Bioresource Technology 175 (2015) 91–96
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Recycling microbial lipid production wastes to cultivate oleaginous yeasts Xiaobing Yang a,b,c, Guojie Jin a, Zhiwei Gong a, Hongwei Shen a, Fengwu Bai b, Zongbao Kent Zhao a,⇑ a
Division of Biotechnology and Dalian National Laboratory for Clean Energy, Dalian Institute of Chemical Physics, CAS, Dalian 116023, China School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, China c University of Chinese Academy of Sciences, Beijing 100049, China b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Spent cell mass hydrolysates
supported cell growth of various oleaginous yeasts. Spent cell mass hydrolysates used as nutrients for lipid production. Recycling both cell mass and spent water improved lipid production efficiency. Spent cell mass hydrolysates improved lipid production from corn stalk hydrolysates. Lipid samples had similar fatty acid compositional profiles to those of vegetable oils.
a r t i c l e
i n f o
Article history: Received 14 August 2014 Received in revised form 3 October 2014 Accepted 5 October 2014 Available online 20 October 2014 Keywords: Microbial lipids Nutrient recycling Oleaginous yeast Spent cell mass Rhodosporidium toruloides
a b s t r a c t To reduce wastes and the costs of microbial lipid production, it is imperative to recycle resources, including spent cell mass, mineral nutrients and water. In the present study, lipid production by the oleaginous yeast Rhodosporidium toruloides was used as a model system to demonstrate resources recycling. It was found that the hydrolysates of spent cell mass were good media to support cell growth of various oleaginous yeasts. When serial repitching experiments were performed using 70 g/L glucose and the hydrolysates alone as nutrients, it produced 16.6, 14.6 and 12.9 g/L lipids, for three successive cycles, while lipid titre remained almost constant when spent water was also recycled. The cell mass hydrolysates could be used as equivalents to the mixture of yeast extract and peptone to support lipid production from corn stalk hydrolysates. Our results showed efficient recycling of lipid production wastes and should be helpful to advance microbial lipid technology. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction The potential of microbial lipids as the feedstock for biodiesel production has been recognized recently (Liu and Zhao, 2007; Meng et al., 2009). However, large-scale production of microbial lipids remains economically challenging. Considerable efforts have ⇑ Corresponding author. Tel./fax: +86 411 84379211. E-mail address: [email protected] (Z.K. Zhao). http://dx.doi.org/10.1016/j.biortech.2014.10.020 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
been devoted to exploring low cost substrates, such as lignocellulosic biomass (Chen et al., 2012; Gong et al., 2013; Hu et al., 2011), industrial wastes (Xue et al., 2008) and crude glycerol (Kiran et al., 2012; Yang et al., 2014a), to reduce the costs for microbial lipid production. Because lipids are intracellular products, microbial lipid technology will co-produce significant amounts of spent cell mass (SCM), which contains polysaccharides, proteins, nutrients, and trace elements (Nguyen et al., 1998). Moreover, there are residual nutrients and trace elements in waste water. Therefore,
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Fig. 1. Flowchart of serial recycling of lipid production-derived resources. (A) Overall processes for microbial lipid production involving resources recycling; (B) detailed operation of each cycle.
it is pivotal to develop processes for efficient utilization of SCM and waste water in order to further advance microbial lipid technology. Interestingly, the recycling spent cell mass has been well known for micro-algae. For example, it has been used as feedstock for the production of bio-hydrogen, methane, bio-ethanol, and even algal biomass (Enquist-Newman et al., 2013; Yang et al., 2011; Zheng et al., 2012). Additionally, spent yeasts from brewery industry have been explored for enhancing docosahexaenoic acid accumulation (Ryu et al., 2013), ethanol fermentation (Suwanapong et al., 2013) and flavor generation (Vieira et al., 2013). We also showed that carbohydrates from SCM of the oleaginous yeast Rhodosporidium toruloides Y4 could be explored as carbon sources for microbial lipid production (Yang et al., 2014b). Two strategies were devised to make full use of SCM in this study. First, SCM hydrolysates (SCH) were prepared and used as nutritious media to cultivate oleaginous yeasts. Alternatively, SCH were employed as nutrient supplements for the serial closed and semi-closed lipid production culture supplemented with carbon sources (Fig. 1). For the closed culture, both SCM and spent water (dashed line) were recycled, while only SCM was reutilized for the semi-closed culture. Our results demonstrated that SCH alone were as good as the nutrient-rich media YEPD to support the growth of various oleaginous yeasts, and that up to 3 successive cycles were possible with no apparent dropping in lipid production efficiency from glucose when both SCM and spent water were reused. Moreover, it was found that SCH could be used as equivalents to the mixture of yeast extract and peptone in terms of supporting lipid production from corn stalk hydrolysates by R. toruloides Y4. Together, this study showed efficient recycling of microbial lipid production-derived resources and should be helpful to advance microbial lipid technology.
2. Methods 2.1. Strain and medium R. toruloides Y4 was a derivative of R. toruloides AS 2.1389 obtained from the China General Microbiological Culture
Collection Center (CGMCC). Lipomyces starkeyi AS 2.1560 and Trichosporon cutaneum AS 2.571 were also from CGMCC. Cryptococcus curvatus ATCC 20509 was from the American Type Culture Collection. All strains were maintained at 4 °C on YPD agar slant containing (g/L) glucose 20, yeast extract 10, peptone 10 and agar powder 15, pH 6.0. The seed medium contained (g/L) glucose 20, yeast extract 10 and peptone 10, pH 6.0. This medium contained 2.4 g/L total nitrogen. Nitrogen-limited lipid production medium contained (g/L): Glucose 70, (NH4)2SO4 0.1, yeast extract 0.75, KH2PO4 1.5, MgSO47H2O 1.5, initial pH 6.0. Trace element solution was added separately to all media after sterilization to a final concentration (mg/L): CaCl22H2O 40, FeSO47H2O 5.5, citric acidH2O 5.2, ZnSO47H2O 1.0, MnSO4H2O 0.76 and H2SO4 1.76. Media for the semi-closed and closed repitching culture with glucose contained (g/L): glucose 70 and SCM 16 for the first cycle, then the resulted SCM (and spent water for the closed culture) was repitched in the next round after glucose loading (Fig 1A). It should be pointed out that SCM in the media formula was referring to that being used for SCH preparation. For lipid production from corn stalk hydrolysates using the semi-closed culture, the media contained (g/L) total reducing sugar (TRS) 41.6 and SCM 5.0, then the following process was conducted as described in Fig 1A. All media were subjected to sterilization by autoclaving at 121 °C for 15 min. The chemicals, if not specified, were of analytical grade and bought locally.
2.2. Preparation of SCM and corn stalk hydrolysates To prepare SCM sample, R. toruloides Y4 cells were cultivated in a 15-L stirred-tank bioreactor (Guoqiang Bioengineering Equipment Co. Ltd., Shanghai, China) as described previously (Li et al., 2007). Briefly, the cultivation conditions were as follows: 10% (v/ v) seed culture, temperature 30 °C, pH 5.6, aeration at 0.9 vvm, dissolved oxygen at 40–50% and started with 7.0 L of media. The initial glucose concentration was 50 g/L and maintained above 10 g/L by regularly feeding 1000 g/L glucose solution. Samples of
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30 mL each were taken at intervals to estimate glucose concentration, cell dry weight and lipid content. Eight cycles of substrate feeding were done to give a final cell mass of 100 g/L and lipid content of 70% after 134 h. Cells were collected and dried. The dry cells were milled, treated with petroleum ethers (bp 60–90 °C) for three times according to a known procedure (Economou et al., 2010). Lipids were recovered from the petroleum ethers layer upon evaporation, and the solid residues were received as the SCM sample. SCH were prepared in 0.5 M sulfuric acid with a solid/liquid ratio of 1/10 (v/w) at 120 °C for 1.5 h. The hydrolysates were neutralized with calcium hydroxide, centrifuged at 8000g for 5 min. This SCH had 27.8 g/L TRS and 2.5 g/L total nitrogen, and was properly diluted when necessary. Corn stalk samples were pretreatment with 0.5 M NaOH as described (Gong et al., 2013). Corn stalk hydrolysates were prepared at 50 °C, pH 4.8, for 48 h, with a solid/liquid ratio of 1/10 (v/w). The enzymes loading for hydrolysis were (per g corn stalk) cellulose 20 FPU, dextranase (40 FPU) and xylanase (5 mg). Typically, the corn stalk hydrolysates had 41.6 g/L TRS and 0.23 g/L total nitrogen, and the sugar compositions were as follows (g/L), glucose 29.9, xylose 9.7, arabinose 1.9, mannose 0.2 and galactose 0.1. 2.3. Batch culture All the cultures were performed with 250-mL Erlenmeyer flasks (loading volume 50 mL) at 30 °C, initial pH 6.0 and a rotary speed of 200 rpm in triplicate. For recycling experiments, a loop of the yeast cells were inoculated in the seed media and cultivated for 24 h, then, the seed culture was re-inoculated with a ratio of 10% (v/v) to the lipid production media. Culture samples (0.2 mL) were withdrawn every 24 h to determine residual glucose or TRS.
To determine fatty acid compositional profile, lipid samples were transmethylated and analyzed by gas chromatography with a 7890F GC instrument (Techcomp Scientific Instrument Co. Ltd., Shanghai, China) equipped with a cross-linked capillary FFAP column (30 m 0.32 mm 0.4 mm) and flame ionization detector, as described (Li et al., 2007). 3. Results and discussion 3.1. SCM recovery and microbial lipids fractionation Microbial lipids were routinely extracted by using a mixture of chloroform and methanol after cells were treated with 4.0 mol/L HCl at 78 °C for 1 h (Li et al., 2007). This chloroform/methanol method facilitated more complete extraction of cellular lipids, but cell mass was digested and difficult to be recycled. To fully recycle SCM, lipids were extracted by using the petroleum ether method (Economou et al., 2010), and 0.39 g of SCM was produced from 1.0 g of R. toruloides Y4 cells with a lipid content of 70% in a typical experiment. Elementary analysis showed that the SCM sample contained carbon 47.4%, nitrogen 3.7% and hydrogen 8.0%. Lipid fractionation results showed that lipid samples extracted with the petroleum ether method had neutral lipids, G + S, and P, respectively, of 98.2%, 1.2% and 0.6%. In contrast, more G + S (8.3–12.4%) and P (4.0–6.6%) were found when the lipids were extracted with the chloroform/methanol method. Similar results were also described elsewhere (Wu et al., 2010). Because petroleum ethers are non-polar solvents, it is less efficient to extract polar lipids including G + S and P. Similar results were observed when hexane was used as the solvent to extract lipids from other oleaginous yeasts (Economou et al., 2010). Importantly, the carrying-over of polar lipids in SCM should be advantageous in terms of providing elementary nutrients such as sulfur and phosphorus for microbial cell growth.
2.4. Analytical methods
3.2. Cultivation of oleaginous yeasts in SCH alone
Glucose was quantified by a glucose analyzer (SBA-50B; Shandong Academy of Sciences, Jinan, China). TRS in corn stalk hydrolysates were quantified according to the 2,4-dinitrosalicylate (DNS) method with a V530 UV/vis spectrophotometer (Jasco; Japan). Sugar compositions were analyzed by ion chromatography as described (Xie et al., 2012). Cell mass was expressed as cell dry weight (CDW). Cells from 30 mL of culture broth were harvested by centrifugation at 8000g for 5 min, washed twice with distilled water, and dried at 105 °C for 24 h to a constant weight. To ensure efficient recovery of both SCM and lipids, cells were treated and extracted with petroleum ethers according to a known procedure (Economou et al., 2010). To determine cellular total lipid contents, cells were extracted by a mixture of chloroform and methanol according to the reported procedure (Li et al., 2007). Lipid content was expressed as g lipid recovered per g CDW. The lipid yield was defined as g lipid produce per g substrate consumed. All values were the average of 3 independent experiments. Nitrogen was determined according to the Kjeldahl method (Morgan et al., 1957). Lipid fractionation was done according to a known procedure with minor modifications (Fakas et al., 2006). Briefly, about 300 mg of lipids in 3 mL of chloroform was loaded a silica gel column. The column was washed successively by 1,2-dichloroethane (300 mL), 1,2-dichloroethane/acetone (200 mL,1:10, v/v), and methanol (150 mL) to receive fractions of neutral lipids (N), glycolipids plus sphingolipids (G + S), and phospholipids (P), respectively. Lipid fractions were determined gravimetrically after evaporation of the solvents.
To test whether SCM could be used to cultivate oleaginous yeasts, it was digested in 1.0 M H2SO4 at 120 °C for 1.5 h, neutralized with calcium hydroxide, centrifuged, and the supernatants were received as SCH. Four oleaginous yeasts, namely R. toruloides Y4, L. starkeyi AS 2.1560, T. cutaneum AS 2.571 and C. curvatus ATCC 20509, were inoculated in SCH and the cultures were held for 24 h. As shown in Fig. 2, C. curvatus produced 12.7 g/L cell mass but L. starkeyi gave only 2.5 g/L. Interestingly, comparable or lower cell mass concentrations were obtained when the YEPD media were used for these strains. Therefore, cell mass differences among those strains were mainly caused by their growth rate differences. These results demonstrated that SCH alone could be used in lieu of the 14 YEPD
SCH
Cell mass (g/L)
12 10 8 6 4 2 0
L.starkeyi
R.toruloides T.cutaneum
C.curvatus
Fig. 2. Cultivation of oleaginous yeasts using YEPD and SCH. Cells were cultivated at 30 °C for 24 h in the YEPD media contained 20 g/L glucose or SCH contained 20 g/L TRS. Error bars denote standard deviation of 3 independent experiments.
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YEPD media to cultivate oleaginous microorganisms and were in line with the fact that spent yeasts from a brewery significantly enhanced cell mass and docosahexaenoic acid production by Aurantiochytrium sp. KRS101 (Ryu et al., 2013). However, as SCH contained relatively rich nutrients including nitrogen, sulfur and phosphorus, it might be not good for lipid production. It is well known that a nutrient-limited yet carbon-rich environment is required to stimulate lipid accumulation when carbohydrates and related materials are used as carbon sources (Papanikolaou and Aggelis, 2011). Nonetheless, SCH may be used as nutrients source for lipid production upon additional carbon sources are supplemented. 3.3. SCH as nutrient source 3.3.1. The combination of SCH and glucose We used 70 g/L glucose as the carbon source and SCH as nutrients to cultivate R. toruloides Y4 cells for lipid production. To determine nutrient recycling efficiency, the semi-closed repitching experiments were performed for 3 cycles. In these experiments, SCM was hydrolyzed and the entire hydrolysates were used in the next cycle. For each cycle, we recorded cell mass, lipid, total nitrogen loaded and nitrogen recovered, and the results were shown in Table 1. For Cycle 1, SCH were derived from 16 g/L SCM that led to the introduction of 0.4 g/L total nitrogen. Because the inoculation with 10 vol% seed culture prepared in the YEPD media introduced about 0.24 g/L total nitrogen, so total nitrogen loaded in the system was 0.64 g/L. Cell mass and lipid were 25.2 g/L and 15.2 g/L, respectively (Table 1, entry 2). Compared to those values of Cycle 1, it was clear that all data decreased for Cycle 2, and further decreasing was observed for Cycle 3 (Table 1, entries 2–4). It was clear that total nitrogen recovered in SCM was much lower than the amount of total nitrogen loaded, indicating that there was substantial nitrogen loss in each cycle. Concurrently, lipid production capacity was also getting worse every cycle as indicated by cell mass and lipid data. The glucose evolution profiles were shown in Fig. 3A, indicating that glucose consumption rates (g/L/h) were 0.59, 0.55 and 0.53, for Cycle 1, 2 and 3, respectively. Compared to the control culture in N-limited media, Cycle 1 obtained higher cell mass and lipid (Table 1, entry 1 vs. 2). This was likely due to the carryover of polar lipids from SCM as well as more nutrients to support cell growth. For the whole semi-closed repitching experiment, overall cell mass yield and lipid yield were 0.30 g/g and 0.19 g/g, respectively, which were comparable to those of the control culture in N-limited media. It should be noted that similar experiments using 70 g/L glucose as the carbon source were also performed without the introduction
of SCH. Under such conditions, nitrogen nutrients were originated solely from the inocula. There was 47.3 g/L glucose left in the culture after 132 h. Compared with results shown in Fig. 3A, it was clear that the application of SCH was necessary to facilitate sugar consumption, and thus cell growth and lipid production. It was found that about 0.16 g/L total nitrogen remained in the medium of Cycle 1 at the end of the culture (Fig. 3B). We also tried using SCM directly without hydrolysis, but there was about 20 g/L glucose unconsumed after 120 h in a typical experiment. Therefore, it was not efficient to use SCM without hydrolysis. This is understandable because red yeast possesses a rigid cell wall structure, which can prevent the releasing of nutritious elements (Jin et al., 2012). Recently, the significance of recycling nutrients from microalgal cell mass has been recognized for biofuel production, and nutrients were released by anaerobic digestion (Bohutskyi et al., 2014) or hydrothermal liquefaction (Zhou et al., 2013). In another aspect, the presence of SCM increased the viscosity of the culture, which may affect the transfer of mass and oxygen, as previous noted (Audet et al., 1996). The closed repitching experiments were performed for 3 cycles to test whether nutrient recycling efficiency could be improved, and the results were shown in Table 1, entries 5–7. In these experiments, both spent water and SCM were reused to prepare the SCH for the next cycle. To start Cycle 1 of the closed culture, the waste water from the Cycle 1 of the semi- closed culture was used. Therefore, total nitrogen loaded was determined as 0.91 g/L (Table 1, entry 5). In sharp contrast to the dropping trend for the results of the semi-closed experiments, there was little variation in terms of cell mass, lipid, total nitrogen loaded and nitrogen recovered. The fact that both total nitrogen loaded and nitrogen recovered were slightly increased suggested that nitrogenous nutrients were fully recycled. Because of this, overall glucose consumption rates were 0.58 g/L/h for all the cycles, and no apparent differences were observed in terms of glucose evolution profile (Fig. 3C), and similar cell mass and lipid were obtained for all cycles. Overall cell mass yield and lipid yield were 0.31 g/g and 0.20 g/g, respectively, which were essentially identical to those of the semi-closed experiments. In addition, roughly 90% water was reused for each cycle, and the rest 10% was lost due to sampling and evaporation. Therefore, these results demonstrated the possibility of low waste water discharge for 3 consecutive batches of lipid production. Admittedly, we noticed that the viscosity of the culture broth increased gradually. It might also lead to the accumulation of some hazardous byproducts that were generated during SCH preparation and cell growth. These effects prevented from successful repitching beyond 3 cycles. Nonetheless, our results clearly demonstrated that the closed repitching strategy could be used for efficient production
Table 1 Results of cell mass, lipid and nitrogen nutrients of different cultures for lipid production by R. toruloides Y4.
a
Entry
Culture trait
Cell mass (g/L)
Lipid (g/L)
Total nitrogen loaded (g/L)
1 2 3 4 5 6 7 8 9 10 11
Control SCR1c SCR2c SCR3c CR1c CR2c CR3c SCR1-CSd SCR2-CSd SCR3-CSd Control-CS
21.8 ± 0.1 25.2 ± 0.6 22.4 ± 0.6 19.6 ± 0.2 23.3 ± 0.0 21.9 ± 0.3 22.1 ± 0.6 12.7 ± 0.2 13.1 ± 0.1 13.2 ± 0.8 11.5 ± 0.4
14.2 ± 0.3 15.2 ± 0.4 14.9 ± 0.4 12.6 ± 0.1 15.3 ± 0.3 13.6 ± 0.2 14.0 ± 0.3 5.9 ± 0.1 5.5 ± 0.1 5.3 ± 0.3 5.4 ± 0.2
– 0.64 ± 0.00 0.63 ± 0.02 0.54 ± 0.01 0.91 ± 0.00 1.03 ± 0.02 1.14 ± 0.01 0.60 ± 0.04 0.68 ± 0.03 0.69 ± 0.05 –
a
Nitrogen recovered (g/L)
b
– 0.43 ± 0.02 0.34 ± 0.01 0.22 ± 0.01 0.30 ± 0.00 0.31 ± 0.01 0.32 ± 0.00 0.27 ± 0.01 0.30 ± 0.01 0.31 ± 0.00 –
Total nitrogen loaded including nitrogen originated from SCH, corn stalk hydrolysates and the inocula. Nitrogen recovered referring nitrogen retained in SCM. Experiments were conducted on the SCH supplemented with glucose for 120 h. d Experiments were performed with SCH and corn stalk hydrolysates for 72 h. SCR: semi-closed recycling; CR: closed recycling; CS: corn stalk hydrolysates directly. Data were presented as the mean ± standard deviation of 3 independent experiments. b
c
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A
SCR 1
70
SCR 2
A
SCR3
50
CS SCR1-CS SCR2-CS SCR3-CS
60
TRS (g/L)
Glucose (g/L)
40 50 40 30
30 20
20
10 10
0
0
Cell mass
60 80 Time (h) Lipid
100
120
0
140
B 16
0.8
Total nitrogen
0.6
20 15
0.4 10 0.2 5 0
10
20
30
40 50 60 Time (h)
Cell mass
Lipid
70
80
90 100
Total nitrogen
0.6 12 0.4 8
0.2
4
Total nitrogen (g/L)
40
Cell mass, Lipid (g/L)
Cell mass, Lipid (g/L)
B 25
20
Total nitrogen (g/L)
0
0.0 0
20
40
60
80
100
120
0.0
0
Time (h)
0
10
20
30
40
50
60
70
Time (h)
C 70
CR 1
CR 2
CR 3
Fig. 4. The time-courses of lipid production from corn stalk hydrolysates by R. toruloides Y4. (A) The evolution of TRS for the semi-closed culture, (B) the evolution of cell mass, total nitrogen and lipid for Cycle 1 of the semi-closed culture. Error bars denote standard deviation of 3 independent experiments.
Glucose (g/L)
60 50 40 30 20 10 0
0
20
40
60
80
100
120
Time (h) Fig. 3. The time-courses of lipid production from glucose by R. toruloides Y4. (A) The evolution of glucose for the semi-closed culture, (B) the evolution of cell mass, total nitrogen and lipid for Cycle 1 of the semi-closed culture, and (C) the evolution of glucose for the closed culture. Error bars denote standard deviation of 3 independent experiments.
of microbial lipids, leading to significantly reduce the consumption of nutrients and water.
3.3.2. The combination of SCH and corn stalk hydrolysates To further demonstrate the usefulness of SCH as microbial nutrients, we used corn stalk hydrolysates as carbon source for lipid production by R. toruloides Y4. It has been demonstrated that this yeast could use corn stalk hydrolysates without additional nutrient supplementation (Hu et al., 2009). Experiments were done in the semi-closed repitching mode for 3 cycles, and the results were shown in Table 1, entries 8–10. For Cycle 1, 1.4 g/L TRS from SCH and 2.0 g/L glucose from the inoculum were also introduced, so the initial carbon source concentration equaled to 45.0 g/L TRS. The total nitrogen loaded in the culture was 0.60 g/L, including 0.23 g/L, 0.13 g/L and 0.24 g/L from corn stalk hydrolysates, SCH and the inoculum, respectively. Therefore, the Cycle 1 culture had a C/N ratio of 35, and led to the production of 12.7 g/L cell mass including 5.9 g/L lipids (Table 1, entry 8). For Cycle 2 and 3,
Table 2 Fatty acid compositional profiles of microbial lipid samples prepared in this study.
a
Entry
Culture trait
C:14-0
C:16-0
C:16-1
C:18-0
C:18-1
C:18-2
C:18-3
1 2 3 4 5 6 7 8 9 10
Controla SCR1a SCR2a SCR3a CR1b CR2b CR3b SCR1-CSb SCR2-CSb SCR3-CSb
1.0 ± 0.1 1.2 ± 0.0 1.4 ± 0.1 1.0 ± 0.2 1.7 ± 0.1 1.3 ± 0.1 1.6 ± 0.1 1.3 ± 0.1 1.5 ± 0.0 1.5 ± 0.1
32.1 ± 0.8 33.6 ± 0.2 40.9 ± 0.4 29.8 ± 0.4 27.5 ± 0.4 25.6 ± 0.2 27.6 ± 0.2 28.2 ± 0.7 30.0 ± 0.3 29..0 ± 1.1
0.2 ± 0.0 0.5 ± 0.1 0.3 ± 0.1 0.4 ± 0.2 0.7 ± 0.1 0.3 ± 0.0 0.5 ± 0.1 0.6 ± 0.0 0.6 ± 0.1 0.8 ± 0.1
12.7 ± 0.2 7.0 ± 0.0 9.8 ± 0.2 7.2 ± 0.2 9.6 ± 0.3 15.4 ± 0.0 12.0 ± 0.0 10.0 ± 0.2 10.6 ± 0.1 10.2 ± 0.1
51.0 ± 1.8 55.0 ± 0.1 46.7 ± 0.2 56.5 ± 0.8 54.0 ± 0.5 50.3 ± 0.3 53.5 ± 0.1 51.2 ± 0.5 51.0 ± 0.2 51.1 ± 0.9
0.9 ± 0.3 2.7 ± 0.1 0.5 ± 0.0 4.5 ± 0.0 4.6 ± 0.1 6.7 ± 0.0 4.7 ± 0.1 6.8 ± 0.1 6.3 ± 0.0 5.9 ± 0.2
0.3 ± 0.2 – – – – – – – – –
Experiments were conducted on the SCH supplemented with glucose for 120 h. Experiments were performed with SCH and corn stalk hydrolysates for 72 h. SCR: semi-closed recycling; CR: closed recycling; CS: corn stalk hydrolysates directly. Data were presented as the mean ± standard deviation of 3 independent experiments. b
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recycled SCM from Cycle 1 and 2, respectively, was hydrolyzed and combined with corn stalk hydrolysates. Compared with Cycle 1, Cycle 2 and 3 produced slightly higher cell mass but lower lipids. Also, sugars were exhausted within 72 h (Fig. 4A), and the TRS consumption rates were 0.63, 0.67 and 0.68 g/L/h for Cycle 1, 2 and 3, respectively, which were higher than those experiments using glucose as carbon source. Apparently, carbon sources were consumed even faster for the culture combined SCH with corn stalk hydrolysates than that with glucose and corn stalk hydrolysates alone. Similarly, about 0.25 g/L total nitrogen remained in the medium of Cycle 1 after 72 h (Fig. 4B). It was interesting to note that a slightly increase in total nitrogen loaded was observed, indicating that recycling SCM was sufficient to balance nitrogen loss due to waste water discharge. However, both cell mass and lipid were lower (Table 1, entry 11) and the overall TRS consumption rate was only 0.45 g/L/h for the experiments with corn stalk hydrolysates alone. Together, these results revealed the feasibility of recycling SCM as nutrients to support microbial lipid production from complex carbon-rich feedstock such as corn stalk hydrolysates.
3.4. Fatty acid compositional profile To analyze the fatty acids profiles, lipid samples were transmethylated and the resulting fatty acid methyl esters were analyzed by GC. Results in Table 2 showed that the presence of long chain fatty acids included oleic acid (C18:1), palmitic acid (C16:0), stearic acid (C18:0), linoleic acid (C18:2), myristic acid (C14:0), palmitoleic acid (C16:1) and linolenic acid (C18:3, present only in the control sample). Little difference was found in terms of the relative fatty acid species regardless of the recycling procedures, and even less discrepancy among the fatty acid profiles within the same repitching sequence (Table 2, entries 2–4, 5–7 and 8–10), indicating that the repitching manipulation had no obvious influence on fatty acid biosynthesis. Specifically, C18:1, C16:0 and C18:0 together accounted for over 90% of the total fatty acids, while C18:1 took 45.1–56.5%. It has been pointed out that methyl oleate make the best candidate for biodiesel production, based on a comprehensive analysis on cetane number, exhaust emissions, combustion heat, low-temperature properties and lubricity (Knothe, 2008). Such fatty acid compositional profiles were of great potential as biodiesel feedstock.
4. Conclusion To facilitate large-scale microbial production of lipids and related chemicals, it is imperative to reutilize resources including spent cell mass, nutrients and water. Our results demonstrated that spent cell mass hydrolysates from the oleaginous yeast R. toruloides could be used as media for cell growth and as nutrients for lipid production from different carbon sources. Moreover, we showed that lipid production could be operated with little efficiency drop for up to 3 cycles by fully recycling the resources. This provided an attractive approach to minimize the input on microbial lipid production.
Acknowledgements This work was supported by the Ministry of Science and Technology of China (No. 2011CB707405), the National Natural Scientific Foundation of China (No. 21325627), Chinese Academy of Sciences (No. KGZD-EW-304-2) and the State Key Laboratory of Motor Vehicle Biofuel Technology, Henan Tianguan Group Co., China (No. 2013001).
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Enquist-Newman, M., Faust, A.M.E., Bravo, D.D., Santos, C.N.S., Raisner, R.M., Hanel, A., Sarvabhowman, P., Le, C., Regitsky, D.D., Cooper, S.R., 2013. Efficient ethanol production from brown macroalgae sugars by a synthetic yeast platform. Nature 505, 239–243. Fakas, S., Papanikolaou, S., Galiotou-Panayotou, M., Komaitis, M., Aggelis, G., 2006. Lipids of Cunninghamella echinulata with emphasis to gamma-linolenic acid distribution among lipid classes. Appl. Microbiol. Biotechnol. 73, 676–683. Gong, Z., Shen, H., Wang, Q., Yang, X., Xie, H., Zhao, Z.K., 2013. Efficient conversion of biomass into lipids by using the simultaneous saccharification and enhanced lipid production process. Biotechnol. Biofuels 6, 36. Hu, C., Wu, S., Wang, Q., Jin, G., Shen, H., Zhao, Z.K., 2011. Simultaneous utilization of glucose and xylose for lipid production by Trichosporon cutaneum. Biotechnol. Biofuels 4, 25. Hu, C., Zhao, X., Zhao, J., Wu, S., Zhao, Z.K., 2009. Effects of biomass hydrolysis byproducts on oleaginous yeast Rhodosporidium toruloides. Bioresour. Technol. 100, 4843–4847. Jin, G., Yang, F., Hu, C., Shen, H., Zhao, Z.K., 2012. Enzyme-assisted extraction of lipids directly from the culture of the oleaginous yeast Rhodosporidium toruloides. Bioresour. Technol. 111, 378–382. Kiran, E.U., Trzcinski, A., Webb, C., 2012. Microbial oil produced from biodiesel byproducts could enhance overall production. Bioresour. Technol. 129, 650–654. Knothe, G., 2008. ‘‘Designer’’ biodiesel: optimizing fatty ester composition to improve fuel properties. Energy Fuels 22, 1358–1364. Liu, B., Zhao, Z.K., 2007. Biodiesel production by direct methanolysis of oleaginous microbial biomass. J. Chem. Technol. Biotechnol. 82, 775–780. Li, Y., Zhao, Z.K., Bai, F., 2007. High-density cultivation of oleaginous yeast Rhodosporidium toruloides Y4 in fed-batch culture. Enzyme Microb. Technol. 41, 312–317. Meng, X., Yang, J., Xu, X., Zhang, L., Nie, Q., Xian, M., 2009. Biodiesel production from oleaginous microorganisms. Renewable Energy 34, 1–5. Morgan, G.B., Lackey, J.B., Gilcreas, F.W., 1957. Quantitative determination of organic nitrogen in water, sewage, and industrial wastes. Anal. Chem. 29, 833– 835. Nguyen, T.H., Fleet, G.H., Rogers, P.L., 1998. Composition of the cell walls of several yeast species. Appl. Microbiol. Biotechnol. 50, 206–212. Papanikolaou, S., Aggelis, G., 2011. Lipids of oleaginous yeasts. Part I: Biochemistry of single cell oil production. Eur. J. Lipid Sci. Technol. 113, 1031–1051. Ryu, B.G., Kim, K., Kim, J., Han, J.I., Yang, J.W., 2013. Use of organic waste from the brewery industry for high-density cultivation of the docosahexaenoic acid-rich microalga Aurantiochytrium sp. KRS101. Bioresour. Technol. 129, 351–359. Suwanapong, S., Khongsay, N., Laopaiboon, L., Jaisil, P., Laopaiboon, P., 2013. Dried spent yeast and its hydrolysate as nitrogen supplements for single batch and repeated-batch ethanol fermentation from sweet sorghum juice. Energies 6, 1618–1631. Vieira, E., Brandão, T., Ferreira, I.M.P.L.V.O., 2013. Evaluation of brewer’s spent yeast to produce flavor enhancer nucleotides: influence of serial repitching. J. Agric. Food Chem. 61, 8724–8729. Wu, S., Hu, C., Jin, G., Zhao, X., Zhao, Z.K., 2010. Phosphate-limitation mediated lipid production by Rhodosporidium toruloides. Bioresour. Technol. 101, 6124–6129. Xie, H., Shen, H., Gong, Z., Wang, Q., Zhao, Z.K., Bai, F., 2012. Enzymatic hydrolysates of corn stover pretreated by the N-methylpyrrolidone/ionic liquids solution for microbial lipid production. Green Chem. 14, 1202–1210. Xue, F., Miao, J., Zhang, X., Luo, H., Tan, T., 2008. Studies on lipid production by Rhodotorula glutinis fermentation using monosodium glutamate wastewater as culture medium. Bioresour. Technol. 99, 5923–5927. Yang, X., Jin, G., Gong, Z., Shen, H., Bai, F., Zhao, Z.K., 2014a. Recycling biodieselderived glycerol by the oleaginous yeast Rhodosporidium toruloides Y4 through the two-stage lipid production process. Biochem. Eng. J. 91, 86–91. Yang, X., Jin, G., Gong, Z., Shen, H., Song, Y., Bai, F., Zhao, Z.K., 2014b. Simultaneous utilization of glucose and mannose from spent yeast cell mass for lipid production by Lipomyces starkeyi. Bioresour. Technol. 158, 383–387. Yang, Z., Guo, R., Xu, X., Fan, X., Luo, S., 2011. Hydrogen and methane production from lipid-extracted microalgal biomass residues. Int. J. Hydrogen Energy 36, 3465–3470. Zheng, H., Gao, Z., Yin, F., Ji, X., Huang, H., 2012. Lipid production of Chlorella vulgaris from lipid- extracted microalgal biomass residues through two-step enzymatic hydrolysis. Bioresour. Technol. 117, 1–6. Zhou, Y., Schideman, L., Yu, G., Zhang, Y., 2013. 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Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Recycling microbial lipid production wastes to cultivate oleaginous yeasts Xiaobing Yang a,b,c, Guojie Jin a, Zhiwei Gong a, Hongwei Shen a, Fengwu Bai b, Zongbao Kent Zhao a,⇑ a
Division of Biotechnology and Dalian National Laboratory for Clean Energy, Dalian Institute of Chemical Physics, CAS, Dalian 116023, China School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, China c University of Chinese Academy of Sciences, Beijing 100049, China b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Spent cell mass hydrolysates
supported cell growth of various oleaginous yeasts. Spent cell mass hydrolysates used as nutrients for lipid production. Recycling both cell mass and spent water improved lipid production efficiency. Spent cell mass hydrolysates improved lipid production from corn stalk hydrolysates. Lipid samples had similar fatty acid compositional profiles to those of vegetable oils.
a r t i c l e
i n f o
Article history: Received 14 August 2014 Received in revised form 3 October 2014 Accepted 5 October 2014 Available online 20 October 2014 Keywords: Microbial lipids Nutrient recycling Oleaginous yeast Spent cell mass Rhodosporidium toruloides
a b s t r a c t To reduce wastes and the costs of microbial lipid production, it is imperative to recycle resources, including spent cell mass, mineral nutrients and water. In the present study, lipid production by the oleaginous yeast Rhodosporidium toruloides was used as a model system to demonstrate resources recycling. It was found that the hydrolysates of spent cell mass were good media to support cell growth of various oleaginous yeasts. When serial repitching experiments were performed using 70 g/L glucose and the hydrolysates alone as nutrients, it produced 16.6, 14.6 and 12.9 g/L lipids, for three successive cycles, while lipid titre remained almost constant when spent water was also recycled. The cell mass hydrolysates could be used as equivalents to the mixture of yeast extract and peptone to support lipid production from corn stalk hydrolysates. Our results showed efficient recycling of lipid production wastes and should be helpful to advance microbial lipid technology. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction The potential of microbial lipids as the feedstock for biodiesel production has been recognized recently (Liu and Zhao, 2007; Meng et al., 2009). However, large-scale production of microbial lipids remains economically challenging. Considerable efforts have ⇑ Corresponding author. Tel./fax: +86 411 84379211. E-mail address: [email protected] (Z.K. Zhao). http://dx.doi.org/10.1016/j.biortech.2014.10.020 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
been devoted to exploring low cost substrates, such as lignocellulosic biomass (Chen et al., 2012; Gong et al., 2013; Hu et al., 2011), industrial wastes (Xue et al., 2008) and crude glycerol (Kiran et al., 2012; Yang et al., 2014a), to reduce the costs for microbial lipid production. Because lipids are intracellular products, microbial lipid technology will co-produce significant amounts of spent cell mass (SCM), which contains polysaccharides, proteins, nutrients, and trace elements (Nguyen et al., 1998). Moreover, there are residual nutrients and trace elements in waste water. Therefore,
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Fig. 1. Flowchart of serial recycling of lipid production-derived resources. (A) Overall processes for microbial lipid production involving resources recycling; (B) detailed operation of each cycle.
it is pivotal to develop processes for efficient utilization of SCM and waste water in order to further advance microbial lipid technology. Interestingly, the recycling spent cell mass has been well known for micro-algae. For example, it has been used as feedstock for the production of bio-hydrogen, methane, bio-ethanol, and even algal biomass (Enquist-Newman et al., 2013; Yang et al., 2011; Zheng et al., 2012). Additionally, spent yeasts from brewery industry have been explored for enhancing docosahexaenoic acid accumulation (Ryu et al., 2013), ethanol fermentation (Suwanapong et al., 2013) and flavor generation (Vieira et al., 2013). We also showed that carbohydrates from SCM of the oleaginous yeast Rhodosporidium toruloides Y4 could be explored as carbon sources for microbial lipid production (Yang et al., 2014b). Two strategies were devised to make full use of SCM in this study. First, SCM hydrolysates (SCH) were prepared and used as nutritious media to cultivate oleaginous yeasts. Alternatively, SCH were employed as nutrient supplements for the serial closed and semi-closed lipid production culture supplemented with carbon sources (Fig. 1). For the closed culture, both SCM and spent water (dashed line) were recycled, while only SCM was reutilized for the semi-closed culture. Our results demonstrated that SCH alone were as good as the nutrient-rich media YEPD to support the growth of various oleaginous yeasts, and that up to 3 successive cycles were possible with no apparent dropping in lipid production efficiency from glucose when both SCM and spent water were reused. Moreover, it was found that SCH could be used as equivalents to the mixture of yeast extract and peptone in terms of supporting lipid production from corn stalk hydrolysates by R. toruloides Y4. Together, this study showed efficient recycling of microbial lipid production-derived resources and should be helpful to advance microbial lipid technology.
2. Methods 2.1. Strain and medium R. toruloides Y4 was a derivative of R. toruloides AS 2.1389 obtained from the China General Microbiological Culture
Collection Center (CGMCC). Lipomyces starkeyi AS 2.1560 and Trichosporon cutaneum AS 2.571 were also from CGMCC. Cryptococcus curvatus ATCC 20509 was from the American Type Culture Collection. All strains were maintained at 4 °C on YPD agar slant containing (g/L) glucose 20, yeast extract 10, peptone 10 and agar powder 15, pH 6.0. The seed medium contained (g/L) glucose 20, yeast extract 10 and peptone 10, pH 6.0. This medium contained 2.4 g/L total nitrogen. Nitrogen-limited lipid production medium contained (g/L): Glucose 70, (NH4)2SO4 0.1, yeast extract 0.75, KH2PO4 1.5, MgSO47H2O 1.5, initial pH 6.0. Trace element solution was added separately to all media after sterilization to a final concentration (mg/L): CaCl22H2O 40, FeSO47H2O 5.5, citric acidH2O 5.2, ZnSO47H2O 1.0, MnSO4H2O 0.76 and H2SO4 1.76. Media for the semi-closed and closed repitching culture with glucose contained (g/L): glucose 70 and SCM 16 for the first cycle, then the resulted SCM (and spent water for the closed culture) was repitched in the next round after glucose loading (Fig 1A). It should be pointed out that SCM in the media formula was referring to that being used for SCH preparation. For lipid production from corn stalk hydrolysates using the semi-closed culture, the media contained (g/L) total reducing sugar (TRS) 41.6 and SCM 5.0, then the following process was conducted as described in Fig 1A. All media were subjected to sterilization by autoclaving at 121 °C for 15 min. The chemicals, if not specified, were of analytical grade and bought locally.
2.2. Preparation of SCM and corn stalk hydrolysates To prepare SCM sample, R. toruloides Y4 cells were cultivated in a 15-L stirred-tank bioreactor (Guoqiang Bioengineering Equipment Co. Ltd., Shanghai, China) as described previously (Li et al., 2007). Briefly, the cultivation conditions were as follows: 10% (v/ v) seed culture, temperature 30 °C, pH 5.6, aeration at 0.9 vvm, dissolved oxygen at 40–50% and started with 7.0 L of media. The initial glucose concentration was 50 g/L and maintained above 10 g/L by regularly feeding 1000 g/L glucose solution. Samples of
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30 mL each were taken at intervals to estimate glucose concentration, cell dry weight and lipid content. Eight cycles of substrate feeding were done to give a final cell mass of 100 g/L and lipid content of 70% after 134 h. Cells were collected and dried. The dry cells were milled, treated with petroleum ethers (bp 60–90 °C) for three times according to a known procedure (Economou et al., 2010). Lipids were recovered from the petroleum ethers layer upon evaporation, and the solid residues were received as the SCM sample. SCH were prepared in 0.5 M sulfuric acid with a solid/liquid ratio of 1/10 (v/w) at 120 °C for 1.5 h. The hydrolysates were neutralized with calcium hydroxide, centrifuged at 8000g for 5 min. This SCH had 27.8 g/L TRS and 2.5 g/L total nitrogen, and was properly diluted when necessary. Corn stalk samples were pretreatment with 0.5 M NaOH as described (Gong et al., 2013). Corn stalk hydrolysates were prepared at 50 °C, pH 4.8, for 48 h, with a solid/liquid ratio of 1/10 (v/w). The enzymes loading for hydrolysis were (per g corn stalk) cellulose 20 FPU, dextranase (40 FPU) and xylanase (5 mg). Typically, the corn stalk hydrolysates had 41.6 g/L TRS and 0.23 g/L total nitrogen, and the sugar compositions were as follows (g/L), glucose 29.9, xylose 9.7, arabinose 1.9, mannose 0.2 and galactose 0.1. 2.3. Batch culture All the cultures were performed with 250-mL Erlenmeyer flasks (loading volume 50 mL) at 30 °C, initial pH 6.0 and a rotary speed of 200 rpm in triplicate. For recycling experiments, a loop of the yeast cells were inoculated in the seed media and cultivated for 24 h, then, the seed culture was re-inoculated with a ratio of 10% (v/v) to the lipid production media. Culture samples (0.2 mL) were withdrawn every 24 h to determine residual glucose or TRS.
To determine fatty acid compositional profile, lipid samples were transmethylated and analyzed by gas chromatography with a 7890F GC instrument (Techcomp Scientific Instrument Co. Ltd., Shanghai, China) equipped with a cross-linked capillary FFAP column (30 m 0.32 mm 0.4 mm) and flame ionization detector, as described (Li et al., 2007). 3. Results and discussion 3.1. SCM recovery and microbial lipids fractionation Microbial lipids were routinely extracted by using a mixture of chloroform and methanol after cells were treated with 4.0 mol/L HCl at 78 °C for 1 h (Li et al., 2007). This chloroform/methanol method facilitated more complete extraction of cellular lipids, but cell mass was digested and difficult to be recycled. To fully recycle SCM, lipids were extracted by using the petroleum ether method (Economou et al., 2010), and 0.39 g of SCM was produced from 1.0 g of R. toruloides Y4 cells with a lipid content of 70% in a typical experiment. Elementary analysis showed that the SCM sample contained carbon 47.4%, nitrogen 3.7% and hydrogen 8.0%. Lipid fractionation results showed that lipid samples extracted with the petroleum ether method had neutral lipids, G + S, and P, respectively, of 98.2%, 1.2% and 0.6%. In contrast, more G + S (8.3–12.4%) and P (4.0–6.6%) were found when the lipids were extracted with the chloroform/methanol method. Similar results were also described elsewhere (Wu et al., 2010). Because petroleum ethers are non-polar solvents, it is less efficient to extract polar lipids including G + S and P. Similar results were observed when hexane was used as the solvent to extract lipids from other oleaginous yeasts (Economou et al., 2010). Importantly, the carrying-over of polar lipids in SCM should be advantageous in terms of providing elementary nutrients such as sulfur and phosphorus for microbial cell growth.
2.4. Analytical methods
3.2. Cultivation of oleaginous yeasts in SCH alone
Glucose was quantified by a glucose analyzer (SBA-50B; Shandong Academy of Sciences, Jinan, China). TRS in corn stalk hydrolysates were quantified according to the 2,4-dinitrosalicylate (DNS) method with a V530 UV/vis spectrophotometer (Jasco; Japan). Sugar compositions were analyzed by ion chromatography as described (Xie et al., 2012). Cell mass was expressed as cell dry weight (CDW). Cells from 30 mL of culture broth were harvested by centrifugation at 8000g for 5 min, washed twice with distilled water, and dried at 105 °C for 24 h to a constant weight. To ensure efficient recovery of both SCM and lipids, cells were treated and extracted with petroleum ethers according to a known procedure (Economou et al., 2010). To determine cellular total lipid contents, cells were extracted by a mixture of chloroform and methanol according to the reported procedure (Li et al., 2007). Lipid content was expressed as g lipid recovered per g CDW. The lipid yield was defined as g lipid produce per g substrate consumed. All values were the average of 3 independent experiments. Nitrogen was determined according to the Kjeldahl method (Morgan et al., 1957). Lipid fractionation was done according to a known procedure with minor modifications (Fakas et al., 2006). Briefly, about 300 mg of lipids in 3 mL of chloroform was loaded a silica gel column. The column was washed successively by 1,2-dichloroethane (300 mL), 1,2-dichloroethane/acetone (200 mL,1:10, v/v), and methanol (150 mL) to receive fractions of neutral lipids (N), glycolipids plus sphingolipids (G + S), and phospholipids (P), respectively. Lipid fractions were determined gravimetrically after evaporation of the solvents.
To test whether SCM could be used to cultivate oleaginous yeasts, it was digested in 1.0 M H2SO4 at 120 °C for 1.5 h, neutralized with calcium hydroxide, centrifuged, and the supernatants were received as SCH. Four oleaginous yeasts, namely R. toruloides Y4, L. starkeyi AS 2.1560, T. cutaneum AS 2.571 and C. curvatus ATCC 20509, were inoculated in SCH and the cultures were held for 24 h. As shown in Fig. 2, C. curvatus produced 12.7 g/L cell mass but L. starkeyi gave only 2.5 g/L. Interestingly, comparable or lower cell mass concentrations were obtained when the YEPD media were used for these strains. Therefore, cell mass differences among those strains were mainly caused by their growth rate differences. These results demonstrated that SCH alone could be used in lieu of the 14 YEPD
SCH
Cell mass (g/L)
12 10 8 6 4 2 0
L.starkeyi
R.toruloides T.cutaneum
C.curvatus
Fig. 2. Cultivation of oleaginous yeasts using YEPD and SCH. Cells were cultivated at 30 °C for 24 h in the YEPD media contained 20 g/L glucose or SCH contained 20 g/L TRS. Error bars denote standard deviation of 3 independent experiments.
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YEPD media to cultivate oleaginous microorganisms and were in line with the fact that spent yeasts from a brewery significantly enhanced cell mass and docosahexaenoic acid production by Aurantiochytrium sp. KRS101 (Ryu et al., 2013). However, as SCH contained relatively rich nutrients including nitrogen, sulfur and phosphorus, it might be not good for lipid production. It is well known that a nutrient-limited yet carbon-rich environment is required to stimulate lipid accumulation when carbohydrates and related materials are used as carbon sources (Papanikolaou and Aggelis, 2011). Nonetheless, SCH may be used as nutrients source for lipid production upon additional carbon sources are supplemented. 3.3. SCH as nutrient source 3.3.1. The combination of SCH and glucose We used 70 g/L glucose as the carbon source and SCH as nutrients to cultivate R. toruloides Y4 cells for lipid production. To determine nutrient recycling efficiency, the semi-closed repitching experiments were performed for 3 cycles. In these experiments, SCM was hydrolyzed and the entire hydrolysates were used in the next cycle. For each cycle, we recorded cell mass, lipid, total nitrogen loaded and nitrogen recovered, and the results were shown in Table 1. For Cycle 1, SCH were derived from 16 g/L SCM that led to the introduction of 0.4 g/L total nitrogen. Because the inoculation with 10 vol% seed culture prepared in the YEPD media introduced about 0.24 g/L total nitrogen, so total nitrogen loaded in the system was 0.64 g/L. Cell mass and lipid were 25.2 g/L and 15.2 g/L, respectively (Table 1, entry 2). Compared to those values of Cycle 1, it was clear that all data decreased for Cycle 2, and further decreasing was observed for Cycle 3 (Table 1, entries 2–4). It was clear that total nitrogen recovered in SCM was much lower than the amount of total nitrogen loaded, indicating that there was substantial nitrogen loss in each cycle. Concurrently, lipid production capacity was also getting worse every cycle as indicated by cell mass and lipid data. The glucose evolution profiles were shown in Fig. 3A, indicating that glucose consumption rates (g/L/h) were 0.59, 0.55 and 0.53, for Cycle 1, 2 and 3, respectively. Compared to the control culture in N-limited media, Cycle 1 obtained higher cell mass and lipid (Table 1, entry 1 vs. 2). This was likely due to the carryover of polar lipids from SCM as well as more nutrients to support cell growth. For the whole semi-closed repitching experiment, overall cell mass yield and lipid yield were 0.30 g/g and 0.19 g/g, respectively, which were comparable to those of the control culture in N-limited media. It should be noted that similar experiments using 70 g/L glucose as the carbon source were also performed without the introduction
of SCH. Under such conditions, nitrogen nutrients were originated solely from the inocula. There was 47.3 g/L glucose left in the culture after 132 h. Compared with results shown in Fig. 3A, it was clear that the application of SCH was necessary to facilitate sugar consumption, and thus cell growth and lipid production. It was found that about 0.16 g/L total nitrogen remained in the medium of Cycle 1 at the end of the culture (Fig. 3B). We also tried using SCM directly without hydrolysis, but there was about 20 g/L glucose unconsumed after 120 h in a typical experiment. Therefore, it was not efficient to use SCM without hydrolysis. This is understandable because red yeast possesses a rigid cell wall structure, which can prevent the releasing of nutritious elements (Jin et al., 2012). Recently, the significance of recycling nutrients from microalgal cell mass has been recognized for biofuel production, and nutrients were released by anaerobic digestion (Bohutskyi et al., 2014) or hydrothermal liquefaction (Zhou et al., 2013). In another aspect, the presence of SCM increased the viscosity of the culture, which may affect the transfer of mass and oxygen, as previous noted (Audet et al., 1996). The closed repitching experiments were performed for 3 cycles to test whether nutrient recycling efficiency could be improved, and the results were shown in Table 1, entries 5–7. In these experiments, both spent water and SCM were reused to prepare the SCH for the next cycle. To start Cycle 1 of the closed culture, the waste water from the Cycle 1 of the semi- closed culture was used. Therefore, total nitrogen loaded was determined as 0.91 g/L (Table 1, entry 5). In sharp contrast to the dropping trend for the results of the semi-closed experiments, there was little variation in terms of cell mass, lipid, total nitrogen loaded and nitrogen recovered. The fact that both total nitrogen loaded and nitrogen recovered were slightly increased suggested that nitrogenous nutrients were fully recycled. Because of this, overall glucose consumption rates were 0.58 g/L/h for all the cycles, and no apparent differences were observed in terms of glucose evolution profile (Fig. 3C), and similar cell mass and lipid were obtained for all cycles. Overall cell mass yield and lipid yield were 0.31 g/g and 0.20 g/g, respectively, which were essentially identical to those of the semi-closed experiments. In addition, roughly 90% water was reused for each cycle, and the rest 10% was lost due to sampling and evaporation. Therefore, these results demonstrated the possibility of low waste water discharge for 3 consecutive batches of lipid production. Admittedly, we noticed that the viscosity of the culture broth increased gradually. It might also lead to the accumulation of some hazardous byproducts that were generated during SCH preparation and cell growth. These effects prevented from successful repitching beyond 3 cycles. Nonetheless, our results clearly demonstrated that the closed repitching strategy could be used for efficient production
Table 1 Results of cell mass, lipid and nitrogen nutrients of different cultures for lipid production by R. toruloides Y4.
a
Entry
Culture trait
Cell mass (g/L)
Lipid (g/L)
Total nitrogen loaded (g/L)
1 2 3 4 5 6 7 8 9 10 11
Control SCR1c SCR2c SCR3c CR1c CR2c CR3c SCR1-CSd SCR2-CSd SCR3-CSd Control-CS
21.8 ± 0.1 25.2 ± 0.6 22.4 ± 0.6 19.6 ± 0.2 23.3 ± 0.0 21.9 ± 0.3 22.1 ± 0.6 12.7 ± 0.2 13.1 ± 0.1 13.2 ± 0.8 11.5 ± 0.4
14.2 ± 0.3 15.2 ± 0.4 14.9 ± 0.4 12.6 ± 0.1 15.3 ± 0.3 13.6 ± 0.2 14.0 ± 0.3 5.9 ± 0.1 5.5 ± 0.1 5.3 ± 0.3 5.4 ± 0.2
– 0.64 ± 0.00 0.63 ± 0.02 0.54 ± 0.01 0.91 ± 0.00 1.03 ± 0.02 1.14 ± 0.01 0.60 ± 0.04 0.68 ± 0.03 0.69 ± 0.05 –
a
Nitrogen recovered (g/L)
b
– 0.43 ± 0.02 0.34 ± 0.01 0.22 ± 0.01 0.30 ± 0.00 0.31 ± 0.01 0.32 ± 0.00 0.27 ± 0.01 0.30 ± 0.01 0.31 ± 0.00 –
Total nitrogen loaded including nitrogen originated from SCH, corn stalk hydrolysates and the inocula. Nitrogen recovered referring nitrogen retained in SCM. Experiments were conducted on the SCH supplemented with glucose for 120 h. d Experiments were performed with SCH and corn stalk hydrolysates for 72 h. SCR: semi-closed recycling; CR: closed recycling; CS: corn stalk hydrolysates directly. Data were presented as the mean ± standard deviation of 3 independent experiments. b
c
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A
SCR 1
70
SCR 2
A
SCR3
50
CS SCR1-CS SCR2-CS SCR3-CS
60
TRS (g/L)
Glucose (g/L)
40 50 40 30
30 20
20
10 10
0
0
Cell mass
60 80 Time (h) Lipid
100
120
0
140
B 16
0.8
Total nitrogen
0.6
20 15
0.4 10 0.2 5 0
10
20
30
40 50 60 Time (h)
Cell mass
Lipid
70
80
90 100
Total nitrogen
0.6 12 0.4 8
0.2
4
Total nitrogen (g/L)
40
Cell mass, Lipid (g/L)
Cell mass, Lipid (g/L)
B 25
20
Total nitrogen (g/L)
0
0.0 0
20
40
60
80
100
120
0.0
0
Time (h)
0
10
20
30
40
50
60
70
Time (h)
C 70
CR 1
CR 2
CR 3
Fig. 4. The time-courses of lipid production from corn stalk hydrolysates by R. toruloides Y4. (A) The evolution of TRS for the semi-closed culture, (B) the evolution of cell mass, total nitrogen and lipid for Cycle 1 of the semi-closed culture. Error bars denote standard deviation of 3 independent experiments.
Glucose (g/L)
60 50 40 30 20 10 0
0
20
40
60
80
100
120
Time (h) Fig. 3. The time-courses of lipid production from glucose by R. toruloides Y4. (A) The evolution of glucose for the semi-closed culture, (B) the evolution of cell mass, total nitrogen and lipid for Cycle 1 of the semi-closed culture, and (C) the evolution of glucose for the closed culture. Error bars denote standard deviation of 3 independent experiments.
of microbial lipids, leading to significantly reduce the consumption of nutrients and water.
3.3.2. The combination of SCH and corn stalk hydrolysates To further demonstrate the usefulness of SCH as microbial nutrients, we used corn stalk hydrolysates as carbon source for lipid production by R. toruloides Y4. It has been demonstrated that this yeast could use corn stalk hydrolysates without additional nutrient supplementation (Hu et al., 2009). Experiments were done in the semi-closed repitching mode for 3 cycles, and the results were shown in Table 1, entries 8–10. For Cycle 1, 1.4 g/L TRS from SCH and 2.0 g/L glucose from the inoculum were also introduced, so the initial carbon source concentration equaled to 45.0 g/L TRS. The total nitrogen loaded in the culture was 0.60 g/L, including 0.23 g/L, 0.13 g/L and 0.24 g/L from corn stalk hydrolysates, SCH and the inoculum, respectively. Therefore, the Cycle 1 culture had a C/N ratio of 35, and led to the production of 12.7 g/L cell mass including 5.9 g/L lipids (Table 1, entry 8). For Cycle 2 and 3,
Table 2 Fatty acid compositional profiles of microbial lipid samples prepared in this study.
a
Entry
Culture trait
C:14-0
C:16-0
C:16-1
C:18-0
C:18-1
C:18-2
C:18-3
1 2 3 4 5 6 7 8 9 10
Controla SCR1a SCR2a SCR3a CR1b CR2b CR3b SCR1-CSb SCR2-CSb SCR3-CSb
1.0 ± 0.1 1.2 ± 0.0 1.4 ± 0.1 1.0 ± 0.2 1.7 ± 0.1 1.3 ± 0.1 1.6 ± 0.1 1.3 ± 0.1 1.5 ± 0.0 1.5 ± 0.1
32.1 ± 0.8 33.6 ± 0.2 40.9 ± 0.4 29.8 ± 0.4 27.5 ± 0.4 25.6 ± 0.2 27.6 ± 0.2 28.2 ± 0.7 30.0 ± 0.3 29..0 ± 1.1
0.2 ± 0.0 0.5 ± 0.1 0.3 ± 0.1 0.4 ± 0.2 0.7 ± 0.1 0.3 ± 0.0 0.5 ± 0.1 0.6 ± 0.0 0.6 ± 0.1 0.8 ± 0.1
12.7 ± 0.2 7.0 ± 0.0 9.8 ± 0.2 7.2 ± 0.2 9.6 ± 0.3 15.4 ± 0.0 12.0 ± 0.0 10.0 ± 0.2 10.6 ± 0.1 10.2 ± 0.1
51.0 ± 1.8 55.0 ± 0.1 46.7 ± 0.2 56.5 ± 0.8 54.0 ± 0.5 50.3 ± 0.3 53.5 ± 0.1 51.2 ± 0.5 51.0 ± 0.2 51.1 ± 0.9
0.9 ± 0.3 2.7 ± 0.1 0.5 ± 0.0 4.5 ± 0.0 4.6 ± 0.1 6.7 ± 0.0 4.7 ± 0.1 6.8 ± 0.1 6.3 ± 0.0 5.9 ± 0.2
0.3 ± 0.2 – – – – – – – – –
Experiments were conducted on the SCH supplemented with glucose for 120 h. Experiments were performed with SCH and corn stalk hydrolysates for 72 h. SCR: semi-closed recycling; CR: closed recycling; CS: corn stalk hydrolysates directly. Data were presented as the mean ± standard deviation of 3 independent experiments. b
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recycled SCM from Cycle 1 and 2, respectively, was hydrolyzed and combined with corn stalk hydrolysates. Compared with Cycle 1, Cycle 2 and 3 produced slightly higher cell mass but lower lipids. Also, sugars were exhausted within 72 h (Fig. 4A), and the TRS consumption rates were 0.63, 0.67 and 0.68 g/L/h for Cycle 1, 2 and 3, respectively, which were higher than those experiments using glucose as carbon source. Apparently, carbon sources were consumed even faster for the culture combined SCH with corn stalk hydrolysates than that with glucose and corn stalk hydrolysates alone. Similarly, about 0.25 g/L total nitrogen remained in the medium of Cycle 1 after 72 h (Fig. 4B). It was interesting to note that a slightly increase in total nitrogen loaded was observed, indicating that recycling SCM was sufficient to balance nitrogen loss due to waste water discharge. However, both cell mass and lipid were lower (Table 1, entry 11) and the overall TRS consumption rate was only 0.45 g/L/h for the experiments with corn stalk hydrolysates alone. Together, these results revealed the feasibility of recycling SCM as nutrients to support microbial lipid production from complex carbon-rich feedstock such as corn stalk hydrolysates.
3.4. Fatty acid compositional profile To analyze the fatty acids profiles, lipid samples were transmethylated and the resulting fatty acid methyl esters were analyzed by GC. Results in Table 2 showed that the presence of long chain fatty acids included oleic acid (C18:1), palmitic acid (C16:0), stearic acid (C18:0), linoleic acid (C18:2), myristic acid (C14:0), palmitoleic acid (C16:1) and linolenic acid (C18:3, present only in the control sample). Little difference was found in terms of the relative fatty acid species regardless of the recycling procedures, and even less discrepancy among the fatty acid profiles within the same repitching sequence (Table 2, entries 2–4, 5–7 and 8–10), indicating that the repitching manipulation had no obvious influence on fatty acid biosynthesis. Specifically, C18:1, C16:0 and C18:0 together accounted for over 90% of the total fatty acids, while C18:1 took 45.1–56.5%. It has been pointed out that methyl oleate make the best candidate for biodiesel production, based on a comprehensive analysis on cetane number, exhaust emissions, combustion heat, low-temperature properties and lubricity (Knothe, 2008). Such fatty acid compositional profiles were of great potential as biodiesel feedstock.
4. Conclusion To facilitate large-scale microbial production of lipids and related chemicals, it is imperative to reutilize resources including spent cell mass, nutrients and water. Our results demonstrated that spent cell mass hydrolysates from the oleaginous yeast R. toruloides could be used as media for cell growth and as nutrients for lipid production from different carbon sources. Moreover, we showed that lipid production could be operated with little efficiency drop for up to 3 cycles by fully recycling the resources. This provided an attractive approach to minimize the input on microbial lipid production.
Acknowledgements This work was supported by the Ministry of Science and Technology of China (No. 2011CB707405), the National Natural Scientific Foundation of China (No. 21325627), Chinese Academy of Sciences (No. KGZD-EW-304-2) and the State Key Laboratory of Motor Vehicle Biofuel Technology, Henan Tianguan Group Co., China (No. 2013001).
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