Optimization of Culture Conditions for Microalgae Treatment Fly Ash Leachate System

Abstract

In order to explore the feasibility of using algae to treat the fly ash leachate from a safe landfill site, leachate samples taken from a certain safe landfill site in Wenzhou City were treated with two different microalgae, Chlorella vulgaris and Scenedesmus obliquus, and the effectiveness of each treatment was evaluated in terms of its efficiency of pollutant removal. The effects of conditions such as pretreatment of leachate by sterilization, the initial concentration of leachate, and the addition of nutrients on pollutant removal efficiency and algae growth were studied. Sterilization of the leachate was found to have a relatively small impact on the growth of C. vulgaris and S. obliquus, as well as the removal of pollutants from the leachate. Therefore, sterilization treatment may not be necessary for engineering applications. Algal growth and the removal of pollutants were optimal when the leachate was used at a concentration of 10%, but when the leachate concentration was 30% or higher, the growth of both algae was weakened. The inclusion of 0.2 g/L K₂HPO₄·3H₂O and 0.06 g/L ammonium ferric citrate in the system led to higher algal growth and pollutant removal. The chlorophyll a levels of C. vulgaris and S. obliquus were 555.53% and 265.15%, respectively, and the nitrogen removal rates were also the highest, reaching 59.51% and 56.69%, respectively. This study optimized the cultivation conditions of a microalgae treatment leachate system, providing technical support and a theoretical basis for the practical engineering of a harmless treatment of leachate.

1. Introduction

In the process of garbage treatment and disposal, the incineration method has been used extensively in the field of garbage treatment, both domestically and internationally, because of its advantages in terms of the efficient reduction of the size and quantity of garbage. However, incineration produces a large amount of fly ash, which contains heavy metals such as Cd, Cr, Pb, Hg, Cu, and Zn, as well as dioxin pollutants [1,2,3,4,5]. At present, the main method for treating incineration fly ash from raw waste in China is to solidify and stabilize it before it enters landfill plants for landfill treatment [6,7,8,9]. During the sanitary landfill process, fly ash generates a large amount of leachate as a result of factors such as rainwater erosion, surface runoff, microbial decomposition, and groundwater infiltration into the landfill area [10]. The components of leachate from fly ash landfill sites are complex and difficult to treat. Improper disposal of leachate can harm the surrounding environment and human health through groundwater, soil, and other means [11,12]. Therefore, establishing a more environmentally friendly way to treat the leachate from fly ash landfill sites is an urgent issue.

Different environmental factors and the different characteristics of landfill waste can lead to very different components in the leachate [13]. Typical landfill leachate mainly contains dissolved organic compounds such as volatile fatty acids, humic acids, and fulvic acids; inorganic components such as ammonia, calcium, magnesium, iron, chlorine, and sulfate ions; various heavy metals such as chromium, nickel, copper, zinc, lead, and cadmium; as well as aromatic hydrocarbons, phenolic resins, chlorinated aliphatic compounds, pesticides, and plasticizers [14,15]. In addition, leachate generally contains high concentrations of chloride and ammonia nitrogen (NH₃-N), coupled with its high conductivity and the presence of toxic heavy metals [16], resulting in acute toxic effects and chronic risks. Through sampling analysis, researchers have detected high levels of conventional pollutants, such as NH₃-N, and chemical oxygen demand (COD) in groundwater near landfills, as well as persistent organic pollutants such as heavy metals and polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and even emerging pollutants such as PPCPs (pharmaceutical and personal care products) [17].

The conventional treatment of landfill leachate usually employs physicochemical methods, membrane filtration, advanced oxidation technologies, and biological methods. Physical and chemical treatments mainly include coagulation/flocculation, carbon adsorption, air stripping, chemical oxidation, and ion exchange, as well as membrane filtration such as nanofiltration and reverse osmosis [18]. These methods are usually used in pretreatment or post-purification in leachate treatment processes. Biological methods include aerobic and anaerobic biological treatments, which mainly rely on the biochemical degradation of organic pollutants by aerobic and facultative microorganisms. The aerobic activated sludge process used for the treatment of leachate mainly includes the sequencing batch reactor (SBR), aerobic membrane bioreactor (MBR), anoxic-aerobic (A/O) process, and biofilm reactor [19]. Many researchers have successfully treated landfill leachate using SBR to remove NH₃-N. The performance of MBR and SBR in the treatment of high-strength landfill leachate has been compared, and MBR was found to be more superior [20]. Zheng et al. found that the multi-stage A/O process can increase the diversity of microorganisms in the system to improve the impact resistance of the system effectively [21]. Based on the above advantages, many researchers have combined the multi-stage A/O process with the MBR process; the results show that this method not only effectively removes nitrogen and phosphorus, but also enhances stability [22]. Anaerobic treatment generally includes anaerobic digestion (AD), anaerobic filtering (AF), an up-flow anaerobic sludge bed (UASB), and anaerobic ammonia oxidation (Anammox). Anaerobic treatment can recover energy from wastewater, but the treatment efficiency is slow and there are requirements for the microbial community in the water. When combined with other technologies, anaerobic treatments can better treat leachate and improve the removal rate of pollutants. Although the conventional methods for treating leachate mentioned above can efficiently remove pollutants such as NH₃-N, the treatment cost is high, the biodegradation of leachate is reduced, the treatment is difficult to perform, and it is very likely to result in secondary pollution. In recent years, researchers have been looking for alternative biological methods to treat landfill leachate, and the use of microalgae seems rather promising, as it can effectively solve the above-mentioned problems. Microalgae-based wastewater treatment technology can not only purify wastewater and remove pollutants but can also utilize nutrients in wastewater to form biomass.

Microalgae are single-celled algae that can be morphologically distinguished under a microscope. “Microalgae” collectively refers to microorganisms that contain chlorophyll a, are capable of photosynthesis, and belong to a type of protists [23]. Microalgae have previously been suggested as a biological system to replace activated sludge in wastewater treatment [24]. A large amount of research, both domestically and internationally, has confirmed the feasibility of using microalgae for treating wastewater [25,26,27]. Currently, microalgae are being applied to the treatment of agricultural wastewater, industrial wastewater, urban wastewater, and harmful wastewater containing pesticides and antibiotics [28,29,30,31,32,33,34]. Examples of microalgal species have been investigated for their ability to treat leachate from a landfill. For example, Chlamydomonas sp. strain SW15aRL is able to grow in various types of leachates, and the growth of the algae can lead to a reduction in the content of NH₃-N in these leachates by as much as 70–100% [35]. Another example is that of Chlorella vulgaris, where the use of this species in the treatment of leachate from a landfill in Tunisia can result in the elimination of NH₃-N by up to 90%, with a maximum COD removal rate of 60% and a maximum lipid production efficiency of 4.74 mg·L⁻¹ per day [36]. Compared to C. vulgaris, Scenedesmus obliquus has a poorer removal effect on pollutants in leachate, and its survival rate is lower in harsh environments. These studies seem to suggest that microalgae clusters are quite effective in treating organic pollutants in landfill leachate. However, there have been very few reports on the application of microalgae in the treatment of fly ash landfill leachate.

In this study, we explored the potential application of microalgae in the treatment of fly ash leachate from safe landfill sites and the effectiveness of such a treatment. The results we obtained could provide technical support and a theoretical basis for the practical engineering of a harmless treatment for leachate.

2. Materials and Methods

2.1. Experimental Materials

The leachate used in this study was taken from a safe landfill site in Wenzhou City, and the sampling point was set 0.5 m below the surface of the leachate in the collection tank. After collection, the leachate was immediately transported back to the laboratory for the measurement of pH, dissolved organic carbon (DOC), NH₃-N, dissolved organic nitrogen (DON), total phosphorus (TP), COD, and other indicators. The samples were stored in a glass bottle at 4 °C. The C. vulgaris (FACHB–8) and S. obliquus (FACHB–14) used in this experiment were purchased from the freshwater algal seed bank of the Chinese Academy of Sciences. The relevant information of experimental reagents and equipment are detailed in Tables S1 and S2.

2.2. Pre Cultivation and Pretreatment of Microalgae

Both C. vulgaris and S. obliquus were cultured using BG11 medium in a light incubator at 25 °C under 2000 Lux light intensity and a 12 h:12 h light–dark cycle. The algal solution was manually shaken three times a day to ensure an even suspension of algal cells [37]. The main ingredient formula of BG11 is shown in

Table 1. Composition of BG11 medium.

Components	Mother Liquor Concentration	Dosage
NaNO3	15.00 g/100 mL dH2O	10 mL/L
K2HPO4	2.00 g/500 mL dH2O	10 mL/L
MgSO4·7H2O	3.75 g/500 mL dH2O	10 mL/L
CaCl2·2H2O	1.80 g/500 mL dH2O	10 mL/L
Citric acid	0.30 g/500 mL dH2O	10 mL/L
Ferric ammonium citrate	0.30 g/500 mL dH2O	10 mL/L
EDTANa2	0.05 g/500 mL dH2O	10 mL/L
Na2CO3	1.00 g/500 mL dH2O	10 mL/L
A5 (trace metal solution) *		1 mL/L

Note: “*” represents BG11 culture medium trace element mixture, trace element composition as shown in Table 2.

and

Table 2. Composition of trace element.

Components	Concentration
H3BO3	2.86 g/L dH2O
MnCl2·4H2O	1.86 g/L dH2O
ZnSO4·7H2O	0.22 g/L dH2O
Na2MoO4·2H2O	0.39 g/L dH2O
CuSO4·5H2O	0.08 g/L dH2O
CO(NO3)2·6H2O	0.05 g/L dH2O

.

A sample of the algal culture was taken from a culture in the logarithmic growth phase (cell density of 10⁶ cells/mL) and placed into a centrifuge tube and centrifuged at 2200× g for 5 min. The supernatant was then discarded, the cells were rinsed with a 15 mg/L NaHCO₃ solution and centrifuged as before, and the supernatant was again discarded. This step was repeated twice to ensure the removal of any nutrients attached to the algal cells. After that, the algal cells were resuspended in ultrapure water and cultured for 24 h, and the algal culture was used in subsequent experiments.

2.3. Experimental Group Setup

2.3.1. Sterilized/Unsterilized

The experimental group was set up according to

Table 3. Experimental group setup.

Group	Algae Species	K2HPO4·3H2O	MgSO4·7H2O	Ammonium Ferric Citrate	Trace Elements	Leachate Gradient	Sterilization Status
A0	C. vulgaris	NA	NA	NA	NA	BG11	NA
A1	C. vulgaris	+	+	−	−	1% leachate	Sterilized
A2	C. vulgaris	−	−	−	+	10% leachate	Non-sterilized
A3	C. vulgaris	+	+	−	+	20% leachate	NA
A4	C. vulgaris	+	−	+	+	25% leachate	NA
A5	C. vulgaris	−	−	−	−	30% leachate	NA
A6	C. vulgaris	−	+	+	+	40% leachate	NA
A7	C. vulgaris	+	−	+	−	50% leachate	NA
A8	C. vulgaris	−	+	+	−	NA	NA
B0	S. obliquus	NA	NA	NA	NA	BG11	NA
B1	S. obliquus	+	+	−	−	1% leachate	Sterilized
B2	S. obliquus	−	−	−	+	10% leachate	Non-sterilized
B3	S. obliquus	+	+	−	+	20% leachate	NA
B4	S. obliquus	+	−	+	+	25% leachate	NA
B5	S. obliquus	−	−	−	−	30% leachate	NA
B6	S. obliquus	−	+	+	+	40% leachate	NA
B7	S. obliquus	+	−	+	−	50% leachate	NA
B8	S. obliquus	−	+	+	−	NA	NA
C0	Algae-free control	NA	NA	NA	NA	NA	NA
C1	Algae-free control	NA	NA	NA	NA	1% leachate	Sterilized
C2	Algae-free control	NA	NA	NA	NA	10% leachate	Non-sterilized
C3	Algae-free control	NA	NA	NA	NA	20% leachate	NA
C4	Algae-free control	NA	NA	NA	NA	25% leachate	NA
C5	Algae-free control	NA	NA	NA	NA	30% leachate	NA
C6	Algae-free control	NA	NA	NA	NA	40% leachate	NA
C7	Algae-free control	NA	NA	NA	NA	50% leachate	NA

Note: “+”represents added, “−” represents not added, and “NA” represents no media.

. Each flask contained either 50 mL of C. vulgaris (Group A) or S. obliquus (Group B) culture (OD₆₈₀ = 1.10 ± 0.01), or 50 mL of ultrapure water in the case of the control (Group C). To each flask, 50 mL of fly ash leachate and 400 mL of ultrapure water were added. The fly ash leachate was either sterilized or non-sterilized before being added to the flask. All flasks were evenly placed in a light incubator and subjected to the same cultivation conditions as described above. The flasks were hand-shaken three times a day to ensure the algal cells were evenly suspended. Different indicators, including OD₆₈₀, chlorophyll a concentration, the optimal photochemical efficiency of PSII in the dark (Fv/Fm), pH, NH₃-N, total nitrogen (TN), TP, DOC, and COD, were measured every two days. The status of the growth of the algae was observed by microscopic examination.

2.3.2. Dilution Ratio of Leachate

The original leachate had a high salt content, which could limit algal growth and was not suitable for the experiment. Therefore, the fly ash leachate was added in different amounts to yield final concentrations of 1%, 10%, 20%, 25%, 30%, 40%, and 50%. The specific experimental setup is shown in Table 3. The volume of algal culture and leachate sample added to the flasks was the same as described in 2.3.1. In addition to the cultures containing fly ash leachate, a control set of cultures was also prepared, which contained just the algae and BG11 medium. All the flasks were evenly placed in a light incubator and incubated under the same conditions as described above for the cultivation of microalgae, and the relevant indicators, including growth, in each culture were measured every two days.

2.3.3. Nutrients

The nutrients that were added to the algal cultures (with or without leachate) were K₂HPO₄·3H₂O, MgSO₄·7H₂O, ammonium ferric citrate, and trace elements. These nutrients are the main ingredients in the BG11 medium, and they were added to the algal cultures to final concentrations of 0.2 g/L (K₂HPO₄·3H₂O), 0.075 g/L (MgSO₄·7H₂O), and 0.06 g/L (ammonium ferric citrate), whereas trace elements were added as 1 mL of trace elements per liter of culture. The different groups in the experimental setup are shown in Table 3.

C. vulgaris or S. obliquus was inoculated into the medium using a ratio of 1 mL algae per 10 mL solution. The cultivation conditions and experimental sampling frequency were consistent with the above experiments.

2.4. Measurement of Indicators

These indicators, Chla, Fv/Fm, OD₆₈₀, pH, and oxidation-reduction potential (ORP) in the leachate, were measured using unfiltered leachate. Chla and Fv/Fm were measured using a phytoplankton fluorescence meter (Zeal Quest Scientific Technology Co., Ltd.). It measures the Chla concentration and Fv/Fm of algae through chlorophyll fluorescence technology. OD₆₈₀ was measured using a UV spectrophotometer (Beijing Puxi General Instrument Co., Ltd.). The pH and ORP were measured using a pH meter (Shanghai Yidian Scientific Instrument Co., Ltd.). After the measurements, the leachate was filtered through a 0.45 μm filter membrane, and the remaining water quality indicators were then determined for the filtered sample. These indicators consisted of TN, TP, DOC, and DON, as well as COD. NH₃-N was measured using Nessler’s reagent spectrophotometry (HJ535—2009) [38]. TN was measured using alkaline potassium persulfate digestion spectrophotometry (HJ636—2012) [39]. TP was measured using ammonium molybdate spectrophotometry (GB11893—89) [40]. DOC and DON were measured using a total organic carbon analyzer (Shimadzu Enterprise Management Co., Ltd.), while COD was measured using a D60 spectrophotometer (Zhejiang Ditexi Technology Co., Ltd.) and precast reagent spectrophotometric method (T/ZJATA0001—2020) [41].

2.5. Data Processing and Analysis

The calculation method for the growth rate of indicators (Chla, OD₆₈₀, Fv/Fm, and DOC) is as follows:

$$Growthrate=\left({\displaystyle \frac{Nt-No}{No}}\right)\times 100\%.$$

In the formula, $Nt$ represents the indicator concentration measured at time t for the experimental group; $No$ is the initial indicator concentration.

The calculation method for the removal rate of indicators (NH₃-N, TN, TP, and DON) is as follows:

$$Removalrate=\left({\displaystyle \frac{Co-Ct}{Co}}\right)\times 100\%$$

Among them, Co represents the concentration of the indicator before treatment, while Ct represents the concentration of the indicator after treatment.

All graphical plots were generated using Origin^® 9.0. All data were expressed as means ± standard deviations.

3. Results and Discussion

3.1. Effect of Sterilization on Algal Treatment of Leachate

3.1.1. Effect of Sterilization of Leachate on Algal Growth

The effects of sterilized versus non-sterilized fly ash leachate on the growth of microalgae are shown in

Table 4. Effects of sterilized and non-sterilized fly ash leachates on the growth of microalgae.

Group	A1	A2	B1	B2
Algae species	C. vulgaris		S. obliquus
Percolate	Sterilized	Non-sterilized	Sterilized	Non-sterilized
Chla growth rate (%)	315.0 ± 14.0	300.3 ± 4.8	77.0 ± 9.4	18.3 ± 6.0
OD680 growth rate (%)	356.3 ± 25.2	326.0 ± 39.2	201.6 ± 36.1	173.7 ± 18.6
Fv/Fm change rate (%)	−24.4 ± 1.0	−25.0 ± 2.3	6.4 ± 2.5	10.2 ± 7

. The sterilization of leachate had an impact on the effectiveness of microalgae treatment of leachate, but the impact was relatively small. In terms of algal growth, both C. vulgaris and S. obliquus displayed a higher growth rate when the cultures were inoculated with sterilized leachate samples. Sterilization could kill off any microorganisms present in the leachate that might compete with the algae for nutrients, and this could result in some inhibition of algal growth and reproduction. Thus, sterilization of the leachate samples ensured better growth and reproduction for the microalgae.

The Fv/Fm in the chlorophyll fluorescence index reflects the potential maximum photosynthetic capacity of plants. The Fv/Fm of C. vulgaris was found to decrease from an initial value of 0.560 ± 0.012 to 0.422 ± 0.005, while the Fv/Fm of S. obliquus showed the opposite trend, increasing from an initial value of 0.560 ± 0.012 to 0.606 ± 0.012, regardless of whether the leachate sample was sterilized or not. During the normal growth and reproduction of algae, the potential maximum photosynthetic capacity decreased, possibly because of environmental stress exerted on C. vulgaris by certain pollutant components in the leachate.

Previous studies have shown that heavy metal stress, e.g., mercury, has a certain inhibitory effect on the growth of C. vulgaris and can significantly reduce its Fv/Fm [42]. The stress of another heavy metal, Cd²⁺, can also lead to a significant decrease in chlorophyll fluorescence parameters such as Fv/Fm [43]. In addition, studies have also shown that when the phosphorus concentration is low, the phosphorus stress on C. vulgaris can lead to an accelerated decrease in Fv/Fm. Referring to the Pollution Control Standards for Landfill of Hazardous Waste (GB 18598—2019) [44], the TN emission concentration limit of the landfill is 30 mg/L, while the TN is 3 mg/L. Using atomic absorption spectrophotometry (GB/T7475—1987) [45], it was found that there were heavy metals, 0.00113 mg/L Cd and 0.06779mg/L Zn, in the leachate. An atomic fluorescence method (HJ694—2014) was used to determine that the leachate contained 0.01422 mg/L Hg [46]. The leachate contained heavy metals, and an abnormal ratio of nitrogen to phosphorus, which could have an impact on algae, causing environmental stress. However, the photosynthetic capacity of S. obliquus was not affected by the presence of leachate, indicating its low sensitivity to heavy metals in the environment.

3.1.2. Effect of Sterilization of Leachate on Pollutant Removal Efficiency

The impacts of the sterilization of fly ash leachate on the efficiency of pollutant removal by the two species of microalgae are shown in

Table 5. Effect of leachate sterilization on the removal effect of leachate pollutants.

Group	A1	A2	B1	B2
Algae species	C. vulgaris		S. obliquus
percolate	Sterilization	Non-sterilized	Sterilization	Non-sterilized
DOC growth rate (%)	125.3 ± 13.7	122.7 ± 19.3	47.9 ± 3.4	43.9 ± 5.3
NH3-N removal rate (%)	67.1 ± 1.3	66.8 ± 0.5	68.1 ± 1.7	67.7 ± 0.9
TN removal rate (%)	55.2 ± 1.0	53.5 ± 0.9	57.0 ± 0.5	56.2 ± 0.8
TP removal rate (%)	88.1 ± 1.0	85.9 ± 0.9	92.7 ± 0.6	92.4 ± 1.7

. There was an increase in the concentration of DOC in both C. vulgaris and S. obliquus cultures. However, the effect that sterilization of the leachate had on the DOC increase was relatively small, despite the increase between C. vulgaris and S. obliquus systems being statistically significant. The rate of DOC increase in the C. vulgaris culture was greater than 100%, while the highest increase detected in the S. obliquus culture was just 51.8%. This may be because C. vulgaris used light energy to convert some inorganic substances in the leachate into organic substances, especially protein substances, and released them into the solution, resulting in an increase in the DOC content in the solution. However, the utilization and conversion efficiency of inorganic carbides in the leachate by S. obliquus were lower, so the change in DOC concentration in the system was relatively small.

The impact of sterilization on the water quality produced by the microalgae was relatively small. The average efficiency for the removal of NH₃-N from the leachate was 67% for C. vulgaris and 68% for S. obliquus, indicating both algae had a similar ability in terms of NH₃-N removal. With unsterilized leachate, the average removal rates of TN and TP for C. vulgaris reached 53.5% and 85.9%, respectively, with TP removal decreasing from the original concentration of 2.70 mg/L to 0.36 mg/L. After sterilization of the leachate, the removal of TN and TP by C. vulgaris became higher compared with those achieved with unsterilized leachate. However, the difference in performance was not very obvious, as only a slight difference was obtained. The result suggested that the sterilization of leachate prior to its treatment with microalgae did not really affect the outcome of the treatment since the effect was insignificant. Therefore, from the perspective of industrial applications, dispensing with the sterilization of leachate prior to algal treatment would actually reduce the cost associated with the sterilization step, making the treatment more economical.

3.2. Effect of Initial Leachate Concentration on Algal Treatment

3.2.1. Algal Growth under Different Concentrations of Leachate

Two indicators, Chla and OD₆₈₀, were measured while characterizing the growth of the two algae. Both species of algae grew best in the absence of fly ash leachate (BG11 medium only: A0 and B0), as shown by the fastest and highest increases in both Chla concentration and OD₆₈₀ (Figure 1a–d). They grew well at low leachate concentrations (1% and 10%), but their growth rates under these leachate concentrations were still far lower than the rates observed in the absence of leachate. When the leachate concentration was 25%, both C. vulgaris (A) and S. obliquus (B) exhibited slow growth, and when the leachate concentration was 30% or above, the two algae could not grow and reproduce normally. In addition, after 16 days of incubation, the rate of increase in the Chla concentration of S. obliquus cultured in BG11 medium (B0) was much higher compared with C. vulgaris (A0) during the same period (Figure 1a,b). Similarly, in the 10% leachate treatment system, the growth rate of S. obliquus was also higher than that of C. vulgaris in the latter half, indicating that the adaptability of S. obliquus was stronger when 10% leachate was used, resulting in more suitable growth conditions in this culture.

The response of algal photosynthetic activity to stress caused by nutrients such as N, P, and Fe or environmental factors such as temperature and light mainly manifested as a decrease in Fv/Fm value [47]. In both C. vulgaris and S. obliquus cultures, the higher the concentration of fly ash leachate, the smaller the Fv/Fm value, indicating that excessive nutrient concentration could also limit the photosynthetic activity of the algae. The Fv/Fm value of C. vulgaris in BG11 medium was relatively stable, maintained between 0.5 and 0.6, while significant fluctuations were observed in the other groups (Figure 1e). The inclusion of leachate at 25% (A4) and 30% (A5) resulted in an initial overall decrease in Fv/Fm values followed by an increase, while other concentrations of leachate resulted in a gradual decrease in Fv/Fm values.

When the concentration of the leachate was higher than 20%, the overall Fv/Fm of the S. oliquus culture was slightly lower, and basically no algal growth was observed when the leachate concentration was increased to 40% and 50% (Figure 1f), so the light energy conversion efficiency was basically zero. At high concentrations of leachate, S. obliquus was not as adaptable as C. vulgaris, and this could be the reason why C. vulgaris displayed higher activity and light energy conversion efficiency when both strains were cultivated under the same conditions. When the concentration of leachate was 1% or 10%, the overall Fv/Fm value of C. vulgaris showed a downward trend (Figure 1e), while that of S. obliquus displayed a smaller fluctuation range, with a higher overall light energy conversion efficiency compared with C. vulgaris. In the 1% concentration leachate, the Fv/Fm of S. obliquus gradually increased in the range of 0.5 to 0.6. At other concentrations, the maximum light energy conversion efficiency first increased and then decreased but became stable (Figure 1f). During the initial stage following the addition of leachate, S. obliquus had strong adaptability and was able to quickly adapt to the growth environment, achieving a high light energy conversion efficiency. As the nutrients in the leachate were being consumed, the energy available for conversion also decreased, leading to decreased efficiency in the conversion of light energy.

3.2.2. Influence of Initial Concentration of Leachate on Pollutant Removal Efficiency

The concentration of NH₃-N in the fly ash leachate was relatively high. The concentration of NH₃-N in each group of algal cultures inoculated with leachate showed a decreasing trend (Figure 2), indicating that the addition of C. vulgaris or S. obliquus had a certain effect on the removal of NH₃-N. In high leachate concentrations, even in cases where the algae grew poorly, the natural consumption of NH₃-N was caused by the microbial activity originating from the leachate itself, and the shaking process that was performed during the experiment probably resulted in a slight decrease in NH₃-N concentration.

When the concentration of the leachate was 1%, the NH₃-N removal rates of C. vulgaris and S. obliquus were the highest, reaching 93.96% and 93.52%, respectively. This was followed by the 10% leachate culture, where C. vulgaris achieved a removal rate of 37.31%, while S. obliquus managed as much as 68.18%. Similarly, for the control culture (BG11 medium), the removal of NH₃-N by S. obliquus reached a rate that was much higher than that attained by C. vulgaris, reaching 85.58%. The result indicate that S. obliquus may better absorb and utilize NH₃-N during growth (

Table 6. NH₃-N removal by algae under different concentrations of fly ash leachate.

Group	A0	A1	A2	A3	A4	A5	A6	A7
Leachate concentration	BG11	1%	10%	20%	25%	30%	40%	50%
Initial concentration (mg/L)	6.37	10.62	42.87	79.38	96.38	110.14	143.64	177.40
The final concentration (mg/L)	4.37	0.67	27.37	49.12	64.38	76.13	100.39	115.64
Removal rate%	23.82	93.96	37.31	35.50	33.85	35.11	29.47	36.58
Group	B0	B1	B2	B3	B4	B5	B6	B7
Leachate concentration	BG11	1%	10%	20%	25%	30%	40%	50%
Initial concentration (mg/L)	7.62	11.62	45.12	80.13	92.38	114.14	151.65	185.15
The final concentration (mg/L)	0.97	0.72	17.08	48.62	66.88	81.63	106.14	124.89
Removal rate%	85.58	93.52	68.18	38.19	27.56	27.33	27.96	31.51

).

The DOC is an indicator that shows the status of photosynthesis in the microalgae, biological metabolism, and bacterial activity in the water. The DOC value fluctuated greatly in the C. vulgaris cultures that contained high concentrations of leachate, with an initial decrease followed by increase that later tended to stabilize (Figure 3a,b). As for the S. obliquus cultures, less fluctuation was observed compared with C. vulgaris cultures. When the leachate concentration was low, 1% in the case of C. vulgaris cultures and 1–10% for S. obliquus cultures, the DOC was rather stable. The fluctuation in DOC may reflect the complex mixture of organic compounds in the cultures and the constant transformation of these compounds by the algae as they grew and multiplied, leading to fluctuations in their concentrations.

During the process of algal growth in BG11 medium, the pH of the solution showed a slight upward trend, while in the presence of different concentrations of leachate, the pH of the algal cultures showed a downward trend. The pH change was most significant at a low leachate concentration (1%), decreasing from a weakly alkaline pH to an acidic one after a slight increase. The lowest pH value reached in the C. vulgaris culture was 4.91. In addition, the pH value among the cultures containing 10% leachate showed significant changes in the range of 7.0–8.5, while the pH value of the cultures containing 20–50% leachate concentration changed very little throughout the entire process, with an overall change in the range of 7.5–8.2 (Figure 3c,d). In addition, it can be seen from the graph that there is a certain relationship between the degree of pH change and the concentration of leachate, with the lower concentration displaying a greater pH change. This could be due to the low concentration of the leachate and the instability of the concentration of each component in the solution, which might be easily affected by the algal activity. Microalgae have a certain absorption effect on inorganic carbon and other substances. At the same time, during the growth process, the algae secreted and produced some extracellular polymers, including some humic acid substances, which could have affected the pH of the cultures.

3.3. Effects of Additional Nutrients on Microalgae Treatment of Leachate System

3.3.1. Growth of Algae in the Presence of Added Nutrients

The removal of nitrogen and phosphorus from wastewater by microalgae is also influenced by nutrient composition and organic load. The addition of nutrients to the algal cultures containing leachate resulted in a higher Chla concentration and optical density compared with cultures with no addition of nutrients (Figure 4a–d), indicating the presence of added nutrients could stimulate algal growth. However, the addition of just trace elements (A2 and B2) did not really result in any appreciable increase in Chla concentration or optical density, suggesting trace elements alone were not adequate for growth stimulation. K₂HPO₄ and ammonium citrate are the main nutrient for algal cell growth. Based on the increase in Chla concentration and changes in optical density, algal growth was optimal when the added nutrients consisted of K₂HPO₄ plus ammonium ferric citrate without or with trace elements, with the addition of trace elements yielding a slightly better result, as seen in the C. vulgaris cultures in the earlier stage, although in the later stage, the absence of trace elements was more effective, with the Chla concentration reaching as high as 555.53%. Under the same nutrient conditions, the growth rate of S. obliquus was slightly lower than that of C. vulgaris, and the differences among the different groups of S. obliquus cultures were relatively small, indicating that C. vulgaris was more sensitive to changes in nutrients in its growth environment. The Chla concentration of S. obliquus cultures containing K₂HPO₄ plus ammonium ferric citrate only increased to 265.15%, which is half the level attained by C. vulgaris cultures.

As shown in Figure 4e,f, in both sets of algal cultures, there was an overall fluctuation in maximum light energy conversion efficiency Fv/Fm in the range of 0.45–0.65, and the difference among the groups was not significant. In the presence of exogenous nutrients from groups 4 and 7, both C. vulgaris and S. obliquus cultures acquired slightly higher maximum light energy conversion efficiency than the other cultures, indicating that under suitable environmental conditions, the algae could grow and reproduce faster, leading to increases in biomass and the corresponding light energy conversion efficiency and ultimately enabling better photosynthesis, absorption, and transformation of nutrients from their environment.

3.3.2. Effect of Added Nutrients on the Efficiency of Pollutant Removal

In the algal cultures containing fly ash leachate and added ammonium ferric citrate (A4, A6–A8, B4, and B6–B8), the initial DOC content was significantly higher compared with those without the addition of this nutrient (A1–A3, A5, B1–B3, and B5) (Figure 5a,b). However, on day 4, all systems had a similar DOC content, so the addition of ammonium ferric citrate to the cultures caused no net increase in organic matter as the incubation progressed.

There was a significant difference in the transformation of dissolved organic matter between the C. vulgaris and S. obliquus cultures. Except for the initial decrease observed in the groups containing added ammonium ferric citrate, the concentration of DOC in the C. vulgaris cultures gradually increased with incubation time. In the S. oblique cultures, the concentration of DOC changed relatively little, with an overall fluctuation only in the range of 14–17 mg/L overall, followed by a slight downward trend and a slight rebound in the later stage. The absorption and transformation of dissolved organic matter occurred in relatively equal proportions in the S. oblique cultures. Changes in the C. vulgaris cultures may be related to the higher protein content in the algal cells. The cellular activities of C. vulgaris during growth may have released more protein substances into the cultures, causing the DOC in the system to increase instead of decrease.

The concentration of total dissolved nitrogen showed a significant downward trend. There was a sharp decrease in the initial stage, which was followed by a gradual decrease in the middle and later stages (Figure 5c,d). The initial stage was marked by the addition of algae to the leachate; at this stage, there was an abundant nitrogen source to promote vigorous algal growth, and the extensive absorption and utilization of nitrogen resulted in a significant decrease in DON content. At the end of the incubation, the test groups containing added K₂HPO₄·3H₂O and ammonium ferric citrate showed the highest increase in algal mass and the lowest final DON concentration. Under these conditions, C. vulgaris and S. obliquus had the highest nitrogen removal rates, reaching 59.51% and 56.69%, respectively.

The addition of exogenous nutrients to the algal cultures in the treatment of leachate also led to changes in the pH of the cultures, but the changes were within the range of pH 7.5–pH 8.6. C. vulgaris is known to have optimum growth at pH 8, whereas the optimum growth of S. obliquus is at pH 9 (Figure 6). Since the changes in pH for the two algal cultures in the treatment of leachate were closer to the pH for optimum C. vulgaris growth, the treatment of leachate without the adjustment of pH would be more suitable for C. vulgaris.

4. Conclusions

The potential of using microalgae in the treatment of fly ash leachate was evaluated for two species of algae. In both cases, the pre-sterilization of fly ash only resulted in a slight improvement (about 2%) in the removal of N and P. The treatment of leachate by C. vulgaris and S. obliquus yielded the best results when the leachate was used at 1% and 10%, respectively. At concentrations above 25%, the leachate became inhibitory to algal growth and reproduction. Energy conversion was higher and more stable in the S. obliquus system with 1% leachate. At 1% leachate, the removal of NH₃-N was also highest, reaching 93.96 and 93.52%, respectively, for C. vulgaris and S. obliquus cultures, while the pH of both systems changed significantly, reaching a low of 4.91. Furthermore, adding additional nutrients (K₂HPO₄·3H₂O and ammonium ferric citrate) to the algae plus leachate system promoted algal growth and pollutant removal, with C. vulgaris showing a much bigger response than S. obliquus. The nitrogen removal rates also peaked in the presence of added nutrients. The chlorophyll a levels of C. vulgaris and S. obliquus were 555.53% and 265.15%, respectively. The treatment of leachate by microalgae described in this study could provide technical support and a theoretical basis for the practical engineering of leachate treatment.