Determination of biochemical and biological activities of Scenedesmus aequicaudatus algae cultivated in wastewater environment

Various wastewaters (domestic, industrial and agricultural) and substrates (dairy waste, cheese and sugar factory waste) are used as algal cultivation medium to preserve existing water reserves and minimize the cost of chemicals used. The basic needs for obtaining biomass through algae cultivation can be listed as high amounts of energy, water, carbon dioxide, macro and micronutrients. In order to obtain economic benefits from algal biomass production, the inputs used in the production process must be low-cost and the targeted raw material must be produced at an affordable price on a commercial scale. It is estimated that the cost varies from approximately $20 to $200 per kilogram, depending on the method used to produce microalgae biomass. For this reason, in order to reduce nutrient costs in recent years, suitable and low-cost sources such as municipal, industrial and agricultural wastewater have begun to be used as alternative nutrient salt sources. Since the nutrients in the wastewater are used by algae, the wastewater will also be cleaned, thus increasing environmental sustainability. Depending on the type of industry, the resulting wastewater may contain various pollutants such as minerals, volatile organic compounds, oil and grease, heavy metals and pesticides. Additionally, industrial and sewage wastewater is generally rich in nutrients. The accumulation of these pollutants in nature and their release into the environment can have serious negative effects on both freshwater and marine ecosystems. On the other hand, nutrients in wastewater serve as a nutrient source for microalgae, enabling the synthesis of biochemical components in their cells. While wastewater-based algae production offers a sustainable approach in terms of energy, it can also bring many advantages such as obtaining products with high economic value, environmental recovery and pollution control. This integrated approach not only supports economic and environmental sustainability, but also helps reduce environmental pollution by contributing to waste management policies. This way of evaluating wastewater offers a more cost-effective and environmentally friendly alternative to traditional treatment methods. Microalgae also have the potential for bioremediation thanks to their ability to absorb and remove some heavy metals and toxic compounds from the environment. In this respect, microalgae-based production systems offer a dual benefit in terms of both waste disposal and the acquisition of valuable products. In addition, recent studies show that microalgae grown in wastewater can be used in different areas such as biofuel, animal feed and bioplastic by optimizing their lipid, protein and carbohydrate contents. Thus, not only the environmental but also the commercial potential of these systems come to the fore. In the future, with the further development of microalgae-based production technologies, it is expected that applications for the evaluation of wastewater will become widespread In this study, we focused on wastewater-supported algae production in order to minimize algae production costs and increase environmental sustainability. It is seen that research is being carried out on biochemical contents, biological activities and biodiesel production potential using various microalgae species on earth. In this study, Scenedesmus aequicaudatus alga, which has not been studied before, was isolated from Sapanca Lake and its growth and development parameters were investigated in the culture medium prepared with wastewater taken from Karaman Wastewater Treatment Plant within the borders of Sakarya province. The collected wastewater samples were filtered using Whatman filter paper and then sterilized by autoclaving at 121°C for 20 minutes. Then it was passed through ordinary filter paper and mixed homogeneously in a 100-liter water tank. Before starting the 10-day measurements, initial chemical analyses (NO2-N, NO3-N, PO4-P) were performed to determine inorganic nutrient removal using the standard methods (APHA, 2012). 6 different culture medium were used as 100% BG11, 80 BG11- 20% Wastewater, 60% BG11- 40% Wastewater, 40% BG11- 60% Wastewater and 20% BG11- 80% Wastewater and 100% Wastewater. Prepared culture media were incubated in the growth cabinet at temperature: 25±1°C, illumination: 93 μmol photon m-2 s-1, photoperiod: 12 hours light: 12 hours dark. 100% BG11 medium was used as the control group in the study. The growth parameters of Scenedesmus aequicaudatus algae (OD680, chl-a, pH) were followed for 10 days in 100% BG11, 80% BG11- 20% Wastewater, 60% BG11- 40% Wastewater, 40% BG11- 60% Wastewater and 20% BG11- 80% Wastewater and 100% Wastewater environments. After the daily measurements were completed, final chemical analyses were performed to compare with the initial chemical analyses. Total protein (Bradford, 1976), total carbohydrate (Kochert, 1978) and chlorophyll-a (chl-a), chlorophyll (chl-b), total carotenoid values were determined from the dry biomass obtained on the 10th day. In addition, total phenolic content, 1,1-diphenyl-2-picrylhydrazine (DPPH) radical scavenging activity and antibacterial activity were determined from ethanolic extracts prepared by the extraction method. Also, the biodiesel potential of this algae was investigated by lipid extraction to determine total lipid and fatty acid content. Initial and final chemical analyses were carried out in a laboratory environment using standard methods (APHA, 2012). OD680 values were determined by spectrophotometric measurements for 10 days. Similarly, kl-a values were obtained using a spectrophotometer for 10 days. pH values were determined using a desktop pH meter (HI 2211pH-HANNA) for 10 days. Total protein values of Scenedesmus aequicaudatus algae grown in 6 different culture media were determined using the Bradford (1976) method. The Phenol-Sulfuric Acid Method (Kochert, 1975) was used to determine total carbohydrate values. Total pigment values (kl-a, kl-b and total carotenoid) were determined by spectrophotometric methods according to Tavakoli et al. (2021). While determining the total phenolic substance amount, 1,1-diphenyl-2-picrylhydrazine (DPPH) radical scavenging activity and antibacterial activity, ultrasound-assisted extraction method was first applied. The samples passed through the ultrasonic bath were filtered with filter paper to separate the solid particles. The obtained liquid samples were rotated in a rotary evaporator under vacuum at 50 ºC to remove the solvent. The extracts obtained after the evaporation process were stored as stock (at a concentration of 10 mg mL-1) at +4 ºC. Total phenolic substance analysis using these extracts was carried out using the Folin-Ciocalteu Method. 1,1-diphenyl-2-picrylhydrazine (DPPH) radical scavenging activity was carried out by the Blois method (Blois, 1958). Antibacterial activity was determined using the disk diffusion method. The maximum values in the growth parameters (OD680, chl-a and pH) of Scenedesmus aequicaudatus grown in six different culture medium were determined as 80% BG11-20% Wastewater, 80% BG11-20% Wastewater, 60% BG11-40% Wastewater (pH=11,61) and 80% BG11-20% Wastewater (pH=11,6), respectively. In our 10-day study, since algae prefer ammonia as their primary nitrogen source, no removal was observed in any culture medium depending on the duration of the experiment in nitrite and nitrate concentrations, while the most effective PO4-P removal was determined in the 100% Wastewater medium. The highest and lowest values in pigment amounts (chl-a, chl-b and total carotenoids) in dry biomass were observed as 60% BG11-40% Wastewater, 80% BG11-20% Wastewater and 40% BG11-60% Wastewater, respectively, after the control group (100% BG11). It was determined that algae grown in culture media diluted with wastewater at certain ratios had the highest biomass amount (80% BG11-20% Wastewater), total protein (60% BG11-40% Wastewater) and total carbohydrate (60% BG11-40% Wastewater) percentages. It was determined that the highest DPPH scavenging effect at all concentrations was in 60% BG11-40% wastewater medium with the same pH, chl-a, total protein and total carbohydrate amount. The total phenolic amount was observed to be the highest in the 40% BG11-60% wastewater environment, similar to the carotenoid amounts, after the control group. Depending on the cetane number, iodine value and saponification amounts, it was determined that biomass obtained from 60%BG11-40% Wastewater and 20%BG11-80% Wastewater environments had biodiesel potential. It was determined that ethanolic extracts prepared from biomass in different environments had no antibacterial effect on the test bacteria used in the study (Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Salmonella typhimurium ATCC 14028, Escherichia coli ATCC 25922 and Staphylococcus epidermidis ATCC 12228). In conclusion, this study has demonstrated the usability of this species isolated from Sapanca Lake in the field of algae production by supporting the wastewater medium with standard nutrient medium in attempts to improve the high costs of algae cultivation, and the biochemical components of this species have been introduced to the literature. In particular, the fatty acid profile and biological activities of S. aequicaudatus have been documented for the first time. S. aequicaudatus has been found to have significant potential for the production of food products such as edible oils, protein and starch.