Using microalga Coelastrella sp. to remove some nutrients from wastewater invitro

: Microalgae have been increasingly used for wastewater treatment due to their capacity to assimilate nutrients. Samples of wastewater were taken from the Erbil wastewater channel near Dhahibha village in northern Iraq. The microalga Coelastrella sp. was used in three doses (0.2, 1, and 2g. l -1 ) in this experiment for 21 days, samples were periodically (every 3 days) analyzed for physicochemical parameters such as pH, EC, Phosphate, Nitrate, and BOD 5 , in addition to, Chlorophyll a concentration. Results showed that the highest dose 2g.l -1 was the most effective dose for removing nutrients, confirmed by significant differences (p≤0.05) between all doses. The highest removal percentage was recorded for ammonium pass 96% followed by NO 3 with 95%, while BOD 5 ranged from 88.7 to 93.5%. Decreases in nutrients coincided with an increase in chlorophyll-a content with the highest biomass on the 17th day of the experiment reaching 1.43 mg. l -1 .


Introduction:
Overuse of water in Erbil Governorate as a result of overpopulation, urbanization, industrialization, and water misuse resulted in a deficiency of this valuable resource, prompting the search for an alternate resource, which turned out to be wastewater 1 . Domestic and industrial wastewater, as well as runoff rainwater, make up urban wastewater 2 . Appropriate treatment of urban wastewater is required to protect human health and the environment. Erbil's wastewater system is a combination of sanitary sewage and stormwater 3 . The amount of sewage discharge changes depending on the season; on average, during the dry season, it reaches 77760 m 3 .day -1 , while during the rainy season, it reaches 108000 m 3 .day -1 4 .
To prevent eutrophication, wastewater contains high quantities of nitrogen and phosphorus, which are routinely removed from the water. Nitrogen is released into the atmosphere by phosphorus precipitation and nitrification-denitrification processes, while phosphorus is wasted to the sludge 5 . Algae are common and necessary part of all stream ecosystems 6,7 . They are the main source of energy for many food web streams, carbon fixing from the atmosphere by photosynthesis and transporting it through the web via consumer pathways. Due to their ecological importance and distinguishing characteristics, algae are suitable as indicators of nutrient pollution and also as assessment endpoints for developing numeric nutrient criteria for water quality management purposes under the Clean Water Act 8,9 . Using microalgae for wastewater treatment is an old idea, and several researchers have devised methods for taking use of the algal rapid growth and nutrient-removal capabilities. The nutrient removal is due to the algae's assimilation of nutrients, although other nutrient stripping events, such as ammonia volatilization and phosphorus precipitation as a result of the algal high pH, also occur 10 . The appropriate nutrient concentration in any system is influenced by a number of factors, including microalgae species, current environmental conditions (light/temperature), the nature of the pilot system, and so on 11 . Treatment methods that combine carbon capture and wastewater treatment while emitting little or no carbon can be considered the most sustainable option 12 . Photosynthesis allows microalgae to produce biomass from light, CO2, nutrients, and water 13 . Microalgae cultivation often generally requires a large number of nutrients that are readily available in home wastewater 14 , and might potentially be obtained for free from wastewater. This can significantly lower the cost of microalgal cultivation, while also reducing pollution and conserving freshwater resources. Microalgal biomass, as a result, is more of a resource than a waste. As a result, utilizing microalgal biomass to treat household waste minimizes the need for sludge disposal and enhances the benefits of using microalgae to treat domestic waste. In order to develop an ecofriendlier wastewater treatment technique, this study attempts to analyze the efficacy of using golden microalga-Coelastrella sp. for nutrient removal in wastewater as a biological treatment.

Material method:
Cultivation of microalgae Coelastrella sp. The microalga, Coelastrella sp., were isolated from Environmental Science and Health Department laboratory according to a previous investigation conducted by 15 . After identification microscopically depending on 16,17 . The cells were coccoid, and elliptical until citriform. They appeared as unicellular microalgae or in few-celled aggregations. Cell size ranged between 8-10 µm in width and 9-14 µm in length 18 . Molecular identification was conducted through algal DNA extraction, amplification, sequencing, and comparing to the GenBank database, it was identified as Coelastrella sp. (MZ801742) 15 . The cells of Coelastrella sp. were cultured in BG11 broth medium in distilled water with light-emitting diode (LED) lamps at ambient temperature.

Experimental design of batch Cultivation
The water sample collection took place in Dhahibah village wastewater channel in Erbil north of Iraq. The wastewater samples were autoclaved for 15 minutes to eliminate bacteria and protozoa. The experiments were carried out in a batch reactor using 2L conical flasks. Each series of experiments began with 1200 mL of wastewater inoculated in flasks with pre-cultured Coelastrella sp. Three different fractions were prepared with different concentrations of air dried Coelastrella sp. to assess the efficiency of nutrient removal by Coelastrella sp. Wastewater with 0.2 g. l -1 Coelastrella sp. (run 1), wastewater with 1 g. l -1 Coelastrella sp. (run 2), wastewater with 2 g. l -1 Coelastrella sp (run 3). The experiments were carried out using municipal wastewater for 21 days at neutral and constant pH (7.5 ± 0.3), constant temperature of 25 ± 1°C, and at constant light intensity (3000 lux) 19 .

Analytical Methods
All of the sampling and measurements took place at the same time of day. The dry weight (air dried) of microalgae produced per liter (g. l -1 ) was used to compute biomass. The following approach was used to calculate the dry cell weight of the microalgal biomass: Before conducting the experiment, untreated wastewater was analyzed for physicochemical parameters. Once a day, 100 mL samples were taken and centrifuged at 3000 rpm. At a 21-day interval, the percentage of NO3, NO2, NH3-N, PO4-P, BOD5 elimination, and growth rate were calculated. Samples were taken from flasks every day at the same time and centrifuged to separate algae. The BOD5 was measured using standard methods at a certified laboratory 20 . All of the other analyses were used photometrically with a spectrometer (UV-Visible 1240; Tecator, Rodgau, Germany). The result of treatment measured as percentage according to this equation 21 . % Treatment=before treatment value-after treatment value/before treatment value×100 Estimation of chlorophyll-a concentration To calculate chlorophyll-a concentration, 10 mL of culture was centrifuged at 3000 rpm for 5 minutes. The pellet was mixed with 5 mL of diethyl ether. The mixture was centrifuged at 3000 rpm for 5 minutes, and the absorbance of the supernatant was measured with a UV spectrometer (UV-Visible 1240; Tecator, Rodgau, Germany) at 660 and 643 nm 22 .
Chlorophyll-a = (9.92* A660) -(0.77*A643) Statistical analysis: Factorial analysis for parameters was performed by using (SPSS version 25) program and Excel spreadsheets. The data were subjected to standard analysis of vari ance (Two-way ANOVA) and the Duncan test was used to compare means at a significant (P≤0.05) level 23 .

Result and discussion:
The micro-alga, Coelastrealla sp. was used for the treatment of wastewaters collected from the Erbil wastewater channel near Dhahibha village. Some physicochemical parameters such as pH, EC, BOD5, NO3, NO2, and PO4 of wastewater were analyzed before treatment as control and after treatment with algae species for 21 days. Before treatment (Control), the mean of initial values of pH, EC, BOD5, PO4, NH4, NO3, and NO2 were 8.09, 912 µS/cm, 775, 23.7, 42.23, 23.7, and 8.94 mg. l -1 , respectively (Figs. [1][2][3][4][5][6]. The pH value of wastewater at all doses (0.2g.l -1 , 1g.l -1 , and 2g.l -1 ) was steadily increased by utilizing Coelastrealla sp., reaching 8.45, 8.52, and 8.65 (Fig. 1) after 21 days of treatment. The enhanced photosynthetic activity of algae or the chemical composition of water may both contribute to the alkaline tendencies [24][25][26] . Photosynthesis-induced increases in pH can accelerate the removal of nutrients via ammonia stripping or phosphorous precipitation , 27 showed that the pH of the wastewater sample treated with Chlorella minutissima was altered from 8.01 to 8.82, while the pH of the wastewater sample treated with Scendesmus sp. was changed from 8.01 to 9.09. pH changes can have a variety of effects on algal metabolism and growth, including changing the equilibrium of inorganic carbon species, modifying the availability of nutrients, and impacting cell physiology directly 27 . High pH values during the treatment period and interpreted the research that the values of the exponent of hydrogen of the medium increase if the ratio of phosphorus absorption into energy that is derived from the process of photosynthesis or respiration as influenced by the absorption of phosphorus by many factors most important of pH, temperature, and light intensity as it was observed that the pH increase by precipitating phosphorus as influenced by the absorption of phosphorus by precipitating phosphorus 28,29 . Highest applied dose (2g.l -1 ) was more effective role for rises pH values than other doses. The amount of substances (salts, minerals, metals, calcium, and other organic and inorganic compounds) that have been dissolved in the liquid is referred to as total dissolved solids (TDS). Total solids levels that are too high or too low can reduce the efficiency of wastewater treatment plants as well as the operation of process industries that use raw water. TDS concentration levels in wastewater typically ranged from 250 to 850 mg. l -1 . TDS variations have the same pattern as EC variations 30 . Salt ions typically affect microalgal cells via the ion homeostasis mechanism 31 . Conductivity is a general indicator of water quality, especially as the number of dissolved salts changes during wastewater treatment processes; changes in total salt concentration cause a change in conductivity 32 . As shown in Fig. 2, the EC values decreased to 353, 368, and 373.5 µS.cm -1 which comprised (59.04, 59.64, and 61.2%) of removal percent respectively on the 21 st day of treatment. TDS ratio decreased from 668 mg. l -1 to 199.16 mg. l -1 run1, 185.7 mg. l -1 runn2, and 173.9 mg. l -1 run3 on the 21 st day. Generally, low level of salts may be facilitated photosynthesis by inorganic nutrient uptake and alkalinity tolerance 31 . The maximum reduction of TDS and EC by Coellastrela sp. occurred at the highest dose (2g.l -1 ) ( Table 1).
On the 21 st day of treatment, the maximal removal percentages of BOD5 were, 93.54, 90.32, and 88.70 % respectively 33 , found that BOD5 removal was 61.38 % on the 10 th day and 78.64 % in natural wastewater, and that percent removal with different algal doses (20g, 40g, 60g, 80g, 120g, and 140g) was 12, 69, 68, 44, 24, and zero, respectively. In contrast to 34 , for pure wastewater the maximum removal of BOD5 by C. vulgaris was 21.63%, while for 50% diluted wastewater, the maximum removal of BOD5 was 30.36%. The lowest dose of algae, 0.2g.l -1 , is the most effective amount for removing BOD5 (Fig. 3). As a result, even a small dose of C. vulgaris is sufficient to efficiently reduce BOD5 in wastewater. The most oxidized form of nitrogen compounds is nitrate. Nitrate determination aids in determining the nature and degree of oxidation in biological processes, as well as enhanced wastewater treatment 35 . In incubation of Coelastrealla sp. in wastewater showed that the minimum and maximum removal of NO3 was occurred on 3 rd day and 21 st day respectively. The lowest reduction of NO3 could be attributed to the nitrification process; algae prefer to assimilate N in the form of ammonia because it is a more passive and energy-efficient method of assimilation than nitrate uptake 26 , or maybe because some nutritional elements, such as nitrate, are gradually consumed as Coelastrealla sp. grows, nitrate concentrations reduce. Results came in accordance with 36 , which showed that Monoraphidium sp. removed NO3 in wastewater by 51% and 95% in 5 days ; 37 , determined that Isochrysis zhanjiangenesis removed 78% active phosphorous and 84.7% nitrate nitrogen in wastewater within 11 days; 38 , showed that Scenedesmus dimorphus, Selenastrum minutum, and Scenedesmus sp. removed phosphate between 91% to 99% in mixed municipal and industrial wastewater ; 25 , who reported that NO3 and PO4 removal were 87.6 %, and 90 %, respectively by Chlorella sp. in wastewater. On the 21st day of treatment, the highest removal percentages of nutrients NO2, NH4, and PO4 were 83.9, 96.6, and 93.1 %, respectively, at a 2g.l -1 dose (Fig. 7). The maximum removal of NO2 occurred on the 21 st day, possibly due to an increase in pH values caused by photosynthesis, which can accelerate the removal of nutrients via ammonia stripping or phosphorous precipitation, which increases phosphate adsorption on microalgal cells [39][40][41][42][43] , In wastewater, NH3-N, nitrate, nitrite, and simple organic nitrogen such as urea, acetic acid, and amino acids can be assimilated by microalgae 44,45 . Because microalgae cells require nitrogen for protein, nucleic acid, and phospholipid synthesis, microalgae growth is thought to be required for nitrogen removal via uptake, degradation, and sedimentation 46 , also the micro-algal denitrification and nitrification processes 47 .
Ammonia was the most preferable form of nitrogen used by microalgae with removal percent pass 96%. Same results were obtained by 48 .        Algae prefer ammonia to nitrate, and nitrate consumption does not occur until the ammonium has been nearly completely consumed. Previous research revealed a ranking of tolerance to high levels of ammonium/ammonia (39-1.2 mM), with the order of tolerance being: Chlorophyceae > Cyanophyceae, Dinophyceae, Bacillariophyceae, and Raphidophyceae 48 . These findings also demonstrate that ammonical nitrogen removal is preferable to nitrite and nitrate nitrogen removal. In comparison to nitrite, the remaining NH4-N concentration vanished quickly. After 9 days, the ammonium depletion from synthetic wastewater by C. vulgaris and Scenedesmus rubescens, respectively was comparable to experiments conducted by 49 , who described ammonia elimination between (79 and 100 %) after 188. 25 hrs. (about 8 days) , and 50 , who recorded ammonium removal efficiencies of 90% from agroindustrial wastewater after 9 days. In the current study, microalgae-Coelastrealla sp. removed more than 93% of PO4 from wastewater, which is a greater removal ratio than that reported by 51 , who found that Chlamydomonas sp. removed 33% of PO4 from wastewater. Because of its critical "light-harvesting" role in photosynthesis, chlorophyll is abundant in nature and critical to the survival of both the plant and animal kingdoms 52 (Fig. 8). On the 21 st day of the experiment, all treatments observe a considerable decline in biomass and chlorophyll-a, indicating that the algae had reached the stage of death, and the number of algal cells decreased to 0.86 mg. l -1 , 1.008 mg. l -1 , and 1.25 mg. l -1 , accordingly. The maximum chlorophyll-a concentration was reported at the end of the treatment period, which could be due to algae's high nutrient consumption for metabolism and growth 53 , with increases in pH values 54 . As the algal cell increases, the rate of nutrient increase, so algal biomass simply increases.

Conclusion:
Finally, algae are increasingly being used to assess the health and ecological quality of aquatic ecosystems because they are relatively inexpensive and easy to measure, and they are sensitive to nutrient pollution, making them a potentially beneficial indicator of ecosystem change. Therefore, algae participate to a significant reduction in the concentrations of all nutrients, accompanied by an increase in their biomass. Also, ammonium nitrogen is a more preferable form of nitrogen for algae and have higher removable percentage than other form of nitrogen. Considerable removal of BOD5 and dissolved salts (EC value) are observed.