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Biochemical Engineering Journal 149 (2019) 107236
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High cell density cultivation of Lipomyces starkeyi for achieving highly efficient lipid production from sugar under low C/N ratio
T
Rezky Lastinov Amzaa,1, Prihardi Kaharb,1, Ario Betha Juanssilferoa,c, Nao Miyamotoa, ⁎ Hiromi Otsukab, Chie Kihirab, Chiaki Oginoa, , Akihiko Kondoa,b a
Graduate School of Engineering, Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe, 657-8501, Japan Graduate School of Science, Technology and Innovation (STIN), Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe, 657-8501, Japan c Research Center for Biotechnology, Indonesian Institute of Sciences (LIPI), Jl. Raya Bogor Km 46, Cibinong, Bogor, 16991, West Java, Indonesia b
H I GH L IG H T S
fermentation of Ls-D35 was carried out at low initial C/N ratio (17.9). • The fermentation of Ls-D35 was optimum at pH 5 for cell and lipid productions. • The • The highest lipid yield was achieved by using single xylose feeding.
A R T I C LE I N FO
A B S T R A C T
Keywords: Oleaginous yeast Lipomyces starkeyi D35 High cell density culture Cell and lipid productions
Oleaginous microorganisms such as oleaginous yeasts are well known for the ability to accumulate intracelular lipid more than 20% w/w in certain conditions. Lipomyces starkeyi D35 (Ls-D35 strain) is one of promising oleaginous yeast for lipid production. The purpose of this research is to establish the optimum condition for cell and lipid production of Ls-D35 strain under low C/N ratio (17.9) by high cell density cultivation. The optimum pH for cell and lipid production was achieved at pH 5 by using batch cultivation. Four different feeding strategies were conducted to observe the cell and lipid production of Ls-D35 strain. The highest cell yield was achieved by mixed glucose and xylose as substrates (0.15 w/w substrate) after 96 h and the highest lipid yield was achieved by single xylose feeding (0.13 w/w substrate) after 120 h. Meanwhile, the two most fatty acids content were C18:1 and C16:0 which are similar to the content of vegetable oil. These results showed that Ls-D35 strain could produce high cell and optimum lipid under low initial C/N ratio medium which can be an alternative for biodiesel production and other industrial products.
1. Introduction The use of microorganisms as oil sources commercially has long history. Lipid from microorganisms which is well known as single cell oil (SCO) has been suggested as alternative energy resource since the second half of 20th century [1]. Since then, SCO has attracted researchers and industrial’s interests for biology production [2,3] and the utilization of lignocellulosic biorefinery [4]. Oleaginous microorganisms are the most well-known organisms for SCO production due to their capability for accumulating lipid in their cells [1]. One of potential microorganism for SCO production is oleaginous yeast [2,3,5]. It has been widely known that oleaginous yeasts are capable to
accumulate lipid more than 20% of their biomass weight or dry cell weight (DCW) [1]. The ability of oleaginous yeast in accumulating high lipid production is predicted to be future trends for biological production, especially biodiesel production [3]. One of robust yeast strain for lipid accumulation is Lipomyces starkeyi (L. starkeyi), which is capable to accumulate high lipid production within cells [5]. For instance, L. starkeyi is capable to utilize single xylose [6,7] and mixed sugar [6–9] as carbon sources for growth and lipid production as well. Lipid accumulation in oleaginous yeasts is usually occurred in nutrient imbalance in the medium [1]. Nutrient starvation and excess of carbon sources become the main factors for high lipid production in oleaginous yeast [1]. Many studies have shown
⁎
Corresponding author at: Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe, 657-8501, Japan. E-mail address: [email protected] (C. Ogino). 1 Rezky Lastinov Amza and Prihardi Kahar contributed equally to this work. https://doi.org/10.1016/j.bej.2019.05.013 Received 17 January 2019; Received in revised form 13 May 2019; Accepted 17 May 2019 Available online 24 May 2019 1369-703X/ © 2019 Elsevier B.V. All rights reserved.
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KH2PO4, 7, and Na2HPO4.2H2O, 5 in. g/L. The medium was supplemented with some trace elements: FeSO4.7H2O, 0.08; ZnSO4.7H2O, 0.01; CaCl2.2H2O, 0.1; MnSO4.4H2O, 0.1; CuSO4.5H2O, 0.002; CoCl2.6H2O, 0.002, in g/L. The pH medium was adjusted at 5.5 with appropiate amount of 1 M H2SO4 and sterilized by filtration.
that the optimum conditions for lipid accumulation in L. starkeyi are mostly under high C/N ratio content (> 20) of fermentation medium [1,6–8,10,11]. Low nitrogen (high C/N ratio) content inside the medium leads to the lipid accumulation by increasing the activity of AMP (adenosine mono phosphate) deaminase to provide the amount of ammonia as nitrogen supply for cells and to inhibit the formation of isocitrate dehydrogenase (ICDH) [5]. This condition provides the transfer of citrate into cytosol for lipid production [5]. In contrast, some studies have shown that L. starkeyi is not able to produce high lipid under high nitrogen (low C/N ratio) [6,11]. High nitrogen content allows citrate to be converted to α-ketoglutarate during the Krebs cycle which is favorable for cell’s growth. Furthermore, the growth of L. starkeyi was not optimum and sugar as carbon source was not totally consumed under high nitrogen (low C/N ratio) condition [6]. Lipomyces starkeyi D35 (Ls-D35) strain has been confirmed being able to accumulate high number of lipids under low nitrogen (high C/N ratio) medium [7] and produce high lipid during chemical compounds as inhibitor [12] in flask scale. This study focused on cells and lipid production in high cell density cultivation of Ls-D35 strain under high nitrogen (low C/N ratio) of initial fermentation medium by using fedbatch strategies. Fed-batch strategies were used in this study with the aim to achieve highly efficient production of both cells and lipid from Ls-D35 strain. Other studies showed that fed-batch strategy was able to improve cell and lipid production in L. starkeyi [6,10] under high C/N ratio condition. However, the objective of this study was to optimize the high cell density cultivation of Ls-D35 strain under initial low C/N ratio for high cell and lipid production. Synthetic sugars (glucose and xylose) were used as carbon sources in this study. Initial low C/N ratio was used to observe the capability of Ls-D35 strain in adjusting the use of pre-treated biomass as for industrial process. Therefore, optimization of pH is important because pre-treated biomass as carbon sources mostly requires acidic condition for further application [13–16]. Other studies have investigated that high cell and lipid production in oleaginous yeast can be achieved by controlling the substrate in fed-batch fermentation mode [8,10,16,17]. Fed-batch fermentation strategy was conducted in order to improve cells and lipid production. Four types of feeding strategy were conducted to compare the cell and lipid production of Ls-D35 strain. Each of single glucose, single xylose, mixed glucose-xylose and biphasic feeding were applied to high cell density cultivation of Ls-D35 strain.
2.3. Optimization of pH fermentation pH optimization was carried out by batch cultivations. The cultivations were performed in 2 L tank bioreactor (ABLE Biott, Japan) with 1 L of working volume. Mixed glucose and xylose was used as carbon sources. Cultivation medium was rich of nitrogen supplied by (NH4)2SO4 (10 g/L) which led to the initial C/N ratio at 17.9. The calculation of C/N ratio was based on Slininger’s patent [18]. pH of fermentation medium was set at 3, 4 and 5 for each cultivation. The conditions were set: intial agitation at 150 rpm; temperature at 30 °C and aeration at 1 vvm. All fermentations were inoculated by prepared seed at a final of 4 (v/v) %. Foaming was controlled using SI (silicone) antifoam solution (Wako, Japan). Samples for further analysis were taken every 24 h. 2.4. Fed-batch cultivations Fed-batch cultivations were performed in a 2 L tank bioreactor (ABLE Biott, Japan) with a one liter of working volume. Fed-batch cultivations were carried out in optimizing the cell and lipid production. Four conditions of cultivation were performed in fed-batch cultivations. The first cultivation was glucose feeding at glucose as single carbon source. The second cultivation was xylose feeding at xylose as single carbon source. The third cultivation is mixed glucose and xylose feeding at initial glucose and xylose as carbon sources. The last cultivation is biphasic fermentation with xylose feeding with glucose as initial carbon source. Feeding was performed manually after carbon source was below 10% of initial concentration. Certain volume of medium was removed in every feeding to maintain the feeding substrate concentration and volume of cultivation. The concentration of sugar feeding stock solutions was 400 g/L with 50 mL of the feeding solutions was fed into the cultivation to maintain ± 20 g/L carbon source in the medium. The conditions for the cultivation was set at 150 rpm of initial agitation and 30 °C of temperature. The initial pH was set at optimum condition and aeration at 1 vvm. All cultivations were performed with low initial C/N ratio at 17.9. Foaming was controlled using 1 (v/v) % of SI (silicone) antifoam solution (Wako, Japan). A 1 M solution of sodium hydroxide (NaOH) and sulfuric acid (H2SO4) were used to control pH. The oxygen for aeration was from oxygen generator instrument (PSA ITO-08-1, IBS Co. Ltd, Japan). The oxygen was filtered by sterilizing filter before it came into the fermentor for aeration. Samples were taken in every feeding time both before and after feeding.
2. Materials and methods 2.1. Microorganism and seed preparation L. starkeyi D35 (Ls-D35) yeast strain (NBRC10381) was selected from NITE Biological Resource Center (NBRC), National Institute of Technology and Evaluation, Japan. Yeast strain was preserved as a glycerol stock at -80 °C and revived by streaking on PDA agar plate. The yeast strain was grown on YMG agar plate (3.0 g/L yeast extract, 3.0 g/ L malt extract, 5.0 g/L peptone, 10.0 g/L glucose and 16.0 g/L agar) for short term storage. Seed cultures were cultivated in 500 mL shake flasks with a 120 mL of YMG medium (10 g/L glucose, 5 g/L peptone, 3 g/L yeast extract, 3 g/L malt extract) at 30 °C and 190 rpm in an orbital shaker incubator (BioShaker BR-43FH MR, TAITEC, Corp., Japan) for 24 h. Cells were collected by centifugation at 12,000 rpm for 4 min and washed two times by deionized water. Cells were resuspended ( ± 50 mL) by fermentation medium for inoculation in fermentor with 1 L working volume.
2.5. Dry cell weight determination Dry cell weight (DCW) was determined gravimetrically from a 1 mL sample of fermentation. Cells were sepearated by centrifugation (12,000 rpm for 4 min), washed two times and dried at -80 °C. Frozen cells were lyophilized by freeze-drying (Freez-one® 4.5 L Freeze Dry System Model 7750020, Labconco®, Kansas City, Missouri, USA) to remove the water content. 2.6. Sugar analysis Sugar analysis were performed by using high performance liquid chromatography (LC-20AB, Shimadzu, Japan). Samples were centrifuged and filtered (0.45 μm) prior to injection (20 μL). Samples were separated using a Coregel-87H3column (7.8 mmI.D. x 300 mm Transgenomic, USA) at the column temperature 80 °C. Mobile phase was 5 mM of H2SO4 with flow rate at 0.6 mL/min. A refractive index
2.2. Nutrient media Fermentation processes were carried out with Nitrogen Mineral Medium (NMM), consisted of carbon sources (glucose, or xylose, or a mixture of both), 100 g/L in single carbon source and 50 g/L of each in mixed-sugar, with yeast extract, 1.5, (NH4)2SO4, 10, MgSO4.7H2O, 1.5, 2
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detector (RID-10A) was used for peak identification. 2.7. Total lipid analysis Total lipid accumulation was measured by gravimetric analysis following the Folch method [19]. Freeze-dried cells (15–20 mg) were transferred into microvial with O-ring sealed cap (Watson, Japan). Glass beads were added into the vial. Lipid was extracted by 1.5 mL of chloroform:methanol 2:1 (v/v) [19]. The cell pulverization was conducted two times with Shake Master Neo ver 1.0 (BMS Tokyo, Japan) at 1500 rpm for 30 min. Cells were centrifuged at 12,000 rpm for 10 min after pulverization and filtrate was disposed after centrifugation. Cells were washed by 1.5 mL of deonized water for second pulverization. Cells were separated again by centrifugation. Filtrate were removed and cells were dried at up to 80 °C to constant weight. At the end, total lipid was measured by the cells’ weight difference at initial and after extraction. 2.8. Fatty acid methyl ester (FAME) analysis The transesterification was conducted by following the protocol from fatty acid methylation kit (Nacalai Tesque, Inc. Japan). The extracted FAME (light phase) was analyzed by using Gas Chromatography-Mass Spectrometer (GC–MS) (Shimadzu) instrument. GC–MS was equipped with DB-23 capillary column 0.25 mm x 30 m (J& W Scientific). The carrier was Helium gas with 0.8 mL/min of flow rate with 1:5 split ratios. The initial column temperature was 250 °C, increased 50 °C for 1 min and then increased 25 °C/min to 190 °C and 5 °C/min to 235 °C for 4 min. The internal standard of C8:0 (caprylic acid) was included in each sample. FAME (%) was calculated as the percentage of each fatty acid to the total number of fatty acids produced upon the fermentation. 2.9. Calculations Lipid content was determined by using Eq. (1).
Lipid content (%, w / w ) =
Fig. 1. Batch fermentation at different pH conditions. The cultivations were conducted by batch fermentation using glucose and xylose at pH 3, pH 4 and pH 5.
weight of cells after extraction (w ) x 100 % weight of cells before extraction (w ) (1)
pH [11]. In this study, the favorable pH of Ls-D35 strain fermentation was determined in order to achieve the optimum condition for cell and lipid production. Three batch fermentations with different pH were performed to determine the optimum pH condition for high cell density cultivation of Ls-D35 strain. Mixed glucose and xylose were used as carbon sources since Ls-D35 strain has been able to accumulate both glucose and xylose to produce higher cell and lipid in this condition [7]. The results of Ls-D35 strain fermentation in three different pH conditions (pH 3, pH 4 and pH 5) are shown in Fig. 1. Glucose and xylose were totally consumed in the end of exponential growth phase from these three different pH cultivations. However, the exponential growth phases ended in different fermentation time. Rapid glucose and xylose consumption occurred in the fermentation at pH 5 in which glucose was totally consumed at 24 h and xylose was at 48 h, respectively. Meanwhile, slower sugar consumption occurred in the fermentation at lower pH (pH 3 and 4) in which glucose and xylose were totally consumed at 48 h and 72 h. The highest dry cell weight (DCW) and lipid production was achieved at the pH 5 of fermentation which were 40.36 g/L and 9.3 g/L (23.12% of DCW) after 24 h, respectively. These results confirm that Ls-D35 strain has optimum condition for cell growth and lipid production at pH 5. The similar results were also found when L. starkeyi was grown in sewage sludge which resulted highest lipid at pH 5 [24] and glucose mineral medium at pH 5 [25]. Furthermore, the optimum growth rate at pH 5 was also found when L. starkeyi was grown in MSG wastewater [26].
Lipid yield was determined by Eq. (2).
amount of lipid (w ) − lipid at initial time (w ) Lipid yield (w /w ) = total carbon sources consumed (w )
(2)
Cell yield was determined by using Eq. (3).
Cell yield (w /w ) =
cells at certain time (w ) − cells at initial time (w ) total carbon sources consumed (w )
(3)
FAME per lipid was determined by using Eq. (4).
FAME per lipid (w /v ) = FAME (%, w /w ) x Lipid (w /v )
(4)
FAME per Dry Cell Weight (DCW) was determined by using Eq. (5).
FAME per DCW (w /v ) =
Lipid (w / v ) x FAME per Lipid (w /v ) DCW (w / v )
(5)
3. Results and discussion 3.1. Optimization of pH in batch culture The effect of pH in yeast fermentation has been studied previously [11,20,21] and specifically in cell and lipid production [22,23]. The optimization of pH is important in cell and lipid production by oleaginous yeast since the use of pretreated biomass for industrial production is mostly at low pH condition [11]. For instance, it has been observed that oleaginous yeast could grow and accumulate lipid at lower 3
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glucose or xylose as single carbon source and produce high cell and lipid at low initial C/N ratio. Furthermore, the use of single xylose as substrate was more preferred for lipid accumulation by L. starkeyi because xylose utilization is more efficient in lipid synthesis by oleaginous yeast through phosphoketolase pathway compared to single glucose [2]. Single glucose feeding resulted 66.64 g/L DCW and 32.67 g/L of lipid with 0.11 (w/w) of lipid yield after 120 h (Fig. 2a). Meanwhile, single xylose feeding resulted 63.41 g/L of DCW and 29.32 g/L of lipid with 0.13 (w/w) of lipid yield after 120 h, respectively (Fig. 2b). In mixed glucose-xylose as carbon sources, Ls-D35 strain assimilated glucose at first which led to the delay of xylose consumption. Xylose consumption was started after total consumption of glucose at 18 h. The delay of xylose when co-fermented with other sugar was also observed in other studies. When L. starkeyi ATCC 56304 which was fermented with glucose and xylose, glucose was completely consumed first at 48 h and then continued by the exhaustion of xylose at 60 h. [6]. Xylose consumption of Ls-D35 strain in flask scale was also started after glucose was exhausted in high C/N ratio fermentation medium (7). The consumption of xylose was also delayed in other L. starkeyi strains when co-fermented with other sugar [8,13,32]. The delay of xylose consumption was caused by the existence of glucose as competitive carbon source to xylose in high concentration [6]. However, glucose and xylose were consumed rapidly before feeding process in this study. Both glucose and xylose were exhausted after 18 h and 36 h, respectively, produced 57.69 g/L of DCW after 96 h of fermentation and 24.25 g/L of lipid (Fig. 2c) with 0.11 (w/w) of lipid yield (Table 1). Biphasic feeding was conducted to minimize the competition between glucose and xylose as substrates and was expected to improve cell and lipid production. Glucose was used as initial sugar for fermentation to gain cell production, since glucose metabolism is favorable for glycolysis which leads to cell production. Xylose was supplied after total consumption of glucose at 24 h and was consumed rapidly until the end of fermentation (120 h). Xylose feeding was supposed to improve lipid production since xylose is preferable for lipid production by pentose phosphate pathway which is easier in generating acetyl-CoA for fatty acid synthesis [1,2]. The similar pattern was also found in another research with slower consumption of glucose as initial sugar in biphasic feeding [6]. However, biphasic feeding could not improve lipid
Determining the optimum pH for cell and lipid production is important since pH value influences lipid accumulation in oleaginous yeasts [27]. L. starkeyi cannot grow well at high pH because of the inhibition of enzyme activity that produce biotin for cell growth [25]. Previous study showed that L. starkeyi can grow well at acidic range of pH [24]. However, the growth rates at lower pH (pH 3 and pH 4) were low due to more acidic condition. It has been shown that more acidic condition can disrupt the membrane of yeast [28] and influence transportation of nutrients [29]. However, in this study, sugar was totally consumed at lower pH (3 and 4), even at slower rate compared to pH 5. Based on these results, it can be confirmed that Ls-D35 strain is able to consume carbon sources and accumulate lipid under low pH especially at acidic condition which can be beneficial for decreasing production cost in controlling pH for industrial process [3] and preventing bacterial contaminations. 3.2. Fed-batch fermentation profile Synthetic glucose and xylose were used in this study as carbon sources both in single and mixed fed-batch fermentation at pH 5 as the optimum pH for Ls-D35 strain. Xylose was used since Ls-D35 strain can consume xylose well both in single and co-fermentation with glucose for high lipid production [7]. This study showed that Ls-D35 strain consumed glucose and xylose entirely at shorter fermentation time at initial low C/N ratio (Fig. 2a and b). Furthermore, the initial consumption of glucose was faster than xylose under low initial C/N ratio (Fig. 2a and b). The late initial consumption of xylose is assumed to be caused by low initial inoculum size (OD600 at around 3, data is not shown) which led in a prolonged lag phase [30]. Previous study of Ls-D35 strain showed that lower inoculum size at initial fermentation of flask scale cultivation will lead to the slower consumption of xylose as well [7]. In comparison, some studies showed that the cultivation of L. starkeyi in high nitrogen medium (low C/N ratio), glucose as single carbon source was not totally consumed and confirmed the lower production of DCW and lipid [6,31]. Slower consumption of single glucose in L. starkeyi under low C/N ratio with lower initial concentration has also been confirmed previously [11]. These comparisons show that Ls-D35 strain has better ability to assimilate
Fig. 2. Fed-batch fermentation profiles under low C/N ratio. Feeding strategies used: A, Fedbatch fermentation with initial single glucose (100 g/L) and feeding with glucose after 24 h (20 g/L) for every 12 h; B, Fed-batch fermentation with initial single xylose (100 g/L) and feeding with xylose after 48 h (20 g/L) for every 12 h; C, Fed-batch fermentation with initial glucose (50 g/L) and xylose (50 g/L), feeding by each glucose (10 g/L) and xylose (10 g/L) for every 12 h; D, Biphasic fed-batch fermentation with initial glucose (100 g/L) and continued by xylose after 24 h (20 g/L) for every 12 h. These data were calculated with triplicate sampling data calculations.
4
5
[6]
NA NAd NAd NAd NAd NAd NAd 0.25 0.21 0.18 0.19
d
c
Dry cell weight at the highest value. Amount of lipid at the highest value time per total consumed carbon source. Amount of lipid produced per liter solution at determined time. Not available/not determined. b
Yarrowiya lipolytica ylXYL + obese-XA Yarrowiya lipolytica W29 Lipomyces starkeyi D35
a
Glucose Glucose Xylose Glucose-Xylose Biphasic Agave bagasse Agave bagasse Glucose Xylose Glucose-Xylose Biphasic
± 13 ± 16 ± 27 ± 38 ± 59 25.8 11.5 66.64 63.41 57.69 58.26
± 4.3 ± 6.8 ± 14 ± 22 ± 36 16.5 2 32.67 29.32 24.25 25.5
34.2 41.8 51.5 57.4 60.1 67 20 49.03 46.25 42.03 43.75
0.08 0.08 0.16 0.17 0.17 0.34 NAd 0.11 0.13 0.11 0.1
0.04 0.06 0.12 0.14 0.14 0.17 0.02 0.27 0.24 0.25 0.26
C18:1 C18:1 C18:1 C18:1 C18:1 NAd NAd C18:1 C18:1 C18:1 C18:1
d
FAME (g/g-DCW)
Table 2 Comparison of cell and lipid production with various substrates under fed-batch fermentations.
Lipid content (%)
Lipid yield (w/w)b
Lipid accumulation rate (g/L h)c
production in Ls-D35 strain compared to single sugar as substrates. In biphasic feeding, the highest cell productions were at stationary phase after 72 h of fermentation (Fig. 2d). This biphasic feeding resulted 58.26 g/L of DCW with 25.49 g/L lipid (43.75%) after 96 h of fermentation (Fig. 2d) and 0.1 (w/w) substrates of lipid yield (Table 1). The highest lipid yield was achieved at xylose feeding: 0.13 (w/w) and the highest cell yield was at mix glucose and xylose feeding: 0.15 (w/w) (Table 2). The highest lipid yield was less than half of the theoretical yield for lipid production in oleaginous yeasts which are 0.32 (w/w) of glucose and 0.34 (w/w) of xylose as substrate [4]. Lipid production could not be improved for higher result, even with biphasic feeding strategies, since the fermentations were carried out with low C/ N ratio which theoretically is favorable for cell production [33]. Theoretically, lipid accumulation inside cell is optimum in nitrogen exhausted medium which triggers the adenosine monophosphate (AMP) deaminase activity. The activity of AMP deaminase leads to reverse reaction to form isocitrate which will be used to the formation of Acetyl-CoA for lipid synthesis. Otherwise, this study used nitrogen rich medium which theoretically is not favorable for lipid production. Citrate was not diverted by citrate-malate translocase (CMT) enzyme out of TCA cycle for lipid biosynthesis since the activity of AMP deaminase was inhibited due to the rich of nitrogen [4]. This condition is assumed to inhibit the lipid accumulation within Ls-D35 strain’s cell in nitrogen rich medium (low C/N ratio). Other studies found that lipid accumulation in L. starkeyi under low C/N ratio was significantly much lower compared to this study [6,21]. It was also found that lipid production was lower under high nitrogen condition in Rhodosporodium TJUWZ4 [34]. This result is also supported by data provided by previous study of Ls-D35 strain in flask scale under high C/N ratio which provided excess sugar for continuation of lipid accumulation compared to cell growth [7]. The comparison of cell and lipid production with other studies is shown in Table 2. L. starkeyi ATCC 56304 showed that it produced the highest lipid ( ± 36 g/L) in biphasic feeding strategy at higher C/N ratio between 80 and 100 [6]. Meanwhile, the engineered Yarrowiya lipolytica strain produced high percentage of lipid content (67%) when was grown in agave bagasse with higher C/N ratio as well [35]. However, as shown in Table 2, Ls-D35 could produce the compatible and even higher amount of cell and lipid production at low initial C/N ratio (17.9) compared to other studies with higher C/N ratio. Furthermore, in this study, as the fermentations were initiated with lower C/N ratio, it was confirmed that the sugar consumption rate of Ls-D35 strain was faster compared to another L. starkeyi strain with higher C/N ratio [6]. Ls-D35 strain could assimilate glucose and xylose even at higher concentration (each 50 g/L) within rapid fermentation time (24–36 h). These results confirm that Ls-D35 strain could produce more cells and lipids at low initial C/N ratio grown in synthetical sugar as carbon sources. It is expected that this study can be an updated reference for lipid production of Ls-D35 strain by using lignocellulosic materials with mostly contain high nitrogen content and limited sugar for carbon sources.
120 120 120 156 255 96 96 120 120 96 96
d
Total lipid accumulation from day 0 per total sugar consumed. Total cell accumulation from day 0 per total sugar consumed. Calculation at 120 h of fermentation. Calculation at 96 h of fermentation.
21 80-100 80-100 80-100 80-100 45 45 17.9 17.9 17.9 17.9
c
0.002 0.001 0.006 0.005
Lipomyces starkeyi ATCC 56304
b
± ± ± ±
Major FAME
a
0.010 0.002 0.011 0.011
Lipid (g/L)
0.12 0.13 0.15 0.13
± ± ± ±
DCW (g/L)a
0.11 0.13 0.11 0.10
Type of Carbon source
Glucosec Xylosec Mixed Glucose and Xylosed Biphasicd
Time (h)
Cell yield (w/w)b
C:N Ratio
Lipid yield (w/w)a
Yeast strain
Feeding type
Ref
Table 1 Lipid and cell yields obtained under different feeding strategies.
[35] [35] This study
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Fig. 3. Profiles of fatty acids produced during fed-batch fermentation. Feeding strategies used: A, single glucose feeding; B, single xylose feeding; C, mix glucose and xylose feeding; D, biphasic feeding.
obtained per DCW in every feeding strategy. Based on the results, the major composition of FAME per lipid and DCW in every feeding process was C18:1. However, the compositions of C16:0, C16:1, and C18:1 per lipid and per DCW were increased by the fermentation time up to 120 h. Ls-D35 strain has been observed as the highest producer of C18:1 among other oleaginous yeasts strain which is ± 75% of total fatty acid [3]. Due to low C/N ratio condition, C18:1 content was not able to be improved in this study since the metabolism of fatty acid synthesis was not optimized as limited pathway for fatty acid synthesis. However, this amount of oleic acid (C18:1) is potential enough for biodiesel production in industrial process. For instance, the production of C16:0 was higher compared to the other study of Ls-D35 strain [36]. These results showed that the modification of C/N ratio in feeding strategies of LsD35 strain is able to optimize the certain fatty acid production. Furthermore, based on previous study in flask scale [7] and this study, LsD35 strain can be recognized as an industrial yeast platform to produce
3.3. FAME profile Fatty Acid Methyl Ester (FAME) profile of each feeding strategies can be seen in Fig. 3. Major fatty acids content of Ls-D35 strain as shown in Fig. 3 are palmitic acid (C16:0), palmitoleic acid (C16:1), stearic acid (C18:0), oleic acid (C18:1) and linoleic acid (C18:2). These fatty acids profiles are closely similar with vegetable oils [3]. Increasing amount of fatty acids for all feeding strategies occurred at palmitic acid (C16:0), palmitoleic acid (C16:1) and oleic acid (C18:1). Specifically, the content of C16:0 and C16:1 in L. starkeyi were higher at this study compared to higher C/N ratio [10]. Furthermore, oleic acid (C18:1) content was more than 40% of the total fatty acids for each feeding strategies. This finding is similar with previous study of Ls-D35 strain in flask scale [7] and other Lipomyces strains [6,8,10]. The comparison of FAME amounts to lipid and DCW were also observed in every feeding process. As shown in Table 3, FAME per lipid was comparable with that
Table 3 Time dependent-changes of Fatty Acid Methyl Ester during the fed-batch fermentation.
6
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large amount of high-valued fatty acid derivates as well.
[11] C.H. Calvey, Y.-K. Su, L.B. Willis, M. McGee, T.W. Jeffries, Nitrogen limitation, oxygen limitation and lipid accumulation in Lipomyces starkeyi, Bioresour. Technol. 200 (2016) 780–788. [12] A.B. Juanssilfero, P. Kahar, R.L. Amza, N. Miyamoto, H. Otsuka, H. Matsumoto, C. Kihira, A. Thontowi, C. Yopi, B. Ogino, A. Prasetya, Kondo, Selection of oleaginous yeasts capable of high lipid accumulation during challenges from inhibitory chemical compunds, Biochem. Eng. J. 137 (2018) 182–191. [13] Z. Yu, M. Zhao, H. Li, H. Zhao, Q. Zhang, C. Wan, H. Li, A comparative study on physiological activities of lager and ale brewing yeasts under different gravity conditions, Biotechnol. Bioprocess Eng. 17 (2012) 818–826. [14] C. Huang, X.-F. Chen, X.-Y. Yang, L. Xiong, X.-Q. Lin, J. Yang, B. Wang, X.-D. Chen, Bioconversion of corncob acid hydrolysate into microbial oil by the oleaginous yeast Lipomyces starkeyi, Appl. Biochem. Biotechnol. 172 (2014) 2197–2204. [15] L. Matsakas, A.-A. Sterioti, U. Rova, P. Christakopoulos, Use of dried sweet sorghum for the efficient production of lipids by the yeast Lipomyces starkeyi CBS 1807, Ind. Crops Prod. 62 (2014) 367–372. [16] K.V. Probst, P.V. Vadlani, Production of single cell oil from Lipomyces starkeyi ATCC 56304 using biorefinery by-products, Bioresour. Technol. 198 (2015) 268–275. [17] M. Han, Z. Xu, C. Du, H. Qian, W.-G. Zhang, Effects of nitrogen on the lipid and carotenoid accumulation of oleaginous yeast Sporidiobolus pararoseus, Bioprocess Biosyst. Eng. 39 (2016) 1425–1433. [18] P.J. Slininger, B.S. Dien, C.P. Kurtzman, B.R. Moser, V. Balan, M. Jin, L.dC. Sousa, E.L. Bakota, US 2016/0265009 A1, United States Patent Application Publication, USA, 2016. [19] J. Folch, M. Lee, G.H. Sloane-Stanle, A simple method for isolation and purification of total lipids from animal tissues, J. Biol. Chem. 226 (1957) 497–509. [20] E. Casey, M. Sedlak, N.W. Ho, N.S. Mosier, Effect of acetic acid and pH on the cofermentation of glucose and xylose to ethanol by a genetically engineered strain of Saccharomyces cereviseae, FEMS Yeast Res. 10 (2010) 385–393. [21] Z.J. Liu, L.P. Liu, P. Wen, N. Li, M.H. Zong, H. Wu, Effects of acetic acid and pH on the growth and lipid accumulation of the oleaginous yeast Trichosporon fermentans, Bioresource 10 (3) (2015) 4152–4166. [22] A. Castelli, G.P. Littarru, G. Barbaresi, Effect of pH and CO2 concentration changes on lipids and fatty acids of Sachharomyces cerevisae, Arch. Mikrobiol. 66 (1969) 34–39. [23] J. Bradenburg, J. Blomqvist, J. Pickova, N. Bonturi, M. Sandgren, V. Passoth, Lipid production from hemicellulose with Lipomyces starkeyi in a pH regulated fed-batch cultivation, Yeast 33 (2016) 451–462. [24] C. Angerbauer, M. Siebenhofer, M. Mittelbach, G.M. Guebitz, Conversion of sewage sludge into lipids by Lipomyces starkeyi for biodiesel production, Bioresour. Technol. Rep. 99 (2008) 3051–3056. [25] Y. Uzuka, T. Naganuma, K. Tanaka, Y. Odagiri, Effect of culture pH on the growth and biotin requirement in a strain of Lipomyces starkeyi, J. Gen. Appl. Microbiol. 20 (1974) 197–206. [26] J.X. Liu, Q.Y. Yue, B.Y. Gao, Z.H. Ma, P.D. Zhang, Microbial treatment of the monosodium glutamate wastewater by Lipomyces starkeyi to produce microbial lipid, Bioresour. Technol. Rep. 106 (2012) 69–73. [27] J.M. Ageitos, J.A. Vallejo, P. Veiga-Crespo, T.G. Villa, Oily yeasts as oleaginous cell factories, Appl. Microbiol. Biotechnol. 90 (2011) 1219–1227. [28] P.W. Piper, Yeast superoxide dismutase mutants reveal a pro-oxidant action of weak organic acid food preservatives, Free Radic. Biol. Med. 27 (1999) 1219–1227. [29] B.E. Bauer, D. Rossington, M. Mollapour, Y. Mamnun, K. Kuchler, P.W. Piper, Weak organic acid stress inhibits aromatic amino acid uptake by yeast, causing a strong influence of amino acid auxotrophies on the phenotypes of membrane transporter mutants, Eur. J. Biochem. 270 (2003) 3189–3195. [30] L.Y. Kun, Bioprocess technology, in: L.Y. Kun (Ed.), Microbial Biotechnology Principles and Applications, 2nd), World Scientific Publishing Co. Pte. Ltd., Singapore, 2006Chapter 2 ISBN : 9812566775. [31] J. Lin, H. Shen, H. Tan, X. Zhao, S. Wu, C. Hu, Z.K. Zhao, Lipid production by Lipomyces starkeyi cells in glucose solution without auxiliary nutrients, J. Biotech. 152 (2011) 184–188. [32] R. Yamada, A. Yamauchi, T. Kashihara, H. Ogino, Evaluation of lipid production from xylose and glucose/xylose mixed sugar in various oleaginous yeasts and improvement of lipid production by UV mutagenesis, Biochem. Eng. J. 128 (2017) 76–82. [33] J.B.M. Rattray, A. Schibeci, D.K. Kidby, Lipids of yeasts, Microbiol. Rev. 39 (3) (1975) 197–231. [34] Q. Wang, Y. Cui, B. Sen, W. Ma, R.L. Zheng, X. Liu, G. Wang, Characterization and robust nature of newly isolated oleaginous marine yeast Rhodosporidiumspp. from coastal water of northern China, AMB Exp. 7 (30) (2017) 1–13. [35] X. Niehus, A.M. Crutz-Le Coq, G. Sandoval, J.M. Nicaud, R. Ledesma-Amaro, Engineering Yarrowiya lipolytica to enhance lipid production from lignocellulosic materials, Biotechnol. Biofuels 11 (11) (2018) 1–10. [36] A. Tanimura, M. Takashima, T. Sugita, R. Endoh, M. Kikukawa, S. Yamaguchi, E. Sakuradani, J. Ogawa, J. Shima, Selection of oleaginous yeasts with high lipid productivity for practical biodiesel production, Bioresour. Technol. 153 (2014) 230–235.
4. Conclusions This study investigated the effect of low initial C/N ratio and feeding strategy for growth and lipid accumulation in L. starkeyi Ls-D35 strain (NBRC 10381). The observation was using low initial C/N ratio (17.9) medium to optimize the cell and lipid production of Ls-D35 strain. The optimum pH for cell and lipid production of Ls-D35 was achieved at pH 5. The use of single xylose feeding achieved the highest lipid yield under low initial C/N ratio medium which was 0.13 (w/w) after 120 h. Although the lipid yield is still less than half of the theoretically yield (0.32 for glucose and 0.34 for xylose), this result can be confirmed as high yield for lipid in wild type strain of L. starkeyi under low initial C/N ratio. Meanwhile, the highest cell yield was achieved at mixed glucose and xylose feeding which was 0.15 (w/w) after 96 h. Furthermore, this study also revealed that the entire sugar substrates were totally assimilated at initial low C/N ratio medium which confirms that Ls-D35 strain is a robust oleaginous yeast due to its capability in utilizing sugar at low C/N ratio for cell and lipid production. Palmitic acid (C16:0) and oleic acid (C18:1) were higher in number of the produced fatty acids. As the comparison, the cell and lipid production in this study were significantly higher compared to the other wild type strain of L. starkeyi studies under low C/N ratio condition. Acknowledgements This work was supported by the International Joint Program, Science and Technology Research Partnership for Sustainable Development (SATREPS) (Innovative Bio-production Indonesia; iBioI) from Japan Science and Technology Agency and the Japan International Cooperation Agency (JST and JICA), Japan. The author is supported by Lembaga Pengelola Dana Pendidikan (LPDP) or Indonesian Endowment Fund for Education for his study in Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, Japan. References [1] C. Ratledge, J.P. Wynn, The biochemistry and molecular biology of lipid accumulation in oleaginous microorganisms, Adv. Appl. Microbiol. 51 (2002) 1–51. [2] S. Papanikolau, G. Aggelis, Lipids of oleaginous yeasts. Part I: biochemistry of single cell oil production, Eur. J. Lipid Sci. Technol. 113 (2011) 1031–1051. [3] I.R. Sitepu, L.A. Garay, R. Sestric, D. Levin, D.E. Block, J.B. German, K.L. BoundyMills, Oleaginous yeast for biodiesel: current and future trends in biology and production, Biotechnol. Adv. 32 (2014) 1336–1360. [4] M. Jin, P.J. Slininger, B.S. Dien, S. Waghmode, B.R. Moser, A. Orjuela, Ld C. Sousa, V. Balan, Microbial lipid-based lignocellulosic biorefinery: feasibility and challenges, Trends Biotechnol. 33 (1) (2015) 43–54. [5] S. Sutanto, S. Zullaikah, P.L. Tran-Nguyen, S. Ismadji, Y.-H. Ju, Lipomyces stakeyi: its current status as a potential oil producer, Fuel Process. Technol. 177 (2018) 39–55. [6] K.V. Probst, P.V. Vadlani, Single cell oil production by Lipomyces starkeyi: biphasic fed-batch fermentation strategy providing glucose for growth and xylose for oil production, Biochem. Eng. J. 121 (2017) 49–58. [7] A.B. Juanssilfero, P. Kahar, R.L. Amza, N. Miyamoto, H. Otsuka, H. Matsumoto, C. Kihira, A. Thontowi, C. Yopi, B. Ogino, A. Prasetya, Kondo, Effect of inoculum size on single-cell oil production from glucose and xylose using oleaginous yeast Lipomyces starkeyi, J. Biosci. Bioeng. 125 (2018) 695–702. [8] A. Anschau, M.C.A. Xavier, S. Hernalsteens, T.T. Franco, Effect of feeding strategies on lipid production by Lipomyces starkeyi, Bioresour. Technol. 157 (2014) 214–222. [9] Z. Gong, Q. Wang, H. Shen, C. Hu, G. Jin, Z.K. Zhao, Co-fermentation of cellobiose and xylose by Lipomyces starkeyi for lipid production, Bioresour. Technol. 117 (2012) 20–24. [10] R. Wild, S. Patil, M. Popovic, M. Zappi, S. Dufreche, R. Bajpai, Lipids from lipomyces starkeyi, Food Technol. Biotechnol. 48 (3) (2010) 329–335.
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Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
High cell density cultivation of Lipomyces starkeyi for achieving highly efficient lipid production from sugar under low C/N ratio
T
Rezky Lastinov Amzaa,1, Prihardi Kaharb,1, Ario Betha Juanssilferoa,c, Nao Miyamotoa, ⁎ Hiromi Otsukab, Chie Kihirab, Chiaki Oginoa, , Akihiko Kondoa,b a
Graduate School of Engineering, Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe, 657-8501, Japan Graduate School of Science, Technology and Innovation (STIN), Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe, 657-8501, Japan c Research Center for Biotechnology, Indonesian Institute of Sciences (LIPI), Jl. Raya Bogor Km 46, Cibinong, Bogor, 16991, West Java, Indonesia b
H I GH L IG H T S
fermentation of Ls-D35 was carried out at low initial C/N ratio (17.9). • The fermentation of Ls-D35 was optimum at pH 5 for cell and lipid productions. • The • The highest lipid yield was achieved by using single xylose feeding.
A R T I C LE I N FO
A B S T R A C T
Keywords: Oleaginous yeast Lipomyces starkeyi D35 High cell density culture Cell and lipid productions
Oleaginous microorganisms such as oleaginous yeasts are well known for the ability to accumulate intracelular lipid more than 20% w/w in certain conditions. Lipomyces starkeyi D35 (Ls-D35 strain) is one of promising oleaginous yeast for lipid production. The purpose of this research is to establish the optimum condition for cell and lipid production of Ls-D35 strain under low C/N ratio (17.9) by high cell density cultivation. The optimum pH for cell and lipid production was achieved at pH 5 by using batch cultivation. Four different feeding strategies were conducted to observe the cell and lipid production of Ls-D35 strain. The highest cell yield was achieved by mixed glucose and xylose as substrates (0.15 w/w substrate) after 96 h and the highest lipid yield was achieved by single xylose feeding (0.13 w/w substrate) after 120 h. Meanwhile, the two most fatty acids content were C18:1 and C16:0 which are similar to the content of vegetable oil. These results showed that Ls-D35 strain could produce high cell and optimum lipid under low initial C/N ratio medium which can be an alternative for biodiesel production and other industrial products.
1. Introduction The use of microorganisms as oil sources commercially has long history. Lipid from microorganisms which is well known as single cell oil (SCO) has been suggested as alternative energy resource since the second half of 20th century [1]. Since then, SCO has attracted researchers and industrial’s interests for biology production [2,3] and the utilization of lignocellulosic biorefinery [4]. Oleaginous microorganisms are the most well-known organisms for SCO production due to their capability for accumulating lipid in their cells [1]. One of potential microorganism for SCO production is oleaginous yeast [2,3,5]. It has been widely known that oleaginous yeasts are capable to
accumulate lipid more than 20% of their biomass weight or dry cell weight (DCW) [1]. The ability of oleaginous yeast in accumulating high lipid production is predicted to be future trends for biological production, especially biodiesel production [3]. One of robust yeast strain for lipid accumulation is Lipomyces starkeyi (L. starkeyi), which is capable to accumulate high lipid production within cells [5]. For instance, L. starkeyi is capable to utilize single xylose [6,7] and mixed sugar [6–9] as carbon sources for growth and lipid production as well. Lipid accumulation in oleaginous yeasts is usually occurred in nutrient imbalance in the medium [1]. Nutrient starvation and excess of carbon sources become the main factors for high lipid production in oleaginous yeast [1]. Many studies have shown
⁎
Corresponding author at: Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe, 657-8501, Japan. E-mail address: [email protected] (C. Ogino). 1 Rezky Lastinov Amza and Prihardi Kahar contributed equally to this work. https://doi.org/10.1016/j.bej.2019.05.013 Received 17 January 2019; Received in revised form 13 May 2019; Accepted 17 May 2019 Available online 24 May 2019 1369-703X/ © 2019 Elsevier B.V. All rights reserved.
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KH2PO4, 7, and Na2HPO4.2H2O, 5 in. g/L. The medium was supplemented with some trace elements: FeSO4.7H2O, 0.08; ZnSO4.7H2O, 0.01; CaCl2.2H2O, 0.1; MnSO4.4H2O, 0.1; CuSO4.5H2O, 0.002; CoCl2.6H2O, 0.002, in g/L. The pH medium was adjusted at 5.5 with appropiate amount of 1 M H2SO4 and sterilized by filtration.
that the optimum conditions for lipid accumulation in L. starkeyi are mostly under high C/N ratio content (> 20) of fermentation medium [1,6–8,10,11]. Low nitrogen (high C/N ratio) content inside the medium leads to the lipid accumulation by increasing the activity of AMP (adenosine mono phosphate) deaminase to provide the amount of ammonia as nitrogen supply for cells and to inhibit the formation of isocitrate dehydrogenase (ICDH) [5]. This condition provides the transfer of citrate into cytosol for lipid production [5]. In contrast, some studies have shown that L. starkeyi is not able to produce high lipid under high nitrogen (low C/N ratio) [6,11]. High nitrogen content allows citrate to be converted to α-ketoglutarate during the Krebs cycle which is favorable for cell’s growth. Furthermore, the growth of L. starkeyi was not optimum and sugar as carbon source was not totally consumed under high nitrogen (low C/N ratio) condition [6]. Lipomyces starkeyi D35 (Ls-D35) strain has been confirmed being able to accumulate high number of lipids under low nitrogen (high C/N ratio) medium [7] and produce high lipid during chemical compounds as inhibitor [12] in flask scale. This study focused on cells and lipid production in high cell density cultivation of Ls-D35 strain under high nitrogen (low C/N ratio) of initial fermentation medium by using fedbatch strategies. Fed-batch strategies were used in this study with the aim to achieve highly efficient production of both cells and lipid from Ls-D35 strain. Other studies showed that fed-batch strategy was able to improve cell and lipid production in L. starkeyi [6,10] under high C/N ratio condition. However, the objective of this study was to optimize the high cell density cultivation of Ls-D35 strain under initial low C/N ratio for high cell and lipid production. Synthetic sugars (glucose and xylose) were used as carbon sources in this study. Initial low C/N ratio was used to observe the capability of Ls-D35 strain in adjusting the use of pre-treated biomass as for industrial process. Therefore, optimization of pH is important because pre-treated biomass as carbon sources mostly requires acidic condition for further application [13–16]. Other studies have investigated that high cell and lipid production in oleaginous yeast can be achieved by controlling the substrate in fed-batch fermentation mode [8,10,16,17]. Fed-batch fermentation strategy was conducted in order to improve cells and lipid production. Four types of feeding strategy were conducted to compare the cell and lipid production of Ls-D35 strain. Each of single glucose, single xylose, mixed glucose-xylose and biphasic feeding were applied to high cell density cultivation of Ls-D35 strain.
2.3. Optimization of pH fermentation pH optimization was carried out by batch cultivations. The cultivations were performed in 2 L tank bioreactor (ABLE Biott, Japan) with 1 L of working volume. Mixed glucose and xylose was used as carbon sources. Cultivation medium was rich of nitrogen supplied by (NH4)2SO4 (10 g/L) which led to the initial C/N ratio at 17.9. The calculation of C/N ratio was based on Slininger’s patent [18]. pH of fermentation medium was set at 3, 4 and 5 for each cultivation. The conditions were set: intial agitation at 150 rpm; temperature at 30 °C and aeration at 1 vvm. All fermentations were inoculated by prepared seed at a final of 4 (v/v) %. Foaming was controlled using SI (silicone) antifoam solution (Wako, Japan). Samples for further analysis were taken every 24 h. 2.4. Fed-batch cultivations Fed-batch cultivations were performed in a 2 L tank bioreactor (ABLE Biott, Japan) with a one liter of working volume. Fed-batch cultivations were carried out in optimizing the cell and lipid production. Four conditions of cultivation were performed in fed-batch cultivations. The first cultivation was glucose feeding at glucose as single carbon source. The second cultivation was xylose feeding at xylose as single carbon source. The third cultivation is mixed glucose and xylose feeding at initial glucose and xylose as carbon sources. The last cultivation is biphasic fermentation with xylose feeding with glucose as initial carbon source. Feeding was performed manually after carbon source was below 10% of initial concentration. Certain volume of medium was removed in every feeding to maintain the feeding substrate concentration and volume of cultivation. The concentration of sugar feeding stock solutions was 400 g/L with 50 mL of the feeding solutions was fed into the cultivation to maintain ± 20 g/L carbon source in the medium. The conditions for the cultivation was set at 150 rpm of initial agitation and 30 °C of temperature. The initial pH was set at optimum condition and aeration at 1 vvm. All cultivations were performed with low initial C/N ratio at 17.9. Foaming was controlled using 1 (v/v) % of SI (silicone) antifoam solution (Wako, Japan). A 1 M solution of sodium hydroxide (NaOH) and sulfuric acid (H2SO4) were used to control pH. The oxygen for aeration was from oxygen generator instrument (PSA ITO-08-1, IBS Co. Ltd, Japan). The oxygen was filtered by sterilizing filter before it came into the fermentor for aeration. Samples were taken in every feeding time both before and after feeding.
2. Materials and methods 2.1. Microorganism and seed preparation L. starkeyi D35 (Ls-D35) yeast strain (NBRC10381) was selected from NITE Biological Resource Center (NBRC), National Institute of Technology and Evaluation, Japan. Yeast strain was preserved as a glycerol stock at -80 °C and revived by streaking on PDA agar plate. The yeast strain was grown on YMG agar plate (3.0 g/L yeast extract, 3.0 g/ L malt extract, 5.0 g/L peptone, 10.0 g/L glucose and 16.0 g/L agar) for short term storage. Seed cultures were cultivated in 500 mL shake flasks with a 120 mL of YMG medium (10 g/L glucose, 5 g/L peptone, 3 g/L yeast extract, 3 g/L malt extract) at 30 °C and 190 rpm in an orbital shaker incubator (BioShaker BR-43FH MR, TAITEC, Corp., Japan) for 24 h. Cells were collected by centifugation at 12,000 rpm for 4 min and washed two times by deionized water. Cells were resuspended ( ± 50 mL) by fermentation medium for inoculation in fermentor with 1 L working volume.
2.5. Dry cell weight determination Dry cell weight (DCW) was determined gravimetrically from a 1 mL sample of fermentation. Cells were sepearated by centrifugation (12,000 rpm for 4 min), washed two times and dried at -80 °C. Frozen cells were lyophilized by freeze-drying (Freez-one® 4.5 L Freeze Dry System Model 7750020, Labconco®, Kansas City, Missouri, USA) to remove the water content. 2.6. Sugar analysis Sugar analysis were performed by using high performance liquid chromatography (LC-20AB, Shimadzu, Japan). Samples were centrifuged and filtered (0.45 μm) prior to injection (20 μL). Samples were separated using a Coregel-87H3column (7.8 mmI.D. x 300 mm Transgenomic, USA) at the column temperature 80 °C. Mobile phase was 5 mM of H2SO4 with flow rate at 0.6 mL/min. A refractive index
2.2. Nutrient media Fermentation processes were carried out with Nitrogen Mineral Medium (NMM), consisted of carbon sources (glucose, or xylose, or a mixture of both), 100 g/L in single carbon source and 50 g/L of each in mixed-sugar, with yeast extract, 1.5, (NH4)2SO4, 10, MgSO4.7H2O, 1.5, 2
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detector (RID-10A) was used for peak identification. 2.7. Total lipid analysis Total lipid accumulation was measured by gravimetric analysis following the Folch method [19]. Freeze-dried cells (15–20 mg) were transferred into microvial with O-ring sealed cap (Watson, Japan). Glass beads were added into the vial. Lipid was extracted by 1.5 mL of chloroform:methanol 2:1 (v/v) [19]. The cell pulverization was conducted two times with Shake Master Neo ver 1.0 (BMS Tokyo, Japan) at 1500 rpm for 30 min. Cells were centrifuged at 12,000 rpm for 10 min after pulverization and filtrate was disposed after centrifugation. Cells were washed by 1.5 mL of deonized water for second pulverization. Cells were separated again by centrifugation. Filtrate were removed and cells were dried at up to 80 °C to constant weight. At the end, total lipid was measured by the cells’ weight difference at initial and after extraction. 2.8. Fatty acid methyl ester (FAME) analysis The transesterification was conducted by following the protocol from fatty acid methylation kit (Nacalai Tesque, Inc. Japan). The extracted FAME (light phase) was analyzed by using Gas Chromatography-Mass Spectrometer (GC–MS) (Shimadzu) instrument. GC–MS was equipped with DB-23 capillary column 0.25 mm x 30 m (J& W Scientific). The carrier was Helium gas with 0.8 mL/min of flow rate with 1:5 split ratios. The initial column temperature was 250 °C, increased 50 °C for 1 min and then increased 25 °C/min to 190 °C and 5 °C/min to 235 °C for 4 min. The internal standard of C8:0 (caprylic acid) was included in each sample. FAME (%) was calculated as the percentage of each fatty acid to the total number of fatty acids produced upon the fermentation. 2.9. Calculations Lipid content was determined by using Eq. (1).
Lipid content (%, w / w ) =
Fig. 1. Batch fermentation at different pH conditions. The cultivations were conducted by batch fermentation using glucose and xylose at pH 3, pH 4 and pH 5.
weight of cells after extraction (w ) x 100 % weight of cells before extraction (w ) (1)
pH [11]. In this study, the favorable pH of Ls-D35 strain fermentation was determined in order to achieve the optimum condition for cell and lipid production. Three batch fermentations with different pH were performed to determine the optimum pH condition for high cell density cultivation of Ls-D35 strain. Mixed glucose and xylose were used as carbon sources since Ls-D35 strain has been able to accumulate both glucose and xylose to produce higher cell and lipid in this condition [7]. The results of Ls-D35 strain fermentation in three different pH conditions (pH 3, pH 4 and pH 5) are shown in Fig. 1. Glucose and xylose were totally consumed in the end of exponential growth phase from these three different pH cultivations. However, the exponential growth phases ended in different fermentation time. Rapid glucose and xylose consumption occurred in the fermentation at pH 5 in which glucose was totally consumed at 24 h and xylose was at 48 h, respectively. Meanwhile, slower sugar consumption occurred in the fermentation at lower pH (pH 3 and 4) in which glucose and xylose were totally consumed at 48 h and 72 h. The highest dry cell weight (DCW) and lipid production was achieved at the pH 5 of fermentation which were 40.36 g/L and 9.3 g/L (23.12% of DCW) after 24 h, respectively. These results confirm that Ls-D35 strain has optimum condition for cell growth and lipid production at pH 5. The similar results were also found when L. starkeyi was grown in sewage sludge which resulted highest lipid at pH 5 [24] and glucose mineral medium at pH 5 [25]. Furthermore, the optimum growth rate at pH 5 was also found when L. starkeyi was grown in MSG wastewater [26].
Lipid yield was determined by Eq. (2).
amount of lipid (w ) − lipid at initial time (w ) Lipid yield (w /w ) = total carbon sources consumed (w )
(2)
Cell yield was determined by using Eq. (3).
Cell yield (w /w ) =
cells at certain time (w ) − cells at initial time (w ) total carbon sources consumed (w )
(3)
FAME per lipid was determined by using Eq. (4).
FAME per lipid (w /v ) = FAME (%, w /w ) x Lipid (w /v )
(4)
FAME per Dry Cell Weight (DCW) was determined by using Eq. (5).
FAME per DCW (w /v ) =
Lipid (w / v ) x FAME per Lipid (w /v ) DCW (w / v )
(5)
3. Results and discussion 3.1. Optimization of pH in batch culture The effect of pH in yeast fermentation has been studied previously [11,20,21] and specifically in cell and lipid production [22,23]. The optimization of pH is important in cell and lipid production by oleaginous yeast since the use of pretreated biomass for industrial production is mostly at low pH condition [11]. For instance, it has been observed that oleaginous yeast could grow and accumulate lipid at lower 3
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glucose or xylose as single carbon source and produce high cell and lipid at low initial C/N ratio. Furthermore, the use of single xylose as substrate was more preferred for lipid accumulation by L. starkeyi because xylose utilization is more efficient in lipid synthesis by oleaginous yeast through phosphoketolase pathway compared to single glucose [2]. Single glucose feeding resulted 66.64 g/L DCW and 32.67 g/L of lipid with 0.11 (w/w) of lipid yield after 120 h (Fig. 2a). Meanwhile, single xylose feeding resulted 63.41 g/L of DCW and 29.32 g/L of lipid with 0.13 (w/w) of lipid yield after 120 h, respectively (Fig. 2b). In mixed glucose-xylose as carbon sources, Ls-D35 strain assimilated glucose at first which led to the delay of xylose consumption. Xylose consumption was started after total consumption of glucose at 18 h. The delay of xylose when co-fermented with other sugar was also observed in other studies. When L. starkeyi ATCC 56304 which was fermented with glucose and xylose, glucose was completely consumed first at 48 h and then continued by the exhaustion of xylose at 60 h. [6]. Xylose consumption of Ls-D35 strain in flask scale was also started after glucose was exhausted in high C/N ratio fermentation medium (7). The consumption of xylose was also delayed in other L. starkeyi strains when co-fermented with other sugar [8,13,32]. The delay of xylose consumption was caused by the existence of glucose as competitive carbon source to xylose in high concentration [6]. However, glucose and xylose were consumed rapidly before feeding process in this study. Both glucose and xylose were exhausted after 18 h and 36 h, respectively, produced 57.69 g/L of DCW after 96 h of fermentation and 24.25 g/L of lipid (Fig. 2c) with 0.11 (w/w) of lipid yield (Table 1). Biphasic feeding was conducted to minimize the competition between glucose and xylose as substrates and was expected to improve cell and lipid production. Glucose was used as initial sugar for fermentation to gain cell production, since glucose metabolism is favorable for glycolysis which leads to cell production. Xylose was supplied after total consumption of glucose at 24 h and was consumed rapidly until the end of fermentation (120 h). Xylose feeding was supposed to improve lipid production since xylose is preferable for lipid production by pentose phosphate pathway which is easier in generating acetyl-CoA for fatty acid synthesis [1,2]. The similar pattern was also found in another research with slower consumption of glucose as initial sugar in biphasic feeding [6]. However, biphasic feeding could not improve lipid
Determining the optimum pH for cell and lipid production is important since pH value influences lipid accumulation in oleaginous yeasts [27]. L. starkeyi cannot grow well at high pH because of the inhibition of enzyme activity that produce biotin for cell growth [25]. Previous study showed that L. starkeyi can grow well at acidic range of pH [24]. However, the growth rates at lower pH (pH 3 and pH 4) were low due to more acidic condition. It has been shown that more acidic condition can disrupt the membrane of yeast [28] and influence transportation of nutrients [29]. However, in this study, sugar was totally consumed at lower pH (3 and 4), even at slower rate compared to pH 5. Based on these results, it can be confirmed that Ls-D35 strain is able to consume carbon sources and accumulate lipid under low pH especially at acidic condition which can be beneficial for decreasing production cost in controlling pH for industrial process [3] and preventing bacterial contaminations. 3.2. Fed-batch fermentation profile Synthetic glucose and xylose were used in this study as carbon sources both in single and mixed fed-batch fermentation at pH 5 as the optimum pH for Ls-D35 strain. Xylose was used since Ls-D35 strain can consume xylose well both in single and co-fermentation with glucose for high lipid production [7]. This study showed that Ls-D35 strain consumed glucose and xylose entirely at shorter fermentation time at initial low C/N ratio (Fig. 2a and b). Furthermore, the initial consumption of glucose was faster than xylose under low initial C/N ratio (Fig. 2a and b). The late initial consumption of xylose is assumed to be caused by low initial inoculum size (OD600 at around 3, data is not shown) which led in a prolonged lag phase [30]. Previous study of Ls-D35 strain showed that lower inoculum size at initial fermentation of flask scale cultivation will lead to the slower consumption of xylose as well [7]. In comparison, some studies showed that the cultivation of L. starkeyi in high nitrogen medium (low C/N ratio), glucose as single carbon source was not totally consumed and confirmed the lower production of DCW and lipid [6,31]. Slower consumption of single glucose in L. starkeyi under low C/N ratio with lower initial concentration has also been confirmed previously [11]. These comparisons show that Ls-D35 strain has better ability to assimilate
Fig. 2. Fed-batch fermentation profiles under low C/N ratio. Feeding strategies used: A, Fedbatch fermentation with initial single glucose (100 g/L) and feeding with glucose after 24 h (20 g/L) for every 12 h; B, Fed-batch fermentation with initial single xylose (100 g/L) and feeding with xylose after 48 h (20 g/L) for every 12 h; C, Fed-batch fermentation with initial glucose (50 g/L) and xylose (50 g/L), feeding by each glucose (10 g/L) and xylose (10 g/L) for every 12 h; D, Biphasic fed-batch fermentation with initial glucose (100 g/L) and continued by xylose after 24 h (20 g/L) for every 12 h. These data were calculated with triplicate sampling data calculations.
4
5
[6]
NA NAd NAd NAd NAd NAd NAd 0.25 0.21 0.18 0.19
d
c
Dry cell weight at the highest value. Amount of lipid at the highest value time per total consumed carbon source. Amount of lipid produced per liter solution at determined time. Not available/not determined. b
Yarrowiya lipolytica ylXYL + obese-XA Yarrowiya lipolytica W29 Lipomyces starkeyi D35
a
Glucose Glucose Xylose Glucose-Xylose Biphasic Agave bagasse Agave bagasse Glucose Xylose Glucose-Xylose Biphasic
± 13 ± 16 ± 27 ± 38 ± 59 25.8 11.5 66.64 63.41 57.69 58.26
± 4.3 ± 6.8 ± 14 ± 22 ± 36 16.5 2 32.67 29.32 24.25 25.5
34.2 41.8 51.5 57.4 60.1 67 20 49.03 46.25 42.03 43.75
0.08 0.08 0.16 0.17 0.17 0.34 NAd 0.11 0.13 0.11 0.1
0.04 0.06 0.12 0.14 0.14 0.17 0.02 0.27 0.24 0.25 0.26
C18:1 C18:1 C18:1 C18:1 C18:1 NAd NAd C18:1 C18:1 C18:1 C18:1
d
FAME (g/g-DCW)
Table 2 Comparison of cell and lipid production with various substrates under fed-batch fermentations.
Lipid content (%)
Lipid yield (w/w)b
Lipid accumulation rate (g/L h)c
production in Ls-D35 strain compared to single sugar as substrates. In biphasic feeding, the highest cell productions were at stationary phase after 72 h of fermentation (Fig. 2d). This biphasic feeding resulted 58.26 g/L of DCW with 25.49 g/L lipid (43.75%) after 96 h of fermentation (Fig. 2d) and 0.1 (w/w) substrates of lipid yield (Table 1). The highest lipid yield was achieved at xylose feeding: 0.13 (w/w) and the highest cell yield was at mix glucose and xylose feeding: 0.15 (w/w) (Table 2). The highest lipid yield was less than half of the theoretical yield for lipid production in oleaginous yeasts which are 0.32 (w/w) of glucose and 0.34 (w/w) of xylose as substrate [4]. Lipid production could not be improved for higher result, even with biphasic feeding strategies, since the fermentations were carried out with low C/ N ratio which theoretically is favorable for cell production [33]. Theoretically, lipid accumulation inside cell is optimum in nitrogen exhausted medium which triggers the adenosine monophosphate (AMP) deaminase activity. The activity of AMP deaminase leads to reverse reaction to form isocitrate which will be used to the formation of Acetyl-CoA for lipid synthesis. Otherwise, this study used nitrogen rich medium which theoretically is not favorable for lipid production. Citrate was not diverted by citrate-malate translocase (CMT) enzyme out of TCA cycle for lipid biosynthesis since the activity of AMP deaminase was inhibited due to the rich of nitrogen [4]. This condition is assumed to inhibit the lipid accumulation within Ls-D35 strain’s cell in nitrogen rich medium (low C/N ratio). Other studies found that lipid accumulation in L. starkeyi under low C/N ratio was significantly much lower compared to this study [6,21]. It was also found that lipid production was lower under high nitrogen condition in Rhodosporodium TJUWZ4 [34]. This result is also supported by data provided by previous study of Ls-D35 strain in flask scale under high C/N ratio which provided excess sugar for continuation of lipid accumulation compared to cell growth [7]. The comparison of cell and lipid production with other studies is shown in Table 2. L. starkeyi ATCC 56304 showed that it produced the highest lipid ( ± 36 g/L) in biphasic feeding strategy at higher C/N ratio between 80 and 100 [6]. Meanwhile, the engineered Yarrowiya lipolytica strain produced high percentage of lipid content (67%) when was grown in agave bagasse with higher C/N ratio as well [35]. However, as shown in Table 2, Ls-D35 could produce the compatible and even higher amount of cell and lipid production at low initial C/N ratio (17.9) compared to other studies with higher C/N ratio. Furthermore, in this study, as the fermentations were initiated with lower C/N ratio, it was confirmed that the sugar consumption rate of Ls-D35 strain was faster compared to another L. starkeyi strain with higher C/N ratio [6]. Ls-D35 strain could assimilate glucose and xylose even at higher concentration (each 50 g/L) within rapid fermentation time (24–36 h). These results confirm that Ls-D35 strain could produce more cells and lipids at low initial C/N ratio grown in synthetical sugar as carbon sources. It is expected that this study can be an updated reference for lipid production of Ls-D35 strain by using lignocellulosic materials with mostly contain high nitrogen content and limited sugar for carbon sources.
120 120 120 156 255 96 96 120 120 96 96
d
Total lipid accumulation from day 0 per total sugar consumed. Total cell accumulation from day 0 per total sugar consumed. Calculation at 120 h of fermentation. Calculation at 96 h of fermentation.
21 80-100 80-100 80-100 80-100 45 45 17.9 17.9 17.9 17.9
c
0.002 0.001 0.006 0.005
Lipomyces starkeyi ATCC 56304
b
± ± ± ±
Major FAME
a
0.010 0.002 0.011 0.011
Lipid (g/L)
0.12 0.13 0.15 0.13
± ± ± ±
DCW (g/L)a
0.11 0.13 0.11 0.10
Type of Carbon source
Glucosec Xylosec Mixed Glucose and Xylosed Biphasicd
Time (h)
Cell yield (w/w)b
C:N Ratio
Lipid yield (w/w)a
Yeast strain
Feeding type
Ref
Table 1 Lipid and cell yields obtained under different feeding strategies.
[35] [35] This study
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Fig. 3. Profiles of fatty acids produced during fed-batch fermentation. Feeding strategies used: A, single glucose feeding; B, single xylose feeding; C, mix glucose and xylose feeding; D, biphasic feeding.
obtained per DCW in every feeding strategy. Based on the results, the major composition of FAME per lipid and DCW in every feeding process was C18:1. However, the compositions of C16:0, C16:1, and C18:1 per lipid and per DCW were increased by the fermentation time up to 120 h. Ls-D35 strain has been observed as the highest producer of C18:1 among other oleaginous yeasts strain which is ± 75% of total fatty acid [3]. Due to low C/N ratio condition, C18:1 content was not able to be improved in this study since the metabolism of fatty acid synthesis was not optimized as limited pathway for fatty acid synthesis. However, this amount of oleic acid (C18:1) is potential enough for biodiesel production in industrial process. For instance, the production of C16:0 was higher compared to the other study of Ls-D35 strain [36]. These results showed that the modification of C/N ratio in feeding strategies of LsD35 strain is able to optimize the certain fatty acid production. Furthermore, based on previous study in flask scale [7] and this study, LsD35 strain can be recognized as an industrial yeast platform to produce
3.3. FAME profile Fatty Acid Methyl Ester (FAME) profile of each feeding strategies can be seen in Fig. 3. Major fatty acids content of Ls-D35 strain as shown in Fig. 3 are palmitic acid (C16:0), palmitoleic acid (C16:1), stearic acid (C18:0), oleic acid (C18:1) and linoleic acid (C18:2). These fatty acids profiles are closely similar with vegetable oils [3]. Increasing amount of fatty acids for all feeding strategies occurred at palmitic acid (C16:0), palmitoleic acid (C16:1) and oleic acid (C18:1). Specifically, the content of C16:0 and C16:1 in L. starkeyi were higher at this study compared to higher C/N ratio [10]. Furthermore, oleic acid (C18:1) content was more than 40% of the total fatty acids for each feeding strategies. This finding is similar with previous study of Ls-D35 strain in flask scale [7] and other Lipomyces strains [6,8,10]. The comparison of FAME amounts to lipid and DCW were also observed in every feeding process. As shown in Table 3, FAME per lipid was comparable with that
Table 3 Time dependent-changes of Fatty Acid Methyl Ester during the fed-batch fermentation.
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large amount of high-valued fatty acid derivates as well.
[11] C.H. Calvey, Y.-K. Su, L.B. Willis, M. McGee, T.W. Jeffries, Nitrogen limitation, oxygen limitation and lipid accumulation in Lipomyces starkeyi, Bioresour. Technol. 200 (2016) 780–788. [12] A.B. Juanssilfero, P. Kahar, R.L. Amza, N. Miyamoto, H. Otsuka, H. Matsumoto, C. Kihira, A. Thontowi, C. Yopi, B. Ogino, A. Prasetya, Kondo, Selection of oleaginous yeasts capable of high lipid accumulation during challenges from inhibitory chemical compunds, Biochem. Eng. J. 137 (2018) 182–191. [13] Z. Yu, M. Zhao, H. Li, H. Zhao, Q. Zhang, C. Wan, H. Li, A comparative study on physiological activities of lager and ale brewing yeasts under different gravity conditions, Biotechnol. Bioprocess Eng. 17 (2012) 818–826. [14] C. Huang, X.-F. Chen, X.-Y. Yang, L. Xiong, X.-Q. Lin, J. Yang, B. Wang, X.-D. Chen, Bioconversion of corncob acid hydrolysate into microbial oil by the oleaginous yeast Lipomyces starkeyi, Appl. Biochem. Biotechnol. 172 (2014) 2197–2204. [15] L. Matsakas, A.-A. Sterioti, U. Rova, P. Christakopoulos, Use of dried sweet sorghum for the efficient production of lipids by the yeast Lipomyces starkeyi CBS 1807, Ind. Crops Prod. 62 (2014) 367–372. [16] K.V. Probst, P.V. Vadlani, Production of single cell oil from Lipomyces starkeyi ATCC 56304 using biorefinery by-products, Bioresour. Technol. 198 (2015) 268–275. [17] M. Han, Z. Xu, C. Du, H. Qian, W.-G. Zhang, Effects of nitrogen on the lipid and carotenoid accumulation of oleaginous yeast Sporidiobolus pararoseus, Bioprocess Biosyst. Eng. 39 (2016) 1425–1433. [18] P.J. Slininger, B.S. Dien, C.P. Kurtzman, B.R. Moser, V. Balan, M. Jin, L.dC. Sousa, E.L. Bakota, US 2016/0265009 A1, United States Patent Application Publication, USA, 2016. [19] J. Folch, M. Lee, G.H. Sloane-Stanle, A simple method for isolation and purification of total lipids from animal tissues, J. Biol. Chem. 226 (1957) 497–509. [20] E. Casey, M. Sedlak, N.W. Ho, N.S. Mosier, Effect of acetic acid and pH on the cofermentation of glucose and xylose to ethanol by a genetically engineered strain of Saccharomyces cereviseae, FEMS Yeast Res. 10 (2010) 385–393. [21] Z.J. Liu, L.P. Liu, P. Wen, N. Li, M.H. Zong, H. Wu, Effects of acetic acid and pH on the growth and lipid accumulation of the oleaginous yeast Trichosporon fermentans, Bioresource 10 (3) (2015) 4152–4166. [22] A. Castelli, G.P. Littarru, G. Barbaresi, Effect of pH and CO2 concentration changes on lipids and fatty acids of Sachharomyces cerevisae, Arch. Mikrobiol. 66 (1969) 34–39. [23] J. Bradenburg, J. Blomqvist, J. Pickova, N. Bonturi, M. Sandgren, V. Passoth, Lipid production from hemicellulose with Lipomyces starkeyi in a pH regulated fed-batch cultivation, Yeast 33 (2016) 451–462. [24] C. Angerbauer, M. Siebenhofer, M. Mittelbach, G.M. Guebitz, Conversion of sewage sludge into lipids by Lipomyces starkeyi for biodiesel production, Bioresour. Technol. Rep. 99 (2008) 3051–3056. [25] Y. Uzuka, T. Naganuma, K. Tanaka, Y. Odagiri, Effect of culture pH on the growth and biotin requirement in a strain of Lipomyces starkeyi, J. Gen. Appl. Microbiol. 20 (1974) 197–206. [26] J.X. Liu, Q.Y. Yue, B.Y. Gao, Z.H. Ma, P.D. Zhang, Microbial treatment of the monosodium glutamate wastewater by Lipomyces starkeyi to produce microbial lipid, Bioresour. Technol. Rep. 106 (2012) 69–73. [27] J.M. Ageitos, J.A. Vallejo, P. Veiga-Crespo, T.G. Villa, Oily yeasts as oleaginous cell factories, Appl. Microbiol. Biotechnol. 90 (2011) 1219–1227. [28] P.W. Piper, Yeast superoxide dismutase mutants reveal a pro-oxidant action of weak organic acid food preservatives, Free Radic. Biol. Med. 27 (1999) 1219–1227. [29] B.E. Bauer, D. Rossington, M. Mollapour, Y. Mamnun, K. Kuchler, P.W. Piper, Weak organic acid stress inhibits aromatic amino acid uptake by yeast, causing a strong influence of amino acid auxotrophies on the phenotypes of membrane transporter mutants, Eur. J. Biochem. 270 (2003) 3189–3195. [30] L.Y. Kun, Bioprocess technology, in: L.Y. Kun (Ed.), Microbial Biotechnology Principles and Applications, 2nd), World Scientific Publishing Co. Pte. Ltd., Singapore, 2006Chapter 2 ISBN : 9812566775. [31] J. Lin, H. Shen, H. Tan, X. Zhao, S. Wu, C. Hu, Z.K. Zhao, Lipid production by Lipomyces starkeyi cells in glucose solution without auxiliary nutrients, J. Biotech. 152 (2011) 184–188. [32] R. Yamada, A. Yamauchi, T. Kashihara, H. Ogino, Evaluation of lipid production from xylose and glucose/xylose mixed sugar in various oleaginous yeasts and improvement of lipid production by UV mutagenesis, Biochem. Eng. J. 128 (2017) 76–82. [33] J.B.M. Rattray, A. Schibeci, D.K. Kidby, Lipids of yeasts, Microbiol. Rev. 39 (3) (1975) 197–231. [34] Q. Wang, Y. Cui, B. Sen, W. Ma, R.L. Zheng, X. Liu, G. Wang, Characterization and robust nature of newly isolated oleaginous marine yeast Rhodosporidiumspp. from coastal water of northern China, AMB Exp. 7 (30) (2017) 1–13. [35] X. Niehus, A.M. Crutz-Le Coq, G. Sandoval, J.M. Nicaud, R. Ledesma-Amaro, Engineering Yarrowiya lipolytica to enhance lipid production from lignocellulosic materials, Biotechnol. Biofuels 11 (11) (2018) 1–10. [36] A. Tanimura, M. Takashima, T. Sugita, R. Endoh, M. Kikukawa, S. Yamaguchi, E. Sakuradani, J. Ogawa, J. Shima, Selection of oleaginous yeasts with high lipid productivity for practical biodiesel production, Bioresour. Technol. 153 (2014) 230–235.
4. Conclusions This study investigated the effect of low initial C/N ratio and feeding strategy for growth and lipid accumulation in L. starkeyi Ls-D35 strain (NBRC 10381). The observation was using low initial C/N ratio (17.9) medium to optimize the cell and lipid production of Ls-D35 strain. The optimum pH for cell and lipid production of Ls-D35 was achieved at pH 5. The use of single xylose feeding achieved the highest lipid yield under low initial C/N ratio medium which was 0.13 (w/w) after 120 h. Although the lipid yield is still less than half of the theoretically yield (0.32 for glucose and 0.34 for xylose), this result can be confirmed as high yield for lipid in wild type strain of L. starkeyi under low initial C/N ratio. Meanwhile, the highest cell yield was achieved at mixed glucose and xylose feeding which was 0.15 (w/w) after 96 h. Furthermore, this study also revealed that the entire sugar substrates were totally assimilated at initial low C/N ratio medium which confirms that Ls-D35 strain is a robust oleaginous yeast due to its capability in utilizing sugar at low C/N ratio for cell and lipid production. Palmitic acid (C16:0) and oleic acid (C18:1) were higher in number of the produced fatty acids. As the comparison, the cell and lipid production in this study were significantly higher compared to the other wild type strain of L. starkeyi studies under low C/N ratio condition. Acknowledgements This work was supported by the International Joint Program, Science and Technology Research Partnership for Sustainable Development (SATREPS) (Innovative Bio-production Indonesia; iBioI) from Japan Science and Technology Agency and the Japan International Cooperation Agency (JST and JICA), Japan. The author is supported by Lembaga Pengelola Dana Pendidikan (LPDP) or Indonesian Endowment Fund for Education for his study in Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, Japan. References [1] C. Ratledge, J.P. Wynn, The biochemistry and molecular biology of lipid accumulation in oleaginous microorganisms, Adv. Appl. Microbiol. 51 (2002) 1–51. [2] S. Papanikolau, G. Aggelis, Lipids of oleaginous yeasts. Part I: biochemistry of single cell oil production, Eur. J. Lipid Sci. Technol. 113 (2011) 1031–1051. [3] I.R. Sitepu, L.A. Garay, R. Sestric, D. Levin, D.E. Block, J.B. German, K.L. BoundyMills, Oleaginous yeast for biodiesel: current and future trends in biology and production, Biotechnol. Adv. 32 (2014) 1336–1360. [4] M. Jin, P.J. Slininger, B.S. Dien, S. Waghmode, B.R. Moser, A. Orjuela, Ld C. Sousa, V. Balan, Microbial lipid-based lignocellulosic biorefinery: feasibility and challenges, Trends Biotechnol. 33 (1) (2015) 43–54. [5] S. Sutanto, S. Zullaikah, P.L. Tran-Nguyen, S. Ismadji, Y.-H. Ju, Lipomyces stakeyi: its current status as a potential oil producer, Fuel Process. Technol. 177 (2018) 39–55. [6] K.V. Probst, P.V. Vadlani, Single cell oil production by Lipomyces starkeyi: biphasic fed-batch fermentation strategy providing glucose for growth and xylose for oil production, Biochem. Eng. J. 121 (2017) 49–58. [7] A.B. Juanssilfero, P. Kahar, R.L. Amza, N. Miyamoto, H. Otsuka, H. Matsumoto, C. Kihira, A. Thontowi, C. Yopi, B. Ogino, A. Prasetya, Kondo, Effect of inoculum size on single-cell oil production from glucose and xylose using oleaginous yeast Lipomyces starkeyi, J. Biosci. Bioeng. 125 (2018) 695–702. [8] A. Anschau, M.C.A. Xavier, S. Hernalsteens, T.T. Franco, Effect of feeding strategies on lipid production by Lipomyces starkeyi, Bioresour. Technol. 157 (2014) 214–222. [9] Z. Gong, Q. Wang, H. Shen, C. Hu, G. Jin, Z.K. Zhao, Co-fermentation of cellobiose and xylose by Lipomyces starkeyi for lipid production, Bioresour. Technol. 117 (2012) 20–24. [10] R. Wild, S. Patil, M. Popovic, M. Zappi, S. Dufreche, R. Bajpai, Lipids from lipomyces starkeyi, Food Technol. Biotechnol. 48 (3) (2010) 329–335.
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