Journal Pre-proof

A complete characterization of microalgal biomass through FTIR/TGA/CHNS

analysis: An approach for biofuel generation and nutrients removal

Muhammad Arif, Yuxi Li, Marwa M. El-Dalatony, Chunjiang Zhang, Xiangkai Li, El-
Sayed Salama

PII: S0960-1481(20)31632-3
DOI: https://doi.org/10.1016/j.renene.2020.10.066
Reference: RENE 14356

To appear in: Renewable Energy

Received Date: 13 June 2020

Revised Date: 25 August 2020
Accepted Date: 15 October 2020

Please cite this article as: Arif M, Li Y, El-Dalatony MM, Zhang C, Li X, Salama E-S, A complete
characterization of microalgal biomass through FTIR/TGA/CHNS analysis: An approach for biofuel
generation and nutrients removal, Renewable Energy, https://doi.org/10.1016/j.renene.2020.10.066.

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© 2020 Published by Elsevier Ltd.

Muhammad Arif and Yuxi Li: Methodology, Visualization, Investigation, Data curation,
Formal analysis, Writing - Original Draft. Marwa M. El-Dalatony: Visualization, Investigation,
Formal analysis. Chunjiang Zhang: Conceptualization, Supervision, Resources, Visualization,
Formal analysis, Writing - Review & Editing. Xiangkai Li: Visualization, Formal analysis,
Writing - Review & Editing. El-Sayed Salama: Conceptualization, Supervision, Resources,
Data Curation, Validation, Visualization, Formal analysis, Writing - Review & Editing, Funding
acquisition, Project administration.

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A complete characterization of microalgal biomass through FTIR/TGA/CHNS analysis: An

approach for biofuel generation and nutrients removal

Muhammad Arifa,1, Yuxi Lia,b,c,1, Marwa M. El-Dalatonya, Chunjiang Zhanga,b,c*, Xiangkai Lia,

El-Sayed Salamad

a
School of Life Sciences, Lanzhou University, Lanzhou 730000, PR China

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b
Key Laboratory of Cell Activities and Stress Adaptations, Ministry of Education, Lanzhou

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University, Lanzhou 730000, PR China
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Gansu Key Laboratory of Biomonitoring and Bioremediation for Environmental Pollution,
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Lanzhou University, Lanzhou 730000, PR China
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Department of Occupational and Environmental Health, School of Public Health, Lanzhou
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University Lanzhou 730000, Gansu, PR China

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*Correspondence author

Chunjiang Zhang (C.-J. Zhang): at School of Life Sciences, Lanzhou University, 222 Tianshui

South Road, Lanzhou 730000, PR China. E-mail: chjzh@lzu.edu.cn

1
Both authors contributed equally to this work, and can be regarded as co-first author.

1
1 A complete characterization of microalgal biomass through FTIR/TGA/CHNS analysis: An

2 approach for biofuel generation and nutrients removal

4 Muhammad Arifa,1, Yuxi Lia,b,c,1, Marwa M. El-Dalatonya, Chunjiang Zhanga,b,c*, Xiangkai Lia,

5 El-Sayed Salamad

6
a
7 School of Life Sciences, Lanzhou University, Lanzhou 730000, PR China

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8 Key Laboratory of Cell Activities and Stress Adaptations, Ministry of Education, Lanzhou

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9 University, Lanzhou 730000, PR China

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Gansu Key Laboratory of Biomonitoring and Bioremediation for Environmental Pollution,
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11 Lanzhou University, Lanzhou 730000, PR China
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12 Department of Occupational and Environmental Health, School of Public Health, Lanzhou
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13 University Lanzhou 730000, Gansu, PR China

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16

17 *Correspondence author

18 Chunjiang Zhang (C.-J. Zhang): at School of Life Sciences, Lanzhou University, 222 Tianshui

19 South Road, Lanzhou 730000, PR China. E-mail: chjzh@lzu.edu.cn

20
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21 Both authors contributed equally to this work, and can be regarded as co-first author.

22

23

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24 Highlights

25 All the microalgae species were able to remove 90%–95% total nitrogen and phosphorus.

26 The highest accumulated protein 46.37% was achieved with T. obliquus.

27 The highest accumulated carbohydrate 36.56%` was attained with C. sorokiniana.

28 Biochemical, CHNS, TGA, FTIR confirmed microalgae potential as a biofuel feedstock.

29 Biodiesel production feasibility was confirmed by the presence of C16/C18 (68%–84%).

30

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31 Abstract

32 A comprehensive bio-composition investigation of microalgal species is essential for

33 categorizing their applications. In this study, nutrients uptake followed by complete biomass

34 characterizations, including proximate, ultimate, and bio-compounds (proteins, carbohydrates,

35 and lipids), were performed along with FTIR and TGA/DTG, analyses of four microalgal strains

36 (Tetradesmus dimorphus GEEL-06, Tetradesmus obliquus GEEL-07, Chlorella sp. GEEL08,

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37 and Chlorella sorokiniana GEEL-09). All microalgae species were able to remove 90%–95%

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38 nitrogen and phosphorus. T. dimorphus GEEL-06 had highest growth with proteins accumulation

39 -p
of 36.63% and 28.33% of lipids. T. obliquus GEEL-07, Chlorella sp. GEEL08, and C.
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40 sorokiniana GEEL-09 accumulated 46.37%, 31.13%, and 39.38% of proteins and 27.94%,

34.61%, and 36.56% of carbohydrates, respectively. FTIR spectra revealed the presence of an
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42 alcohol, carboxyl, and amino groups, while biomass pyrolysis showed 67%–80% decomposition.
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43 Biodiesel feasibility was confirmed by the presence of 68%–84% C16/C18. These results
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44 indicate the potential applications of T. obliquus GEEL-07, C. sorokiniana GEEL-09, and T.

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45 dimorphus GEEL-06/Chlorella sp. GEEL08 for production of higher alcohols, bioethanol,

46 biodiesel/oil for, respectively, along with nutrients removal.

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49

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51

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53 Keywords: Microalgae; Nutrient removal; Characterization; Pyrolysis; Fatty acids; TGA/DTG

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54 1. Introduction

55 Microalgae are promising candidates for biofuel generation because of their high biomass

56 yields, high biochemical content, low cultivation costs, and non-competition for agricultural

57 cropland [1]. These benefits make microalgae suitable for environmentally friendly large-scale

58 biofuel production. Microalgae are photosynthetic, autotrophic, or heterotrophic unicellular

59 microscopic microorganisms that grow in freshwater and marine environments [2]. There are

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60 three main bio-compounds present in microalgae that account for more than 50% of their total

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61 biomass: 25%–70% proteins/amino acids, 8%–65% carbohydrates/sugars, and 0%–45%

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lipids/fatty acids [3]. The amino acids found in microalgae proteins can be supplied in the human
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63 diet and animal feed as ingredients with high nutritional value [4] and also be used as feedstock

for higher alcohols [5]. Carbohydrates, such as polysaccharides and starch, are converted into
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65 bioethanol through fermentation [6]. Fatty acids, present in microalgal lipids, can be trans-
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66 esterified to produce biodiesel; however, they also have food and feed supplement applications
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67 (such as omega-3 fatty acids) [7]. Microalgae can dramatically increase their biocomponent
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68 content in days or even hours via carbon partitioning, which appears to be species specific [8].

69 To follow these rapid changes, a simple, cost-effective, and fast technique is needed for real-time

70 monitoring of microalgal cultures, the rapid selection of strains with high productivity, and the

71 understanding of metabolic behavior under various growth parameters and nutrient-deficiency

72 conditions. Major challenges during cultivation of microalgae for high biomass production are

73 environmental compatibility, temperature and pH fluctuation, light intensity, antagonistic effect.

74 High concentration of nutrients such as nitrogen and phosphorus may also result in lower

75 biomass production sometimes pretreatment of wastewater is preferred. Recently, promising

76 processes has been developed for bulk biomass production with different wastewater treatment

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77 result in cost effective of microalgae cultivation. An integrated approach for coupling of high

78 microalgal biomass production with nutrients removal is highly prominent and required to

79 identify robust microalgae strains which tolerable to high nutrient toxicity [9].

80 It has been suggested that economically feasible microalgal biofuel production can be

81 achieved only through the exploration of its coproducts [10]. This can be achieved by isolating

82 and screening native and/or novel microalgae, followed by their complete biochemical and

83 physiochemical characterization. Most commonly, proximate analysis is being used to

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84 characterize the role of microalgae in coal and other fuels [11]. Ultimate analysis is the

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85 determination of elements such as carbon and hydrogen, as well as sulfur, nitrogen, and oxygen,

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in a substance by analyzing its gaseous products after complete combustion [12]. Determining
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87 the whole biomass composition, including elements and minerals, is also helpful in
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88 understanding microalgae as biofuel feedstock [13]. Fourier transform infrared (FTIR)

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89 spectroscopy is useful to identify functional groups of biological samples, such as microalgal

90 biomass and intra- and extracellular metabolites [8]. Thermogravimetric analysis (TGA) has
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91 been employed as a proven technique for studying microalgae pyrolysis [14]. The TGA and FTIR
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92 analysis of microalgal biomass might be helpful for identification of volatile products and

93 chemical nature of microalgal biocomponents [15].

94 Climate change might cause an evolutionary variation in microalgae, resulting in

95 adaptability to different environmental stress conditions [16]. Therefore, monitoring and

96 screening of potential microalgal strains should be a continuous work. Previous research work on

97 microalgae was focused on characteristics of a single bio-compounds (i.e., lipids, carbohydrates

98 or proteins) of the isolates, ignoring the complete biomass characterization and co-products

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 99 generation. Recently, researchers are interested in complete analysis of microalgal species before

100 their use for any applications [17].

101 Northwestern region of China has not been studied for microalgal diversity and their

102 applications. Therefore, in this study, monitoring and screening of various microalgae from that

103 region was carried out. The growth kinetics and nutrients (N, P) removal efficiency of the

104 isolated microalgal strains was evaluated. Harvested microalgal biomass was fully characterized

105 for bio-components (including lipids/fatty acids, proteins, and carbohydrates), proximate, and

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106 ultimate properties. Fourier transform infrared (FTIR) spectroscopy, thermogravimetric analysis

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107 (TGA) followed by biodiesel properties were analysed to deeply understand the applications of

108 individual microalgae.

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2. Materials and Methods
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109

2.1. Screening and identification of microalgal strains

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111 Fresh water samples were collected from Yantang Park and Five Spring Park in Lanzhou

112 City, China. Yantang Park had longitude of 103.511° East and latitude of 36.335° North located
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113 at Tianshui North Road, Chengguan District, Lanzhou City, China. Five Spring Park was located

114 at Wuquan South Road, Chengguan District, Lanzhou City, China (Fig. 1). The physicochemical

115 properties of collected samples were shown in Table 1. For isolation of microalgae, the collected

116 water samples were enriched in Bold’s basal medium (BBM) prepared in fresh water and kept in

117 a shaker (MQD S3R, Minquan Instruments, Shanghai, China) under a continuous white light

118 tube (Philips Master tl-d eco 51w/54–765) with an intensity of 40 µmol m−2s−1 for 4 weeks [18,

119 19]. To obtain pure microalgal culture for identification, a single cell method was followed [20].

120 In brief, serial dilutions (1, 2, 4, and 6 dilution fractions) of enriched cultures were performed

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121 and 50 µL samples from each dilution were inoculated to a 0.25 L of BBM. Followed by, sub-

122 culturing of 50 µL from the cultivated cultures on BBM Petri-plates (1.5% w/v bacteriological

123 agar) at 25 °C under continuous light for three weeks. The culture’s purity was confirmed by

124 repeated culturing and microscopic examinations. Botanical approaches were used for

125 morphological identification, while molecular markers using 18S rRNA gene sequences was

126 used for species identification [20]. The phylogenetic tree was constructed using the neighbor-

127 joining (NJ) Kimura’s two-parameter algorithm (Fig. 2), as implemented within the MEGA5

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128 program package [21].

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129 2.2. Microalgal growth and nutrients removal -p
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130 Microalgal culture (at day 10 taken from log phase) with a 0.1 absorbance at 680nm was
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131 used as an inoculum, and 15 mL (10%) of each species was added to 250 mL Erlenmeyer flasks

containing BBM (150 mL). The main nutrients in BBM are NaNO3 (250 mg L−1) as nitrogen
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133 source while K2HPO4 (75 mg L−1) and KH2PO4 (175 mg L−1) as phosphorus source. The pH of
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134 culture media was adjusted to be 6.8 in all flasks. The flasks were incubated at 150 rpm, 25±2
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135 °C, and under continuous illumination of 40 µmol m−2s−1 intensity for 28 days to obtain

136 sufficient biomass for further analyses [22]. Microalgal growth was evaluated by measuring the

137 optical density at 680nm with a spectrophotometer (UV 5500, Metash, China) after 2 days.

138 Microalgal suspensions with an optical density (OD680) higher than 1.0 were diluted to the range

139 of 0.1–1.0. The overall specific growth rate (µ) was calculated by Equation 1:

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 ×₁− ×₀
μ=
₁− ₀

140 where ×1 is the final biomass concentration, ×0 is the initial biomass concentration at optical

141 density (OD680nm), t1 is the final time, t0 is the initial time, and μ is the specific growth rate

142 (day−1). The specific growth rate was calculated after 2 days.

143 2.3. Characteristics of microalgal biomass

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144 The moisture content in biomass was assessed by heating the samples at 105 °C for 24 h.

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145 The volatile and ash content were calculated using the Leco TGA 701 instrument according to

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EN 14776 by heating the sample for 2.5 hr at 550 °C under a constant air supply [23]. The
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147 percentage of elemental composition (including carbon, nitrogen, hydrogen, sulfur, and oxygen)
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148 in the microalgal freeze-dried biomass cultivated in BBM was determined using the Elementar
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149 Vari EL Cube (Germany). The higher heating value (HHV) of microalgal biomass was

150 calculated from C, H, N, and S using the Dulong equation [24].

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151 ¯ = . + . − (2)

152 2.4. Analyses of biocomponents

153 Microalgal biomass cultivated in BBM was harvested after 28 days as per protocol

154 describe by Abou-Shanab [22]. The lipid extraction of freeze-dried microalgal biomass was

155 carried out using the protocol reported by Bligh and Dyer [25] with a few modifications. The

156 samples were evaporated in a dry oven at 50 °C and the crude lipid content was weighed

157 gravimetrically. The carbohydrate content in the microalgal biomass was assessed using the

158 phenol-sulfuric acid method [26]. The carbohydrate concentrations were measured by

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159 determining the optical density at 490nm using a spectrophotometer. A glucose standard was used

160 to measure the carbohydrate concentration. Microalgal protein content was evaluated by the

161 Lowry method [27]. The freeze-dried algal biomass (1 mg mL−1) was diluted with 5 mL Lowery

162 reagent and 0.5 mL Folin-Ciocalteu reagent (Sigma Co., St. Louis, MO, USA). The protein was

163 measured at 660nm using a spectrophotometer. The values were compared to a bovine serum

164 albumin standard.

165 A modified method of Lepage and Roy [28] was used to analyze fatty acids (FAs); 1 mL

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166 methanol and 0.3 mL H2SO4 were added to the 1 mL crude lipid layer. The mixture was vortexed

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167 for 3–5 min and incubated at 100 °C for 10 min. Then 1 mL dH2O was added, vortexed for 3–5

168
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min, and then centrifuged at 4000 rpm for 10 min. Fatty acid methyl esters (FAMEs) were
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169 evaluated with a gas chromatograph equipped with a flame ionization detector GC-(FID) (Foli
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170 Instruments, China) and a KB-FFAP (30 m × 0.32 mm) column. For FAME, the conditions of
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171 the inlet and detector were 240 °C and 260 °C, respectively, and the oven was set at 100 °C (2

172 min), raised by 4 °C min−1 to 180 °C (10 min), then raised by 5 °C min−1 to 235 °C (9 min).
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173 Then, 1 µL sample was injected using nitrogen as the carrier gas. The FAMEs standard, Supelco
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174 ® 37-component (Sigma-Aldrich, USA), was used for analyzing fatty acids in the extracted lipid.

175 Other reagents used were analytical grade. All the samples were analyzed in triplicates and the

176 data were represented as mean ± standard deviation (SD).

177 The properties of the biodiesel were calculated based on the FAME characteristics

178 according to the following calculations:

179 !" = ∑$%"&' + × ("&' ) (3)

-. ×/%
180 *+ = ∑ , 1 (4)
%

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181 2 = ∑$ ) (5)

- -
182 3 = .. + − . -×2 (6)
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183 6 57 = . × .: + .-× : + × : + .- × : + × :

184 (7)

185 799 = . : × 6 57 − .. :: (8)

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186 where DU is the unsaturation degree (%), MUFA is monounsaturated fatty acids, PUFA is

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polyunsaturated fatty acids, N% is the percentage of each fatty acid, M represents the molecular

188 weight of the fatty acid, D is the number of double bonds, SV is saponification value (mg KOH
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189 g−1), IV is iodine value (gI2/100 g oil), CN is the cetane number, LCSF is the long-chain
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190 saturation factor, and C16:0, C18:0, C20:0, C22:0, and C24:0 represent the weight percentage of
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191 the corresponding fatty acids. Cold filter plugging points (CFPP) (°C) were estimated according
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192 to [29].

Fourier transform infrared (FTIR) spectroscopy was used to predict the structural and
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194 chemical bonding (targeting functional groups) between different biomolecules in the microalgal

195 biomass. Freeze-dried microalgal biomass was analyzed by a MAGNA 550 Nicolet (Madison,

196 USA), equipped with a mercury cadmium telluride detector, and the spectra were recorded in the

197 frequency range of 500–4000 cm−1 at a resolution of 4 cm−1 [24]. For thermogravimetric analysis

198 (TGA), microalgal biomass (5 mg) was placed in aluminum crucibles and transferred to a

199 thermal analyzer. The biomass was pyrolyzed at 10 °C min−1 for 3 hours to 800 °C (Linseis

200 Messgerate GmbH, Germany) with a continuous supply of nitrogen gas (100 mL min−1) to ensure

201 an inert environment. The proposed design for the pyrolytic and proximate properties of the

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202 microalgal biomass are represented in Fig. 3. TGA data were converted to differential

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203 thermogravimetric (DTG) data using the conversion rate ;=
(where da is microalgal mass

204 reduction and dt is time) versus temperature [30].

205 3. Results and Discussion

206 3.1. Microalgal identification and growth kinetics

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207 Isolation, identification, and screening of potential microalgal species are the initial and

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208 crucial phase before their selection for any biotechnological applications [17]. The isolated

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strains were identified as Tetradesmus dimorphus GEEL-06, Tetradesmus obliquus GEEL-07,
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210 Chlorella sp. GEEL-08, and Chlorella sorokiniana GEEL-09 based on molecular
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211 characterization. The genome sequences of the isolates were published in the NCBI databases

212 (Fig. 2). The growth of selected microalgae species (including T. dimorphus GEEL-06, T.
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213 obliquus GEEL-07, Chlorella sp. GEEL-08, and C. sorokiniana GEEL-09) increased with the
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214 cultivation time, and the greatest growth was obtained for T. dimorphus GEEL-06, followed by
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215 T. obliquus GEEL-07 (Fig. 4A). The strains were selected on the basis of high abundance in

216 natural environment due to its high growth. T. dimorphus GEEL-06 had higher growth (2.210

217 OD680nm) than previously reported S. pectinatus (1.632 OD600nm) and C. minutissima (1.595

218 OD600nm) cultivated in BBM for 29 days [31]. The pattern of the specific growth rate (µ) was also

219 initially high (0.1–0.8 day−1), constant during the log phase, and then growth decreased during

220 the declining phase (Fig. 4B), which might be due to the ideal environmental conditions (such as

221 active inoculum and less competition among the cells) and availability of nutrients in the lag

222 phase, which accelerates microalgal growth. Nayak [32] reported that the µ for Chlorella sp. and

223 Scenedesmus sp., was 0.0744 and 0.0743 day−1, respectively. The difference in specific growth

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224 values among the microalgal strains can be attributed to the alterations in cultivation time,

225 culture medium, and cultivation conditions (including initial algal inoculum concentration,

226 working volume, light/dark cycle, temperature, and media pH) [33].

227 The regularly increased in pH for all microalgae species during the cultivation period,

228 and the highest pH was observed with T. dimorphus GEEL-06, indicating its ability for high

229 photosynthetic activity and biomass production. The initial pH of the medium was 6.8 and

230 reached to 9.2–10.84 on the last day (Fig. 4C). The media pH of C. sorokiniana str. SLA-04 was

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231 increased (up to 10) without showing inhibitory effects on the growth [34]. It has been reported

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232 that as microalgae grow fast in the exponential phase, the photosynthetic activity increases, and

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there is high uptake of CO2 from the medium in the form of HCO3 by inorganic-carbon (Ci)
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234 transporters to the chloroplasts. A proton (H+) is used during the conversion of HCO3– to CO2,
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235 and this CO2 is fixed by the enzyme RuBisCO during photosynthesis and releases OH– in the

cell, and this has to be neutralized by H+ uptake from the extracellular environment. The
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237 decrease of H+ in the medium unavoidably leads to an increased pH [35]. Such an increase in pH
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238 of the media during microalgal growth supports the approach of nutrient recovery.
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239 3.2. Nutrients removal during microalgal growth

240 Microalgal strains are required for coupling of biomass production and efficient removal

241 of nutrients such as nitrogen (N) and phosphorus (P) [36]. In this study, the microalgal strains

242 effectively removed N and P from the BBM. The N and P concentration in the culture medium T.

243 dimorphus GEEL-06 showed highest removal efficiency as compared to other strains (Fig. 5).

244 Three different strains of Chlorella sorokiniana eliminate (90-95%) N from the BBM after 25

245 days. The high concentration of nutrients such as N and P could reduce the growth of microalgae

246 due high concentration of NO3−2 and PO4−3 which assimilated slowly [37]. The primary

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247 mechanism involved in total nitrogen (TN) and total phosphorus (TP) elimination might be

248 microalgal biomass uptake, and the uptake rate could be different during the cultivation period

249 [38]. The initial concentrations of N and P in BBM were 40.92 mg L−1 and 51.46 mg L−1 and

250 reduced to 0.046 mg L−1 to 0.099 mg L−1, respectively by T. dimorphus GEEL-06 (Fig. 5A). N is

251 a vital component for microalgal growth that comprises 1%–10% of the total cell weight. P is

252 essential for growth and the functions of deoxyribonucleic acid (DNA), such as energy transfer

253 and biosynthesis [39]. Utilization of nutrients such as N and P by microalgae enhanced their

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254 growth and decreased the N and P concentrations in the culture media (Fig. 5B). N and P

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255 removal efficiency indicated that the isolated microalgae might be potential candidates for

256
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wastewater treatment as the physical-chemical characteristics of BBM (such as N, P speciation
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257 and concentration) are comparable to various wastewaters [40, 41].
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258 3.3. Microalgal biocomposition

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259 3.3.1. Proximate, ultimate, and biocompounds

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260 Microalgal species with a high level of bio compounds (proteins, carbohydrates or lipids)
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261 are an ideal source for many applications, such as biofuels generation, food production, food

262 additives, and nutraceuticals [42], and this could make large-scale microalgae cultivation

263 economically feasible [24]. Therefore, comprehensive analyses (proximate, ultimate, and bio

264 compounds) of harvested microalgal biomass were performed in this study (Table 2). Proximate

265 analysis of C. sorokiniana GEEL-09 showed the highest volatile content (94.13%) of total solids

266 in comparison to other strains. Ash content was 5.86%–12.24% of the total dry cell weight

267 (DCW) (Table 2), the higher inorganic and mineral content in microalgal biomass might be

268 result in higher ash content and reflecting lesser biomass energy production at higher

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269 temperatures. The ultimate analysis showed that all microalgal species cultivated in BBM had a

270 carbon, oxygen, nitrogen, and sulfur content of 49.31%–53.91%, 31.95%–35.67%, 5.32%–

271 7.01%, and 0.17%–0.55%, respectively. T. obliquus GEEL-07 had high nitrogen contributing to

272 high protein content (Table 2). The available nitrogen in culturing medium might affect cellular

273 protein level in microalgal biomass. High protein content and C/N ratio in these microalgae

274 distinguish them from lignocellulosic feedstocks, which usually have <1% N [43]. The carbon

275 and hydrogen contents of microalgae were 49.31%–53.91% and 7.46%–7.74%, respectively.

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276 This remarkably high carbon and hydrogen content makes them potential candidates for biofuel

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277 production as compared to other lignocellulosic feedstocks and fossil fuels (coal) [44]. High

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levels of nitrogen and sulfur in the biomass have been reported to cause enhanced emission of
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279 toxic oxides such as nitrogen oxides (NO×) and sulfur oxides (SO×) [45]. The emission of SO× is
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280 negligible in case of microalgae because of a lower sulfur content. The N content (5.32–7.01%)
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281 of microalgae was similar (6.64%–7.72%) to the N content reported by Pandey [17] because of

282 the high availability of TN in the culture medium. The lower oxygen content (31.95%) of
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283 Chlorella sp. GEEL-08 resulted in a higher heating value (HHV) of 23.52 MJ kg−1 DCW, which
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284 was higher than in the previously reported study [46]. These findings predicted that because of

285 the presence of high carbon content, high HHV, low N and S in T. dimorphus GEEL-06, T.

286 obliquus GEEL-07, Chlorella sp. GEEL-08, and C. sorokiniana GEEL-09, these species can be

287 used as a potential feedstock for biodiesel production.

288 Previous studies were mainly focused on the isolation of microalgae and analyzed only

289 the lipid content for biodiesel production [47] and ignored other major bio components, such as

290 protein and carbohydrates, which are primary feedstocks for production of ethanol and higher

291 alcohols. In this study, all the important biocomponents that could be used for biofuel

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292 production, such as proteins, carbohydrates, and lipids/fatty acids, were analyzed (Table 3). T.

293 obliquus, Chlorella sp., and C. sorokiniana accumulated 46.37%, 31.13%, and 39.38% proteins

294 and 27.94%, 34.61%, and 36.56% carbohydrates, respectively, indicating their potential for

295 production of higher alcohols and ethanol. T. dimorphus accumulated proteins (36.63%) and

296 lipids (28.33%), also indicating their potential use for production of higher alcohols and

297 biodiesel.

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298 3.3.2. Microalgal fatty acid composition and its biodiesel properties

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299 Microalgal lipids are composed of saturated fatty acids (SFAs), monosaturated fatty acids

300
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(MUFAs), and polyunsaturated fatty acids (PUFAs) (Table 3). SFAs content (41.01%–54.27%),
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301 MUFAs content (14.98%–32.99%), and PUFAs content (13.67%–40.08%) are shown in Table 3.
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302 The quality of biodiesel depends on the content of oleic and palmitic acids. The major fatty acids

were palmitic acid (23.23%–26%), oleic acid (8.46%–20.54%), and linoleic acid (13.43%–
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304 37.90%). Higher content of these two fatty acids in microalgal biomass suggest that it can be a
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305 high-quality feedstock for biodiesel production because high palmitic acid imparts a higher
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306 oxidative stability and cetane number (CN), in addition to lower NOX emissions [48]. Oleic acid

307 content in oils offers a reasonable balance of fuel properties such as ignition quality, combustion

308 heat, cold filter plugging point (CFPP), oxidative stability, viscosity, and lubricity [43]. In this

309 study, T. dimorphus GEEL-06 had (PUFAs content of 40.08%, useful for biodiesel in colder

310 regions to avoid blockage in engine filters. C. sorokiniana GEEL-09 has a high total SFAs

311 content (54.27%); the biodiesels derived from such feedstocks are more desirable for use in

312 temperate regions and provide oxidative stability [49].

313 The assessment of biodiesel properties is important in order to understand the

314 performance and quality of biodiesel in engines. Biodiesel properties, such as degree of

15
315 unsaturation (UD), saponification value (SV), iodine value (IV), cetane number (CN), long-chain

316 saturation factors (LCSFs), and cold filter plugging point (CFPP) have been determined (Table

317 4). Most of the microalgal-derived biodiesel characteristics comply with international standards

318 such as ASTM 6751−03 and the EN 14214. Chlorella sp. GEEL-08 and C. sorokiniana GEEL-

319 09 had lower LCSF, as shown by a study supporting the presence of low LCSFs and noting that

320 the biodiesel derived from SFAs could not be easily oxidized and could be stored for a long time

321 [29]. T. dimorphus GEEL-06 had high PUFAs content (40.08%), resulting in lower CFPP (-6

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322 .49°C) and could be suitable for biodiesel fuel in colder regions [50]. For all microalgal-derived

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323 biodiesel, other properties, such as CN (≥51) and IV (≤120 value g I2 100 g−1 oil), comply with

324 the international standards guidelines [51].

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3.4. Spectroscopic analysis by FTIR
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325

The FTIR spectra of the microalgal biomass represented the alcohol group (OH), carboxyl
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326

327 group (COOH), amino (NH2), and other groups associated with organic compounds (Fig. 6). The
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328 peak in the high intensity region of 3550–3200 cm−1 indicated the presence of lipids in biomass,
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329 suggesting that the lipid peak in this region is due to the symmetrical and asymmetrical

330 stretching vibration of CH2 [52]. Different peaks of alkane groups at 3100–2800 cm−1, aldehyde

331 groups at 2830–2695 cm−1, ester groups at 1800–1700 cm−1, and carboxylic groups at 1420–1330

332 cm−1 were recorded. The presence of saturated and unsaturated hydrocarbons was marked by the

333 peaks of alkane and alkene groups. Abundance of carbohydrate, protein, and lipid was predicted

334 by the corresponding peaks in functional groups such as aldehyde, carboxyl, and ester groups.

335 Peaks in the regions of 1,750–1720 cm−1 and 1163–1210 cm−1 showed the presence of esters in

336 the microalgal biomass, indicating a distinct peak for triglycerides (lipids) and fatty acids,

337 respectively. A study reporting the peaks at 1,700–1,800 cm−1 confirmed the higher

16
338 concentration of fatty acids [53]. The FTIR spectra of the microalgae samples showed similar

339 results as found in other studies [54].

340 3.5. Thermogravimetric analysis (TGA/ DTG) of microalgal biomass

341 The analysis of pyrolytic properties of the microalgal biomass by thermogravimetric

342 analysis (TGA) is shown in Figure 7. The thermal decomposition behavior of microalgal biomass

343 had three main phases, and most of the moisture content and highly volatile compounds were lost

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344 in the first phase up to a temperature of 150 °C. Maximum biomass reduction was observed

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345 during the second phase at temperatures of 150–550 °C. The decomposition of major organic

346
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compounds of microalgal biomass, such as lipids, proteins, and carbohydrates, occurred at this
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347 temperature and that is why this stage is also called the active pyrolytic zone. During the last
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348 phase, thermally stable compounds were decomposed at 600–800 °C and biochar formation

occurred. The microalgal biomass TGA curve represented 5%–7% loss of biomass in first the
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349

350 phase, 67%–80% decomposition in the second phase, and 3%–5% in the final stage (Fig. 7). The
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351 microalgal biomass decomposition (%) at the second stage was higher than previously reported
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352 [55]. The differential thermogravimetric (DTG) spectra for all microalgal biomass represent a

353 devolatilization peak at temperature 270–310 °C, indicating the maximum decomposition of

354 biomass (Fig. 7). Two other important peaks to the right and left of the main peaks are also

355 observed at temperature ranges from 150–200°C and 370–400 °C, respectively. The temperature

356 variation for all the peaks showed the differences in microalgal biomass, similar to the results of

357 another study [17].

358 4. Conclusions

17
359 Comprehensive bio-composition investigation of microalgal species is essential for

360 categorizing their applications. The proximate and ultimate analyses of microalgae showed

361 highly volatile content (87.75%–94.13%) and carbon (49.31%–53.91%). The highest amounts of

362 proteins, carbohydrates, and lipids were 46.37%, 36.56%, and 28.33% DCW accumulated by T.

363 obliquus, C. sorokiniana, and T. dimorphus, respectively with > 90% N, P removal. Major fatty

364 acids that present in the microalgal species were palmitic, oleic, and linoleic acids. This study

365 demonstrated that T. obliquus, C. sorokiniana, and T. dimorphus/Chlorella sp., can be potential

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366 candidates for higher alcohols, bioethanol, biodiesel production, respectively, along with

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367 nutrients removal.

368 Acknowledgments
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369 This work was supported by the startup fund for the construction of the double first-class project
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370 (No. 561119201), Lanzhou University, China

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371
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372 References
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536 169.

537

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538 Figure Captions

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539

540 -p
Fig. 1. Map of Lanzhou city showing the collection sites.

541 Fig. 2. Cultural purification, microscopic, and molecular characterization of microalgae.

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542 Fig. 3. Schematic analysis procedures for proximate and pyrolytic analyses of dried microalgal
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543 biomass.
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544 Fig. 4. Variation of growth (A) and specific growth rate (B) according to growth of the four
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545 microalgal species cultivated in culture media. Changes in pH of culturing medium during

546 microalgae cultivation (C).

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547 Fig. 5. Removal of total nitrogen (A) and phosphorus (B) from the culture media as a result of

548 microalgal growth.

549 Fig. 6. FTIR spectra of microalgal biomass with a distinct fingerprint that represents different

550 bio-components.

551 Fig. 7. Thermogravimetric analysis (TGA) and differential thermogravimetric (DTG) dataset the

552 microalgal species.

553 Table legends

554 Table 1. Physicochemical analysis of the various collected samples.

26
555 Table 2. Proximate and ultimate analyses of the selected microalgal species.

556 Table 3. Detailed bio-components (including proteins, carbohydrates, and lipids/fatty acids) of

557 microalgal species.

558 Table 4. The estimated properties of biodiesel microalgae compared with those previously

559 reported.

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Table 1. Physiochemical analysis of the various collected samples.
Parameters Five Spring Park Yan Tang Park
pH 7.3 7.48
TDS (mg L-1) 621±4.73 661±2.160
Conductivity (µS cm-1) 995±2.64 1326±5.715
Oxidation/reduction potential (mV) N/D -29.433±0.169
COD (mg L-1) 209.2±7.5 55.87±1.256
TP (mg L-1) 5.6±1.13 9.6±1.067
TN (mg L-1) 18±0.13 29.283±6.43
TSS (mg L-1) 6.00±2.83 20.00±2.83
-1
Chromium (mg L ) 0.069 ND

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Iron (mg L ) 2.149 ND
-1
Nickle (mg L ) 0.055 ND

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Copper (mg L-1) 0.054 ND
TDS= total dissolved solids, COD = chemical oxygen demand, TP= total phosphorus, TN= total
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nitrogen, TSS= total suspended solid
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Table 2. Proximate and ultimate analyses of the selected microalgal species.
Parameters T. dimorphus T. obliquus Chlorella sp. C. sorokiniana
GEEL-06 GEEL-07 GEEL-08 GEEL-09
Proximate analysis
Total solid (wt.%) 93.52±1.223 94.77±0.80 92.71±2.570 91.92±1.299
Volatile solid (wt.% dry) 90.30±3.229 88.35±4.048 87.75±5.960 94.13±3.191
Moisture (wt.% dry) 6.48±1.223 5.23±0.080 7.29±2.570 8.08±1.299
Ash (wt.% dry) 9.695±2.944 11.645±3.893 12.247±4.002 5.865±2.198
Ultimate analysis
C (wt.% dry) 49.94±0.029 49.31±0.040 53.91±0.34 52.80±0.44
H (wt.% dry) 7.58±0.008 7.46±0.007 7.71±0.10 7.74±0.054
N (wt.% dry) 6.60±0.018 7.01±0.012 6.01±0.09 5.32±0.03
S (wt.% dry) 0.52±0.003 0.55±0.003 0.43±0.03 0.17±0.01

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O2 (wt.% dry) 35.35±0.047 35.67±0.054 31.95±0.31 33.79±0.53
C/N ratio 7.56±0.021 7.03±0.005 8.99±0.19 9.93 ±0.02

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C/H ratio 6.59±0.005 6.61±0.005 6.99±0.04 6.82±0.01
-1
HHV (MJ Kg ) 21.39 20.94 23.52 22.86
HHV= High heating value -p
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Table 3. Detailed bio-components (including proteins, carbohydrates, and lipids/fatty acids) of microalgal species.
Biocomponents (% WW-1) Chemical formula T. dimorphus T. obliquus Chlorella sp. C. sorokiniana
GEEL-06 GEEL-07 GEEL-08 GEEL-09
Carbohydrates C1H1.67O0.83 26.15±0.454 27.94±2.588 34.61±1.458 36.56±0.825
Proteins C1H1.56O0.3N0.26S0.006 36.63±1.905 46.37±3.001 31.13±1.322 39.38±2.863
Lipids C1H1.83O0.17N0.0031P0.006S0.0014 28.33±3.146 18.33±1.909 21.66±2.602 15.83±1.909
Fatty acids (% WW-1)
Palmitic acid C16:0 25.11±1.777 23.23±0.054 26.00±1.271 24.27±0.846
Palmitoleic acid C16:1 0.42±0.052 0.40±0.022 0.93±0.013 0.26±0.26

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Heptadecanoic acid C17:0 0.39±0.004 0.38±0.064 1.60±0.067 2.09±0.005

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Cis-10-Heptadecenoic C17:1 2.65±0.326 4.95±0.0133 3.78±0.189 4.32±0.111

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Stearic acid C18:0 - 12.61±0.117 - -

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Trans-9-Elaidic acid C18:1 5.35±0.107 - 4.62±0.236 4.99±0.143
Oleic acid C18:1 8.92±0.934 8.46±0.091 20.54±0.961 15.41±0.446

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Linolelaidic acid C18:2 0.75±0.113 0.54±0.025 - 0.80±0.002
Linoleic acid C18:2 37.90±0.979 33.54±0.057 13.43±0.655 15.13±0.462

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γ-Linolenic acid C18:3n6 - - 0.53±0.035 0.73±0.057
α-Linolenic acid C18:3 - - 0.66±0.036 0.42±0.420

na
Others - 18.51 15.89 27.93 31.56
ΣC16-C18 81.48 84.10 72.07 68.44
ΣSFAs (saturated fatty acids)
ΣMUFAs (monounsaturated fatty acids) ur 41.01
18.9
50.34
14.98
50.45
32.99
54.27
27.04
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ΣPUFAs (polyunsaturated fatty acids) 40.08 13.67 16.54 18.68

3
Table 4. The estimated properties of biodiesel microalgae compared with those previously
reported.
Parameters DU SV IV CN LCSF CFPP Reference
(wt.%) (mg (g I2 100 g-1 (wt.%) (°C)
KOHg-1) oil)
C. sorokiniana 58 197.5 71.82 57.85 3.58 -5.22 [56]
P. hussii - 184.06 57.03 63.12 - - [57]
Chlorella 74.1 217.8 65 56.7 6.7 4.5 [58]
S. obliquus 67.8 217.5 68.2 65.1 11.9 20.8
N. oceanica 57.4 200 94.7 52.3 6.7 4.7 [29]
NIOF15/001
N. oceanica 805 86.8 213 105.7 54.5 4.2 3.3
Chlorella 89.61 123.2 53.91 78.47 6.56 4.14 [59]
S. obliquus 94.09 93.64 40.97 95.37 7.46 6.96

of
T. dimorphus 102.529 216.389 89.171 51.459 3.177 -6.496 This study
GEEL-06

ro
T. obliquus 84.398 205.346 71.954 56.689 11.801 20.598
GEEL-07
Chlorella sp.
GEEL-08
C. sorokiniana
65.08

61.297
221.126

231.696
58.269

52.984
-p 57.872

57.935
14.046

10.833
27.65

17.557
re
GEEL-09
Palm - - 54 61.9 17.20 9 [60]
lP

Corn - - 101.0 55.7 17.80 -8

Sunflower - - 128.7 51.1 18.10 -2
Jatropha - - 109.5 55.7 18.30 -
na

Rapeseed - - 116.1 53.3 17.90 -12

DU - Unsaturation degree
SV - Saponification value
ur

CN - Iodine value= IV, Cetane number

LCSF - Long-chain saturated factor
CFPP - Cold filter plugging point
Jo

Standard recommendation for CN of biodiesel – [EN-14214 and ASTM D675140: ≥47, Australian standard: ≥51,
National Petroleum Agency in Brazil (ANP 255): ≥45]. EN-14214 recommends the iodine value should be ≤120
[29].

4
 Fig. 1.
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Highlights

All the microalgae species were able to remove 90%–95% total nitrogen and phosphorus.

The highest accumulated protein 46.37% was achieved with T. obliquus.

The highest accumulated carbohydrate 36.56%` was attained with C. sorokiniana.

Biochemical, CHNS, TGA, FTIR confirmed microalgae potential as a biofuel feedstock.

Biodiesel production feasibility was confirmed by the presence of C16/C18 (68%–84%).

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1
Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be
considered as potential competing interests:

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