3.1 Dry cell weight in mixotrophic/heterotrophic growth under six media types
Figure 1 The variations in (a) dry cell weight (DCW) (b) specific growth rate (K) of Scenedesmus quadricauda FACHB-1297 under different media in mixotrophic cultivation, and (c) dry cell weight (DCW) (d) specific growth rate (K) in heterotrophic cultivation
For assessing the mixotrophic and heterotrophic growth characters of Scenedesmus quadricauda under six different media types, diverse nitrogen and phosphorus concentrations in light conditions for mixotrophic culture simultaneously at the lack of light for heterotrophic culture were probed the effect of nitrogen and phosphorus for the development characteristics of Scenedesmus quadricauda (Fig. 1). Figure 1 (a) and (b) manifested that Scenedesmus quadricauda under six different media types entered the logarithmic growth phase after two days of adaptive phase, which was correspond with the research of Song et al (2018). Under the condition of mixotrophic culture, the specific growth rate of all the experimental groups reached the maximum on the sixth day, the reason was from the sixth day Scenedesmus quadricauda started to enter the period of stabilization, which was earlier than the Scenedesmus obliquus, that needed approximately 40–45 days to reach a plateau (Yang et al. 2014). This suggested that xylose was well tolerated by our earlier domesticated Scenedesmus quadricauda.
In the conditions of mixotrophic modes, the biomass productivity under different nitrogen and phosphorus concentrations were 137.45 mg/L/d (N&P), 95.03 mg/L/d (N&P−), 83.45 mg/L/d (N&P0), 83.83 mg/L/d (N0&P), 80.04 mg/L/d (N0&P−), and 75.01 mg/L/d (N0&P0) respectively (Table 2). The highest biomass productivity was achieved when nitrogen and phosphorus were sufficient, being 1.8-fold of the nitrogen and phosphorus deficiency group, which was accorded with the maximum value of dry cell weight (1.10 g/L) and the fastest particular growth velocity (0.14 d− 1) under N&P mode. This phenomenon can be explained that nitrogen and phosphorus were essential nutrients for the development of microalgae (Jiang et al. 2017; Chu et al. 2013; Han et al. 2014; Li et al. 2021).
Figure 1 (c) and (d) showed that the highest dry cell weight (0.60 g/L) and specific growth rate (0.13 d− 1) for Scenedesmus quadricauda were received at the N&P mode under heterotrophic cultivation. All of these values were lower than those obtained in the light under N&P mode, this phenomenon could be explained by the report of Manhaeghe et al. (2020), the mixotrophic development rate of microalgae was faster than those of the photoautotrophic mode and the heterotrophic mode. Furthermore, the highest biomass productivity of Scenedesmus quadricauda (137.45 mg/L/d) was not only more than the value of 61.11 mg/L/d got in the dark, but also more than that of 37.8 mg/L/d got by the Chlorella (PCH90) fed on xylose in mixotrophic condition (Leite et al. 2016). The reason why Scenedesmus quadricauda grew best under the mixotrophic conditions with nitrogen and phosphorus abundant was that in the mixotrophic culture, carbon sources could provide energy other than light and be synchronously absorbed for ATP yield (Pang and Chen 2017).
Figure 1 showed that the biomass and the specific growth rate of Scenedesmus quadricauda increased under mixotrophic and heterotrophic modes in diverse nitrogen and phosphorus content, which indicated that xylose could be absorbed and utilized by Scenedesmus quadricauda. The xylose was assimilated by microalgae via the cell membrane into the cells, in the mean time, the inducible hexose derivatives also facilitated xylose to entry the microalgae cells (Zheng et al. 2014). The endocellular xylose afterwards entranced the pentose phosphate pathway was decomposed via the NADPH-linked xylose reductase and NADP+-linked xylitol dehydrogenase in two steps to prepare for biomass and fatty acid synthesis (Alper and Stephanopoulos 2009). The energy and coenzymes produced by photosynthesis of microalgae under light could facilitate the above processes, so the biomass of microalgae in mixotrophic modes was more than that in heterotrophic modes.
Table 2
Biomass productivity, Lipid content and Lipid productivity of six treatment groups after 8-day cultivation in mixotrophic/heterotrophic cultivation
|
Cultivation conditions
|
N&P
|
N&P−
|
N&P0
|
N0&P
|
N0&P−
|
N0&P0
|
Biomass productivity (mg/L/d)
|
Mixotrophic
|
137.45
|
95.03
|
83.45
|
83.83
|
80.04
|
75.01
|
Heterotrophic
|
61.11
|
72.68
|
64.38
|
61.77
|
61.01
|
60.25
|
Lipid content (%)
|
Mixotrophic
|
21.00
|
27.22
|
31.42
|
41.50
|
36.93
|
20.37
|
Heterotrophic
|
32.23
|
17.34
|
14.40
|
35.72
|
34.01
|
16.70
|
Lipid productivity (mg/L/d)
|
Mixotrophic
|
28.86
|
25.66
|
26.25
|
34.37
|
28.82
|
15.00
|
Heterotrophic
|
22.29
|
12.60
|
9.27
|
22.06
|
20.75
|
10.06
|
3.2 TN, TP and xylose changes under six media types in light/light-free
Figure 2 Assimilation profiles of xylose (a), TN (b), TP (c) by Scenedesmus quadricauda FACHB-1297 in mixotrophic cultivation, and xylose (d), TN (e), TP (f) in heterotrophic cultivation
As shown in Fig. 2, the removal rate of TN and xylose were a bit slow, nevertheless the removal rate of TP was faster especially in the first two days (60%-92%) and almost all of the TP was removed by Scenedesmus quadricauda on the eighth day. Under mixotrophic mode, the TP content of phosphorus sufficient assays rapidly reduced from 37.00 to 0 mg/L in 8 days (100%), and these of phosphorus restrictive groups likewise lessened from 4.00 to 0 mg/L in 8 days (100%). However, the assimilate efficiency of TP in heterotrophic mode was not as high as under the condition of mixotrophic culture, which was 60% of phosphorus sufficient assays and 90% of phosphorus restrictive conditions. The rapid absorption of TP in the beginning may be due to the surface area of microalgae, and the higher uptake efficiency of TP in the mixotrophic culture may be caused by photosynthesis (Song et al. 2014; Song et al. 2018).
Whether there was light or not, the removal rate of TN decreased with the phosphorus concentration decreased by N&P, N&P− and N&P0, which was also consistent with the varations of maximum dry cell weight of 1.10 g/L, 0.76 g/L, 0.66 g/L, of N&P, N&P− and N&P0 group, successively. In the heterotrophic mode, the removal rates of TN achieved at 99% in the N&P trial, following by N&P− of 95% and N&P0 of 74%, those were much higher than that got under mixotrophic mode with the value of 43%, 24% and 21% under N&P, N&P−, N&P0 conditions, successively. The xylose removal rate of 96% under heterotrophic mode was also better than that under mixotrophic conditions. However, the biomass of heterotrophic mode were lower than that of mixtrophic mode, the absorption of xylose might promote the absorption of nitrogen, and the adsorbed nitrogen and xylose might be transeferred to lipid (Leite et al. 2016), and similar to the loss of biomass under the conditions of heterotrophic condition also appeared in the report of Miao and Wu (2006). Those suggested that Scenedesmus quadricauda could use xylose both in and out of the light, which was in line with the report of Leite et al. (2016), who showed that only Scenedesmus quadricauda could use xylose to grow in mixotrophic and heterotroph modes.
3.3 Lipid accumulation in mixotrophic/heterotrophic growth under six media types
Figure 3 Lipid content and the production variations of lipid under six treatment during 8-day cultivation in (a) mixotrophic cultivation and (b) heterotrophic cultivation
Figure 4 The mechanism of the effect of xylose assimilation and nitrogen starvation on the lipid accumulation in mixotrophic/heterotrophic cultivation
Table 2 Biomass productivity, lipid content and lipid productivity of six treatment groups after 8-day cultivation in mixotrophic/heterotrophic cultivation
When Scenedesmus quadricauda was cultured under nitrogen-deficient conditions, the maximum oil percentage (about 41%) was obtained on 5th day (heterotrophic cultivation) and 8th day (mixotrophic cultivation) under the nitrogen starved and phosphorus sufficient (N0&P) condition, which was about 2-fold compared with the initial lipid content under nitrogen-sufficient condition (control) (Fig. 3). The groups with the highest oil production had similar results with oil content, which achieved at 8 days of mixotrophic culture (274.97 mg/L) and 5 days of heterotrophic culture (193.77 mg/L) under N0&P groups (Fig. 3). It was interesting to find that the highest lipid content was reached faster under heterotrophic conditions, in addition, this result was accorded with the faster absorption rate of xylose under heterotrophic conditions (30%-96%). Figure 3 (b) showed that the lipid content began to decrease slowly from the fifth day, which illustrated Scenedesmus quadricauda began consuming lipid after the fifth day in the dark. However, the maximum lipid production was obtained in the mixotrophic cultivation under N0&P condition (274.97 mg/L) but not in the heterotrophic cultivation (178.34 mg/L), which suggested that the presence of light was beneficial for Scenedesmus quadricauda to absorb xylose to synthesize biomass, so that the lipid productivity reached high. Similar conclusions have been proved by Perez-Garcia et al. (2011), that the photosource was beneficial to oil synthesis of microalgae in the heterotrophic mode. It could be attributed to the photosynthesis provided enough energy for Acyl-CoA to malonyl coenzyme A (Mayl-CoA) for formation of fatty acids.
Another finding of this experiment was that in both mixotrophic and heterotrophic modes, the maximum lipid content were received in nitrogen starvation media. The general content of lipid in microalgae perhaps was on a scale of 1–85% (Chisti 2007), when nutrition was restricted, it could usually reach more than 40%, which was consistent with the lipid content (41%) obtained by restricting nitrogen in this study. And this data (41%) was more than the oil content (37%) of the accumulation of Chlorella vulgaris SDEC-3M in nitrogen lack of conditions within NSE nutrient solution (Qi et al. 2016). As shown in Fig. 4, when the microalgae was under stress, such as nitrogen deficiency, salt stress, light limiting and other conditions, some stress markers like superoxide dismutase/reactive oxygen species (SOD/ROS) would be produced, which stimulated triglyceride (TAG) production in reply to environmental force (Yu et al. 2018). In addition to that, the carbon storage mechanism was triggered by the restricted conditions of nitrogen to adapt to the continuous consumption of carbon and the growth of cells, which could inhibit the protein synthesis and transfer redundant carbon to amylum and/or lipid to accelerate oil accumulation (Farooq et al. 2022). To sum up (according to Fig. 4), conditions of xylose and nitrogen stress co-promoted fatty synthesis. One of the reasons was nitrogen restriction increased the microalgae content of fatty acid acetyl-CoA (Takagi et al. 2000; Qi et al. 2016), which was a crucial ferment and the precursor of certain energy-storing substances. In the meantime, when xylose was absorbed by microalgae, the substance (xylulose-5-phosphat) involved in the regulation of lipogenesis genes synthesis was also produced, which also stimulated more oil synthesis (Leite et al. 2016). However, there are few studies on the use of nitrogen and phosphorus in xylose mixotrophic cultivation and heterotrophic cultivation to produce biodiesel.
As shown in Fig. 3, the lipid percentage reduced from 41–20% with the phosphorus content decreased under nitrogen deficiency in mixotrophic cultivation, the similar pattern was discovered under heterotrophic conditions (with the decrease of phosphorus content, the lipid percentage decreased from 35–16%). This result demonstrated that phosphorus promoted lipid accumulation in the case of nitrogen starvation under mixotrophic cultivation and heterotrophic cultivation. In the case of insufficient nitrogen, phosphorus played a vital part during the lipid production capacity of microalgae in autotrophic growth cultivation (Chu et al. 2013; Shen et al. 2020). During the growth of microalgae, phosphorus in the culture solution was converted into Poly-P and stored in the microalgae cells (Chu et al. 2013). Soto et al. (2019) demonstrated that Poly-P as an energy storage material, could accumulate in large quantities at the condition of nutrient lack. In the case of nitrogen lack, the increase of phosphorus content led to the increase of Poly-P, which promoted the accumulation of lipid.
3.4 Relationship between xylose conversion factor and lipid accumulation
Table 3 Biomass yield (YB/X), Lipid yield (YL/X) and Nitrogen depletion (YN/X) of six treatment groups after 8-day cultivation in mixotrophic/heterotrophic cultivation
Table 3
Biomass yield (YB/X), Lipid yield (YL/X) and Nitrogen depletion (YN/X) of six treatment groups after 8-day cultivation in mixotrophic/heterotrophic cultivation
|
Cultivation conditions
|
N&P
|
N&P−
|
N&P0
|
N0&P
|
N0&P−
|
N0&P0
|
YL/X(g·g− 1)
|
Mixotrophic
|
0.63
|
0.57
|
0.36
|
0.83
|
0.73
|
0.17
|
Heterotrophic
|
0.67
|
0.59
|
0.38
|
0.93
|
0.75
|
0.19
|
YB/X(g·g− 1)
|
Mixotrophic
|
0.85
|
0.61
|
0.48
|
0.36
|
0.25
|
0.16
|
Heterotrophic
|
0.73
|
0.56
|
0.32
|
0.28
|
0.22
|
0.13
|
YN/X(g·g− 1)
|
Mixotrophic
|
0.31
|
0.23
|
0.13
|
-
|
-
|
-
|
Heterotrophic
|
0.24
|
0.25
|
0.27
|
-
|
-
|
-
|
For further research the relationship between the xylose assimilation, lipid production, nitrogen removal and development characteristics, the YL/X, YN/X and YB/X were calculated (Table 3). The highest transfer coefficient of xylose to the lipid (YL/X) under mixotrophic cultivation in N0&P mode was 0.83 g/g, which was 5 times more than the conversion factor of 0.17 g/g without nitrogen and phosphorus addition, and this maximum value was also 1.4 times higher than the factor of 0.63 g/g in the light under N&P group. These indicated that nitrogen deficiency was an important factor to promoting lipid accumulation. This result was correspond with that observed by Breuer et al. (2012) for Chlorella vulgaris, Chlorella zofingiensis, Neochloris oleoabundans, and Scenedesmus obliquus, which synthesized over 35% of their dry weight as triglycerides under nitrogen starvation condition. Similar result was obtained under heterotrophic condition, the highest YL/X (0.93 g/g) was acquired in N0&P condition as well.
According to Table 3, it could be seen that under mixotrophic cultivation the maximum values of YL/X (0.83 g/g) and YN/X (0.31 g/g) were both lower than YL/X (0.92 g/g) and YN/X (0.47 g/g) under heterotrophic condition, but the value of YB/X (0.73 g/g) under heterotrophic mode was lower than YB/X (0.85 g/g) under mixtrophic mode. These conversion coefficients were consistent with higher nitrogen and xylose absorption rates under heterotrophic condition, which also indicated that heterotrophic condition was more conducive to nitrogen absorption by microalgae and xylose conversion to oil. In the dark, microalgae only relied on absorbing xylose as carbon source, while under light conditions, microalgae still obtained energy through photosynthesis. Therefore, the conversion factor of xylose into lipid was higher under dark than that in light. But in the fact, in terms of total oil production, microalgae accumulated more lipid and biomass under light, owing to the energy and the coenzyme (NADPH) produced by photosynthesis could promote xylose metabolism (Zheng et al. 2014), which could facilitate the expression of ACCase to induce the synthesis of fatty acids (Song and Pei 2018).
By comparing the data obtained by the experiment, the highest lipid percentage (41%) was received faster under heterotrophic condition (5 day) than mixotrophic condition (8 day), which suggested that in order to obtain lipid, microalgae was cultured more economically under heterotrophic conditions. We could get the same content of lipid in a short period of cycle. In these two cultural modes, the energy supplied by light and/or xylose was assimilated by Scenedesmus quadricauda and then converted to ATP for various energy requirements within cells. Yang et al. (2000) certified that the ATP production rate under mixotrophic and heterotrophic conditions were 12% and 18% respectively, this could explain the cause of this interesting phenomenon. In heterotrophic culture, due to the conversion capacity to ATP (18%) and the transforming factor of xylose to oil (0.92 g/g) were both higher than that of mixotrophic culture, these factors had jointly caused faster lipid accumulation under heterotrophic condition.
3.5 Implications of this work
It was reported that the discharge of papermaking wastewater would cause certain pollution to the environment (An et al. 2022), while there was a lot of lignocellulosic biomass such as xylose in the waste liquid of papermaking, and the yield of xylose could reach 21.91–57.15 g/L (Shi et al. 2021) by using the existing technology. The cost of extracting bio-oil from lignocellulosic biomass was about $47/ton (Huang et al. 2013). According to the highest oil yield obtained in this experiment (274.97 mg/L), it consumed about 14.55 tons of xylose to synthesize one ton of bio-oil, so the cost of producing one ton of bio-oil was about $1100-$ 2860, which had a great advantage in price than employing glucose ($3400/ton) to produce oil (Singh et al. 2022). Therefore, this study could not only economically utilize xylose in papermaking wastewater, but also produced microalgae oil, which had double economic benefits.