Different carbon sources on the lipids production characteristics of R-ZL2
Strains can adapt to different carbon sources and exhibit distinct substrate preferences (Lin and Wu 2015). To investigate the ability of R-ZL2 to utilise and assimilated different carbon sources, we compared the biomass, lipids production, sugar consumption, and lipids yield of strains cultured at 96 h in different concentrations of sucrose, glucose and xylose to determine the optimal carbon source and the best dosage.
Our results showed that the biomass of strains increased with increasing sugar concentration for sucrose and glucose (Fig. 1a). When the concentration of sucrose and glucose was 5%, the biomass reached the maximum value (10.77 g/L and 9.85 g/L, respectively), suggesting that the carbon sources satisfied the growth needs of the strains. Comparing the effects of different carbon sources with the same mass fraction on the biomass accumulation of strains, sucrose was found to be most effective. When the xylose concentration was 1-3%, the biomass increased with increasing sugar mass fraction, but when the xylose concentration was > 3%, the biomass did not increase significantly (p > 0.05). In summary, sucrose as the carbon source was most conducive to biomass accumulation in R-ZL2.
There was a correlation between lipids production and the concentration of the carbon source (Fig. 1b). When the amount of sucrose added was 4% and 5%, there was no significant difference in lipids production (p > 0.05), indicating that excess carbon source was not effectively converted into lipids by the strains. The fermentation trends for glucose and xylose were similar, when the carbon source was < 3%, accumulation of lipids and biomass was low, and when the sugar concentration was 3-5%, there was no significant difference in lipids production (p > 0.05), indicating that excess carbon source was not used for lipids accumulation. It can be concluded that carbon source concentrations > 3% were optimal for accumulation of lipids, and sucrose was the best sugar substrate.
Sugar consumption reflects a strain’s ability to transform and utilise carbon sources. As shown in Figure 1c, when sucrose addition was < 3%, sugar consumption was low, possibly due to carbon source inhibition; when sucrose addition was > 3%, the difference in sugar consumption was not significant (p > 0.05), indicating that sugar consumption maybe limited within the specified time. Compared with sucrose, strains were better able to consume glucose, and sugar consumption of different concentrations of glucose was higher than sucrose fermentation. The ability to consume xylose was similar to sucrose.
The lipids yield is the amount of lipids produced per unit mass of carbon source, which can further verify whether the carbon source is effectively converted. As shown in Figure 1d, when sucrose was the carbon source, the lipids yield with 4% was the highest; when glucose was the carbon source, the lipids yield with 3% was the highest; when xylose was the carbon source, the lipids yield with 5% was the highest.
In summary, the preference of R-ZL2 for three carbon sources was ordered sucrose > glucose > xylose. In addition, sufficient carbon is needed for growth, but excess carbon sources do not promote lipids production. Therefore, we chose 4% sucrose as the carbon source for follow-up experiments.
Different C/N ratios on the lipids production characteristics of R-ZL2 with ammonium sulphate as the nitrogen source
Ammonium sulphate is widely used in the cultivation of oleaginous yeast, providing the nitrogen source for yeast growth and lipids production. However, the concentration of the nitrogen source had a great influence on both the growth and lipids production of the strains. Therefore, we used 4% sucrose as the carbon source and ammonium sulphate as the nitrogen source to study the effect of different C/N ratios (20:1, 40:1, 80:1, 160:1, 200:1 and ∞ (∞ indicate that no nitrogen source was added to the medium)) on the lipids production characteristics of R-ZL2.
With increasing fermentation duration, the biomass of strains in media with different C/N ratios displayed an upward trend, then tended to be stable, while further extension of the fermentation time did not change the biomass significantly (p > 0.05; Figure 2a). When the C/N ratio was ∞, the minimum biomass was 6.8 g/L, when the C/N ratio is 20:1 and 40:1, the maximum biomass was 13.07 g/L and 12.94 g/L, respectively. The results show that the nitrogen source content in the range of 1-2 g/L was more conducive to biomass accumulation.
The lipids production of strains in different C/N ratio media gradually increased with increasing fermentation duration until the 8th day of fermentation, at which point lipids production reached a peak (Fig. 2b). When the C/N ratio was 20:1, lipids production was the lowest (2.15 g/L); when the C/N ratio was 160:1 and 200:1, lipids production was the highest (4.88 g/L and 6.32 g/L, respectively). From this, we concluded that 4% sucrose as the carbon source, ammonium sulphate as the nitrogen source, and a C/N ratio of 200:1 were the optimal conditions for lipids production.
The effect of different C/N ratios on the lipids content of the strains is shown in Figure 2c. During the entire fermentation process, the lipids content of the experimental group with a C/N ratio of 200:1 was consistently significantly higher than other groups (p < 0.05), and the lipids content reached the maximum value on the 7th day (59.73%), indicating that the strains had a strong ability to produce intracellular lipids. With a C/N ratio of 160:1, the lipids content was 43.26%; and when the C/N ratios was 20:1, the lipids content was only 16%, significantly lower than other groups (p < 0.05). When the C/N ratio was ∞, the lipids content of the strains was increased to a certain extent, but lipids content is not the highest. The above results indicate that the nitrogen source is indispensable for lipids accumulation of the strains, and effective control of the C/N ratio important for lipids accumulation.
Different C/N ratios on the lipids production characteristics of R-ZL2 with ammonium nitrate as the nitrogen source
Ammonium nitrate is a good nitrogen source. In order to investigate the effect of ammonium nitrate on yeast growth and lipids accumulation, and to compare with yeast cultured with ammonium sulphate as the nitrogen source, we explored using 4% sucrose as the carbon source, ammonium nitrate as the nitrogen source, and assessed the effect of different C/N ratios on the lipids production characteristics of yeast.
With increasing fermentation duration, the biomass of yeast increased continuously (Figure 3a). The biomass was the lowest when the C/N ratio was ∞, and the biomass reached the maximum value on the 7th day (6.82 g/L). When the C/N ratio was 20:1, the biomass was slightly higher than that when the C/N ratio was ∞, but lower than other experimental groups. When the C/N ratio was 40:1, the biomass reached the maximum value on the 9th day (10.16 g/L). The biomass of the experimental groups with a C/N ratio of 80:1, 160:1 and 200:1 was always higher than previous three groups, at the end of fermentation, the biomass of them was 12.35 g/L, 12.56 g/L and 11.88 g/L, respectively. Therefore, when ammonium nitrate as the nitrogen source, effective control of the amount of nitrogen source added could promote the accumulation of biomass. Comparing the biomass with a C/N ratio of 200:1 and 20:1 showed that the biomass was increased by 1.8-fold. When ammonium sulphate as the nitrogen source, the biomass was the highest when the C/N was 20:1 (13.07 g/L). The above results indicate that the strains have differences in their utilisation of different nitrogen sources, and there are significant differences in the biomass of strains cultured with different C/N ratios.
C/N ratio of 200:1 achieved the highest lipids production, significantly higher than other groups during the entire fermentation stage (p < 0.05, Figure 3b), and reached the maximum value on the 8th day (8.25 g/L). By comparison, a C/N ratio of 160:1 reached 7.85 g/L, a C/N ratio of 80:1 and 40:1 achieved intermediate amounts of lipids production, and a C/N of 20:1 recorded the lowest lipids production. When the C/N was ∞, lipids production was slightly higher than 20:1, but significantly lower than other experimental groups (p < 0.05). The above results show that when ammonium nitrate as the nitrogen source, increasing the C/N ratio could promote the accumulation of lipids, but when the nitrogen source is not added at all, lipids production was not favoured. Thus, the optimal C/N ratio was determined to be 200:1. This result is consistent with the results for ammonium sulphate as the nitrogen source. When the C/N ratio was 200:1, lipids production using ammonium sulphate or ammonium nitrate as the nitrogen source was 6.32 g/L and 8.25 g/L, respectively. Therefore, ammonium nitrate was more advantageous when mixed with sucrose to culture R-ZL2 to produce lipids.
The effects of different C/N ratios on the lipids content of strains are shown in Figure 3c. When the C/N ratio was 200:1, the lipids content reached the maximum value on the 8th day (65.64%). When the C/N ratio was 160:1, the lipids content was slightly lower than ratio of 200:1, and the maximum value was also reached on the 8th day. When the C/N ratio was 20:1, the lipids content of the strains was the lowest (~15%). When the C/N ratio was ∞, the lipids content of the strains was slightly higher than 20:1, but significantly lower than when the C/N was 200:1, 160:1 or 80:1 (p < 0.05). These results show that using an appropriate carbon source and C/N ratio can increase the lipids content.
Evaluation of the performance of biodiesel in the fatty acid composition of lipids produced by R-ZL2 under different nutrient conditions
Using sucrose as the carbon source and ammonium sulphate as the nitrogen source, we determined the fatty acid composition of lipids produced by R-ZL2 cultured with different C/N ratios (Table 1). Fatty acids produced by yeast cultured with different C/N ratios were C14:0, C16:0, C18:0, C18:1, C18:2 and C18:3, among which C16:0, C18:1 and C18:2 was the most abundant. With increasing C/N ratio in the medium, the ability of yeast to produce C16:0 gradually increased. When the C/N ratio was 160:1, the accumulated C16:0 content was the highest, and continue to increase the C/N ratio, the change in C16:0 was not significant (p > 0.05). In addition, with an increase in C/N ratio in the medium, the ability of yeast to produce C18:1 weakened. When the C/N ratio was 20:1, the content of C18:1 was the highest. Continue to increase the C/N ratio, the content of C18:1 was significantly decreased (p < 0.05). C18:2 as most abundant when the C/N ratio was 40:1. The C18:3 content was the lowest when the C/N ratio was 20:1. The abundance of C18:3 did not differ significantly between different culture conditions. In summary, a change in the C/N ratio in the medium can alter the fatty acid composition of the lipids produced.
The fatty acid results when using sucrose as the carbon source and ammonium nitrate as the nitrogen source are shown in Table 2. The identified fatty acids were composed of C14:0, C16:0, C18:0, C18:1, C18:2 and C18:3, and the most abundant were C16:0, C18:1 and C18:2 (as observed for ammonium sulphate as the nitrogen source). C16:0 was the most abundant when the C/N ratio was 80:1. C18:1 had the highest content when the C/N ratio was 40:1, and the lowest content when the C/N ratio was 80:1. Levels of C18:2 was the highest when the C/N ratio was 20:1, and levels decreased with increasing C/N ratio. The content of C18:3 was the lowest when the C/N ratio was 20:1, but it did not differ much between other conditions, indicating minimal effect on the synthesis of C18:3.
We evaluated whether the lipids produced under different C/N ratios could be used as biodiesel based on the content of each fatty acid (Table 3), through comparison of viscosity, specific gravity, turbidity, cetane number, iodine value and calorific value. When the carbon source was 4% sucrose and the nitrogen source was ammonium sulphate, the unsaturation of the lipids produced by yeast at C/N ratios of 40:1 and 80:1 was the highest, resulting in iodine values greater than the maximum limit of the national standard, which is unsuitable for the production of biodiesel.
Using 4% sucrose as the carbon source (Table 4), ammonium nitrate as the nitrogen source, and different C/N ratios, the lipids produced by yeast were found to be suitable for the production of biodiesel. In addition, we found that the fatty acids produced by yeast under different C/N ratios contained 60-70% unsaturated fatty acids and 25-40% saturated fatty acids. This shows that R-ZL2 has a strong ability to produce unsaturated fatty acids when using ammonium nitrate as the nitrogen source.