Growth of microalgae adapted to high-light temperature conditions in different types of wastewater

There are several processes for urban wastewater treatment including the conventional activated sludge (Elkamah et al. 2011), membrane bioreactor (Abdel-Shafy & Abdel-Shafy 2017), biofilm reactor (Elkamah et al. 2016), sequencing batch reactor (Ozgun et al. 2013), as well as other simpler techniques such as constructed wetlands (Abdel-Shafy et al. 2022), sewage land infiltration (Abdel-Shafy & Mansour 2020), oxidation ponds, and oxidation ditches (Abdel-Shafy & Salem 2007; Arita et al. 2015). All these processes need a high capital expenditure, energy consumable, sludge management costs, as well as operation and maintenance costs (Wang et al. 2017). In addition, the treatment processes are not completely efficient in removing the nutrient elements including nitrogen (as total nitrogen (TN)) and total phosphorus (TP) from wastewater which remain at elevated concentrations even in the treated wastewater. Likewise, in China's wastewater treatment plants, only 37% of ammonium-nitrogen (⁠⁠) was removed during 2012, leaving enough amounts of to cause eutrophication problems (Ozgun et al. 2013). Therefore, the removal of such nutrient elements (i.e. nitrogen and phosphorus) is the key to eutrophication prevention and water pollution control. Presently, the implemented processes for the removal of nitrogen and phosphorus from wastewater are based essentially on physical, chemical, and biological treatment. All these processes are very costly, and can only decrease but not eliminate such nutrients elements from wastewater (Abdel-Shafy et al. 2011; Yang et al. 2020).

The use of microalgae for wastewater treatment has many advantages in removing the nutrient elements compared with the conventional wastewater treatment processes. The advantages are (i) lower cost as well as higher removal of such pollutants (Whitton et al. 2015), (ii) lower greenhouse gas emissions (Clarens et al. 2010), (iii) higher added value (Zamolloa et al. 2012; Raheem et al. 2015), and (iv) higher efficiency of autotrophic carbon fixation (Raheem et al. 2015; Sukačová et al. 2015).

A municipal wastewater treatment process using microalgae can be established in photo-bioreactors and/or high-rate algal ponds (Fayed et al. 1983; Whitton et al. 2015). The wastewater treatment via high-rate algal ponds requires a longer hydraulic retention time (HRT) of more than 1 day, to reach a satisfactory effluent quality as a result of the slow growth rate of such microalgae (Winkler & Straka 2019). However, decreasing HRT in a high-rate algal pond process from 6 to 2 days resulted in a decrease in the removal rate of and from 29 and 96% to 9 and 15%, respectively (Arcila & Buitrón 2016). It is worth mentioning that the presence of heterotrophic bacteria in domestic wastewater competes with the microalgae to consume organic substrates and nutrients (Zhang et al. 2019). Therefore, the bacterial contaminated micro-algal biomass has often low carbohydrate and lipid contents, thus lowering its value as a precursor for higher-value chemical production and/or livestock food (Li et al. 2018; Mu et al. 2021). To obtain high amounts of microalgae in comparison to bacteria, the algae species used for wastewater treatment must be adapted to these conditions. These are mainly ruled by the type of wastewater to treat but in the Mediterranean also by high temperature and high solar radiation.

The aim of the present investigation is to obtain microalgae which allow mass cultivation with treated wastewater as a culture medium at high-light intensity and temperature prevailing in the Mediterranean. A sample from the Nile River was used to identify and isolate respective strains. These were subsequently cultivated in laboratory in different types of wastewater. For comparison, high-light temperature (HLT) and low-light temperature (LLT) conditions were established in cultures in the laboratory. Growth rates, changes in pH values, and uptake of nutrients were followed over a period of 15 days to determine applicability for tertiary treatment of wastewater.

To obtain microalgae that are adaptable to high light and high temperature (HLT) and wastewater conditions, freshwater was collected from the River Nile during spring (May 2021) from the El-Giza Water Works in a plastic container of 5 L.

To isolate microalgae adapted to HLT, the sample was cultivated in 400-mL transparent glass bottles. LED lamps were used to maintain photosynthetic active radiation (PAR 400–700 nm) of 1,000 μmol photons m⁻² s⁻¹. Temperature was kept at 35 °C and at a constant purging with a mixture of air and CO₂ of 5 vol.%. Nutrient concentrations at the beginning of the cultivation were established at 300 mg N L⁻¹ and 50 mg P L⁻¹. For comparison, cultivations at LLT conditions were done at room temperature ∼25 °C under cool white fluorescent lamps at 24.3 μmol photons m⁻² s⁻¹, a constant purging with a mixture of air and 5 vol.% of CO₂ and nutrient concentrations of 300 mg N L⁻¹ and 50 mg P L⁻¹.

Samples taken daily from the HLT and LLT cultures were examined under an OLYMPUS CX41 microscope at 40× magnification. Species composition and dominance in the sample were thus semi-quantitatively determined during an incubation period of 15 days. Algal identification was carried out according to the main references used in phytoplankton identification (Streble & Krauter 2006). Scenedesmus arcuatus which became dominant under HLT conditions was isolated and used in growth experiments with treated wastewater as a culture medium. For isolation, batch sub-culturing was done by single cells of S. arcuatus that were picked from the culture and transferred into a 150-mL flask containing (EPA 2002) growth media for further cultivation for 2 days until exponential growth started. After two steps of batch sub-culturing, it was found that S. arcuatus was the only remaining species.

During the growth experiments, different types of primary treated wastewater (grey, black, and sewage) were used as culture medium. They were obtained from the pilot treatment plant of municipal wastewater at the Training Demonstration Centre (TDC) of the National Research Centre (NRC), Cairo, Egypt (Abdel-Shafy et al. 2019). For the present growth experiments, black water (BW) was collected from a piping system which samples the wastewater of the toilets of five apartments of a residential building. Grey water (GW) was sampled from a separate piping system of the same building which collects wastewater from baths, showers, hand wash basins, washing machines, dishwashers, and kitchen sinks. Sewage water was derived from a piping system of another residential building close by which collects and mixes all the wastewater produced. Prior to be used as a culture medium, the different types of wastewater were all subjected to sedimentation as a primary treatment to remove the particles (Table 1). Settling particles were removed from the wastewater by sedimentation. This was done in sedimentation chambers each of 1 m height, 1.0 m depth, and 0.9 m width, holding a volume of 0.7 m³. Three chambers were coupled in a way that the water passed the chambers successively within a period of 3 h (Abdel-Shafy et al. 2022).

HLT cultivation was done in triplicates in a cultivation system described by Krimech et al. (2022). The system consists of transparent glass tubes (D = 50 mm, L = 400 mm) holding 300 mL kept at a constant temperature of 32–35 °C established by a thermostat and a constant aeration with air enriched with ∼5 vol.% of CO₂. LED lamps were used to maintain photosynthetic active radiation (PAR 400–700 nm) of 1,000 μmol photons m⁻² s⁻¹. LLT cultivation was done in the same cultivation system at room temperature ∼25 °C, illumination with cool white fluorescent lamps at 24.3 μmol photons m⁻² s⁻¹ and aeration without the addition of CO₂. Culturing was done both for a period of 15 days and samples of about 10 mL were taken daily for analysis as described in the following.

Algae biomass was determined as dry weight after filtration of subsample on a glass fibre filter of a pore size of 0.45 μm and drying for 24 h at 55 °C.

Differences in growth were statistically analysed by two tailed t-test.

Nutrient consumption of the algae was determined by the percentage decrease of concentration of and inorganic N species (⁠⁠, ⁠, ⁠) determined according to APHA (2017) in the culture medium at the start of the experiment and algae biomass reached its maximum. COD was determined according to APHA (2017) and the elimination of COD was calculated from the difference in concentration between the start and the minimum COD concentration reached in the respective culture. Organic N (N_org) was calculated from total N (TN) determined after Kjeldahl digestion (APHA 2017) by subtraction of the inorganic N. The pH values were measured and monitored daily throughout the entire incubation period using (a Genway pH meter). For all analysis, standard deviations were calculated from the measurements obtained in triplicates.

In the water from the Nile River used as inoculum for the HLT and LLT experiments three algal groups were identified with 24 different species. Eleven species belonged to the Chlorophyta, three species to Cyanophyta, and 10 species to Bacillariophyta (Tables 2 and 3). During HLT and LLT cultivation, the community structure changed differently coupled to a very low increase of biomass from 0.007 to 0.14 g dw L⁻¹ and 0.007 to 0.01 g dw L⁻¹, respectively. During HLT, Bacillariophyta of the species Diatomae longatum, Melosira granulate, and Synedra ulna remained at high numbers during the first 4 days. At day 5, S. arcuatus and Scenedesmus obliquus started to overgrow other species and became the dominant species until the end of the experiment at day 15. In contrast, changes in species composition under LLT conditions were marked by a diverse algal species community during the whole period of cultivation of 15 days (Table 3). Dominance was observed only for the species of the Bacillariophyta, namely D. longatum, M. granulate, S. ulna, Nitzschia acicularis while species of the chlorophytae remained at lower numbers. From the experiments, it cannot be decided whether the differences in microalgae dominance established during HLT and LLT conditions were controlled by light or by temperature (Basu et al. 2013; Ling et al. 2020). For doing so, further studies are needed which should at least include the opposite combination (high light with low temperature and low light with high temperature).

Thus, for outdoor mass cultivation of microalgae, species are needed which grow both under high light and high temperature. Further studies on growth with wastewater as the culture medium are described in the following. The studies werefocused on S. arcuatus isolated during the HLT experiment.

The elimination of dissolved organic matter present in the different types of wastewater by microalgae was determined by the decrease in COD (Figure 2). The highest elimination percentages were coupled to the highest loads of COD and amounted to SW of up to 88%. At LLT, elimination of dissolved organic substances was lower than at HLT and reached between 50 and 86% in different wastewaters. These findings indicate that S. arcuatus is very efficient in mixotrophic growth and eliminates dissolved organic matter present in wastewater (Bakuei et al. 2015; Nowicka-Krawczyk et al. 2022).

During 9 days of incubation, ammonia and nitrite present in the different wastewaters were completely eliminated in the S. arcuatus cultures both at HLT and LLT (Table 5). Although bacterial processes like nitrification cannot be ruled out, the low concentrations of nitrite below 0.45 mg NO₂-N L⁻¹ during the start of experiments indicate that this was a minor process and that most of the ammonia was assimilated by the microalgae. Phosphorus and nitrate elimination at HLT reached up to 96 and 100% in GW and SW and was markedly lower in BW with 51 and 81%, respectively. These differences can be explained as follows: nutrients uptake in BW (blackwater) may be inhibited by the higher amounts of coloured substances, which might restrict the supply of light. In addition, the coloured substances can complex the nutrients, making them unavailable to the algae (Gonçalves et al. 2014).

Table 5

Consumption of nutrients from different types of wastewaters (GW = grey water, BW = black water, and SW = sewage water) during cultivation of Scenedesmus arcuatus under HLT and LLT conditions

.	Initial concentrations (mg L−1) .					Final concentrations (mg L−1) .					Elimination (%) .
Experiment .	.	.	Norg .	.	.	.	.	Norg .	.	.	NO3 .	PO4 .	Norg .
GW/HLT	64 ± 3	4.4 ± 0.2	40 ± 2.0	0.4 ± 0.02	275 ± 13	9.3 ± 0.5	0.00	34 ± 2	0.00	96 ± 5	85	65	15
GW/LLT	63 ± 3	4.6 ± 0.2	41 ± 2.1	0.45 ± 0.02	278 ± 14	19 ± 0.9	0.00	36 ± 2	0.00	238 ± 12	70	14	12
BW/HLT	12 ± 0.6	5.0 ± 0.2	105 ± 5.3	0.35 ± 0.018	25 ± 1.3	5.7 ± 0.3	0.00	83 ± 4	0.00	3.5 ± 0.2	51	86	21
BW/LLT	12 ± 0.7	6.3 ± 0.3	108 ± 5.4	0.38 ± 0.019	28 ± 1.3	8.1 ± 0.4	0.00	86 ± 4	0.00	6.5 ± 0.3	33	77	20
SW/HLT	3.1 ± 0.2	3.0 ± 0.1	95 ± 4.75	0.05 ± 0.002	6.9 ± 0.4	0.14 ± 0.01	0.00	81 ± 4	0.00	0.0	96	100	15
SW/LLT	3.8 ± 0.2	4.8 ± 0.2	97 ± 4.86	0.08 ± 0.004	7.8 ± 0.4	3.1 ± 0.2	0.00	83 ± 4	0.00	0.3 ± 0.015	19	96	14

.	Initial concentrations (mg L−1) .					Final concentrations (mg L−1) .					Elimination (%) .
Experiment .	.	.	Norg .	.	.	.	.	Norg .	.	.	NO3 .	PO4 .	Norg .
GW/HLT	64 ± 3	4.4 ± 0.2	40 ± 2.0	0.4 ± 0.02	275 ± 13	9.3 ± 0.5	0.00	34 ± 2	0.00	96 ± 5	85	65	15
GW/LLT	63 ± 3	4.6 ± 0.2	41 ± 2.1	0.45 ± 0.02	278 ± 14	19 ± 0.9	0.00	36 ± 2	0.00	238 ± 12	70	14	12
BW/HLT	12 ± 0.6	5.0 ± 0.2	105 ± 5.3	0.35 ± 0.018	25 ± 1.3	5.7 ± 0.3	0.00	83 ± 4	0.00	3.5 ± 0.2	51	86	21
BW/LLT	12 ± 0.7	6.3 ± 0.3	108 ± 5.4	0.38 ± 0.019	28 ± 1.3	8.1 ± 0.4	0.00	86 ± 4	0.00	6.5 ± 0.3	33	77	20
SW/HLT	3.1 ± 0.2	3.0 ± 0.1	95 ± 4.75	0.05 ± 0.002	6.9 ± 0.4	0.14 ± 0.01	0.00	81 ± 4	0.00	0.0	96	100	15
SW/LLT	3.8 ± 0.2	4.8 ± 0.2	97 ± 4.86	0.08 ± 0.004	7.8 ± 0.4	3.1 ± 0.2	0.00	83 ± 4	0.00	0.3 ± 0.015	19	96	14

Organic N was calculated from total N minus the sum of nitrate, nitrite, and ammonia (APHA 2017). Standard deviation (SD) values were calculated from triplicates.

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In accordance with the literature, elimination of nitrate and phosphorus at LLT was lower than at HLT and reached 70 and 96%, respectively (Sutherland et al. 2015). However, results reveal that even at LLT when autotrophic growth was limited by CO₂ and was mainly heterotrophic, N elimination of the inorganic nutrients could be nearly complete. This is because Scenedesmus was found to have a high capacity for heterotrophic growth (Msanne et al. 2020). Even more, recent literature work reveals that persistent micropollutants are eliminated in Acutodesmus obliquus cultures (former S. obliquus) by photolysis and microalgae activity (Reymann et al. 2020). In contrast, in the present study, elimination of the organic nitrogen (N_org) remained between 12 and 20% in all experiments and all types of wastewater. This elimination was much lower than the 65 and 90% of removal by biological processes reported in the literature (Zheng et al. 2021). Together with the results from COD elimination, it can be thus concluded that the wastewater samples taken for the study contained high amounts of nitrogen-rich organic substances which were persistent in degradation.

The green alga S. arcuatus was isolated under HLT conditions by dominance which ensured that the species was highly adapted to the climate conditions in the Mediterranean and was thus fit for outdoor cultivation. Its ability to grow in wastewater samples was tested by using GW, BW, and SW. No differences in growth were observed, and it could be thus concluded that all types of wastewaters favoured growth similarly at high rates when CO₂ was added as a nutrient. Without CO₂, growth was 1.5- to 2.7-fold lower and was maintained by the COD present in the wastewaters which was thus eliminated. Inorganic N and P present in the wastewater were efficiently eliminated by up to 100%. However, most of the organic N remained unaffected and elimination during incubation with S. arcuatus was below 20%. This study thus revealed that wastewater can be efficiently treated with algae to remove inorganic N, phosphorus, and COD. The present study indicates advantages in the reuse of treated wastewater for microalgae cultivation which allows both high production and a tertiary water treatment.

The work was funded within the projects ‘Development of the frame conditions for the establishment of an innovative water technology which couples anaerobic wastewater treatment and biomass production in a bioreactor in the Mediterranean region’ – number (31319)-FRAME, ERANETMED3-75 and ‘Towards Innovative and Green Water Reuse with Integrated Constructed Wetlands and Ferrate(VI) Treatment’ – number (42688) – supported in whole and in part by NAS and USAID, respectively.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.