Simultaneous Treatment of Swine and Furfural Wastewater Integrated with Lipid Production of Chlorella pyrenoidosa

Abstract

Adding organic compounds to wastewater can improve the carbon/nitrogen ratio and benefit microalgae growth. We studied microalgal growth, nutrient removal and lipid accumulation of Chlorella pyrenoidosa cultured in a mixture of swine wastewater (SW) and furfural wastewater (FW). The mix ratio of SW:DFW (diluted furfural wastewater) had a significant effect on microalgae growth. As the mix ratio of SW:DFW decreased from 1:0.5 to 1:19, the maximum microalgal biomass increased, while the specific growth rate initially increased and then decreased. The efficiency of nutrient removal also depended on the mix ratio of wastewater. The highest chemical oxygen demand (COD) removal efficiency (57.30%) occurred at the mix ratio of SW:DFW = 1:3. The highest removal efficiencies of total phosphorous (TP) reached 61.93% when the mix ratio of SW:DFW was 1:9. Wastewater at the mix ratio of SW:DFW = 1:19 had a maximum lipid productivity of 49.48 mg L⁻¹ d⁻¹, which was 4.9 times higher than that at a mix ratio of SW:DFW = 1:0.5. These results showed that C. pyrenoidosa can be used to remove nutrients from mixed wastewater sources and simultaneously produce algal lipids.

1. Introduction

Vast amounts of industrial and agricultural wastewater are annually generated and discharged worldwide [1]. To reduce environmental pollution, effective wastewater treatment must be completed before wastewater discharge. For wastewater treatment, conventional physicochemical approaches (screening, floatation and oxidation) and biological methods (activated sludge, biological filters and constructed wetlands) are used [2]. Microalgae-based wastewater treatment has attracted attention because it is a high efficiency, low cost, sludge-free process, with substantial nutrient recovery and recycling [1]. Microalgae cultured in wastewater can achieve wastewater purification and also transform the nutrients in wastewater into microalgal biomass and bio-products such as lipids, polysaccharides and carotenoids [3]. The use of wastewater as a culture medium can reduce the cost of microalgae production, which is important for expanding the scale of microalgae application [4].

Swine wastewater has high chemical oxygen demand (COD), total nitrogen (TN), ammonia nitrogen (NH₃-N) and total phosphorus (TP) [5]. Many studies have been conducted on swine wastewater treatment using phycoremediation [6,7,8]. Most of these studies showed effective microalgal growth and nutrient removal [9]. Likewise, many types of industrial wastewater, such as brewery, textile, pharmaceutical and pesticide wastewaters, have been used to culture microalgae and these have resulted in high removal efficiencies of contaminants [2]. Many microalgae strains are able to acclimatize to wastewater environments and can tolerate high levels of harmful pollutants [10,11].

For microalgae grown in wastewater, the mixotrophic growth mode is involved since most wastewater has organic matter [12,13]. However, several common types of wastewater, such as swine, municipal and domestic wastewater, have high nitrogen concentration, thus leading to the low C/N ratio (carbon to nitrogen source ratio) which is not beneficial to the mixotrophic growth of microalgal cells [13]. Therefore, the addition of an exogenous organic carbon source to wastewater has been proposed to enhance the mixotrophic growth of microalgae. This can increase the growth rate of microalgae compared to photoautotrophic cultivation [7]. In addition, microalgae mixotrophically grown in wastewater with addition of organic matter had a greatly increased nutrient removal rate and efficiency [14]. Liu et al. [15] showed that the addition of sodium acetate significantly stimulated algal growth and also increased lipid productivity when Scenedesmus obliquus was cultured in municipal wastewater.

Several kinds of organic compounds are potential carbon sources for algal mixotrophic culture in wastewater. These include glucose, glycerol and sodium acetate [2,13]. However, the addition of these compounds increases the treatment cost and is not cost-efficient and sustainable [16]. The use of an organic carbon source from wastewater has been considered as an option. Tan et al. [13] reported that enhancements of lipid productivity and nutrient removal were achieved by adding acidified starch wastewater to the mixotrophic culture of Chlorella pyrenoidosa. There are several other types of wastewater that contain organic acids. Furfural wastewater (FW) is a highly acidic wastewater generated by depolymerizing lignocellulose crop stalks with sulfuric acid at high temperature [17]. Large amounts (4–6 million tons annually) of furfural wastewater are produced in China [17], and this furfural wastewater typically requires costly treatment.

Wastewater treatment using microalgae integrated with biodiesel production is an environmentally friendly and cost effective way to achieve resource recovery and recycling [4,5,18,19]. Oleaginous microalgae cultured in wastewater can purify wastewater and also reduce the cost of microalgal lipid production [20]. Many studies have evaluated microalgae-based wastewater treatment integrated with algal lipid production. These have focused on microalgae species screening [21], wastewater type testing [18,22], culture process optimization [23], trophic mode comparison [24] and lipid composition analysis [13]. For example, Nguyen et al. [22] found that the initial biomass concentration significantly influenced lipid accumulation when Chlorella vulgaris was cultivated using seafood wastewater effluent. Liu et al. [24] confirmed that simultaneously adding optimal amounts of iron and acetate to municipal wastewater increased the biomass concentration of Chlorella pyrenoidosa, lipid content and lipid productivity, respectively, by 185%, 248% and 671%. These results indicated that iron and acetate supplements can greatly improve lipid productivity. C. pyrenoidosa is often used for wastewater treatment, such as swine wastewater, municipal and industrial wastewater [24,25,26]. Moreover, several studies demonstrated that C. pyrenoidosa has a strong capability to remove the COD, TN, NH₃-N and TP from wastewater, and simultaneously accumulate a large amount of lipid [27,28].

In the present study, we mixotrophically cultivated C. pyrenoidosa in a mixture of swine wastewater (SW) and diluted furfural wastewater (DFW) to evaluate the feasibility of simultaneous treatment of swine and furfural wastewater combined with microalgal lipid production. Microalgae were cultivated in wastewater with different mix ratios of swine to furfural wastewater, and microalgal growth, nutrient removal and lipid accumulation were evaluated.

2. Materials and Methods

2.1. Microalgal Strain

C. pyrenoidosa were used to simultaneously treat swine and furfural wastewater. C. pyrenoidosa was obtained from the Freshwater Algae Culture Collection of the Institute of Hydrobiology, Chinese Academy of Sciences (FACHB collection, Wuhan, China), and maintained with BG-11 medium [6].

2.2. Wastewater Collection and Pretreatment

SW was obtained from a swine farm belonging to Ningbo Mingsheng Agricultural Technology Development Co., Ltd. located at Fenghua District, Ningbo, Zhejing Province, China. FW was collected from a furfural production plant in Puyang, Henan Province, China. Before the experiments, the furfural and swine wastewaters were pretreated by sedimentation for 24 h, and then sediments were removed. The remaining slightly clear wastewater was filtered by filter paper to further remove the particulate solids. The supernatants were stored in a 4 °C refrigerator before being used for microalgae culture.

2.3. Wastewater Treatment by Microalgae Cultivation

Batch cultivation of microalgae was conducted using a mixture of SW and DFW to evaluate microalgal growth, nutrients removal and algal lipid production. Different mixture ratios of the two types of wastewaters were tested. Microalgae seeds for wastewater treatment were prepared in 500 mL flasks using BG-11 medium at 25 °C with a light intensity of 100 μmol m⁻² s⁻¹. The pretreated FW was first diluted 10 times with sterile tap water to decrease its high concentration of COD. Subsequently, the DFW was further mixed with the SW to prepare the wastewater mixture for microalgae cultivation. The different mixture ratios (SW:DFW, v/v) were 1:0.5, 1:3, 1:9 and 1:19. The mixed wastewater was autoclaved at 121 °C for 20 min after the pH was adjusted to 7.1. When the temperature of mixed wastewater was cooled to room temperature, the inoculum was then inoculated into 500 mL flasks containing 200 mL of mixed wastewater with a ratio of inoculum to mixed wastewater 1:10 (v/v). The flasks were placed in a shaker with a rotation speed of 150 rpm to cultivate microalgae for 8 days. Culture temperature and light intensity were controlled at 25 °C and 200 μmol m⁻² s⁻¹. All of the experiments were conducted in triplicate. During the culture process, microalgal biomass, COD, TN and TP were measured daily, while the lipid content of microalgae was measured at the beginning and end of the culture process.

2.4. Analytical Methods

2.4.1. Determination of Microalgal Concentration and Growth Rate

Twenty milliliters of microalgal culture were collected and then centrifuged at 6000× g for 10 min. The precipitate was washed with distilled water once and centrifuged again. The microalgal slurry was transferred to a pre-weighed glass plate and dried at 105 °C in a hot air circulating oven to a constant weight. The weight of the glass plate containing dried microalgae was measured to determine microalgal biomass concentration that indicated by the dry cell weight per volume (X, g L⁻¹),

$$X=\frac{W_{end}-W_0}{V}$$

(1)

where W_end was the weight of the glass plate containing microalgal cells (g), W₀ was the weight of the blank glass plate (g), and V was the volume of microalgal culture for measurement (L).

The algal growth rate (R, g L⁻¹ d⁻¹) and specific growth rate of microalgae (μ, d⁻¹) were calculated by the following equations:

$$R=\frac{X_2-X_1}{t_2-t_1}$$

(2)

$$\mu =\frac{Ln\left(X_2\right)-Ln\left(X_1\right)}{t_2-t_1}$$

(3)

where X₂ (g L⁻¹) and X₁ (g L⁻¹) were the microalgal biomass concentrations at culture times t₂ (d) and t₁ (d), respectively.

2.4.2. Determination of Water Quality Indexes

Microalgal culture and wastewater samples were collected and centrifuged at 9000× g for 5 min, and the supernatants were filtered through 0.45 µm cellulose acetate membranes. The filtered supernatant was subsequently used to determine the concentration of COD, TN and TP. The concentrations of COD and TP were measured using a Water Quality Tester (Lianhua company, Shanghai, China). The TN content was determined by the alkaline potassium persulfate digestion-UV spectrophotometric method [29]. The pH of wastewater was measured using a portable multiparameter analyzer DZB-718 (Shanghai INESA Scientific Instrument Co., Ltd., Shanghai, China). The carbon/nitrogen ratio of the wastewater was calculated by dividing COD by TN. Nutrient removal efficiency (NRE, %) was expressed as

$$NRE=\frac{(C_i-C_f)}{C_i}\cdot 100\%$$

(4)

where C_i (mg L⁻¹) and C_f (mg L⁻¹) were the initial and final concentrations of nutrients in the wastewater, respectively.

The nutrient removal rate (NRR, mg L⁻¹ h⁻¹) was calculated as follows

$$NRR=\frac{C_i-C_f}{t}$$

(5)

where t was the culture period (8 days).

2.4.3. Determination of Lipid Content

The total lipid content of the microalgae was measured using the improved method described by Bligh and Dyer [30]. Specifically, 50 mL of microalgal culture was sampled and then centrifuged to obtain the microalgal slurry. Then all slurry was mixed with 5 mL chloroform/methanol (1:1, v/v) in the dark for 45 min. The mixture was centrifuged at 4000 rpm for 10 min, the residual biomass was then repeatedly extracted five times, and the supernatant was collected in a rotary evaporation bottle. Subsequently, the rotary evaporation bottle with organic solvent was steamed on the rotary evaporation instrument with a pressure of 0.1 MPa until the organic solvent was completely volatilized. The rotating evaporation bottle containing the light-yellow oil was then placed in an oven at 80 °C to dry to a constant weight. The lipid content of the microalgae (C_Lipid, %) was determined as follows:

$$C_{Lipid}=\frac{(L_2-L_1)}{V_S·X}·100\%,$$

(6)

where L₂ represented the weight of the rotary evaporation bottle containing lipids (g), L₁ was the weight of the blank rotary evaporation bottle (g), V_S represented the sample volume of microalgal culture (L) and X indicated the microalgal biomass concentration (g L⁻¹).

The lipid productivity (LP, mg L⁻¹ d⁻¹) was calculated according to the Equation (7)

$$LP=\frac{X_f\cdot C_{Lipid\_f}-X_i{C}_{Lipid\_i}}{t}\cdot 1000$$

(7)

where X_f and X_i were the final and initial microalgal biomass concentrations of culture experiment (g L⁻¹), respectively. C_{Lipid_f} and C_{Lipid_i} were lipid contents of the microalgal biomass obtained at the end of the culture period and the inoculum (%), respectively. t was the culture period (8 days).

2.4.4. Statistical Analysis

Analysis of variance (ANOVA) and LSD post hoc test were implemented via SPSS software (ver. 19.0). A confidence level of 95% was selected to determine the significance of treatment differences. There was a statistically significant difference when p < 0.05. The experimental values were expressed as the mean ± standard deviation (SD).

3. Results and Discussion

3.1. Composition Analysis of Swine and Furfural Wastewater

Before microalgal cells were grown in the mixed wastewater, the essential parameters of the stock wastewater were measured (

Table 1. Physicochemical characteristics of swine wastewater (SW), furfural wastewater (FW), and the mixed wastewater at different ratios of SW to DFW. Data are shown as mean ± SD, n = 3.

Parameters	Swine Wastewater (SW)	Furfural Wastewater (FW)	SW:DFW a = 1:0.5	SW:DFW = 1:3	SW:DFW = 1:9	SW:DFW = 1:19
COD (mg L−1)	4316.40 ± 396.56	50,978.67 ± 544.95	4394.00 ± 207.12	5026.01 ± 65.91	5056.67 ± 42.34	4996.33 ± 21.91
TN (mg L−1)	425.41 ± 17.82	33.47 ± 3.52	249.55 ± 11.73	147.56 ± 4.71	63.73 ± 2.34	24.92 ± 1.67
TP (mg L−1)	14.18 ± 1.56	1.01 ± 0.04	8.88 ± 0.06	3.68 ± 0.03	1.52 ± 0.01	1.00 ± 0.01
pH	6.52 ± 0.12	3.10 ± 0.51	7.1 ± 0.20	7.1 ± 0.21	7.1 ± 0.18	7.1 ± 0.20
COD/TN ratio	10 ± 1	1540 ± 157	18 ± 1	34 ± 3	79 ± 5	202 ± 20

^a DFW means the diluted furfural wastewater. The furfural wastewater was diluted by 10 times with water to obtain the DFW.

). The concentrations of COD, TN and TP in SW were 4316.40, 425.41 and 14.18 mg L⁻¹, respectively. The FW had a high COD concentration, reaching 50,978.67 mg L⁻¹. This was approximately 12 times higher than the COD level in the SW. However, the concentrations of TN and TP in the furfural wastewater were 33.47 and 1.01 mg L⁻¹, which were lower than those in the SW. The pH value of the FW was only 3.1 because it contained many organic acid substances such as acetic acid [17]. The FW was not suitable to directly culture microalgae not only because of its excessive COD concentration, but also because of its low pH value.

However, it was possible to obtain appropriate concentrations of nutrients for microalgae growth by mixing the two types of wastewater. To achieve the appropriate TN and TP concentration of wastewater, the FW and SW were mixed together for microalgae cultivation. The physicochemical characteristics of the mixed wastewater of SW and FW at different mixing ratios are shown in Table 1. With the increase of the proportion of FW, the concentrations of TN and TP decreased, showing more characteristics of FW. The COD/TN ratio of mixed wastewater increased with the decrease in the SW:DFW ratio, which was beneficial to the microalgae growth in high-strength ammonium wastewater [25]. An increase of carbon/nitrogen can eliminate the ammonia toxicity of SW. Zheng et al. [16] found that the cell growth and viability of C. vulgaris were significantly improved when the carbon/nitrogen ration increased from 17:20 to 5:1, 25:1 and 125:1 by addition of glycerol into manure-free piggery wastewater with an initial ammonium concentration of 220 mg L⁻¹ that was harmful to cells and inhibited microalgal growth. The possible reason was that the enhancement of carbon/nitrogen ratios promoted cell division and stimulated the absorption of ammonia, which facilitated the growth of cells and mitigated the ammonia toxicity. Lu et al. [31] also observed that the addition of exogenous alpha-ketoglutarate, citric acid and glucose to high strength ammonia wastewater greatly promoted ammonia assimilation and cell growth of Chlorella sp. Compared to alpha-ketoglutarate and citric acid, glucose was a better carbon source because it generated more energy and hydride donors that are critical to ammonia assimilation. This was an efficient way to alleviate ammonia toxicity in wastewater, accelerating ammonia assimilation by adding a carbon source for algae cultivation.

An appropriate nutrient environment is an essential prerequisite for microalgae growth. Excessive concentrations of nutrients in wastewater can inhibit microalgal cells growth [26], while deficient concentrations are unable to support long-term growth of microalgae, leading to a low removal efficiency of nutrients from the wastewater [7].

3.2. Growth Performance of Microalgae Cultured in the Mixed Wastewater

Figure 1 shows that the growth performance of C. pyrenoidosa varied greatly at different mix ratios of SW to DFW. Microalgae grew poorly at the mix ratio of SW:DFW = 1:0.5, which was possibly due to the high concentration of TN (249.55 mg L⁻¹). Ammonia nitrogen is toxic to microalgal cells and can inhibit microalgae growth [26]. In this study, although the ammonia nitrogen concentration of the mixed wastewater was not measured, it is probable that the mixed wastewater had a high ammonia nitrogen concentration. This is because the nitrogen in the mixed wastewater was mainly sourced from the SW, which had a large proportion of ammonia nitrogen [16].

Microalgae grew well in the mixed wastewater at the other mix ratios. In general, microalgal biomass concentration increased rapidly, after cells acclimatized to the environment of the mixed wastewater, until to a maximum point and then slightly decreased, or were maintained at a stable level. The maximum biomass concentrations at mixing ratios of SW:DFW = 1:3, 1:9 and 1:19 were 0.87, 0.93 and 0.99 g L⁻¹, respectively, which were approximately three times higher than at mixing ratios of SW:DFW = 1:0.5. The specific growth rate strongly decreased when the mix ratio of SW:DFW was 1:0.5. In comparison with the other experimental groups, the TN concentration at the mix ratio of SW:DFW = 1:0.5 was much higher. Thus, the low specific growth rate may be caused by the high concentration of ammonia nitrogen in the mixed wastewater due to its toxicity to the microalgae cells [16,26,31].

The increased proportion of FW in the mixed wastewater can promote the growth of microalgae because FW contains organic acids. The addition of organic matter into wastewater in a certain range is beneficial to the mixotrophic growth of microalgae [15,32]. However, we observed no significant differences in the biomass concentration and algal growth rate at the mix ratios of SW:DFW = 1:9 and 1:19. The specific growth rate of microalgae was even decreased slightly with an increase of FW proportion in the mixed wastewater, suggesting that the further increase in organic matter in the mixed wastewater cannot further promote microalgae growth. A high carbon/nitrogen ratio can decrease the microalgae growth rate and reduce cell viability due to substrate inhibition [33,34]. In addition, the N/P ratios of wastewaters mix ratios of SW:DFW = 1:0.5, 1:3, 1:9 and 1:19 were 28, 40, 41 and 25, respectively. All these values were greater than the value of Redfield ratio for N/P (16:1), showing that microalgae growth may be subjected to the P limitation. It may be the one of reasons why the microalgae stopped growing during the culture process, and why the final microalgal biomass concentrations of each group are all not greater than 1 g L⁻¹.

3.3. Influence of Mix Ratio of Wastewaters on Nutrient Removal

During the culture process, microalgae were able to assimilate and utilize organic matter, nitrogen and phosphorus in the wastewater to synthesize algal biomass [11]. Therefore, the process of nutrient removal from the mixed wastewater by microalgae cultivation was studied. Variations in the COD, TN and TP concentrations in the mixed wastewaters at different mix ratios of SW to DFW are presented in Figure 2. All of the nutrients in the mixed wastewaters gradually decreased with increased culture time. The reduction degrees and rates were significantly dependent on the mix ratios of SW to DFW. For example, the removal efficiencies of COD in the wastewaters at the mix ratios of SW:DFW = 1:0.5, 1:3, 1:9 and 1:19 were 5.11%, 57.30%, 49.43% and 49.21%, respectively (Figure 3). With an increase in the proportion of FW, the COD removal efficiency initially increased and then slightly decreased. This suggested an optimal mix ratio of 1:3 (SW: DFW).

The COD values of mixed wastewaters at the mix ratios of SW:DFW = 1:3, 1:9 and 1:19 reached a stable level and varied only slightly after 6 d. However, we cannot confirm whether the residual COD are recalcitrant COD or not. The main composition of the remaining COD may be the recalcitrant organic substances that cannot be easily absorbed and utilized by microalgae. Another possibility may be associated with microalgae growth. Microalgae did not constantly grow or even died at the late stage of culture process (Figure 1). Thereby, microalgae cells are not able to efficiently utilize organic substance in the wastewater. Thus, even if the biodegradable matters (COD) existed in the mixed wastewater, microalgal cells would be unable to use them. On possible factor limiting microalgae growth at a late stage is the low gas-liquid mass transfer. With the increase of biomass concentration, microalgal cells need more dissolved oxygen or carbon dioxide to grow mixotrophically [35]. However, the gas-liquid mass transfer coefficient is low for flasks without aeration. Thus, microalgae growth is likely hindered by low concentrations of dissolved oxygen or carbon dioxide. Another reason may be the extremely low TP concentrations in the experimental groups with ratios of SW:DFW = 1:3 and 1:9. The deficiency of TP resulted in the limitation of microalgae growth.

For TN and TP removals, similar tendencies as for COD removal were observed. There was poor removal of TN and TP from the mixed wastewater at the mix ratio of SW:DFW = 1:0.5. The highest removal efficiencies of TN and TP were 33.76% and 61.93%, respectively, both at the mix ratio of SW:DFW = 1:9. The mixed wastewater with a low (18:1) or high (202:1) COD/TN ratio had low removal efficiency of COD, TN and TP (Figure 3). Yan et al. [36] also observed that nutrient removal was relatively low in synthetic domestic sewage when the concentration of the carbon source was low (low COD/TN) or the nitrogen source was insufficient (high COD/TN). Zheng et al. [16] studied the effect of carbon/nitrogen ratio on nutrient removal from manure-free piggery wastewater. They found that a low or high carbon/nitrogen ratio (indicated by TC/TN) restricted nutrient removal due to the inhibition of ammonium or glycerol. In the present study, the low amount of organic carbon added to the mixed wastewater at the mixing ratio of SW:DFW = 1:05 (low COD/TN) possibly limited microalgae growth, resulting in low removal efficiency of nutrients. For the wastewater with a high COD/TN ratio, the deficiency of nitrogen and phosphorus or the accumulation of toxic substances such as furfural sourced from the FW were possible reasons for hindering the microalgae growth at the late stage of the culture process. Toxic substances such as alcohol, formaldehyde and phenols can inhibit algal growth and decrease nutrient removal efficiency in the treatment of the anaerobic digestate of sludge combined with acidified starch wastewater [13].

At the end of the culture process, the mixed wastewater with a dilution rate of SW:DFW = 1:19 achieved the best water quality. The corresponding concentrations of COD, TN and TP were 2538, 18 and 0.4 mg L⁻¹, respectively. The TN and TP concentration reached the discharge threshold values of 80 and 8 mg L⁻¹. Unfortunately, the COD concentration exceeded 400 mg L⁻¹ which is a maximum discharge requirement of livestock and poultry pollutants in China [37]. For the FW discharge, the COD and NH₃-N concentrations should be lower than 60 and 15 mg L⁻¹ according to discharge standards of water pollutants for the furfural industry of Heilongjiang province, China. However, the mixed wastewater after microalgae treatment did not achieve these discharge requirements. Additional work must be done to decrease the nutrient concentrations and meet the corresponding values of the discharge threshold. In terms of the carbon/nitrogen ratio, more mixture ratios of SW to DFW should be investigated, such as SW:DFW = 1:1, 1:2. Better mixture ratios of wastewater may be found to achieve high nutrient removal. On the other hand, improvement of gas-liquid mass transfer could be realized by increasing shaker speed or providing aeration. Thus, the oxygen or CO₂ concentration in the culture medium will be increased, which is critical to microalgae growing mixotrophically.

3.4. Lipid Production of Microalgae Cultured in the Mixed Wastewater

There were no significant differences in the lipid contents of the experimental groups (Figure 4). The lipid content of microalgae only slightly increased with an increased proportion of FW. The possible reasons were the relatively lower nitrogen concentration or the higher organic carbon source in the wastewater when the mix ratio of SW to DFW decreased. Nitrogen deprivation is an effective approach to stimulate lipid accumulation in microalgal cells [38]. However, the final TN concentrations of wastewater at mixture ratios of 1:0.5, 1:3, 1:9 and 1:19 were 242.34, 99.06, 42.09 and 18.03 mg L⁻¹, respectively. This indicated that there was sufficient TN in the mixed wastewater, which did not provide a nitrogen-depletion condition for microalgae cultivation. Therefore, the lipid content of the microalgae was not significantly different between each experimental group.

The addition of an organic carbon source to wastewater can also affect lipid accumulation [13]. The influence of carbon sources on lipid accumulation in microalgal cells was dependent on the amounts of carbon sources in the culture medium under the mixotrophic culture mode. Tan et al. [13] observed that a small amount of organic carbon source in the medium could not promote the accumulation of lipids in algal cells. Li et al. [39] also found that there was no significant increase in the lipid content of Chlorella sorokiniana cultured mixotrophically with low glucose addition. However, a relatively high concentration of an organic carbon source added to the wastewater can significantly stimulate lipid accumulation in microalgal cells [24,40,41].

Some microalgae species, such as Nannochloropsis oculata [42], Chlorella sorokiniana [39], Chlorella pyrenoidosa [24] and Chlamydomonas reinhardtii [41], cultured in mixotrophical mode, can not only achieve a high microalgal biomass but also accumulate more lipids within cells in comparison with autophototrophic mode. Therefore, it is possible to improve algal lipid content and the productivity of microalgae grown in wastewater by adding an organic carbon source. Liu et al. [39] found that the biomass concentration of C. pyrenoidosa increased from 0.218 g L⁻¹ to 0.518 g L⁻¹ when actual municipal wastewater (MW) was added with 2.0 g L⁻¹ NaAC, and the corresponding lipid content was increased by 1.48 times. Acetyl-CoA is a well-known substantial material for lipid accumulation of microalgae [39]. In addition, lipid accumulation is an energy storage process that requires ATP and NADPH. The biosynthesis of triacylglycerol (TAG) in the green alga Chlorella is preceded by a large increase in acetyl CoA via upregulation of plastidic pyruvate dehydrogenase [42]. Lin et al. [43] believed that the acetyl CoA was likely produced by acetyl CoA synthase when microalgal culture medium was added by acetate as a substrate, which supplies sufficient acetyl CoA for algal growth and lipid synthesis in mixotrophic conditions. Compared to photoautotrophic cultivation, acetate supplies sufficient carbon molecules for algal mixotrophic growth. This leads to the reduction of ATP and NADPH that are used to fix CO₂ as a carbon source, resulting in more ATP and NADPH available for lipid synthesis [43]. Hence, the addition of organic carbon such as acetate can promote lipid accumulation and increase lipid productivity. In the present study, acetate was indirectly added to the SW by mixing with the FW that contained large amounts of acetate and which may improve the lipid accumulation of microalgae. However, some microalgal species can accumulate more lipid in photoautotrophic than in mixotrophic cultivation [27]. Thus, lipid accumulation behavior in the different trophic modes may be strain dependent.

The lipid production was significantly affected by the mix ratio of SW to DFW. The lipid productivity of microalgae increased considerably with an increase in the mix proportion of FW. A large increase in lipid productivity was observed at the mixing ratio of SW:DFW = 1:19. This reached 49.48 mg L⁻¹ d⁻¹, which was 4.9 times greater than that obtained in the wastewater at the mix ratio of SW:DFW = 1:0.5. Compared to photoautotrophic cultivation, algal biomass concentration was markedly enhanced using the mixotrophic culture of microalgae, and this resulted in higher lipid productivity [15,24,41,44]. Therefore, the combination of SW and FW used to culture microalgae had a distinct advantage in the production of microalgal lipids. Microalgae cultured in a mixture of SW and FW appears to be a sustainable and cost-effective process for simultaneously treating these two types of wastewaters and efficiently producing algal lipids for biofuel production. However, it should be noted that the present study is a preliminary exploration of treating SW and FW using microalgae. There are many technical issues to be solved before commercialization of this method, such as the low efficiencies of nutrient removal, complex wastewater pretreatment (including sterilization), stability of culture performance at a large scale. The evaluation of microalgae culture in mixed wastewater after pre-treatment without sterilization should be carried out, and algae-bacteria symbiotic systems should be tested by adding additionally microorganism and applied to improve the nutrient removal efficiency. Other work including process optimization, scale up, pilot experiment, special culture system design and demonstration all need to be carried out step by step in the future to move this technology towards the real application.

4. Conclusions

We evaluated the production of microalgae biomass and lipids as well as nutrient removal by cultivation of C. pyrenoidosa in a mixture of SW and FW at different mix ratios. The mix ratio of SW to DFW significantly affected microalgae growth, which further influenced the efficiency of nutrient removal. The mixture of wastewater at a mix ratio of SW:DFW=1:19 achieved the best water quality. The corresponding TN and TP concentrations reached the discharge threshold values of 80 and 8 mg L⁻¹, respectively. In addition, the production of algal lipids also depended significantly on the mix ratio of these two types of wastewater. The highest lipid productivity of 49.48 mg L⁻¹ d⁻¹ occurred at the mix ratio of SW:DFW=1:19. Further improvement of nutrient removal efficiency for mixed wastewater could be achieved by optimization of the wastewater mixture ratio, enhancement of the gas-liquid ratio and addition of a pretreatment. This approach would allow for the simultaneous treatment of SW and FW integrated with lipid production in the future.