Bioresource Technology 157 (2014) 149–153
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Lipid and carotenoid production by Rhodotorula glutinis under irradiation/high-temperature and dark/low-temperature cultivation Zhiping Zhang, 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 Investigated the effects of temperature and irradiation on biomass and production. The optimum C/N ratio for lipid and carotenoid synthesis was 90. Concentrated broth enhanced products yields and reduced the residual sugar. A new two-stage cultivation method significantly improved the yields of products.
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Article history: Received 12 November 2013 Received in revised form 7 January 2014 Accepted 10 January 2014 Available online 21 January 2014 Keywords: Rhodotorula glutinis Irradiation Two-stage cultivation Lipid Carotenoid
a b s t r a c t The capacity of lipid and carotenoid production by Rhodotorula glutinis was investigated under different irradiation conditions, temperatures and C/N ratios. The results showed that dark/low-temperature could enhance lipid content, while irradiation/high-temperature increased the yields of biomass and carotenoid. The optimum C/N ratio for production was between 80 and 100. A two-stage cultivation strategy was used for lipid and carotenoid production in a 5 L fermenter. In the first stage, the maximum biomass reached 28.1 g/L under irradiation/high-temperature. Then, the cultivation condition was changed to dark/low-temperature, and C/N ratio was adjusted to 90. After the second stage, the biomass, lipid content and carotenoid reached 86.2 g/L, 26.7% and 4.2 mg/L, respectively. More significantly, the yields of biomass and lipid were 43.1% and 11.5%, respectively. Lipids contained 79.7% 18C and 16.8% 16C fatty acids by GC analysis. HPLC quantified the main carotenoids were b-carotene (68.4%), torularhodin (21.5%) and torulene (10.1%). Crown Copyright Ó 2014 Published by Elsevier Ltd. All rights reserved.
1. Introduction The third generation biodiesel is considered as processing oils from microbial lipid (Nigam and Singh, 2011), and the crude lipids are mainly conducted through batch cultivation by oleaginous microorganisms (Meng et al., 2009). Compared with the feedstock of the second generation biodiesel (animal fat and vegetable oil), microbial lipid has several advantages in short fermentation time requirement, low space demand and independence of location, seasons and climates (Xue et al., 2006). Among oleaginous microorganisms, yeast has an advantage over bacterial, moulds and alga, due to its unicellular relatively high growth rate and rapid lipids accumulation ability in discrete lipids bodies (Saenge et al., 2011). It has been reported (Xue et al., 2008; Galafassi et al., 2012) that as an oleaginous red yeast, Rhodotorula glutinis could ⇑ Corresponding authors. Address: No. 15 Beisanhuan East Road, Chaoyang District, Beijing 100029, PR China. Tel./fax: +86 10 64448962. E-mail addresses: [email protected] (X. Zhang), [email protected]. edu.cn (T. Tan). http://dx.doi.org/10.1016/j.biortech.2014.01.039 0960-8524/Crown Copyright Ó 2014 Published by Elsevier Ltd. All rights reserved.
accumulate both lipids and carotenoids, wherein b-carotene was the main component (Somashekar and Joseph, 2000). Carotenoids are commercially used as colorant for food, feed, and cosmetic products (Malisorn and Suntornsuk, 2008), and b-carotene has similar biological functions to vitamin A, such as antioxidant, anticancer and immunomodulatory activity (Granado et al., 2001). However, no commercial application of biodiesel production from microbial lipid has been found due to the high cost of the process (Schnider et al., 2013). Particularly, the cost of carbon source for lipid production from oleaginous microorganisms contributes more than 80% to the cultivation stage (Fontanille et al., 2012). Thus, reduction of the cost is crucial in promoting microbial lipid for biodiesel. In addition, carotenoids production by fermentation could become industrially feasible if the cost of production could be minimized by enhancing the productivity and utilizing the cheap sources (Aksu and Eren, 2007). In previous studies and papers, several approaches have been proposed, including the utilization of cheap and abundant carbon source (Xue et al., 2006, 2010; Chatzifragkou et al., 2011; Marova et al., 2012) and process optimizations. The process optimizations include of controlling
Z. Zhang et al. / Bioresource Technology 157 (2014) 149–153
2.1. Microorganism and culture conditions R. glutinis (CGMCC No. 2258) was supplied by the China National Research Institute of Food and Fermentation Industries and kept on yeast extract, peptone and dextrose (YPD) agar slant at 4 °C. The strain was incubated in 500 mL flask contained 150 mL culture medium for 40 h in a rotary shaker at 180 rpm. Because the aim of the reported method in this study mainly faced the industrial production, the relative temperatures (higher temperature of 30 °C and lower temperature of 24 °C) were used to culture the oleaginous yeast. The initial pH value of the pre-culture medium was 5.5 and the medium contained (w/v): glucose, 4%; yeast extract, 0.15%; (NH4)2SO4, 0.2%; KH2PO4, 0.7%; MgSO4, 0.1%; Na2SO4, 0.2%. The inoculums were cultured for 24 h at 30 °C with rotation speed of 180 rpm, and then, inoculated into a 5 L fermentor (BIOTECH-2002, Shanghai Baoxing, China) with 10% (v/v). The fermentor was exposed to light by three LED lamps with 800 lmol/m2 s of one LED lamp. The light intensity was measured by a light-meter (TES-1339, Taiwan Taishi) on the outside surface of fermentor. 2.2. Measurement of glucose and biomass Glucose concentration was detected using a glucose biosensor (SBA 40C, Shandong Academy of Sciences, China). Biomass was measured by the dry cell weight method (Yen and Zhang, 2011). The culture sample was diluted and measured the turbidity at 600 nm (OD600), using a standard curve of absorbance against dry cell mass concentration (Biomass (g/L) = 1.052 OD600 dilution factor + 0.041).
3.1. The effects of irradiation on biomass and productions Three different irradiation conditions (without light, two LEDs and three LEDs) were used for R. glutinis cultivation in the 500 mL flask trials. The results showed that irradiation significantly enhanced the biomass concentration (Fig. 1A). The maximum biomasses of three groups reached 15.9 g/L, 17.4 g/L and 17.7 g/L, respectively. The increase of biomass in the batch with irradiation was consistent to the decrease of residual glucose concentration measured in the broth. However, the lipid content in the group without irradiation was higher than those of two irradiation groups, measuring 33.8%, 27.1% and 24.8%, respectively (Fig. 1B). This was possible due to the rapid glucose consumption in the groups with irradiation (Yen and Zhang, 2011). Notably, the total amounts of lipid production were very similar in three groups
A
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2.3. Lipid, carotenoid extraction and analysis Total carotenoid (mg/L)
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The lipid content was determined by sulfo-phospho-vanillin method (Izard and Limber, 2003). Lipid was extracted as previously reported (Xue et al., 2008). The lipid components were analyzed by the GC-2010 gas-chromatograph (Shimadzu, Japan). The operation parameters of GC-2010 analysis were described as follows: flame ionization detector (FID) 350 °C; column Rt-wax (Agilent, USA), 30 m (length) 0.25 mm (inner diameter) 0.1 lm (thickness); PTV sample entrance (33 cm/s); diffluent ratio 1:5; carrier gas: N2 (Cheng et al., 2013). Total carotenoids were extracted, and then measured by spectrophotometrically (Buzzini and Martini, 2000), as mg of equivalent torulene/L of culture fluid. The composition of major carotenoids (b-carotene, torularhodin, torulene) was quantified by High Performance Liquid Chromatography (HPLC) (Agilent 1260 Infinity, USA) (Schnider et al., 2013).
Biomass
without light 2 LED 3 LED
Total lipid (g/L)
2. Methods
3. Results and discussion
Biomass & Glucose content (g/L)
nutrients to improve lipid yield (Mujtaba et al., 2012), controlling carbon concentration by fed-batch cultivation (Zheng et al., 2012), expanding the product types (Buzzini and Martini, 2000) and improving the production value (Rawat et al., 2013). But even if these strategies have improved production efficiency and reduced production cost somewhat, no approach has reached commercial realization yet due to the high cost of the process, low production yield and production value. In order to overcome the conversion rate constraint and reduce the cost, the red yeast R. glutinis was selected for the present study through combined production of lipid and high-value carotenoid. A new two-stage cultivation method was developed based on investigating the effects of controllable operational factors (light irradiation, temperature and carbon–nitrogen ratio) on biomass yield, lipid content and carotenoid productivity. In the first stage, the biomass and carotenoid concentrations were maximized and then lipid content and lipid productivity were optimized in the second stage.
Lipid content (%)
150
2.0 1.6 1.2 0.8 0.4 10
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Time (hour) Fig. 1. Effects of different light irradiation on the biomass (A), lipid content, total lipid (B) and total carotenoid (C) of R. glutinis.
Z. Zhang et al. / Bioresource Technology 157 (2014) 149–153
3.2. The effects of temperature on the biomass and productions
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35 Lipid content (%)
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1.0 0.8 0.6 0.4 0.2 10
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Time (hour) Fig. 2. Effects of different incubation temperature on the biomass (A), lipid content (B), total lipid and total carotenoid (C) of R. glutinis.
(Fig. 1B) in spite of the lipid content decreased, that was due to the increase of biomass growth. Besides the enhancement of biomass growing, the increase of carotenoid formation was also observed in the irradiation groups (Fig. 1C). The maximum carotenoid concentration reached 2.6 mg/L in the three LED lamps batch, which increased nearly by 1.4 mg/L compared with the control group (1.2 mg/L). Carotenoids are important in protecting against photo-oxidative damage (Marova et al., 2012). Many non-phototrophic yeast and bacteria rely on carotenoids for protection when growing in light (Yen and Yang, 2012). Therefore, the application of irradiation during cultivation could not only increase the yields of the biomass and carotenoid, but also short the culture time. Moreover, cultivation with irradiation could potentially expand to the mixed culture of yeast and microalgae due to the effects of irradiation on both of species (Cheirsilp et al., 2011).
Temperature was another influential factor for the growth and products accumulation of R. glutinis. In this study, R. glutinis was cultivated in 500 mL flask under two different temperatures (24 °C and 30 °C). The results indicated that the maximum biomass was 17.9 g/L in a relatively higher temperature group at 30 h (Fig. 2A), while that was 15.8 g/L in a relatively lower temperature group at 45 h. Meanwhile, higher carotenoid productivity of 1.2 mg/L was obtained at 30 °C than that of 0.9 mg/L at 24 °C (Fig. 2C). Under normal conditions, a higher temperature of 44 °C and lower temperatures of 18 °C severely inhibited the specific growth rate, biomass production rate and nutrient utilization rate (Sheng et al., 2011). The effect of temperature on the growth rate has also been observed in other species, especially on microalgae. The higher growth rates with temperature from 25 to 30 °C for Chaetoceros sp. were observed by Renaud et al. (2002). Sayegh and Montagnes (2011) reported that four Isochrysis galbana strains increased their growth rates with temperature from 15 to 30 °C. Several enzymes involved in carotenoid biosynthesis in prokaryotic and eukaryotic cells, for example, phytoene desaturase, b-carotene hydroxylase and lycopene cyclase, and the enzyme activities are stimulated at a higher temperature of 30 °C (Malisorn and Suntornsuk, 2008). However, the result of lipid content was different from the reported by Malisorn and Suntornsuk (2008). They obtained the high lipid content at a higher temperature of 30 °C. In this research, the lipid content and lipid production of two groups were similar before 45 h (Fig. 2B). When reached the stable growth phase, the low temperature batch had the maximum lipid content of 38.8% and the maximum total lipid of 6.2 g/L, which were higher than those of high temperature batch (32.5% and 5.1 g/L, respectively). In particular, temperature affects the quality and quantity of the fatty acids attached to the glycerol backbone of membrane lipids (Murata, 1989). An increase in the growth temperature from 25 to 35 °C led to a decrease in the lipid content of some oleaginous microorganisms from 32.9% to 29.6% (Wu et al., 2013). In the other hand, the increased lipid content in lower temperature batch could be attributed to a higher residual glucose and absence of nitrogen sources (Roleda et al., 2013). It was inferred that the machinery for lipid accumulation remained active for a long time even cell growth related activity was abolished (Lin et al., 2011). Therefore, it would be possible to attempt two-stage cultivation using different temperatures for increasing the yields of lipid and carotenoid. 3.3. The optimal C/N ratio for products synthesized The optimal C/N ratio for lipid and carotenoid synthesized was determined in 500 mL flask trials. After the logarithmic growth phase, the C/N ratios of media were adjusted to a series of concentration gradients from 30 to 150 by the ratio of glucose to ammonium sulfate. After the production biosynthesis stage for 48 h (Fig. 3), the results demonstrated that the optimum C/N ratio for lipid biosynthesis was between 80 and 100, and the maximum lipid content was 29.7%, while that for carotenoid accumulation was from 70 to 100, and the maximum carotenoid production was 1.9 mg/L. Combination of both results, it was concluded that the optimum C/N ratio for lipid and carotenoid accumulation was found to be between 80 and 100. The results are very similar to those reported by Wang et al. (2013) in batch cultures of Chlorella protothecoides at different C/N ratios. According to the report by Saenge et al. (2011), both lipid and carotenoid required a medium with an excess of carbon source and limited other nutrients, especially nitrogen, but cell propagation could be inhibited. The mechanism is that lipid accumulation serves as an energy sink and b-carotene serves as an electron sink
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Lipid content (%) & Total carotenoid (mg/L)
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30
yields might be low due to the decrease of cell number. Thus, it is important to not only look at the product content but also at the yields of biomass and products. Moreover, microbial growth and lipid accumulation processes could be spatially separated (Lin et al., 2011). Therefore, in order to obtain higher yields of the products, a high biomass should be achieved firstly, and then the optimum C/N ratio could be adjusted for enhancing the contents of lipid and carotenoid.
Lipid content Carotenoid
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3.4. New two-stage strategy for R. glutinis cultivation
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Lipid conten (%) , Total lipid (g/L) & Total carotenoid (mg/L)
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Lipid content (%), Tolal lipid (g/L) & Total carotenoid (mg/L)
Fig. 3. The optimum C/N ratio for products accumulation by R. glutinis.
Based on the above results, the fermentation process of R. gutinis could be divided into two stages: microorganism growth stage and products accumulation stage. During microorganism growth stage, R. gutinis grew fast to obtain high biomass of 28.1 g/L (Fig. 4) under irradiation/high-temperature at 60th hour. In order to reduce the amount of glucose to the broth, about 35% of the broth was filtered out by a micro-filtration combined system. Glucose of 20 g and ammonium of 1.5 g were added to the broth reaching a C/N of 90 (Fig. 4A). The control group was not concentrated, but additional carbon source was needed for products accumulation (Fig. 4B). Then, the environment of cultivation system was shifted to dark/low-temperature condition and continued to cultivation for products accumulation. The results of microfiltration group were shown in Fig. 4A, and it indicated that not only lipid content was significantly increased from 20.3% to 26.7%, but also lipid yield was improved up to 11.3 g/L at 84th hour. The biomass and carotenoid production were not affected in this stage. The comprehensive data of microfiltration and no microfiltration groups was shown in Table 1. The biomass, lipid content, carotenoid yields of two groups were maintained the same level, but the residual glucose of microfiltration group was greatly reduced from 30 to 2 g/L. The biomass and lipid coefficients were increased from 38.7 to 43.1 and 10.4 to 11.5, respectively. Finally, the lipid yield was significantly improved from 7.25 to 11.5 g/L, which was increased by nearly 58.6%. Therefore, there were two advantages in the new two-stage cultivation using microfiltration to reduce the broth volume: firstly, the lipid yield was greatly improved when consuming the same amount of carbon source; secondly, the residual sugar was greatly reduced at the end of fermentation, which could improve the utilization of glucose and benefit for the treatment of broth. Moreover, as a nonphototrophic species, the biomass and production content of R. gutinis were greatly influenced by irradiation in our studied. It is possible that by mixing cultivation R. gutinis of and microalgae may eventually lead to higher productivities. There are synergistic effects on gas, substance exchange and pH adjustment under irradiation in the mixed culture system of oleaginous yeast and microalgae in theory (Cheirsilp et al., 2011).
Fig. 4. Two-stage culture of R. glutinis for lipid and carotenoid production.
3.5. Lipid and carotenoid compositions when microorganisms are exposed to an energy imbalance caused by nutrient limitation (Klok et al., 2013). In addition, the lipid yield is relevant to both biomass concentration and lipid content. Although a high content of production was obtained, production
Lipid extracted from the cells of R. glutinis which were cultured under two-stage cultivation system was analyzed by gas-chromatograph (GC). The results showed that the main components of the lipids were oleic acid (C18:1; 51.1% in total lipid), linoleic
Table 1 The comprehensive of parameters in the second stage cultivation.
1 2
Group1
Final biomass (g)
Residual glucose (g/L)
Total sugar consumption (g)
Max. lipid content (%)
Max. total lipid (g/L)
Max. total carotenoid (mg/L)
Biomass coefficient2
Lipid coefficient2
A B
81.3 86.2
30 2
210 200
26.8 26.7
7.3 11.5
4.1 4.2
38.7 43.1
10.4 11.5
A and B were the groups of no microfiltration and microfiltration, respectively. The coefficients of biomass and lipid refer to conversion the quality (g) of the biomass or lipid from 100 g of glucose.
Z. Zhang et al. / Bioresource Technology 157 (2014) 149–153 Table 2 The composition and content of R. glutinis lipid. Lipid compositions
Lipid content (%)
Oleic acid (C18:1n9c) Linoleic acid (C18:2n6c) Palmitic acid (C16:0) Stearic acid (C18:0) Twenty-four carbonate (C24:0) a-linolenic acid (C18:3n3) Myristic acid (C14:0) Palmitoleic acid (C16:1n7) Behenic acid (C22:0) DPA (C22:5)
51.3 21.6 16.5 3.7 1.4 2.9 0.7 0.4 0.5 0.3
acid (C18:2; 21.6% in total lipid), palmitic acid (C16:0; 16.4% in total lipid) and stearic acid (C18:0; 3.7% in total lipid) (Table 2). The fatty acids of C18 and C16 accounted for 79.7% and 16.8% in total lipid, respectively. Therefore, the compositions of lipid fermented in two-stage cultivation were similar to those of vegetable seed lipids and soybean lipids (Xue et al., 2006), which could be used for biodiesel and jet fuel production because of a high content of fatty acid of C16 and C18 (96.6% of total lipid) obtained (Sivakumar et al., 2012). The carotenoids were extracted from the cells of R. glutinis and the main compounds were measured by HPLC. The results showed that the main carotenoids were identified as b-carotene, torularhodin, torulene, which contents in total carotenoids were 68.4%, 21.5% and 10.1%, respectively. This result was similar to the report by Somashekar and Joseph (2000) but different from the result of other reports (Schnider et al., 2013; Buzzini and Martini, 2000; Aksu and Eren, 2007), where the main pigment was torularhodin. Presumably, changed temperature and light inhibited some enzymes, which were used to catalyze torularhodin and torulene synthesized (Malisorn and Suntornsuk, 2008). 4. Conclusion This work investigated the effects of different of temperatures, irradiation conditions and C/N ratios on biomass growth, lipid content and carotenoid productivity. A new strategy of two-stage cultivation was developed for lipid and carotenoid production by R. glutinis. Especially concentrated broth before the second stage, the yields of lipid and carotenoid were significantly improved and the residual sugar concentration was reduced. This strategy as well as might be used for mixing cultivation of yeast and alga. Acknowledgements Authors acknowledge the financial support from the National High Technology Research and Development 863 Program of China (Grant No. 2013AA050702, 2012AA022304). References Aksu, Z., Eren, A.T., 2007. Production of carotenoids by the isolated yeast of Rhodotorula glutinis. Biochem. Eng. J. 35, 107–113. Buzzini, P., Martini, A., 2000. Production of carotenoids by strains of Rhodotorula glutinis cultured in raw materials of agro-industrial origin. Bioresour. Technol. 71, 41–44. Chatzifragkou, A., Makri, A., Belka, A., Bellou, S., Mavrou, M., Mastoridou, M., 2011. Biotechnological conversions of biodiesel derived waste glycerol by yeast and fungal species. Energy 36, 1097–1108. Cheng, P., Ji, B., Gao, L., Zhang, W., Wang, J., Liu, T., 2013. The growth, lipid and hydrocarbon production of Botryococcus braunii with attached cultivation. Bioresour. Technol. 138, 95–100. 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.
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Lipid and carotenoid production by Rhodotorula glutinis under irradiation/high-temperature and dark/low-temperature cultivation Zhiping Zhang, 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 Investigated the effects of temperature and irradiation on biomass and production. The optimum C/N ratio for lipid and carotenoid synthesis was 90. Concentrated broth enhanced products yields and reduced the residual sugar. A new two-stage cultivation method significantly improved the yields of products.
a r t i c l e
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Article history: Received 12 November 2013 Received in revised form 7 January 2014 Accepted 10 January 2014 Available online 21 January 2014 Keywords: Rhodotorula glutinis Irradiation Two-stage cultivation Lipid Carotenoid
a b s t r a c t The capacity of lipid and carotenoid production by Rhodotorula glutinis was investigated under different irradiation conditions, temperatures and C/N ratios. The results showed that dark/low-temperature could enhance lipid content, while irradiation/high-temperature increased the yields of biomass and carotenoid. The optimum C/N ratio for production was between 80 and 100. A two-stage cultivation strategy was used for lipid and carotenoid production in a 5 L fermenter. In the first stage, the maximum biomass reached 28.1 g/L under irradiation/high-temperature. Then, the cultivation condition was changed to dark/low-temperature, and C/N ratio was adjusted to 90. After the second stage, the biomass, lipid content and carotenoid reached 86.2 g/L, 26.7% and 4.2 mg/L, respectively. More significantly, the yields of biomass and lipid were 43.1% and 11.5%, respectively. Lipids contained 79.7% 18C and 16.8% 16C fatty acids by GC analysis. HPLC quantified the main carotenoids were b-carotene (68.4%), torularhodin (21.5%) and torulene (10.1%). Crown Copyright Ó 2014 Published by Elsevier Ltd. All rights reserved.
1. Introduction The third generation biodiesel is considered as processing oils from microbial lipid (Nigam and Singh, 2011), and the crude lipids are mainly conducted through batch cultivation by oleaginous microorganisms (Meng et al., 2009). Compared with the feedstock of the second generation biodiesel (animal fat and vegetable oil), microbial lipid has several advantages in short fermentation time requirement, low space demand and independence of location, seasons and climates (Xue et al., 2006). Among oleaginous microorganisms, yeast has an advantage over bacterial, moulds and alga, due to its unicellular relatively high growth rate and rapid lipids accumulation ability in discrete lipids bodies (Saenge et al., 2011). It has been reported (Xue et al., 2008; Galafassi et al., 2012) that as an oleaginous red yeast, Rhodotorula glutinis could ⇑ Corresponding authors. Address: No. 15 Beisanhuan East Road, Chaoyang District, Beijing 100029, PR China. Tel./fax: +86 10 64448962. E-mail addresses: [email protected] (X. Zhang), [email protected]. edu.cn (T. Tan). http://dx.doi.org/10.1016/j.biortech.2014.01.039 0960-8524/Crown Copyright Ó 2014 Published by Elsevier Ltd. All rights reserved.
accumulate both lipids and carotenoids, wherein b-carotene was the main component (Somashekar and Joseph, 2000). Carotenoids are commercially used as colorant for food, feed, and cosmetic products (Malisorn and Suntornsuk, 2008), and b-carotene has similar biological functions to vitamin A, such as antioxidant, anticancer and immunomodulatory activity (Granado et al., 2001). However, no commercial application of biodiesel production from microbial lipid has been found due to the high cost of the process (Schnider et al., 2013). Particularly, the cost of carbon source for lipid production from oleaginous microorganisms contributes more than 80% to the cultivation stage (Fontanille et al., 2012). Thus, reduction of the cost is crucial in promoting microbial lipid for biodiesel. In addition, carotenoids production by fermentation could become industrially feasible if the cost of production could be minimized by enhancing the productivity and utilizing the cheap sources (Aksu and Eren, 2007). In previous studies and papers, several approaches have been proposed, including the utilization of cheap and abundant carbon source (Xue et al., 2006, 2010; Chatzifragkou et al., 2011; Marova et al., 2012) and process optimizations. The process optimizations include of controlling
Z. Zhang et al. / Bioresource Technology 157 (2014) 149–153
2.1. Microorganism and culture conditions R. glutinis (CGMCC No. 2258) was supplied by the China National Research Institute of Food and Fermentation Industries and kept on yeast extract, peptone and dextrose (YPD) agar slant at 4 °C. The strain was incubated in 500 mL flask contained 150 mL culture medium for 40 h in a rotary shaker at 180 rpm. Because the aim of the reported method in this study mainly faced the industrial production, the relative temperatures (higher temperature of 30 °C and lower temperature of 24 °C) were used to culture the oleaginous yeast. The initial pH value of the pre-culture medium was 5.5 and the medium contained (w/v): glucose, 4%; yeast extract, 0.15%; (NH4)2SO4, 0.2%; KH2PO4, 0.7%; MgSO4, 0.1%; Na2SO4, 0.2%. The inoculums were cultured for 24 h at 30 °C with rotation speed of 180 rpm, and then, inoculated into a 5 L fermentor (BIOTECH-2002, Shanghai Baoxing, China) with 10% (v/v). The fermentor was exposed to light by three LED lamps with 800 lmol/m2 s of one LED lamp. The light intensity was measured by a light-meter (TES-1339, Taiwan Taishi) on the outside surface of fermentor. 2.2. Measurement of glucose and biomass Glucose concentration was detected using a glucose biosensor (SBA 40C, Shandong Academy of Sciences, China). Biomass was measured by the dry cell weight method (Yen and Zhang, 2011). The culture sample was diluted and measured the turbidity at 600 nm (OD600), using a standard curve of absorbance against dry cell mass concentration (Biomass (g/L) = 1.052 OD600 dilution factor + 0.041).
3.1. The effects of irradiation on biomass and productions Three different irradiation conditions (without light, two LEDs and three LEDs) were used for R. glutinis cultivation in the 500 mL flask trials. The results showed that irradiation significantly enhanced the biomass concentration (Fig. 1A). The maximum biomasses of three groups reached 15.9 g/L, 17.4 g/L and 17.7 g/L, respectively. The increase of biomass in the batch with irradiation was consistent to the decrease of residual glucose concentration measured in the broth. However, the lipid content in the group without irradiation was higher than those of two irradiation groups, measuring 33.8%, 27.1% and 24.8%, respectively (Fig. 1B). This was possible due to the rapid glucose consumption in the groups with irradiation (Yen and Zhang, 2011). Notably, the total amounts of lipid production were very similar in three groups
A
40
Glucose
35 30
without light 2 LED 3 LED
25 20 15 10 5 0 0
10
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Time (hour)
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40 35 30
16
Total lipid without light 2 LED 3 LED
Lipid content without light 2 LED 3 LED
14 12
25
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8
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0
0 10
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Time (hour)
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2.8 Without light 2 LED 3 LED
2.3. Lipid, carotenoid extraction and analysis Total carotenoid (mg/L)
2.4
The lipid content was determined by sulfo-phospho-vanillin method (Izard and Limber, 2003). Lipid was extracted as previously reported (Xue et al., 2008). The lipid components were analyzed by the GC-2010 gas-chromatograph (Shimadzu, Japan). The operation parameters of GC-2010 analysis were described as follows: flame ionization detector (FID) 350 °C; column Rt-wax (Agilent, USA), 30 m (length) 0.25 mm (inner diameter) 0.1 lm (thickness); PTV sample entrance (33 cm/s); diffluent ratio 1:5; carrier gas: N2 (Cheng et al., 2013). Total carotenoids were extracted, and then measured by spectrophotometrically (Buzzini and Martini, 2000), as mg of equivalent torulene/L of culture fluid. The composition of major carotenoids (b-carotene, torularhodin, torulene) was quantified by High Performance Liquid Chromatography (HPLC) (Agilent 1260 Infinity, USA) (Schnider et al., 2013).
Biomass
without light 2 LED 3 LED
Total lipid (g/L)
2. Methods
3. Results and discussion
Biomass & Glucose content (g/L)
nutrients to improve lipid yield (Mujtaba et al., 2012), controlling carbon concentration by fed-batch cultivation (Zheng et al., 2012), expanding the product types (Buzzini and Martini, 2000) and improving the production value (Rawat et al., 2013). But even if these strategies have improved production efficiency and reduced production cost somewhat, no approach has reached commercial realization yet due to the high cost of the process, low production yield and production value. In order to overcome the conversion rate constraint and reduce the cost, the red yeast R. glutinis was selected for the present study through combined production of lipid and high-value carotenoid. A new two-stage cultivation method was developed based on investigating the effects of controllable operational factors (light irradiation, temperature and carbon–nitrogen ratio) on biomass yield, lipid content and carotenoid productivity. In the first stage, the biomass and carotenoid concentrations were maximized and then lipid content and lipid productivity were optimized in the second stage.
Lipid content (%)
150
2.0 1.6 1.2 0.8 0.4 10
20
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60
Time (hour) Fig. 1. Effects of different light irradiation on the biomass (A), lipid content, total lipid (B) and total carotenoid (C) of R. glutinis.
Z. Zhang et al. / Bioresource Technology 157 (2014) 149–153
3.2. The effects of temperature on the biomass and productions
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High temperature Low temperature
Biomass (g/L)
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0
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30 40 Time (hour)
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16
Lipid content High temperature Low temperature
35 Lipid content (%)
10
14 12
Lipid production High temperature Low temperature
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8 6
20
Total lipid (g/L)
0
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Time (hour)
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1.4 High temperature Low temperature
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Total carotenoid (mg/L)
151
1.0 0.8 0.6 0.4 0.2 10
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Time (hour) Fig. 2. Effects of different incubation temperature on the biomass (A), lipid content (B), total lipid and total carotenoid (C) of R. glutinis.
(Fig. 1B) in spite of the lipid content decreased, that was due to the increase of biomass growth. Besides the enhancement of biomass growing, the increase of carotenoid formation was also observed in the irradiation groups (Fig. 1C). The maximum carotenoid concentration reached 2.6 mg/L in the three LED lamps batch, which increased nearly by 1.4 mg/L compared with the control group (1.2 mg/L). Carotenoids are important in protecting against photo-oxidative damage (Marova et al., 2012). Many non-phototrophic yeast and bacteria rely on carotenoids for protection when growing in light (Yen and Yang, 2012). Therefore, the application of irradiation during cultivation could not only increase the yields of the biomass and carotenoid, but also short the culture time. Moreover, cultivation with irradiation could potentially expand to the mixed culture of yeast and microalgae due to the effects of irradiation on both of species (Cheirsilp et al., 2011).
Temperature was another influential factor for the growth and products accumulation of R. glutinis. In this study, R. glutinis was cultivated in 500 mL flask under two different temperatures (24 °C and 30 °C). The results indicated that the maximum biomass was 17.9 g/L in a relatively higher temperature group at 30 h (Fig. 2A), while that was 15.8 g/L in a relatively lower temperature group at 45 h. Meanwhile, higher carotenoid productivity of 1.2 mg/L was obtained at 30 °C than that of 0.9 mg/L at 24 °C (Fig. 2C). Under normal conditions, a higher temperature of 44 °C and lower temperatures of 18 °C severely inhibited the specific growth rate, biomass production rate and nutrient utilization rate (Sheng et al., 2011). The effect of temperature on the growth rate has also been observed in other species, especially on microalgae. The higher growth rates with temperature from 25 to 30 °C for Chaetoceros sp. were observed by Renaud et al. (2002). Sayegh and Montagnes (2011) reported that four Isochrysis galbana strains increased their growth rates with temperature from 15 to 30 °C. Several enzymes involved in carotenoid biosynthesis in prokaryotic and eukaryotic cells, for example, phytoene desaturase, b-carotene hydroxylase and lycopene cyclase, and the enzyme activities are stimulated at a higher temperature of 30 °C (Malisorn and Suntornsuk, 2008). However, the result of lipid content was different from the reported by Malisorn and Suntornsuk (2008). They obtained the high lipid content at a higher temperature of 30 °C. In this research, the lipid content and lipid production of two groups were similar before 45 h (Fig. 2B). When reached the stable growth phase, the low temperature batch had the maximum lipid content of 38.8% and the maximum total lipid of 6.2 g/L, which were higher than those of high temperature batch (32.5% and 5.1 g/L, respectively). In particular, temperature affects the quality and quantity of the fatty acids attached to the glycerol backbone of membrane lipids (Murata, 1989). An increase in the growth temperature from 25 to 35 °C led to a decrease in the lipid content of some oleaginous microorganisms from 32.9% to 29.6% (Wu et al., 2013). In the other hand, the increased lipid content in lower temperature batch could be attributed to a higher residual glucose and absence of nitrogen sources (Roleda et al., 2013). It was inferred that the machinery for lipid accumulation remained active for a long time even cell growth related activity was abolished (Lin et al., 2011). Therefore, it would be possible to attempt two-stage cultivation using different temperatures for increasing the yields of lipid and carotenoid. 3.3. The optimal C/N ratio for products synthesized The optimal C/N ratio for lipid and carotenoid synthesized was determined in 500 mL flask trials. After the logarithmic growth phase, the C/N ratios of media were adjusted to a series of concentration gradients from 30 to 150 by the ratio of glucose to ammonium sulfate. After the production biosynthesis stage for 48 h (Fig. 3), the results demonstrated that the optimum C/N ratio for lipid biosynthesis was between 80 and 100, and the maximum lipid content was 29.7%, while that for carotenoid accumulation was from 70 to 100, and the maximum carotenoid production was 1.9 mg/L. Combination of both results, it was concluded that the optimum C/N ratio for lipid and carotenoid accumulation was found to be between 80 and 100. The results are very similar to those reported by Wang et al. (2013) in batch cultures of Chlorella protothecoides at different C/N ratios. According to the report by Saenge et al. (2011), both lipid and carotenoid required a medium with an excess of carbon source and limited other nutrients, especially nitrogen, but cell propagation could be inhibited. The mechanism is that lipid accumulation serves as an energy sink and b-carotene serves as an electron sink
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Lipid content (%) & Total carotenoid (mg/L)
152
30
yields might be low due to the decrease of cell number. Thus, it is important to not only look at the product content but also at the yields of biomass and products. Moreover, microbial growth and lipid accumulation processes could be spatially separated (Lin et al., 2011). Therefore, in order to obtain higher yields of the products, a high biomass should be achieved firstly, and then the optimum C/N ratio could be adjusted for enhancing the contents of lipid and carotenoid.
Lipid content Carotenoid
25 20 15 10
3.4. New two-stage strategy for R. glutinis cultivation
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Biomass Glucose Lipid content Total lipid Total carotenoid
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30 20
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72
96
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Biomass Glucose Lipid content Total lipid Total carotenoid
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40 15
30 20
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Time (hour)
0 120
Lipid conten (%) , Total lipid (g/L) & Total carotenoid (mg/L)
Biomass (g/L) & Glucose (g/L)
A
Lipid content (%), Tolal lipid (g/L) & Total carotenoid (mg/L)
Fig. 3. The optimum C/N ratio for products accumulation by R. glutinis.
Based on the above results, the fermentation process of R. gutinis could be divided into two stages: microorganism growth stage and products accumulation stage. During microorganism growth stage, R. gutinis grew fast to obtain high biomass of 28.1 g/L (Fig. 4) under irradiation/high-temperature at 60th hour. In order to reduce the amount of glucose to the broth, about 35% of the broth was filtered out by a micro-filtration combined system. Glucose of 20 g and ammonium of 1.5 g were added to the broth reaching a C/N of 90 (Fig. 4A). The control group was not concentrated, but additional carbon source was needed for products accumulation (Fig. 4B). Then, the environment of cultivation system was shifted to dark/low-temperature condition and continued to cultivation for products accumulation. The results of microfiltration group were shown in Fig. 4A, and it indicated that not only lipid content was significantly increased from 20.3% to 26.7%, but also lipid yield was improved up to 11.3 g/L at 84th hour. The biomass and carotenoid production were not affected in this stage. The comprehensive data of microfiltration and no microfiltration groups was shown in Table 1. The biomass, lipid content, carotenoid yields of two groups were maintained the same level, but the residual glucose of microfiltration group was greatly reduced from 30 to 2 g/L. The biomass and lipid coefficients were increased from 38.7 to 43.1 and 10.4 to 11.5, respectively. Finally, the lipid yield was significantly improved from 7.25 to 11.5 g/L, which was increased by nearly 58.6%. Therefore, there were two advantages in the new two-stage cultivation using microfiltration to reduce the broth volume: firstly, the lipid yield was greatly improved when consuming the same amount of carbon source; secondly, the residual sugar was greatly reduced at the end of fermentation, which could improve the utilization of glucose and benefit for the treatment of broth. Moreover, as a nonphototrophic species, the biomass and production content of R. gutinis were greatly influenced by irradiation in our studied. It is possible that by mixing cultivation R. gutinis of and microalgae may eventually lead to higher productivities. There are synergistic effects on gas, substance exchange and pH adjustment under irradiation in the mixed culture system of oleaginous yeast and microalgae in theory (Cheirsilp et al., 2011).
Fig. 4. Two-stage culture of R. glutinis for lipid and carotenoid production.
3.5. Lipid and carotenoid compositions when microorganisms are exposed to an energy imbalance caused by nutrient limitation (Klok et al., 2013). In addition, the lipid yield is relevant to both biomass concentration and lipid content. Although a high content of production was obtained, production
Lipid extracted from the cells of R. glutinis which were cultured under two-stage cultivation system was analyzed by gas-chromatograph (GC). The results showed that the main components of the lipids were oleic acid (C18:1; 51.1% in total lipid), linoleic
Table 1 The comprehensive of parameters in the second stage cultivation.
1 2
Group1
Final biomass (g)
Residual glucose (g/L)
Total sugar consumption (g)
Max. lipid content (%)
Max. total lipid (g/L)
Max. total carotenoid (mg/L)
Biomass coefficient2
Lipid coefficient2
A B
81.3 86.2
30 2
210 200
26.8 26.7
7.3 11.5
4.1 4.2
38.7 43.1
10.4 11.5
A and B were the groups of no microfiltration and microfiltration, respectively. The coefficients of biomass and lipid refer to conversion the quality (g) of the biomass or lipid from 100 g of glucose.
Z. Zhang et al. / Bioresource Technology 157 (2014) 149–153 Table 2 The composition and content of R. glutinis lipid. Lipid compositions
Lipid content (%)
Oleic acid (C18:1n9c) Linoleic acid (C18:2n6c) Palmitic acid (C16:0) Stearic acid (C18:0) Twenty-four carbonate (C24:0) a-linolenic acid (C18:3n3) Myristic acid (C14:0) Palmitoleic acid (C16:1n7) Behenic acid (C22:0) DPA (C22:5)
51.3 21.6 16.5 3.7 1.4 2.9 0.7 0.4 0.5 0.3
acid (C18:2; 21.6% in total lipid), palmitic acid (C16:0; 16.4% in total lipid) and stearic acid (C18:0; 3.7% in total lipid) (Table 2). The fatty acids of C18 and C16 accounted for 79.7% and 16.8% in total lipid, respectively. Therefore, the compositions of lipid fermented in two-stage cultivation were similar to those of vegetable seed lipids and soybean lipids (Xue et al., 2006), which could be used for biodiesel and jet fuel production because of a high content of fatty acid of C16 and C18 (96.6% of total lipid) obtained (Sivakumar et al., 2012). The carotenoids were extracted from the cells of R. glutinis and the main compounds were measured by HPLC. The results showed that the main carotenoids were identified as b-carotene, torularhodin, torulene, which contents in total carotenoids were 68.4%, 21.5% and 10.1%, respectively. This result was similar to the report by Somashekar and Joseph (2000) but different from the result of other reports (Schnider et al., 2013; Buzzini and Martini, 2000; Aksu and Eren, 2007), where the main pigment was torularhodin. Presumably, changed temperature and light inhibited some enzymes, which were used to catalyze torularhodin and torulene synthesized (Malisorn and Suntornsuk, 2008). 4. Conclusion This work investigated the effects of different of temperatures, irradiation conditions and C/N ratios on biomass growth, lipid content and carotenoid productivity. A new strategy of two-stage cultivation was developed for lipid and carotenoid production by R. glutinis. Especially concentrated broth before the second stage, the yields of lipid and carotenoid were significantly improved and the residual sugar concentration was reduced. This strategy as well as might be used for mixing cultivation of yeast and alga. Acknowledgements Authors acknowledge the financial support from the National High Technology Research and Development 863 Program of China (Grant No. 2013AA050702, 2012AA022304). References Aksu, Z., Eren, A.T., 2007. Production of carotenoids by the isolated yeast of Rhodotorula glutinis. Biochem. Eng. J. 35, 107–113. Buzzini, P., Martini, A., 2000. Production of carotenoids by strains of Rhodotorula glutinis cultured in raw materials of agro-industrial origin. Bioresour. Technol. 71, 41–44. Chatzifragkou, A., Makri, A., Belka, A., Bellou, S., Mavrou, M., Mastoridou, M., 2011. Biotechnological conversions of biodiesel derived waste glycerol by yeast and fungal species. Energy 36, 1097–1108. Cheng, P., Ji, B., Gao, L., Zhang, W., Wang, J., Liu, T., 2013. The growth, lipid and hydrocarbon production of Botryococcus braunii with attached cultivation. Bioresour. Technol. 138, 95–100. 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.
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