The growth and lipid accumulation of Scenedesmus quadricauda under nitrogen starvation stress during xylose mixotrophic/heterotrophic cultivation

In order to conquer the block of high cost and low yields which limit to realize the commercialization of microalgal biodiesel, the mixotrophic and heterotrophic cultivation of Scenedesmus quadricauda FACHB-1297 fed on xylose was separately studied employing six forms of media: phosphorus sufficient, phosphorus restricted, and phosphorus starvation were combined with nitrogen sufficient and nitrogen starvation conditions. The maximum lipid content (about 41% of dry weight) was obtained on the 5th day (heterotrophic cultivation) and 8th day (mixotrophic cultivation) under the nitrogen starved and phosphorus sufficient (N0&P) conditions, which was about twofold in comparison to the final lipid content on the sufficient nitrogen condition (control). Under mixotrophic and heterotrophic modes, the highest lipid production was achieved in the N0&P trial, with the value of 274.96 mg/L and 193.77 mg/L, respectively. Xylose utilization rate of 30–96% under heterotrophic modes was apparently higher than that of 20–50% in mixotrophic modes. In contrast, phosphorus uptake rate of 100% under mixotrophic cultivation was significantly more than that of 60–90% in heterotrophic cultivation. Furthermore, under the condition of heterotrophic cultivation using xylose as a carbon source, the phosphorus had a positive impact on microalgae cell synthesis and the lipid content enhanced with the augmentation in phosphorus concentrations. We suggested that sufficient phosphorus should be supplied for obtaining higher microalgal lipid production in the lack of nitrogen under xylose heterotrophic/mixotrophic condition. This was a highly effective way to obtain efficient microalgae lipid production.


Introduction
The deteriorating living environment and the fossil energy crisis on the verge of exhaustion are the two most serious and urgent problems confronting the world today (Rosli et al. 2020;Peter et al. 2022).It is urgent for humanity to accelerate the search for economic and environmental protection methods for waste recycling.In the meantime, studies are still required to generate renewable new energy.Compared to other biodiesel feedstocks, microalgae has become the focus of current attention due to its superior properties, such as its rich lipid content, ease of cultivation, large yield per unit area, and not competing with agriculture for land (Yin et al. 2020;Rosli et al. 2020;Chu et al. 2021;Siddiki et al. 2022).

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At present, the high cost and low energy conversion rate of microalgae lipid production limit its large-scale industrial production.Consequently, how to enhance the biomass and lipid productivity and lessen the cost has become the focus of current research (Marangon et al. 2021;Chhandama et al. 2021).Microalgae fed with wastewater is a promising approach in the field of environmental protection and energy development.Due to the limitation of light, temperature, and other factors (Brennan and Owende 2010), it was hard to realize the full-scale culture of microalgae only by the autotrophic culture (Perez-Garcia et al. 2011;Castillo et al. 2021).The autotrophic mode of microalgae converts light energy into biomass through photosynthesis.As the culture progresses, a biofilm will form on the surface of the microalgae to reduce light transmittance and influence the development of microalgae.The heterotrophic culture of microalgae utilizes an external organic carbon source to synthesize biomass in the absence of light, which can eliminate the influence of light.Mixotrophic cultivation is comparable to associating heterotrophic with autotrophic cultures, and the interaction of these two processes will likewise enhance the multiplication of microalgae.According to reports, the biomass and oil yield of autotrophic culture were both lower than those of heterotrophic and mixotrophic modes (Miao and Wu 2004;Pang et al. 2019;Gomaa and Ali 2021).But in fact, the consumption of organic carbon source took up approximately 80% of the general culture medium consumption (Katiyar et al. 2017;Liang 2013).To reduce the expenditure of high organic carbon, it is imminent to search for a cheap substitute for carbon sources.
Most of the studies were concentrated on the impact of organic carbon sources such as glucose (Shen et al. 2020;Ray et al. 2021), acetate (Arora et al. 2020), glycerol (Rzechonek et al. 2019), galactose (Suwal et al. 2019) on the growth and lipid accumulation of algae.Ma et al. (2022) reported that the disadvantages of the carbon source mentioned above were higher price (glucose, acetate) and less effective in promoting cell growth (glycerol, galactose).In recent years, the utilization of lignocellulose has become the focus of researchers' attention.Xylose, as a product of lignocellulosic hydrolysis, is not only a renewable resource, but also has great promising development in the manufacture of chemicals and fuels.Studies have shown that pulp and paper wastewater contained 70% xylose and xylan (Wang et al. 2014).As a hemicellulose component of pulp and paper waste liquid, it was a kind of pentose, which was proven to have toxic effects on some microalgae (Giuliano Dragone 2022).Song and Pei (2018) have reported that Scenedesmus quadricauda FACHB-1297 had the ability to utilize xylose for mixotrophic and heterotrophic growth in xylose-rich papermaking wastewater.Xylose could promote lipid synthesis by microalgae in a short period (6 to 12 h) (Leite et al. 2016).In addition, xylose can be taken from papermaking wastewater, which is more economical and environmentally friendly, thereby reducing costs and increasing lipid productivity.
Many studies have shown that nitrogen deficiency could promote lipid accumulation in algae, for example, Chlorella vulgaris, Chlorella zofingiensis, Neochloris oleoabundans, and Scenedesmus obliquus, with lipid content exceeding in 35% of their dry weight under nitrogen stress situation proved by Breuer et al. (2012).Morales-Sánchez et al. (2013) discovered that the lipid content of Neochloris oleoabundans added up to 28% in the absence of nitrogen.Shen et al. (2015) investigated that Chlorella vulgaris NIES-227 nourished with glucose obtained a large yield of fatty acids and a high percentage of fatty acid methyl ester under heterotrophic condition with nitrogen deficiency.Another research showed that after 20 days, both lipid and carbohydrate content of the chosen microalgae in the effluent with deprived nitrogen enhanced about 2 and 1.5-fold, respectively, than those under nitrogen sufficient condition (Nagappan and Kumar 2021).In summary, many studies have demonstrated the feasibility of using nitrogen stress to increase the lipid content of microalgae.However, the characteristics of Scenedesmus quadricauda under different nitrogen and phosphorus content have not been studied using xylose as a carbon source.
In our previous work, it had been proved that Scenedesmus quadricauda could tolerate xylose and the optimum concentration of xylose for the lipid synthesis was 4 g/L, which proved that Scenedesmus quadricauda was suitable for further research to use waste streams for biofuel production.In our research, the dominant energy microalgae (Scenedesmus quadricauda FACHB-1297) was chosen as the research object and xylose was selected as the organic carbon source for the development of Scenedesmus quadricauda.In order to probe the lipid accumulation of this dominant algae species under nitrogen starvation stress, six forms of media: phosphorus sufficient, phosphorus-restricted, and phosphorus starvation combined with nitrogen sufficient and nitrogen starvation conditions in mixotrophic/heterotrophic cultures were set up.The removal properties of xylose, nitrogen and phosphorus, and lipid accumulation properties of Scenedesmus quadricauda FACHB-1297 in six medias were determined.

Species and medium
The superior energy microalgal species Scenedesmus quadricauda (FACHB-1297) applied during the experiment was purchased from Freshwater Algae Culture Collection at the Institute of Hydrobiology, China (FACHB-collection, China).
Xylose with 4 g/L was selected as the external organic carbon source; the BG-11 medium-deprived nitrogen and phosphorus component were prepared for the basic medium.The growth stages of microalgae mainly include adaptation stage, exponential growth phase, stable stage, and decline stage.The seed liquid in logarithmic stage was selected in the experiment (the initial dry weight of microalgae was 0.4-0.5 g/L).After centrifugation, the microalgae liquid was mixed together with the culture medium, which was placed in the light incubator.In order to avoid contamination of microalgae cultivation, the operations of our sampling were performed on ultra-net platforms, and the culture medium used for microalgae was sterilized.The sample was microscopically examined daily to see if it was contaminated with bacteria.The culture temperature was 25 ± 1℃, the light intensity was 3000 l × for 12 h light and 12 h dark photoperiod, and one culture cycle was set as 8 days.Each set of tests was in triplicate.

Experimental scheme
Six medias of enough nitrogen and enough phosphorus (N&P), sufficient nitrogen and inadequate phosphorus (N&P − ), enough nitrogen and phosphorus starved (N&P 0 ), nitrogenstarved and enough phosphorus (N 0 &P), nitrogen-starved and insufficient phosphorus (N 0 &P − ), and simultaneously nitrogen and phosphorus-starved (N 0 &P 0 ) (other nutrients were the same as BG11) were applied during this experiment (Table 1).Algal inoculum was centrifugally suspended in the above six cultures.The dry weight, pH, TN, TP, and xylose concentrations were measured every 2 days.The lipid content was measured on the days of 0, 2, 5, and 8.

Growth measurement of Scenedesmus quadricauda
The Scenedesmus quadricauda culture solution was extracted every 2 days for biomass concentration as reported in Song et al. (2013): algal liquid was filtered through 0.45-μm filter membranes that have been dried to constant weight, and then the filter membranes with algal mud were baked in an oven at 50℃ to constant weight to determine the increase of biomass concentration (mg/L).
The specific growth rate (K) of microalgae in exponential stage was figured up as the formula below (Pandey et al. 2020): where C 1 and C 2 (mg/L) represented the biomass concentration on the day T 1 and T 2 .
Biomass productivity during the exponential period of microalgal growth was acquired through the below equation (Song et al. 2013): where DM represented the biomass concentration (mg/L) in the last stage of the exponential period.

Lipid determination of Scenedesmus quadricauda
The total lipid content was measured and analyzed by the chloroform/methanol extraction method (Chiu and Kuo 2020).Firstly, about 0.1 g of algal powder was transferred to the mixture of chloroform and methanol (v:v of 2:1) for ultrasonic crushing for 10 min.After the concentration at 4000 rpm for 10 min, the supernatant was extracted for the phase separation with the addition of 0.9% sodium chloride solution (1:5 by volume).The mixture was stated for 15 min after homogeneous mixing.Then the lower phase liquid sampled in the 10 mL glass tube was dried with a nitrogen blower and weighed according to the formula below to calculate the lipid content: where LC was the lipid content according to the dry weight, m 2 was the dry weight of the algae powder, m 1 was the dry weight of 10 mL glass tube, m 3 was the dry weight of 10-mL tube and the total lipids, and v was the overall bulk of the low phase liquid.
The lipid productivity (PL) was computed through the below equation (Song et al. 2013): where PL represented the lipid productivity during the exponential growth stage.LC was the lipid content according to the dry weight.

Removal of total nitrogen and total phosphorus
Before the determination of total nitrogen (TN) and total phosphorus (TP), microalgal samples were centrifuged at 4000 rpm for 10 min (4℃), and the supernatant liquor was collected for TN and TP detection after the 0.45-µm filtration.TN was (1) determined by peroxide potassium sulfate-ultraviolet spectrophotometry (GB11894-89).TP was determined by potassium persulfate oxidation-molybdenum antimony anti-spectrophotometry (GB11893-89) (Han et al. 2014).

Xylose concentration analysis
The DNS solution was added to the separated supernatant after the centrifugation of algal sample and boiled at 90℃ for 15 min in a water bath (Miller 1959;Fetyan et al. 2022).Then the system was cooled down quickly.The absorbance of the xylose at 630 nm was received using an UV-visible spectrophotometer (TU-1810PC, Persee, Beijing) and in comparison with the calibration curve made in the same modes (Leite et al. 2015).

Calculation of conversion coefficient of xylose
The biomass yield coefficient from xylose (Y B/X ), the lipid yield coefficient from xylose (Y L/X ), and the coefficient by a unit (g) of nitrogen consumption to a unit (g) of xylose intake (Y N/X ) were received on the basis of the equation below (Freitas et al. 2017) of ( 4)-( 6): where B 0 , L 0 , N 0 , and X 0 stood for the original values of biomass, lipid, nitrogen, and xylose content, successively.B max and L max stood for the maximal values of the biomass and lipid content, successively.N f and X f represented the end values of nitrogen and xylose content, successively.Under mixotrophic mode, ΔB ctrl , ΔL ctrl , and ΔN ctrl were the increment of biomass content, the increase in lipid accumulation, and the consumption of nitrogen content in the control group without xylose in the autotrophic culture, successively.As for heterotrophic mode, the group without xylose in the dark was chosen as the control group.

Statistical analysis
The statistical analysis was carried out using the SPSS 25.0 software, and the values with difference between treatments (ANOVA) were indicated in the shape of P < 0.05. (5)

Dry cell weight in mixotrophic/heterotrophic growth under six medias with different nitrogen and phosphorus
For assessing the mixotrophic and heterotrophic growth characters of Scenedesmus quadricauda under six different medias, the effect of diverse nitrogen and phosphorus concentrations on the development characteristics of the microalge in mixotrophic and heterotrophic culture was determined (Fig. 1). Figure 1a and b manifest that Scenedesmus quadricauda under six different medias entered the logarithmic growth phase after 2 days of adaptive phase, which was consistent with the research of Song and Pei (2018).In the conditions of mixotrophic modes, the biomass productivity under different nitrogen and phosphorus concentrations was 137.45 mg/L/d (N&P), 95.03 mg/L/d (N&P − ), 83.45 mg/L/d (N&P 0 ), 83.83 mg/L/d (N 0 &P), 80.04 mg/L/d (N 0 &P − ), and 75.01 mg/L/d (N 0 &P 0 ) respectively (Fig. 1).The highest biomass productivity obtained under N&P group was 1.8-fold of the N 0 &P 0 group, which was accorded with the maximum value of dry cell weight (1.10 g/L) and the highest specific growth rate (0.14 day −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 1d and e show that under heterotrophic cultivation, the highest dry cell weight (0.60 g/L) and specific growth rate (0.13 day −1 ) for Scenedesmus quadricauda were got under N&P mode.All of these values were lower than those under N&P mode obtained in mixotrophic cutivation, this phenomenon could be explained by the report of Manhaeghe et al. (2020), and 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/day) was not only more than the value of 61.11 mg/L/day got in the dark, but also more than that of 37.8 mg/L/day got by the Chlorella (PCH90) fed on xylose in mixotrophic condition (Leite et al. 2016).The reason that Scenedesmus quadricauda grew best under the mixotrophic conditions with nitrogen and phosphorus abundant was that in the mixotrophic culture, nitrogen and phosphorus were involved in photosynthesis to synthesis biomass, and carbon sources could provide energy in addition to light and be synchronously absorbed for ATP yield (Pang and Chen 2017;Nigam et al. 2022).
Figure 1 show 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 enter the microalgae cells (Portillo et al. 2022).The endocellular xylose afterwards entranced the pentose phosphate pathway that 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;Patel et al.2021).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.

TN, TP, and xylose changes under six medias in light sufficient/absent
As shown in Fig. 2, the removal rate of TN and xylose was a bit slower; nevertheless, the removal rate of TP was faster, especially in the first 2 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 to 0 mg/L in 8 days (100%), and these of phosphorus restrictive groups likewise lessened from 4 to 0 mg/L in 8 days (100%).However, the assimilation efficiency of TP in heterotrophic mode was not as high as in mixotrophic condition, which was 60% in 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;Xing et al. 2022).
Whether light was sufficient or absent, the removal rate of TN decreased with decreasing phosphorus concentration in the N&P, N&P − , and N&P 0 groups, which was also consistent with the variations 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&P 0 group, successively.In the heterotrophic mode, the removal rates of TN achieved at 99% in the N&P trial, followed by N&P − of 95% and N&P 0 of 74%, which were much higher than those achieved in mixotrophic mode with the value of 43%, 24%, and 21% under N&P, N&P − , and N&P 0 conditions, successively.The xylose removal rate of 96% under heterotrophic mode was also better than that under mixotrophic conditions.Nevertheless, the biomass of heterotrophic trials was lower than those of mixtrophic modes, the absorption of xylose might promote the absorption of

Lipid accumulation in mixotrophic/heterotrophic growth under six medias
Recent studies of lipid accumulation in microalgae under different conditions were shown in Table 2. Most of these studies focused on stimulating lipid production by altering photosynthetic carbon flow under environmental stress, which included nitrogen stress, salt stress, ultrasonic stress, adding plant hormones and so on (Xie et al. 2020).It could be seen from Table 2 that the lipid productivity of 34.4 mg/L in this study was comparable to most of studies.
When Scenedesmus quadricauda was cultured under nitrogen-deficient conditions, the maximum lipid percentage (about 41%) was obtained on the 5th day (heterotrophic cultivation) and the 8th day (mixotrophic cultivation) under the nitrogen starved and phosphorus sufficient (N 0 &P) condition, which was about twofold to the initial lipid content under nitrogen-sufficient condition (control) (Fig. 3).The highest lipid production also achieved at the 8th day with 274.96 mg/L in mixotrophic culture and the 5th day with 193.77 mg/L in heterotrophic culture under N 0 &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 3b shows 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 N 0 &P condition (274.96mg/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 Yu et al. (2022) that the lipid production and the growth of the microalgae in mixotrophic mode were both better than that under 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 was received in nitrogen starved media.The general content of lipid in microalgae perhaps was on a scale of 20-50% (Zhu et al. 2022), 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 lipid content (37%) of Chlorella vulgaris SDEC-3 M 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;Wei et al. 2022).In addition, 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 lipid accumulation (Farooq et al. 2022).To sum up (according to Fig. 4), the conditions of xylose and nitrogen stress co-promoted fatty synthesis.One of the reasons was nitrogen restriction increased the content of fatty acid acetyl-CoA of the microalgae (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 Fig. 4 The mechanism of the effect of xylose assimilation and nitrogen starvation on the lipid accumulation of Scenedesmus quadricauda FACHB-1297 in mixotrophic/heterotrophic cultivation microalgae, the substance (xylulose-5-phosphat) involved in the regulation of lipogenesis genes synthesis was also produced, which also stimulated more lipid 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 content reduced from 41 to 20% with the phosphorus content decreased under nitrogen deficiency in mixotrophic cultivation on the eighth day; the similar pattern was discovered under heterotrophic conditions (with the decrease of phosphorus content, the lipid percentage decreased from 35 to 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;Xing et al. 2022;Zahed et al. 2022).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 shortage, the increase in phosphorus content led to an increase in Poly-P, which promoted more energy material for lipid accumulation (Chu et al. 2013).

Relationship between xylose conversion factor and lipid accumulation
For further research of the relationship between the xylose assimilation, lipid production, nitrogen removal and development characteristics, the Y L/X , Y N/X , and Y B/X were calculated (Table 3).The highest transfer coefficient of xylose to the lipid (Y L/X ) under mixotrophic cultivation got in N 0 &P trial was 0.53 g/g, which was 7.5 times more than the conversion factor of 0.07 g/g in N 0 &P 0 trial, and this maximum value was also 1.2 times higher than the factor of 0.43 g/g in the light under N&P group.This indicated that nitrogen deficiency played an important factor in promoting lipid accumulation.This result was consistent with the report of Breuer et al. (2012) that the microalgae of Chlorella vulgaris, Chlorella zofingiensis, Neochloris oleoabundans, and Scenedesmus obliquus synthesized triglycerides over 35% of dry weight under nitrogen starvation condition.Similar result was obtained under heterotrophic condition; the highest Y L/X (0.55 g/g) was acquired in N 0 &P group as well.
According to Table 3, it could be seen that under mixotrophic cultivation the maximum values of Y L/X (0.53 g/g) and Y N/X (0.24 g/g) were both lower than Y L/X (0.55 g/g) and Y N/X (0.27 g/g) under a heterotrophic condition, but the value of Y B/X (0.73 g/g) under heterotrophic mode was lower than Y B/X (0.85 g/g) under a 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 convert xylose to lipid.Xylose assimilation accelerated the expression of acetyl coenzyme A carboxylase (ACCase), which could stimulate lipid accumulation by converting acetyl coenzyme A (Acyl-CoA) to malonyl coenzyme A (Mayl-CoA) (Song and Pei 2018).In the dark, microalgae only relied on xylose as a carbon source, while under light conditions, microalgae still obtained energy through photosynthesis.As a result, the conversion factor of xylose into lipid was higher in the dark than in the light.But in fact, in terms of total lipid production, microalgae accumulated more lipid and biomass under light, owing to the energy and the coenzyme (NADPH) produced by photosynthesis could promote xylose metabolism (Portillo et al. 2022), 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 content (41%) was received faster under heterotrophic condition (Day 5) than that under mixotrophic condition (Day 8), which suggested that in order to obtain lipid, microalgae was cultured more economically under heterotrophic conditions.We could get the same lipid content in a short period of time.In both cultural modes, the energy supplied by light and xylose was assimilated by Scenedesmus quadricauda and then converted into ATP for various energy requirements within cells.Yang et al. (2000) certified that the ATP production rate under mixotrophic and heterotrophic conditions was 12% and 18%, respectively.This could explain the cause of this interesting phenomenon.As the production rate of ATP (18%) and the transforming factor of xylose to lipid (0.55 g/g) in heterotrophic culture were both higher than those under mixotrophic culture with the ATP production rate of 12% and conversion factor of xylose to lipid of 0.53 g/g, the lipid content under heterotrophic condition accumulated faster than that under mixotrophic culture.

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 (Shi et al. 2021).The cost of extracting bio-oil from lignocellulosic biomass was about $47.10/ton and each ton of lignocellulose contains 0.45 ton of total sugar (70% of total sugar was xylose) (Huang et al. 2013;Song and Pei 2018).Consequently, the cost of lipid production from xylose can be calculated to be $149.78/ton.According to the highest Y L/X (0.55 g/g), it took 1.82 tons of xylose to produce 1 ton of biodiesel.It can be calculated that it costs $272.60 to produce one ton of lipid from xylose by regulating nitrogen and phosphorus content in this study, which had a great advantage in price than employing glucose ($3400/ton) to produce lipid (Singh et al. 2022).Therefore, this study can not only economically utilize xylose in papermaking wastewater, but also produce microalgae biodiesel, which has double economic benefits.

Conclusion
This research indicated that nitrogen starvation with mixotrophic/heterotrophic conditions fed on xylose seemed to be a promising way for Scenedesmus quadricauda to accumulate lipid.The maximum lipid content (41%) was obtained in the N 0 &P group on the fifth day of heterotrophic and the eighth day of mixotrophic culture, respectively.In terms of lipid production, the mixotrophic culture was more dominant, with a value of 274.96 mg/L, compared to 193.77 mg/L in the heterotrophic mode.But heterotrophic condition could shorten the culture cycle to achieve the highest lipid content (41%).The maximum absorption rate of xylose was 96% (in heterotrophic mode), and the maximum phosphorus removal rate was 100% (in mixotrophic mode).Phosphorus played a critical part in increasing the lipid synthesis of Scenedesmus quadricauda under the nitrogen depletion condition.As the phosphorus content increased, the lipid content increased by 21% in mixotrophic and 19% in heterotrophic conditions.
This study demonstrated the large perspectives of Scenedesmus quadricauda to efficiently produce algal lipids in heterotrophic/mixotrophic culture using xylose in wastewater combined with nitrogen stress.

Fig. 1
Fig. 1 The variations in a dry cell weight (DW), b specific growth rate (K), c biomass productivity of Scenedesmus quadricauda FACHB-1297 under different medias in mixotrophic cultivation, and

Fig. 3
Fig. 3 The variations of lipid content (the dotted bar) and lipid production (the dotted line) of Scenedesmus quadricauda FACHB-1297 under six treatments during 8-day cultivation in a mixotrophic cultivation and b heterotrophic cultivation

Table 1
Initial concentrations of nitrate and phosphate in the six medias

Table 2
Comparison of studies on lipid accumulation under different culture conditions of microalgae