# Utilization of Wastewater as a Nutritional Source for the Production of Algal Biomass

Authors: Sami-ullah Rather; MubarakAli Davoodbasha; Hisham S. Bamufleh; Hesham Alhumade; Usman Saeed; Aqeel Ahmad Taimoor; Aliyu Adebayo Sulaimon; Walid Al-Alaya; Azim M. Shariff
Journal: International Journal of Energy Research
 (2023)

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## Abstract

In general, sludge wastewater treatments from plants are often disposed of in landfills (60%) rather than recycled, and this eventually affects the environment. Microalgae cultivation in wastewater has recently emerged as an alternate method for successfully treating wastewater in an ecofriendly way. The present study explores the possibility of growing microalgae in sludge effluent derived from industries. Industrial sludge acts as the only nutritional supplement for algal growth. The amount of organic carbon might increase the amount of protein and carbohydrates used for the production of lipids. The batch culture of different ratios of sludge wastewater was compared. The features of algal growth and biodiesel generation were studied, as well as the nitrogen and phosphate removal rates. The lipid levels ofChlorella sp. produced in this medium were clearly superior to those grown on the BG11 medium. Furthermore, this study indicated that the removal of industrial sludge and wastewater, in the absence of other nutritional supplies, allows for an efficient culture of Chlorella sp., followed by biodiesel generation. This has significant research and industrial implications since it will enable Chlorella sp. production in a mixed waste culture medium without the need for additional nutritional sources.


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## Body

## 1. Introduction

Wastewater treatment is a global problem that cannot be controlled by merely a simple technology due to the wide range of scales and types of contaminants and regional circumstances involved [1, 2]. While water scarcity can affect entire regions, the most vulnerable and poor are the ones that suffer the most. As a result, water scarcity is often considered in terms of both physical and economic limits. The discharge of effluent from a variety of industries has been identified as a significant source of pollution in rivers and other bodies of water [3, 4]. Inorganic compounds found in wastewater include sulfur, arsenic, chloride, sodium, phosphate, bicarbonate, magnesium, calcium, and ammonium ions, as well as lipids, proteins, volatile acids, carbohydrates, and amino acids. Significant obstacles develop when a microbiological organism, biological BOD, toxicity, and nutritional availability are discovered altogether, and the removal of these substances occurs in multiple steps at a traditional wastewater treatment facility. Treatment of wastewater is a crucial step in lowering pollutants and harmful compounds in wastewater, and wastewater treatment pollutants are now frequently utilized in urban settings by physical, biological, and chemical methods processed at various stages to break down organic waste and minimize pathogen burdens [5]. Copper is generally found in larger amounts in wastewater because it is the most precious and widely utilized metal in a variety of industrial applications, such as metal polishing, electroplating, plastics, and etching [6].In recent decades, the amount of sludge produced has grown dramatically. By the end of 2013, China had 3508 wastewater treatment facilities, producing vast amounts of sewage sludge; however, only twenty-five percent of the sludge produced was correctly handled. In 2016, China had 5300 wastewater treatment plants, growing roughly thirty million tons of wet sludge (eighty percent moisture content) each year [7]. The use of liquid-phase biofuels in the transportation industry has grown rapidly in recent years, owing primarily to the reduction of greenhouse gas emissions. Biodiesel has now come to refer to a highly precise chemical modification of natural oils. Oilseed crops, including rapeseed and soybean oil, have been thoroughly researched as biodiesel sources by a number of scientists. Microalgal products range from human nutrition used in organic substances in livestock, feed used in medicines, dyes, and a variety of others as well as industrial use and implementation of energy. They grow quickly and offer a lipid fraction for biodiesel production. Most of the lipids found in microalgae are neutral lipids with modest unsaturation. As a reason, microalgae lipids may be able to take the place of fossil fuels in the future [8, 9].Microalgae have excellent nutrient removal efficiency for the quantities of nitrogen and phosphorus found in many wastewaters. Microalgae seem to be a better biodiesel source than existing plants since they are photosynthetic organisms that grow the fastest. Furthermore, they can endure extreme temperature conditions and pH levels and utilize CO2 more efficiently in their photosynthesizing activity. Microalgae do not need to be grown on agricultural land, but rather they can be grown on less appropriate agricultural land, and they can produce more biofuel oil than oilseed crops using less land and water [10]. Microalgae are unicellular, photosynthetic organisms that provide a rich food supply under watery settings. Their size can range from a few micrometers to a few hundred micrometers. Because of their rapid growth rate, short life cycle, and wide range of growing environments, microalgae are the most prevalent feedstock for biofuel production [11–13].They are well known for their capacity to efficiently absorb nutrients from wastewater, for their cultivation necessitates a lot of nitrogen and phosphorus. In addition, microalgae biomass efficiently treats wastewater, and it is a possible source of precious chemicals and other products [14, 15]. Furthermore, many microalgae have significantly greater lipid productivity than the best-producing oil crops, implying that algae make the most biodiesel and may be able to produce up to 200 times more energy per unit of surface area than crops do. Microalgal biomass technology is still in its infancy, and no industrialization systems capable of generating algal oil at the scale needed for biodiesel production exist at the moment [16, 17]. Using concentration gradient treatments, a method for growing microalgae in industrial sludge effluent is proposed in this work. The goal was to explore Chlorella sp. performance without the addition of other nutrients, to specify biomass and lipid productivity, and to measure nutrient removal capabilities. For the large-scale growth of algae, this innovative methodology might present a cost-effective and environmentally beneficial solution.


## 2. Materials and Methods

### 2.1. Culture Collection and Maintenance

The microalgal samples were obtained at the School of Life Sciences, B. S. Abdur Rahman Crescent Institute of Science and Technology, Chennai, Tamil Nadu, India. The collected samples were cultured in a BG11 medium for biomass production. The culture purity and morphological identification were examined under a microscope.


### 2.2. Treatment Strategies for Sludge Water

A quantum of sludge dissolved in water and green algal culture was taken at the OD value of 2.0. The sludge and culture were mixed with various ratios such as 95 : 5, 75 : 25, 50 : 50, and 25 : 75. The OD value (600 nm) was measured periodically using a UV-Vis spectrophotometer. The change of sludge color and odor was investigated every two days up to 20 days at the appropriate ratio. Further investigation and water analysis were carried out for comparison. The treatment strategy is described schematically in Figure1.Figure 1
Schematic representation of the sludge treatment strategy adopting microalgae.


### 2.3. Analytical Study on Sludge Treatment Using Microalgae

After the treatment of sludge with microalgae at various concentrations, an appropriate day of incubation was chosen for further analytical investigation. Preliminarily, water analysis was carried out with treated and untreated sludge water. Multiple parameters were considered for the investigation of industrial water, and their admissible limits as per the standard are tabulated in Table1. Nutrient removal was evaluated by analyzing N and P, using a water quality analyzer (Hach Co., CO, USA).Table 1
Various parameters used for the analysis of wastewater at admissible limits.
ParametersIndustrial sludge waterAdmissible limitTemperature68°C95°CColorBrown, yellowishBlackpH69TSS155 mg L−1330 mg L−1TDS250 mg L−1850 mg L−1Chloride745.25 ppm or mg L−140 to 50 mg L−1Sulfate250 mg L−1500 mg L−1DO6.5-8 mg L−14-5 ppmCOD100 mg L−160,000 mg L−1BOD10 mg L−1300 mg L−1


### 2.4. Extraction of Lipids from the Microalgae

For extracting the lipid from the microalgae, the culture was mixed with methanol/chloroform in the ratio of (1 : 2). After diffusion, the sample was kept in an orbital shaker for 10-15 min at room temperature. Then, the whole sample was heated up to 75°C for 1 hr using a water bath. The sample was filtrated using a filter paper. The collected mixture solvent was washed with 0.9% NaCl solution. After being shaken well, the sample was transferred into a separating funnel and stored for 20 min. After that, the sample was divided into two different phases, the upper and lower phases. The lipid part at the lower phase, finally, the extracted lipid, was collected in a Petri plate, and the solvent was evaporated; then, the lipid collection from the sample was weighed using an electronic balance [18].


### 2.5. Transesterification by the Conventional Method

150 mg of culture was taken in a test tube, and 3 ml of a catalyst such as saturated potassium hydroxide (KOH-CH3OH) in methanol solution was added; then, the sample was heated at 75°C for 10 min. After that, an acidic catalyst such as sulfuric acid (H2SO4) was added and heated at 75°C for another 10 min. After that, by using ethyl acetate, aqueous layers were formed; finally, FAME was collected and dried in a Petri dish, and then, the sample was characterized by gas chromatography mass spectroscopy (GC-MS) [19–21].


### 2.6. FAME Composition Analysis

After extracting the lipid from the microalgae, the transesterification procedure for the generation of biodiesel was carried out utilizing this microalgal lipid. The amount of FAME was created during the biodiesel manufacturing process following the transesterification process. Then, the ethyl acetate was used to extract FAME [22]. FAME offers favorable characteristics for usage as biofuels. The results revealed that the composition of FAME was influenced by nutrient types as well as culture methods [23]. The prepared FAME samples were analyzed by gas chromatography (GC; QP 2022, Agilent) using a flame ionization detector. Briefly, 1 μL of the sample was injected into an Ag-8890 column (Agilent, USA) (30 m and hold for 5 min). Helium (20 cm s−1) was the carrier gas, detector temperature was at 250°C, and split ratio was 5 : 1.


## 2.1. Culture Collection and Maintenance

The microalgal samples were obtained at the School of Life Sciences, B. S. Abdur Rahman Crescent Institute of Science and Technology, Chennai, Tamil Nadu, India. The collected samples were cultured in a BG11 medium for biomass production. The culture purity and morphological identification were examined under a microscope.


## 2.2. Treatment Strategies for Sludge Water

A quantum of sludge dissolved in water and green algal culture was taken at the OD value of 2.0. The sludge and culture were mixed with various ratios such as 95 : 5, 75 : 25, 50 : 50, and 25 : 75. The OD value (600 nm) was measured periodically using a UV-Vis spectrophotometer. The change of sludge color and odor was investigated every two days up to 20 days at the appropriate ratio. Further investigation and water analysis were carried out for comparison. The treatment strategy is described schematically in Figure1.Figure 1
Schematic representation of the sludge treatment strategy adopting microalgae.


## 2.3. Analytical Study on Sludge Treatment Using Microalgae

After the treatment of sludge with microalgae at various concentrations, an appropriate day of incubation was chosen for further analytical investigation. Preliminarily, water analysis was carried out with treated and untreated sludge water. Multiple parameters were considered for the investigation of industrial water, and their admissible limits as per the standard are tabulated in Table1. Nutrient removal was evaluated by analyzing N and P, using a water quality analyzer (Hach Co., CO, USA).Table 1
Various parameters used for the analysis of wastewater at admissible limits.
ParametersIndustrial sludge waterAdmissible limitTemperature68°C95°CColorBrown, yellowishBlackpH69TSS155 mg L−1330 mg L−1TDS250 mg L−1850 mg L−1Chloride745.25 ppm or mg L−140 to 50 mg L−1Sulfate250 mg L−1500 mg L−1DO6.5-8 mg L−14-5 ppmCOD100 mg L−160,000 mg L−1BOD10 mg L−1300 mg L−1


## 2.4. Extraction of Lipids from the Microalgae

For extracting the lipid from the microalgae, the culture was mixed with methanol/chloroform in the ratio of (1 : 2). After diffusion, the sample was kept in an orbital shaker for 10-15 min at room temperature. Then, the whole sample was heated up to 75°C for 1 hr using a water bath. The sample was filtrated using a filter paper. The collected mixture solvent was washed with 0.9% NaCl solution. After being shaken well, the sample was transferred into a separating funnel and stored for 20 min. After that, the sample was divided into two different phases, the upper and lower phases. The lipid part at the lower phase, finally, the extracted lipid, was collected in a Petri plate, and the solvent was evaporated; then, the lipid collection from the sample was weighed using an electronic balance [18].


## 2.5. Transesterification by the Conventional Method

150 mg of culture was taken in a test tube, and 3 ml of a catalyst such as saturated potassium hydroxide (KOH-CH3OH) in methanol solution was added; then, the sample was heated at 75°C for 10 min. After that, an acidic catalyst such as sulfuric acid (H2SO4) was added and heated at 75°C for another 10 min. After that, by using ethyl acetate, aqueous layers were formed; finally, FAME was collected and dried in a Petri dish, and then, the sample was characterized by gas chromatography mass spectroscopy (GC-MS) [19–21].


## 2.6. FAME Composition Analysis

After extracting the lipid from the microalgae, the transesterification procedure for the generation of biodiesel was carried out utilizing this microalgal lipid. The amount of FAME was created during the biodiesel manufacturing process following the transesterification process. Then, the ethyl acetate was used to extract FAME [22]. FAME offers favorable characteristics for usage as biofuels. The results revealed that the composition of FAME was influenced by nutrient types as well as culture methods [23]. The prepared FAME samples were analyzed by gas chromatography (GC; QP 2022, Agilent) using a flame ionization detector. Briefly, 1 μL of the sample was injected into an Ag-8890 column (Agilent, USA) (30 m and hold for 5 min). Helium (20 cm s−1) was the carrier gas, detector temperature was at 250°C, and split ratio was 5 : 1.


## 3. Results and Discussion

### 3.1. Isolation and Purification of Microalgae

The isolated microbial biomass was used for the wastewater treatment process. Generally, cyanobacteria produced from effluent could yield biomass for high-value compounds with the low-price performance of microalgae-based biodiesel production having been improved. Furthermore, the various uses of microalgae for pollutants, correct bioenergy generation, and precious component removal may assist in lowering the cost of traditional wastewater treatment. Wastewater is a complicated mixture of natural and artificial molecules, and its components can support microalgal growth via ammonium, phosphate, carbon, and magnesium, among others. Different types of wastewaters have been investigated for microalgal cultivation since the 1960s [24].During the experimentation period from 0 to 25 days of treatment time, the 16th day was found to be the optimum treatment time for biomass productivity and sludge treatment. The concentrations of microalgae biomass, pH, COD, phosphorous, nitrate, carbon, and oxygen were all measured. The volumetric biomass attentiveness of microalgae was at a rate of roughly 50 mg/L per day for the 125 mg/L and 250 mg/L samples and at 80 mg/L per day for the 500 mg/L samples. Microalgae require the addition of different carbons for development. As a result, all microalgae concentrations are predicted to rise because they were all given a carbon addition, glucose.Chlorella vulgaris can thrive in heterotrophic environments depending on the type of carbon availability. Some research claimed a high volumetric biomass growth rate of 2-4 days, while others reported a 14-day culture duration. Previous research has shown that a 14-day treatment using microalgae could remove 93.9% of ammonia, 89.1% of whole nitrogen, 80.9% of total phosphorus, and 90.8% of Chemical Oxygen Demand (COD) from raw wastewater [25]. For five days, wastewater treatment using microalgae was studied. In this test, three duplicates of microalgae wastewater were employed, and then, the pH was examined [26]. The effectiveness of the wastewater treatment process depends on the amount of carbon-based (or organic) chemicals present, particle solids that can dissolve or suspend in wastewater, targeted nutrients, and other physical characteristics. The Biochemical Oxygen Demand (BOD), Chemical Oxygen Demand (COD), Total Organic Carbon (TOC), and Oil and Grease (O&G) tests are used to assess the concentration of organic compounds, whereas particulate particles are evaluated by measuring Total Solids (TS), Total Suspended Solids (TSS), Total Dissolved Solids (TDS), Total Volatile Solids (TVS), and Total Fixed Solids (TFS). By monitoring the nitrogen and phosphorus content in the wastewater, it is possible to identify the targeted nutrients that influence the eutrophication’s acceleration. The effectiveness of wastewater treatment operations is also evaluated by measuring additional physical attributes like temperature, color, pH, turbidity, and odor.


### 3.2. Assessment of Microalgal Growth Cultivated in Sludge Wastewater

Different ratios were followed for analyzing the sludge that has been utilized by microalgae at various treatment times (0-20 days). On day zero, SW was 17.64 and S+C 75 : 25 was 36.11%. On day 2, SW was 17.28 and S+C 75 : 25 was 22.01%. On day 4, SW was 17.20 and S+C 75 : 25 was 32.64%. On day 8, SW was 17.91 and S+C 75 : 25 was 51.42%. On day 12, SW was 17.36 and S+C 75 : 25 was 51.20%. On day 16, SW was 17.15 and S+C 75 : 25 was 61.92%. Day 16 shows the better result in all concentrations.Chlorella has to owe to its great photosynthetic organization and great nutritional value. Chlorella sp. have demonstrated their excellent biosorption capabilities and effectiveness in removing contaminants from diverse aqueous solutions on several occasions. Chlorella vulgaris had a total phosphorous (TP) removal efficiency of roughly 85% and a total nitrogen (TN) removal efficiency of around 89%. Chlorella minutissima and Chlorella sorokiniana were demonstrated to remove up to 41 and 34% of TN, respectively, and up to 70% of the TP overall. Chlorella is a popular genus of microalgae for wastewater treatment because it adapts well to a wide range of wastewater and is particularly effective at eliminating a wide range of contaminants [27]. The outcomes of sludge treatment graphed in percent (%) are shown in Figure 2.Figure 2
Assessment of microalgal growth cultivated in the sludge wastewater.The graph depicts the percentage of water that has been cleansed or utilized by microalgae (day 1–day 20). On day zero, SW was 18.36 and S+C 75 : 25 was 28.05%. On day 2, SW was 18.89 and S+C 75 : 25 was 31.99%. On day 4, SW was 18.65 and S+C 75 : 25 was 34.64%. On day 8, SW was 17.91 and S+C 75 : 25 was 44.42%. On day 12, SW was 18.36 and S+C 75 : 25 was 59.20%. On day 16, SW was 16.15 and S+C 75 : 25 was 63.92%. On day 18, SW was 17.01 and S+C 75 : 25 was 70.48%. On day 20, SW was 17.03 and S+C 75 : 25 was 74.63%. Day 20 showed superior performance in all concentrations, but day 20 and day 16 are closer in ratio, so we chose day 16. As a result, day 16 is better for treating wastewater. Microalgae were shown to grow in various forms of wastewater, with high growth rates and biomass output. Due to the reasonably stable culture conditions and enough nutrient loadings (N and P) for microalgal growth, high biomass of microalgal cultures was achieved. According to the findings, PS concentration may be significantly involved in the growth of microalgal biomass, with the incremental growth in biomass most likely being brought on by the addition of N and P to the culture environment. The concentration of organics and metal ions, among many other things, can affect the growth of algae. The growth of microalga in sludge is significantly influenced by trace elements, including Mn, Fe, Zn, and Cu. [28–30].In this research, both 5% and 10% liquor media stimulated RTC growth, resulting in more significant cell application, biomass, and specific growth rate than those of the basal medium. The 10% liquor medium produced the most growth, with a final OD of 750 and biomass being 25.52% and 30.11% excessive, respectively, than that of the basal medium [24]. Raphidocelis subcapitata was effective in the elimination of E2 and diethylstilbestrol (DES), with removal efficiencies of 74.6% and 54.1%, respectively, after 96 hr at a starting concentration of 1.5 mg/L. Biodegradation/transformation was revealed to be the predominant removal mechanism for hormones by microalgae; however, it had no effect on microalgal cell development. This is because hormones primarily work in mammals and aquatic species (e.g., fish), both of which can be adversely influenced by hormones. Hormones, on the other hand, have no substantial inhibitory impact on microalgal development. However, biomagnification by the accumulation of altered products in microalgae is a significant challenge [31].The best sludge and algal ratios for successful sludge water utilization and biomass production were discovered to be 25 : 75 (C : S) because the sludge water should have a lower concentration (75 : 25 S+C) chosen. Therefore, microalgae were cleaned or used 67% of the water. According to this study, including microalgae production in wastewater treatment can result in a cost-effective and environmentally friendly wastewater treatment solution. Figure3 shows the determination of microalgae and sludge ratio for the effective treatment of algal growth. Based on these results and the experiment, the ratios (S+C 75 : 25) were similar to those found on day zero (S+C 75 : 25 was 29.99% and S+C 75 : 25 was 27.58%). Following these, on day 4, S+C 75 : 25 was 29.15% and S+C 75 : 25 was 28.67%; on day 8, S+C 75 : 25 was 45.74% and S+C 75 : 25 was 50.47%; and on day 16, S+C 75 : 25 was 67.04% and S+C 75 : 25 was 66.39%. The experiments revealed that microalgae could thrive in aquaculture effluents. The first trial was conducted in batch mode and lasted 33 days, ending when all nitrogen and phosphorus supplies were used and a decrease in the optical density of the cultures was detected. The goals of this experiment were studied, and the two microalgal species performed the best in terms of remediation and biomass production. Microalgae C. vulgaris and S. obliquus were able to accomplish restoration in 22 days, but the other microalgae took 33 days. When compared to the control, the biomass concentration attained after 22 days for the microalga Chlorella vulgaris cultured in aquaculture effluent (3.22±0.04 g L-1) was substantially greater (1.36±0.39 g L−1). The water analysis study was performed, and the results are shown in Table 2.Figure 3
Determination of microalgal and sludge ratio for the effective treatment of algal growth C+S (blue bar) 25 : 75 and C+S (red bar) 75 : 25.Table 2
Examination results of biochemical analysis in treated water.
ParametersIndustrial sludge waterAdmissible limitControlThis studyTemperature68o95°C——ColorBrown, yellowishBlackDarkGreenpH698.228.02Hardness155 mg/L330 mg/L10289TDS250 mg L−1850 mg L−1616221088Chloride745.25 ppm or mg/L40 to 50 mg/L——Sulfate250 mg/L500 mg/L4644DO6.5-8 mg/L4-5 ppm1.71.2COD100 mg/L60,000 mg/L160130.2BOD10 mg/L300 mg/L180110


### 3.3. Estimation of Lipid Content

A modified Folch technique was used to extract lipids from the microalgae consortia. Lipid was obtained by evaporating the solvent, and the total lipid collected from the sample was quantified using an electronic balance; then, the lipid content was weighed at26.66±0.5 mg/g.


### 3.4. FAME Profiling

FAME composition was determined and quantified by comparing retention periods and peak regions from the GC data. The results revealed that the composition of FAMEs was influenced by nutrient types as well as growing methods. The GC-MS profiling was identified and quantified by comparing the abundance percentage of the 14 different compounds of methyl ester, such as 2,4-di-tert-butylphenol; methyl tetradecanoate; methyl 13-methyltetradecanoate; neophytadiene; 2-pentadecanone,6,10,14-trimethyl; 3,7,11,15-tetramethyl-2-hexadecen-1-ol; (Z)-methyl hexadec-11-enoate; hexadecanoic acid; cyclopropanebutanoic acid,2-[[2-[[2-[(2-pentylcyclopropyl); phytyl, 2-methylbutanoate; 7,10-octadecadienoic acid, methyl ester; 10-octadecenoic acid; and heptadecanoic acid, 16-methyl, and their properties are mentioned in Tables3 and 4. Particularly, FAME with a high concentration of heptadecanoic acid has favorable qualities for usage as biofuels. This finding suggests that using microalgal biomass as an alternate carbon source can enhance both the quantity and quality of biodiesel. The fatty acid composition is based on saturation form. The aspect of biodiesel quality is connected to saturated bonds and carbon chain length. The quality of biodiesel improves as the proportion of polyunsaturated fatty acids (PUFA) decreases. Because of their high concentration of saturated and monounsaturated fatty acids, Chlorella sp. cells cultivated under autotrophic conditions seem to be a viable option for the generation of biodiesel (Figure 4). Taken together, our work has established a successful scenario in which algae combined with industrial sludge can be employed as a biodiesel-manufacturing technique. A further benefit of using Chlorella sp. culture is that it is a technology that can control microalgal waste through biological recycling in addition to producing biodiesel at a low cost. In addition, by using sludge waste as an alternative carbon source, agricultural land and freshwater resources might be considerably preserved. In light of the carbon cycle, Chlorella sp. can convert carbon sources into lipids with high lipid productivity using CO2 from air/flue gas and organic carbon from sludge waste.Table 3
FAME analysis and their properties were identified using GC-MS.
S. no.Methyl esterCarbon no.Abundance (%)CAS no.Type(1)UndecaneC1157.11120-21-4SFA(2)2,4-Di-tert-butylphenolC1419.196-76-4—(3)Methyl tetradecanoateC1574.0124-10-7SFA(4)Methyl 13-methyltetradecanoate74.0SFA(5)NeophytadieneC2068.1504-96-1—(6)2-Pentadecanone,6,10,14-trimethylC1858.0502-69-2—(7)3,7,11,15-Tetramethyl-2-hexadecen-1-olC2081.0102608-53-7—(8)(Z)-Methyl hexadec-11-enoateC1755.0822-05-9FAME(9)Hexadecanoic acid, methyl esterC1774.0112-39-0SFA(10)Cyclopropanebutanoic acid,2-[[2-[[2-[(2-pentylcyclopropyl)C2574.156051-53-7—(11)Phytyl, 2-methylbutanoate85.1—(12)7,10-Octadecadienoic acid, methyl esterC19101.056554-24-6SFA(13)10-Octadecenoic acid, methyl esterC1955.113481-95-3SFA(14)Heptadecanoic acid, 16-methyl, methyl esterC1974.15129-61-3SFATable 4
FAME analysis and their properties were identified using GC-MS.
S. no.Compound nameLinear formulaMolecular weight (g/mol)(1)UndecaneCH3(CH2)9CH3156.31(2)2,4-Di-tert-butylphenol[(CH3)3C]2C6H3OH206.32(3)Methyl tetradecanoateCH3(CH2)12COOCH3242.40(5)NeophytadieneC20H38278.5(6)2-Pentadecanone,6,10,14-trimethylC18H36O268.5(7)3,7,11,15-Tetramethyl-2-hexadecen-1-olC20H40O296.5(8)(Z)-Methyl hexadec-11-enoateC17H32O2268.4(9)Hexadecanoic acid, methyl esterCH3(CH2)14CO2CH3270.45(10)Cyclopropanebutanoic acid,2-[[2-[[2-[(2-pentylcyclopropyl)C25H42O2374.6(12)7,10-Octadecadienoic acid, methyl esterC19H34O2294.5(13)10-Octadecenoic acid, methyl esterC19H36O2296.5(14)Heptadecanoic acid, 16-methyl, methyl esterC19H38O2298.5Figure 4
GC analysis showing the fatty acid methyl esters at various carbon lengths and their derivatives.


## 3.1. Isolation and Purification of Microalgae

The isolated microbial biomass was used for the wastewater treatment process. Generally, cyanobacteria produced from effluent could yield biomass for high-value compounds with the low-price performance of microalgae-based biodiesel production having been improved. Furthermore, the various uses of microalgae for pollutants, correct bioenergy generation, and precious component removal may assist in lowering the cost of traditional wastewater treatment. Wastewater is a complicated mixture of natural and artificial molecules, and its components can support microalgal growth via ammonium, phosphate, carbon, and magnesium, among others. Different types of wastewaters have been investigated for microalgal cultivation since the 1960s [24].During the experimentation period from 0 to 25 days of treatment time, the 16th day was found to be the optimum treatment time for biomass productivity and sludge treatment. The concentrations of microalgae biomass, pH, COD, phosphorous, nitrate, carbon, and oxygen were all measured. The volumetric biomass attentiveness of microalgae was at a rate of roughly 50 mg/L per day for the 125 mg/L and 250 mg/L samples and at 80 mg/L per day for the 500 mg/L samples. Microalgae require the addition of different carbons for development. As a result, all microalgae concentrations are predicted to rise because they were all given a carbon addition, glucose.Chlorella vulgaris can thrive in heterotrophic environments depending on the type of carbon availability. Some research claimed a high volumetric biomass growth rate of 2-4 days, while others reported a 14-day culture duration. Previous research has shown that a 14-day treatment using microalgae could remove 93.9% of ammonia, 89.1% of whole nitrogen, 80.9% of total phosphorus, and 90.8% of Chemical Oxygen Demand (COD) from raw wastewater [25]. For five days, wastewater treatment using microalgae was studied. In this test, three duplicates of microalgae wastewater were employed, and then, the pH was examined [26]. The effectiveness of the wastewater treatment process depends on the amount of carbon-based (or organic) chemicals present, particle solids that can dissolve or suspend in wastewater, targeted nutrients, and other physical characteristics. The Biochemical Oxygen Demand (BOD), Chemical Oxygen Demand (COD), Total Organic Carbon (TOC), and Oil and Grease (O&G) tests are used to assess the concentration of organic compounds, whereas particulate particles are evaluated by measuring Total Solids (TS), Total Suspended Solids (TSS), Total Dissolved Solids (TDS), Total Volatile Solids (TVS), and Total Fixed Solids (TFS). By monitoring the nitrogen and phosphorus content in the wastewater, it is possible to identify the targeted nutrients that influence the eutrophication’s acceleration. The effectiveness of wastewater treatment operations is also evaluated by measuring additional physical attributes like temperature, color, pH, turbidity, and odor.


## 3.2. Assessment of Microalgal Growth Cultivated in Sludge Wastewater

Different ratios were followed for analyzing the sludge that has been utilized by microalgae at various treatment times (0-20 days). On day zero, SW was 17.64 and S+C 75 : 25 was 36.11%. On day 2, SW was 17.28 and S+C 75 : 25 was 22.01%. On day 4, SW was 17.20 and S+C 75 : 25 was 32.64%. On day 8, SW was 17.91 and S+C 75 : 25 was 51.42%. On day 12, SW was 17.36 and S+C 75 : 25 was 51.20%. On day 16, SW was 17.15 and S+C 75 : 25 was 61.92%. Day 16 shows the better result in all concentrations.Chlorella has to owe to its great photosynthetic organization and great nutritional value. Chlorella sp. have demonstrated their excellent biosorption capabilities and effectiveness in removing contaminants from diverse aqueous solutions on several occasions. Chlorella vulgaris had a total phosphorous (TP) removal efficiency of roughly 85% and a total nitrogen (TN) removal efficiency of around 89%. Chlorella minutissima and Chlorella sorokiniana were demonstrated to remove up to 41 and 34% of TN, respectively, and up to 70% of the TP overall. Chlorella is a popular genus of microalgae for wastewater treatment because it adapts well to a wide range of wastewater and is particularly effective at eliminating a wide range of contaminants [27]. The outcomes of sludge treatment graphed in percent (%) are shown in Figure 2.Figure 2
Assessment of microalgal growth cultivated in the sludge wastewater.The graph depicts the percentage of water that has been cleansed or utilized by microalgae (day 1–day 20). On day zero, SW was 18.36 and S+C 75 : 25 was 28.05%. On day 2, SW was 18.89 and S+C 75 : 25 was 31.99%. On day 4, SW was 18.65 and S+C 75 : 25 was 34.64%. On day 8, SW was 17.91 and S+C 75 : 25 was 44.42%. On day 12, SW was 18.36 and S+C 75 : 25 was 59.20%. On day 16, SW was 16.15 and S+C 75 : 25 was 63.92%. On day 18, SW was 17.01 and S+C 75 : 25 was 70.48%. On day 20, SW was 17.03 and S+C 75 : 25 was 74.63%. Day 20 showed superior performance in all concentrations, but day 20 and day 16 are closer in ratio, so we chose day 16. As a result, day 16 is better for treating wastewater. Microalgae were shown to grow in various forms of wastewater, with high growth rates and biomass output. Due to the reasonably stable culture conditions and enough nutrient loadings (N and P) for microalgal growth, high biomass of microalgal cultures was achieved. According to the findings, PS concentration may be significantly involved in the growth of microalgal biomass, with the incremental growth in biomass most likely being brought on by the addition of N and P to the culture environment. The concentration of organics and metal ions, among many other things, can affect the growth of algae. The growth of microalga in sludge is significantly influenced by trace elements, including Mn, Fe, Zn, and Cu. [28–30].In this research, both 5% and 10% liquor media stimulated RTC growth, resulting in more significant cell application, biomass, and specific growth rate than those of the basal medium. The 10% liquor medium produced the most growth, with a final OD of 750 and biomass being 25.52% and 30.11% excessive, respectively, than that of the basal medium [24]. Raphidocelis subcapitata was effective in the elimination of E2 and diethylstilbestrol (DES), with removal efficiencies of 74.6% and 54.1%, respectively, after 96 hr at a starting concentration of 1.5 mg/L. Biodegradation/transformation was revealed to be the predominant removal mechanism for hormones by microalgae; however, it had no effect on microalgal cell development. This is because hormones primarily work in mammals and aquatic species (e.g., fish), both of which can be adversely influenced by hormones. Hormones, on the other hand, have no substantial inhibitory impact on microalgal development. However, biomagnification by the accumulation of altered products in microalgae is a significant challenge [31].The best sludge and algal ratios for successful sludge water utilization and biomass production were discovered to be 25 : 75 (C : S) because the sludge water should have a lower concentration (75 : 25 S+C) chosen. Therefore, microalgae were cleaned or used 67% of the water. According to this study, including microalgae production in wastewater treatment can result in a cost-effective and environmentally friendly wastewater treatment solution. Figure3 shows the determination of microalgae and sludge ratio for the effective treatment of algal growth. Based on these results and the experiment, the ratios (S+C 75 : 25) were similar to those found on day zero (S+C 75 : 25 was 29.99% and S+C 75 : 25 was 27.58%). Following these, on day 4, S+C 75 : 25 was 29.15% and S+C 75 : 25 was 28.67%; on day 8, S+C 75 : 25 was 45.74% and S+C 75 : 25 was 50.47%; and on day 16, S+C 75 : 25 was 67.04% and S+C 75 : 25 was 66.39%. The experiments revealed that microalgae could thrive in aquaculture effluents. The first trial was conducted in batch mode and lasted 33 days, ending when all nitrogen and phosphorus supplies were used and a decrease in the optical density of the cultures was detected. The goals of this experiment were studied, and the two microalgal species performed the best in terms of remediation and biomass production. Microalgae C. vulgaris and S. obliquus were able to accomplish restoration in 22 days, but the other microalgae took 33 days. When compared to the control, the biomass concentration attained after 22 days for the microalga Chlorella vulgaris cultured in aquaculture effluent (3.22±0.04 g L-1) was substantially greater (1.36±0.39 g L−1). The water analysis study was performed, and the results are shown in Table 2.Figure 3
Determination of microalgal and sludge ratio for the effective treatment of algal growth C+S (blue bar) 25 : 75 and C+S (red bar) 75 : 25.Table 2
Examination results of biochemical analysis in treated water.
ParametersIndustrial sludge waterAdmissible limitControlThis studyTemperature68o95°C——ColorBrown, yellowishBlackDarkGreenpH698.228.02Hardness155 mg/L330 mg/L10289TDS250 mg L−1850 mg L−1616221088Chloride745.25 ppm or mg/L40 to 50 mg/L——Sulfate250 mg/L500 mg/L4644DO6.5-8 mg/L4-5 ppm1.71.2COD100 mg/L60,000 mg/L160130.2BOD10 mg/L300 mg/L180110


## 3.3. Estimation of Lipid Content

A modified Folch technique was used to extract lipids from the microalgae consortia. Lipid was obtained by evaporating the solvent, and the total lipid collected from the sample was quantified using an electronic balance; then, the lipid content was weighed at26.66±0.5 mg/g.


## 3.4. FAME Profiling

FAME composition was determined and quantified by comparing retention periods and peak regions from the GC data. The results revealed that the composition of FAMEs was influenced by nutrient types as well as growing methods. The GC-MS profiling was identified and quantified by comparing the abundance percentage of the 14 different compounds of methyl ester, such as 2,4-di-tert-butylphenol; methyl tetradecanoate; methyl 13-methyltetradecanoate; neophytadiene; 2-pentadecanone,6,10,14-trimethyl; 3,7,11,15-tetramethyl-2-hexadecen-1-ol; (Z)-methyl hexadec-11-enoate; hexadecanoic acid; cyclopropanebutanoic acid,2-[[2-[[2-[(2-pentylcyclopropyl); phytyl, 2-methylbutanoate; 7,10-octadecadienoic acid, methyl ester; 10-octadecenoic acid; and heptadecanoic acid, 16-methyl, and their properties are mentioned in Tables3 and 4. Particularly, FAME with a high concentration of heptadecanoic acid has favorable qualities for usage as biofuels. This finding suggests that using microalgal biomass as an alternate carbon source can enhance both the quantity and quality of biodiesel. The fatty acid composition is based on saturation form. The aspect of biodiesel quality is connected to saturated bonds and carbon chain length. The quality of biodiesel improves as the proportion of polyunsaturated fatty acids (PUFA) decreases. Because of their high concentration of saturated and monounsaturated fatty acids, Chlorella sp. cells cultivated under autotrophic conditions seem to be a viable option for the generation of biodiesel (Figure 4). Taken together, our work has established a successful scenario in which algae combined with industrial sludge can be employed as a biodiesel-manufacturing technique. A further benefit of using Chlorella sp. culture is that it is a technology that can control microalgal waste through biological recycling in addition to producing biodiesel at a low cost. In addition, by using sludge waste as an alternative carbon source, agricultural land and freshwater resources might be considerably preserved. In light of the carbon cycle, Chlorella sp. can convert carbon sources into lipids with high lipid productivity using CO2 from air/flue gas and organic carbon from sludge waste.Table 3
FAME analysis and their properties were identified using GC-MS.
S. no.Methyl esterCarbon no.Abundance (%)CAS no.Type(1)UndecaneC1157.11120-21-4SFA(2)2,4-Di-tert-butylphenolC1419.196-76-4—(3)Methyl tetradecanoateC1574.0124-10-7SFA(4)Methyl 13-methyltetradecanoate74.0SFA(5)NeophytadieneC2068.1504-96-1—(6)2-Pentadecanone,6,10,14-trimethylC1858.0502-69-2—(7)3,7,11,15-Tetramethyl-2-hexadecen-1-olC2081.0102608-53-7—(8)(Z)-Methyl hexadec-11-enoateC1755.0822-05-9FAME(9)Hexadecanoic acid, methyl esterC1774.0112-39-0SFA(10)Cyclopropanebutanoic acid,2-[[2-[[2-[(2-pentylcyclopropyl)C2574.156051-53-7—(11)Phytyl, 2-methylbutanoate85.1—(12)7,10-Octadecadienoic acid, methyl esterC19101.056554-24-6SFA(13)10-Octadecenoic acid, methyl esterC1955.113481-95-3SFA(14)Heptadecanoic acid, 16-methyl, methyl esterC1974.15129-61-3SFATable 4
FAME analysis and their properties were identified using GC-MS.
S. no.Compound nameLinear formulaMolecular weight (g/mol)(1)UndecaneCH3(CH2)9CH3156.31(2)2,4-Di-tert-butylphenol[(CH3)3C]2C6H3OH206.32(3)Methyl tetradecanoateCH3(CH2)12COOCH3242.40(5)NeophytadieneC20H38278.5(6)2-Pentadecanone,6,10,14-trimethylC18H36O268.5(7)3,7,11,15-Tetramethyl-2-hexadecen-1-olC20H40O296.5(8)(Z)-Methyl hexadec-11-enoateC17H32O2268.4(9)Hexadecanoic acid, methyl esterCH3(CH2)14CO2CH3270.45(10)Cyclopropanebutanoic acid,2-[[2-[[2-[(2-pentylcyclopropyl)C25H42O2374.6(12)7,10-Octadecadienoic acid, methyl esterC19H34O2294.5(13)10-Octadecenoic acid, methyl esterC19H36O2296.5(14)Heptadecanoic acid, 16-methyl, methyl esterC19H38O2298.5Figure 4
GC analysis showing the fatty acid methyl esters at various carbon lengths and their derivatives.


## 4. Conclusions

In this study, the production of biomass and lipid byChlorella sp. using inexpensive nutrient sources from industrial sludge wastewater was investigated. The aim of this microalgal production for wastewater treatment is to provide a low cost and environmentally friendly alternative method while reducing greenhouse gas emissions for wastewater treatment. The sludge and microalgal ratio 75 : 25 was determined from the 16th day as the best treatment time for biomass productivity and sludge treatment over the trial period of 20 days. The culture of Chlorella sp. in a mixotrophic environment resulted in high cell density and lipid productivity of 26.66±0.5 mg g−1. Furthermore, its fatty acids included a high concentration of heptadecanoic acid, with a molecular weight of 298.5 g/mol, which is advantageous for biodiesel quality. These findings suggested that microalgal biomass for the cultivation of Chlorella sp., as well as industrial sludge effluent, might be used as a cost-effective alternative carbon source.



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*Source: 9939252-2023-03-22.xml*