthetic mixed culture. Bioresour. Technol. 148, 474–479. Hama, S., Yamaji, H., Kaieda, M., Oda, M., Kondo, A., Fukuda, H., 2004. Effect of fatty acid membrane composition on whole-cell biocatalysts for biodiesel-fuel production. Biochem. Eng. J. 21, 155–160. Izard, J., Limber, R., 2003. Rapid screening method for quantitation of bacterial cell lipids from whole cells. J. Microbiol. Methods 55, 411–418. Li, X., Hu, H.Y., Gan, K., Sun, Y.X., 2010. Effects of different nitrogen and phosphorus concentrations on the growth nutrient uptake, and lipid accumulation of a freshwater microalga Scenedesmus sp.. Bioresour. Technol. 101, 5494–5500.
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