Assessment of inorganic nitrogen and phosphorus compounds removal efficiency from different types of wastewater using microalgae cultures

Artificial wastewater with different loads of nitrogen and phosphorus was used in this study (Table 1). The composition of the synthetic wastewater was based on a modified formulation of the BG-11 medium. NH₄Cl and KH₂PO₄ were added to the culture medium for microalgae in varying amounts to obtain typical concentrations of ammonium nitrogen and phosphate found in wastewater (Hence et al. 2002; Rawat et al. 2011; Acevedo et al. 2017).

Cultures of microalgae in the late exponential phase of growth (18-day culture) were used to prepare the inoculum. The ratio of inoculum to wastewater was 1:10. Test vessels were flasks with a volume of 500 cm³. Microalgal cultures were incubated in wastewater for 14 days under artificial lighting (using Philips TL-D 18W 54-765 G13 daylight fluorescent lamps). The light intensity was 50–54 μmol photons m⁻² s⁻¹; light regime – 16 h of light and 8 h of darkness. The pH and temperature of the test solutions were monitored throughout the exposure period. The pH of the solutions remained in the range of 7.5 to 8.2 and did not change during the exposure period by more than 0.7 units in any test; the temperature of the solutions was 25.0–26.2°C.

The concentration of ammonium nitrogen and phosphate in water was analyzed according to Arsan et al. (2006).

The nutrient removal efficiency was calculated as follows: Removal efficiency(%)=C0−CfCn×100user1{Removal}\,\user1{efficiency}(\% ) = \frac{{C_0 - C_f }}{{C_n }} \times \user1{100} where: C₀ – initial concentration, C_f – final concentration.

The functional activity of the cultures under the studied conditions was assessed based on changes in dry weight and the content of photosynthetic pigments (chlorophyll a and carotenoids). For the respective analyses, the biomass of microalgae was concentrated by filtering the cell suspension through cellulose nitrate membrane filters (“Sartorius”, pore size 0.45 μm) under low vacuum conditions. Samples for analysis were collected at the beginning and at the end of the experiment.

To determine the dry weight, the filters with concentrated microalgae were dried in a thermostat at a temperature of 105ºC to constant weight. Dry matter content was calculated according to the formula (Lim et al. 2010): Dry weight=Weight of filter with algal cells−Weight of blank filters (mg)Volume of algal culture(L){\text{Dry}}\,{\text{weight}} = \frac{{{\text{Weight}}\,{\text{of}}\,{\text{filter}}\,{\text{with}}\,{\text{algal}}\,{\text{cells}} - {\text{Weight}}\,{\text{of}}\,{\text{blank}}\,{\text{filters}}\,(mg)}}{{{\text{Volume}}\,{\text{of}}\,{\text{algal}}\,{\text{culture}}(L)}}

The specific growth rate (μ) of microalgae was calculated using the equation (Krzemińska et al. 2014): μ=ln(N2N1)t2−t1(day−1)\mu = \frac{{\user1{ln}\left( {\frac{{N_2 }}{{N_1 }}} \right)}}{{t_2 - t_1 }}\left( {{\text{day}}^{ - 1} } \right) where N₁ and N₂ are biomass at time 1 (t₁) and time 2 (t₂), respectively.

The content of pigments was analyzed by an extractive spectrophotometric method (SCOR-UNESCO, 1966). For extraction of pigments, the filter with algae was thoroughly homogenized with the addition of quartz sand and 90% acetone. The resulting extract was separated by centrifugation. In samples of green microalgae, which contained chlorophyll a, chlorophyll b, and carotenoids (Rowan, 1989), the optical density of extracts was determined at 647, 664, and 480 nm, respectively. In the culture of blue-green microalgae, containing only chlorophyll a and carotenoids (Rowan, 1989), the optical density of extracts was determined at 664 and 480 nm, respectively. Non-specific light absorption by extracts at a wavelength of 750 nm was also measured. Chlorophyll a concentration was calculated according to the equations of Jeffrey & Humphrey (1975), and the total carotenoid content was calculated according to the formula of Parsons & Strickland (1963).

All measurements were conducted in triplicate. Statistical processing of the research results was performed using SPSS Statistics software (version 17). The Dunnett test was used to assess the differences between the values of the indicators. A difference was considered significant at p ≤ 0.05. All data were presented as mean ± standard deviation (M ± SD).

During the cultivation period, the efficiency of ammonium nitrogen and phosphate removal by Ch. reinhardtii in the experiment variant with their lowest concentrations (30 mg N-NH₄⁺ l⁻¹ + 7 mg P-PO₄³⁻ l⁻¹) was 63% and 18%, respectively (Table 2). Ch. reinhardtii is distinguished by high efficiency of nitrogen removal at its significantly higher concentrations in wastewater, 50, 90 and 140 mg l⁻¹ (removal efficiency of 60%, 56% and 49%, respectively). As for P-PO₄³⁻, the efficiency of its removal by microalgae was low and at higher concentration of P-PO₄³⁻ did not exceed 7%.

For O. neglecta in the experiment variant with 30 mg N-NH₄⁺ l⁻¹ + 7 mg P-PO₄³⁻ l⁻¹, the efficiency of their removal on day 14 was 60% and 55%, respectively, while in the variant with 50 mg N-NH₄⁺ l⁻¹ + 14 mg P-PO₄³⁻ l⁻¹ – 61% and 43%, respectively (Table 2). The results obtained indicate the ability of the blue-green algae culture to significantly reduce both nitrogen and phosphorus concentrations in the analyzed wastewater. However, at higher concentrations of nutrients, the percentage of their removal by O. neglecta decreases rapidly. In the experiment variant with 140 mg N-NH₄⁺ l⁻¹ + 41 mg P-PO₄³⁻ l⁻¹, an increase in the concentrations of nitrogen and phosphorus was observed on day 14 compared to their initial values. This is obviously due to the fact that the culture under the studied conditions died and was lysed.

A reliable indicator of the metabolic activity of microalgal cells is the growth rate. Figure 1 shows the changes in dry weight of Ch. reinhardtii and O. neglecta during the period of their cultivation in wastewater with different loads of ammonium nitrogen and phosphate. The maximum specific growth rate of Ch. reinhardtii (0.091 day⁻¹) was observed at a concentration of 140 mg N-NH₄⁺ l⁻¹ and 41 mg P-PO₄³⁻ l⁻¹, with slightly lower rates (0.087–0.082 day⁻¹) at lower nitrogen and phosphorus concentrations. Thus, as the concentration of nutrients increases, the growth rate of Ch. reinhardtii also increases.

Figure 1

An opposite trend was observed for the blue-green alga O. neglecta: as the concentration of N-NH₄⁺ and P-PO₄³⁻ in the aquatic environment increases, the dry matter content decreases. The maximum specific growth rate of O. neglecta was observed at a concentration of 30–50 mg N-NH₄⁺ l⁻¹ + 7–14 mg P-PO₄³⁻ l⁻¹, reaching 0.083–0.084 day⁻¹. The minimum values of this indicator (0.054 day⁻¹) were observed when microalgae were grown in wastewater with 140 mg N-NH₄⁺ l⁻¹ + 41 mg P-PO₄³⁻ l⁻¹.

When microalgae are used for wastewater treatment, their high photosynthetic activity is of paramount importance, because the rate of photosynthetic processes in algal cells determines the rate of biomass accumulation, nutrient assimilation, and photosynthetic aeration (Solovchenko et al. 2013). An important characteristic of the photosynthetic apparatus, determining its functional activity, is the content of photosynthetic pigments, chlorophyll a and carotenoids in particular (Nezbrytskaya et al. 2015). The highest content of these pigments in the biomass of Ch. reinhardtii was recorded at the maximum concentration of N-NH₄⁺ (140 mg l⁻¹) and P-PO₄³⁻ (41 mg l⁻¹; Table 3). The results obtained indicate that the content of photosynthetic pigments and dry weight increase in Ch. reinhardtii with increasing concentration of nutrients, which indicates the intensification of the functional activity of the microalga and its resistance to a high load of inorganic nitrogen and phosphorus compounds in the aquatic environment. In the experiment variants with 30–90 mg N-NH₄⁺ l⁻¹ + 7–41 mg P-PO₄³⁻ l⁻¹, the content of chlorophyll a and carotenoids was almost at the same level (p > 0.05, Dunnett’s test) in terms of dry weight of algae.

In the biomass of the O. neglecta culture, a high content of chlorophyll a and carotenoids was recorded in the experiment variant with 30–50 mg N-NH₄⁺ l⁻¹ + 7–14 mg P-PO₄³⁻ l⁻¹ (Table 4). With increasing concentration of nutrients in blue-green algae, a noticeable decrease in the content of the main photosynthetic pigments (P ≤ 0.05, Dunnett’s test) was observed, which is consistent with a change in the growth rate and removal of nutrients. At the maximum concentration of nitrogen and phosphorus, the values of indicators of the content of chlorophyll a and carotenoids were even lower than their initial values, which indicates the death of culture cells.