Characteristics of S. segobiensis DSM 27193 fermentation using fed‑batch culture
Fig. 1 displays the cell growth and lipid accumulation of S. segobiensis DSM 27193 using fed‑batch culture with glucose as sole carbon source. The fermentation process of general oleaginous yeast was usually be divided into two phases: cell growth phase and lipid accumulation phase [23]. Surprisingly, S. segobiensis DSM 27193 kept the cell growth during whole the fermentation time. At the end of the first phase, 9 g/L of biomass with only 5.78% of lipid has been obtained. Nitrogen depletion induced the lipid accumulation phase at ~50 h. Finally, 28.6 g/L of dry biomass and 19.2% per dry biomass of lipid has been obtained, corresponding to 5.49 g/L of lipids. Fatty acid profile analysis revealed that S. segobiensis DSM 27193 contained 13.8% of POA in its intracellular lipid. At present, high content of POA is usually reported in Kluyveromyces polysporus and S. cerevisiae, however, the total lipid content in these non-oleaginous yeasts is much lower than S. segobiensis DSM 27193[24]. In contrast, oleaginous yeasts like R. glutinis and Trichosporum cutaneum can accumulate higher lipid amounts, but their POA content is much lower, as <5% of POA have been detected in their intracellular lipid [24]. The high POA content in the lipid profile of S. segobiensis DSM 27193 endows it promising potential in developing POA commercial production.
In addition to intracellular lipids, 6.26 g/L of ethanol has been detected as the main by-product in the fermentation process. As shown in Fig 1, ethanol began to accumulate at ~48 h, when pO2 decreased to 0%. The limited oxygen availability might have stimulated the production of ethanol, characterizing this yeast as Crabtree negative [25], contrary to Crabtree positive yeast like S. cerevisiae, which only produces ethanol under the condition of excess carbon source [26]. Ethanol production might lower the carbon flux toward lipid production. Therefore, process optimization strategies should focus on DO regulation to improve lipid and POA production and to decrease ethanol accumulation.
Effect of dissolved oxygen concentration on cell growth and lipid accumulation
DO concentration plays a key role in the production of lipid and is also reflected by cell growth during the fermentation process. In the early phase of cultivation, pO2 of the medium decreased rapidly even to zero and maintained at a low level throughout the exponential phase (data not known), which inhibited cell growth and lipid production, while promoting ethanol formation[13]. Therefore, sufficient oxygen supply in the early phase (approximately at 48 h) should be helpful for enhancing cell growth and lipid accumulation.
In this study, three different pO2 levels of pO2>20%, pO2>40% and without pO2 control (constant agitation at 600 rpm) were carried out through dynamic agitation regulation. The initial agitation speeds were set at 600 rpm. Fast cell proliferation led to rapid pO2 decrease. Within 30 h and 72 h cultivation, pO2 level decreased to 40% and 20%, respectively. Subsequently, the dynamic agitation regulation was launched. After around 2 days of cultivation, 850 rpm and 750 rpm were necessary to maintain pO2>40% and pO2>20%, respectively. In terms of cell growth, 33.9% improvement in cell growth has been achieved by maintaining pO2>40% compared to no pO2 control, indicating the limitation of DO in the medium during the experiment without pO2 control; however, no obvious difference has been found between pO2>20% (28.6 g/L) and without pO2 control (29.55g/L) (Fig. 2A).
Fig. 2C showed the lipid content variation under different pO2 conditions. 19.2%, 21.7% and 24.4% of lipid content per dry biomass has been achieved without pO2 control, pO2>20% and pO2>40%, respectively, corresponding to lipid concentrations of 5.49 g/L, 6.41g/L and 9.34 g/L, respectively (Fig. 2D) (Table.1). Lipid composition analysis determined that the main fatty acids in S. segobiensis DSM 27193 are palmitic acid (C16:0), POA (C16:1), oleic acid (C18:1), linoleic acid (C18:2), linolenic acid (C18:3) and eicosanoic acid (C20:0) regardless of the oxygen supply strategy (Table.2). Thereof, POA and C18:1 were the major components constituting 13.8% and 60.1% of the total lipid, respectively. POA ratio could be increased from 13.8% to 16.4% and 19.9% when maintaining pO2>20% and pO2>40% compared with the without pO2 control, respectively. In contrast, C18:1 decreased from 60.1% to 54.7% when keeping pO2>40%. We also noted that C16:0, the precursor of POA slightly increased from 12.95% to 16.4% once keeping pO2>40%. The increase of DO concentration regulated by agitation might prevent the transformation of C16:0 to C18:0, so that more POA could be accumulated. The phenomenon that variation in pO2 levels affects the fatty acid profile has also been observed in Lipomyces starkeyi, Y. lipolytica and Rhodotorula gracilis [15, 27, 28]. While in these studies, fatty acid desaturases were considered to be oxygen-related enzymes and oxygen-rich conditions reflected relative activity of desaturases and finally led to an increase in the degree of unsaturation of fatty acids.
Table 1. Product titers and yields obtained at different pO2 levels
pO2
|
Biomass
(g/L)
|
Lipid
content (%per CDW)
|
lipid
production (g/L)
|
POA
ratio
(%)
|
POA
production (g/L)
|
YL/g
(g lipid/g glucose)
|
YL/X (g lipid/g biomass)
|
600 rpm
|
28.6±1.53
|
19.2±0.45
|
5.49±0.42
|
13.8±1.05
|
0.76±0.12
|
0.04
|
0.19
|
pO2 >20%
|
29.5±2.12
|
21.7±0.73
|
6.41±0.68
|
16.7±1.61
|
1.07±0.22
|
0.04
|
0.22
|
pO2 >40%
|
38.3±3.16
|
24.4±0.23
|
9.34±0.86
|
19.94±1.10
|
1.86±0.27
|
0.07
|
0.24
|
YL/g (g lipid/g glucose) was estimated by the formula XL/g= Lipid production/glucose consumption
YL/X (g lipid/g biamass) was estimated by the formula XL/X= Lipid production/biomass
Table.2 Fatty acid composition profile in % of total fatty acids of Scheffersomyces segobiensis DSM 27193 under different pO2 levels
Fatty acid species
|
600 rpm
|
pO2 >20%
|
pO2 >40%
|
C16:0
|
12.9±1.14
|
14.1±0.90
|
16.4±0.50
|
POA
|
13.8±1.05
|
16.4±1.64
|
19.9±1.10
|
C18:0
|
0.7±0.07
|
0.6±0.22
|
0.4±0
|
C18:1
|
60.7±0.78
|
57.7±0.61
|
54.7±0.88
|
C18:2
|
10.8±1.54
|
9.7±1.14
|
7.6±0.30
|
C20:0
|
1.1±0.04
|
1.5±0.24
|
1.0±0
|
In terms of ethanol production, high DO concentration lowered ethanol production, as the highest ethanol concentrations of 6.26 g/L, 5.76 g/L and 2.3 g/L have been detected under the conditions of constant agitation at 600 rpm (no pO2 control), pO2>20% and pO2>40%, respectively (Fig. 2B). Interestingly, the highest ethanol accumulation was found in the middle period of fermentation process, and the produced ethanol was reassimilated in the later fermentation stage in all experiments. Speculatively, on one hand, the decrease of oxygen demand in the later stage of fermentation led to the DO increase in the fermentation medium, therefore, the prerequisites of DO limitation for ethanol production has been rescinded, hence limiting the further production of ethanol; while on the other hand, the increased oxygen supplied to medium caused an increasing oxidation of the ethanol produced even in the presence of glucose in the fermentation medium. The same ethanol reassimilation phenomenon has also been described in Candida tropicalis and Pachysolen tannophilus [29, 30]. Despite the fact that high DO control decreased the ethanol production, 2.3 g/L of ethanol was still produced at 69 h under the condition of pO2>40%. Therefore, more considerate DO regulation strategy was required.
Effect of aeration rate on cell growth and POA production
Aeration control is another important DO regulation strategy. Compared with agitation, aeration regulation imposes lower shear stress to the cells and energy consumption. In this study, three different aeration rates of 0.1 vvm, 1 vvm and 2 vvm were tested with a constant agitation speed of 600 rpm. Fig.3A shows the cell growth under different aeration conditions. When aerating with 0.1 vvm, S. segobiensis DSM 27193 grew slowly, and only 8.8 g/L of dry biomass were obtained at the end of fermentation process. In contrast, high aeration invigorated cell growth throughout the fermentation process, and maximal dry biomass concentration of 46.8 g/L was obtained under the condition of 2 vvm, whereas 33 g/L of dry biomass has been reached with 1 vvm aeration.
As expected, higher aeration rates did also increase the lipid accumulation, as 18.91%, 20.05% and 28.26% lipid per cell dry mass was reached when employing aeration rates of 0.1 vvm, 1 vvm and 2 vvm, respectively (Fig. 3C). In addition, lipid accumulation was accelerated when using the highest aeration rate. Lipid composition analysis revealed, that the accumulation of POA and C18:2 was slightly higher at the lowest aeration rate of 0.1 vvm, while the amount of C16:0 and C18:1 decreased from 12.95% to 7.91% and from 60.10% to 52.47%, respectively (Fig. 5A). Low DO was speculated to induce the expression of Δ-9 fatty acid desaturase, which is responsible to form a double bond in saturated fatty acyl-CoA substrates, thereby increase the unsaturation level of lipid, which has been confirmed in yeast S. cerevisiae [19]. However, POA and linoleic acid content was more or less constant regardless of the aeration rate.
In terms of ethanol production, high ethanol levels up to 20 g/L were produced with the lowest aeration rate (0.1 vvm) (Fig.3B), which seems not to be reassimilated in the later fermentation stages. Instead, it stagnated at a high level. This might be due to the low oxygen availability at the lowest aeration rate used. A similar phenomenon is known for P. stipites, where the volumetric oxygen transfer coefficient (KLa) of less than 11.7 h-1 inhibited ethanol assimilation [31]. However, even at higher aeration rates, about 6 g/L (1 vvm) and 7,4 g/L (2 vvm) of ethanol was produced around 120 h fermentation time, indicating DO limitation. Therefore, higher applying aeration rates are seemingly not sufficient for decreasing ethanol production. Further combination of both aeration and agitation regulation might not only favor cell growth and lipid production, but might also decrease ethanol production, and was therefore tested in the next experiments.
Two-stage agitation regulation to enhance POA production
Considering that higher agitation speed did significantly improve cell growth and lipid production without exerting damaging stress to the yeast cells, and the high DO demand in the early fermentation period, two high agitation regulation strategies were applied: ① constant high agitation speed of 1000 rpm; ② two-stage agitation regulation with 600 rpm in the early fermentation phase which was switched to 1000 rpm after 48 h to avoid reduction of cell growth. For both strategies 1vvm was selected as the aeration rate, when taking all factors (cell growth, lipid and POA production, ethanol production) into consideration.
As shown in Fig. 4A, high agitation speed had a positive effect on cell growth as expected, as the biomass concentration of 44.8 g/L and 42.1 g/L were obtained under the condition of 1000 rpm and the two-stage agitation regulation, respectively. Additionally, nearly no ethanol was detected throughout the fermentation when using constantly high agitation speed of 1000 rpm (Fig. 4B). In contrast, with the two-stage agitation regulation ~2 g/L of ethanol were produced at 45 h, which was quickly reassimilated after the agitation speed increase. Constant agitation at 1000 rpm could keep pO2 level always >40%, while in the two-stage agitation regulation, pO2 was decreased to 10% during the early fermentation period.
In terms of lipid production, 1000 rpm and two-stage agitation regulation increased lipid content to 30.07% and 25.3%, respectively (Fig. 4C). In this case, high pO2 condition had both positive effects on cell growth and lipid accumulation for S. segobiensis DSM 27193. In addition, POA content was greatly improved from 13.8% to 21.78% with constant high agitation of 1000 rpm compared to the control condition (Fig. 5B), and the final POA production of 2.93g/L was achieved, corresponding to a 2-fold increase.
The synthesis pathway of POA in S. segobiensis DSM 27193 was displayed in Fig.6A. Transcription analysis of the key enzymes in ethanol production pathway (ALDH1, ADH1, PDC1) and POA synthetic pathway (ACC, DGAT1, DGAT2, OLE1, OLE2) in the early fermentation phase were displayed in Fig.6B. ALDH1, ADH1, PDC1 were strongly induced at 600 rpm compared with high agitation condition. In detail, the transcription levels of ADH1 at 600 rpm was 3.6 times higher than that at 1000 rpm; Also, the transcription of PDC1 under the condition of 600 rpm displayed 3 times higher than that achieved in 1000 rpm, indicating ethanol synthetic pathway was significantly activated at low DO condition in the early fermentation phase. Unsurprisingly, the transcription of the key enzymes such as DGAT1 and OLE2 responsible for lipid synthesis and desaturation in S. segobiensis DSM 27193 were increased at higher DO condition in the early fermentation phase. In specific, the transcription of DGAT1 was up-regulated by 5.4 times and OLE2 was up-regulated by 3.76 times under the condition of 1000 rpm. This may explain why increasing the agitation speed can effectively increase the total lipid production and the proportion of POA.