Bioremediation of urban river wastewater using Chlorella vulgaris microalgae to generate biomass with potential for biodiesel production

The production of biofuels through microalgae biomass represents a new generation of raw materials from renewable sources to meet society’s clamors and growing insertion in the market of fuels from products that could grant the planet a sustainable future. The present study assesses the biomass obtained from microalgae Chlorella vulgaris when grown in urban wastewater, extracting the lipids from the biomass and performing Gas Chromatography analysis of Fatty acid Methyl Esters (FAME) composition after submitting the lipids through the transesterification process. The microalgae cultivation was monitored through chlorophyll Research, Society and Development, v. 9, n. 7, e823974882, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i7.4882 2 (a) analysis and the highest cell growth was 845.8 μg L using urban wastewater as growth medium. The nutrients of interest were monitored for primary concentration of 8.06 ± 0.06 mg L of ammoniacal nitrogen, 12.27 ± 0.27 mg L of nitrate and 21.22 ± 0.85 mg L of phosphate, reducing about 99% of ammoniacal nitrogen and nitrate, along with reducing 87% of phosphate. The lipid constitution extracted from 3.7 g of dry biomass of Chlorella vulgaris after cultivation using urban wastewater, was 7.7%. The lipids extracted from the Chlorella vulgaris biomass are suitable biodiesel production regarding the amounts of FAMEs identified, after the analysis carried out, the comparison of the results obtained with other studies and the hypotheses evaluation.

conditions for this species of microalgae (Lam et al., 2017). To analyses the efficiency of removal of phosphate, ammoniacal nitrogen and nitrate, was made through by Equation 1 and 2, whereas Nf refers to the final reading the total quantity of nitrogen in the sample and Ni the initial reading. Pf refers to the final reading of the total quantity of phosphorus and the Pi refers to initial reading (Nayak et al., 2016). (2)

Measurement microalgae growth
The monitoring microalgae growth, was determined the pigment concentration (chlorophyll (a)) using spectrophotometer (Agilent UV-VIS) to record the absorbance values with selected 630, 647, 664 and 750nm wavelength by Standard methods -10200 H (Jenkins, 1982;APHA, 2012). The monitoring was planned in six-time intervals being the 1 st day, 6 th day, 9 th day, 15 th day, 21 st day and 30 th day, because is the importance of monitoring the nutrients removal, nitrogen and phosphorus, to wastewater treatment and also check what day will have the microalgae growth.

Collecting microalgae biomass
After the Chlorella vulgaris microalgae reached the stationary growth phase, the experiment was stopped, and the samples were centrifuged with a Baby I model 206 BL (Fanem) centrifuge at 2800 rpm for 15 minutes. The supernatant was removed using a Pasteur pipette for further analysis. The accumulated biomass collected in the test tubes was stored under refrigeration for freezing until the start of the cold drying phase.

Biomass drying
Using a L108 model (Liotop) lyophilizer, with a total capacity of 8.0 Kg and 5.0 Kg of ice/24h, the previously frozen samples were subjected to the process of freeze-drying during the period of 5 days, when, after removed from the freeze-dryer, were weighted, totalizing a mass of 3.7 g, and stored in a dissector for the lipid extraction step. Fisatom heating blanket, model 22E. To obtain a continuous flow of biomass washing with the reagent, a refrigeration system using condensers was used.

Oil extraction of dry biomass
Using a temperature of 70 ºC, the extraction lasted 3 hours. After extraction, the flask was taken to a Buchi rotary evaporator, model R-210, to remove excess reagent.
After drying, the oil was collected and transferred to a 1.5 ml vial previously weighed and deposited in the hood, where it remained for 24 hours, so that any remaining reagent could be evaporated. At the end of 24 hours, the flask containing the extracted oil was weighed again to quantify the mass of the oil. After extraction, the lipid content was calculated using the following Equation 5:

Analysis of lipid extracted
To evaluate the composition of the fatty acid methyl esters (FAME) was performed by gas chromatography. The oil extracted from the dry biomass sample, went through a transesterification process which uses as a catalyst a solution of 2 molar NaOH dissolved in methanol, creating the ion methoxide necessary for the reaction that allows in the production of biodiesel. The alcohol used in the reaction was Heptane (

Statistical analysis
The experiment was carried out in triplicate with mean, standard deviation and one-way analysis of variance (ANOVA) with a significance level of p<0.05 was used for statistical analysis. The analyses were conducted using BioEstat 5.3 software. The chart was built in Microsoft Excel Software (2010)

Nutrients removal of urban river wastewater
The values of the initial and final concentrations of nutrients phosphate, ammoniacal nitrogen and nitrate from urban rivers wastewater, analyzed with mean in triplicate and standard deviation (Table 1). Table 1 show that, at the end of the experiment, the concentrations of ammoniacal nitrogen and nitrate in urban wastewater diluted in distilled water reached the final concentration <0.05 mg L -1 . Research, Society and Development, v. 9, n. 7, e823974882, 2020 (CC BY 4. This proportion was initially analyzed by Redfield (1958) who claims that there is an absorption ratio phosphate and nitrogen for microorganisms of 1 P: 16 N moles. Other researchers have evaluated the variation of this proportion for a good treatment of urban wastewater using microalgae, contribute to a variation between 1 P: 9 N and 1 P: 18 N moles (Xin et al., 2010). In this research, where the phosphate presented higher concentrations in relation to nitrogen, 1 P: 0, 58N was evaluated, a proportion well below the recommended which explains the non-removal of 100% phosphate. The research carried out by Khanzada (2020) highlights that it is required to have a balance between the concentrations of nutrients and synthetic forms can be added, this process significantly improved the growth of microalgae in wastewater.
In the research by Álvarez-Díaz et al. (2017) seven different species of microalgae were grown in urban wastewater to assess the potential for nutrient removal and biodiesel generation.

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The species Chlorella vulgaris was the first to completely remove the phosphate (about 1.5 mg L -1 ) compared to the others. As phosphate concentrations were less than ideal, total nitrogen removal has not been achieved, so it is indicated that it can be complemented with other wastewater with higher phosphate concentrations (Álvarez-Díaz et al., 2017). The initial value of ammoniacal nitrogen in wastewater from contaminated urban rivers was 8.06 ± 0.06 mg L -1 , reaching a final result of <0.05 mg L -1 , which would be below the detection limit of the equipment. This removal of 100% ammoniacal nitrogen from the wastewater was achieved in just 9 days of cultivation with microalgae for concentrations of 0%, 25% and 50% of urban waste water. Already the concentrations of 75% and 100% of urban waste water, the ammoniacal nitrogen was completely removed in 15 days of cultivation ( Figure   2). This removal was also observed in the prodution of Iasimone et al. (2018) cultivated microalgae in sewage and ammoniacal nitrogen removal has been reported to present as a result of removal in about 80% of an initial concentration of 30.2 ± 1.7 mg L -1 .
The absorption of ammoniacal nitrogen and nitrate occurs during the exponential growth phase and is not necessarily influenced by light intensity, but rather by the initial concentration of these nutrients (Iasimone et al., 2018). In the research by Mujtaba & Lee (2017), the efficiency of removing ammoniacal nitrogen from wastewater was around 80% where concentrations were between 13 to 21 mg L -1 , the same species was used for the treatment, Development, v. 9, n. 7, e823974882, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i7.4882 13 proving that this microalgae is capable of treating urban wastewater and consequently bioremediation of contaminated urban rivers.
The initial nitrate value for urban wastewater was 12.27 ± 0.27 mg L -1 . After 15 days of microalgae cultivation in urban wastewater, total removal of nitrate occurred, reaching <0.05 mg L -1 (Figure 3). Other studies have achieved nitrate removal around 67% in 11 days of microalgae cultivation in urban wastewater, where the need to increase the cultivation time to optimize removal efficiency has been observed (Kumar et al., 2019). The concentrations of 0% and 25% of urban wastewater, the nitrate removal efficiency was around 94% and 89%, respectively, in just 9 days of cultivation.
Microalgae have priority in the removal of ammoniacal nitrogen, as it is the reduced form of nitrogen and its assimilation is easier, which justifies the faster removal of ammoniacal nitrogen in relation to nitrate (Delgadillo-Mirquez et al., 2016). Figure 3 shows a slight increase in the concentration of nitrate in the experiment containing 75% of urban wastewater on the 9th day of cultivation. This increase is explained by the fact that microalgae in the intracellular metabolic process oxidize nitrogen to form nitrate (Sanz-Luque et al., 2015). Anyhow, shortly after, nitrate was completely consumed in 15 days. It is worth mentioning that the nitrifying bacteria that make this biological process in urban wastewater, however, all samples were sterilized in an autoclave, avoiding the existence of any type of bacteria, which highlights the exclusive action of microalgae. Source: Authors, 2020. Research, Society and Development, v. 9, n. 7, e823974882, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i7.4882 For treatments, experiments containing 0%, 25% and 50% of urban wastewater, this process was not evident, since all ammoniacal nitrogen had already been removed, leaving only the nutritive nitrate consumed. Other studies considered the removal of ammoniacal nitrogen and nitrate with efficiency in 11 days as well, but reinforce that to obtain a complete treatment with greater efficiency it is necessary to extend the cultivation process. The duration of treatment also depends on the type of microalgae species used and the specific growth rate (Samorì et al., 2013). In general, the microalgae were efficient in removing the nutrients proposed in this research, proving its use for the treatment of urban wastewater as a way of bioremediation and cleaning up urban rivers in cities that are important mainly for public health.
The response to the removal of nitrogen and phosphorus by the microalgae is already achieved in 7 days, presenting about 21% despite noting that the longer the cultivation time the better and more efficient is the removal of nutrients by the microalgae (Marques et al., 2017).

Microalgae growth and biomass production
The growth monitoring of the microalgae Chlorella vulgaris was done during 30 days of experiment, through the analysis of the chlorophyll pigment concentration (Figure 4). The first days corresponds to the phase of acclimatization of the microalgae with the new culture medium, since when they were propagated it was in the stationary phase. The greatest growth observed was in 100% of urban wastewater reaching the maximum of cultivation with 845.82