Journal Pre-proof

Isolation and identification of Chlorella sorokiniana HDMA-16

and its cultivation potential in flax retting wastewater

Meiqi Wang, Jing Xu, Jialin Zou, Hongzhi Ling, Jingping Ge,
Yimeng Lin, Wenxiang Ping

PII: S2211-9264(23)00394-6
DOI: https://doi.org/10.1016/j.algal.2023.103361
Reference: ALGAL 103361

To appear in: Algal Research

Received date: 31 July 2023

Revised date: 6 December 2023
Accepted date: 8 December 2023

Please cite this article as: M. Wang, J. Xu, J. Zou, et al., Isolation and identification of
Chlorella sorokiniana HDMA-16 and its cultivation potential in flax retting wastewater,
Algal Research (2023), https://doi.org/10.1016/j.algal.2023.103361

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© 2023 Published by Elsevier B.V.

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Isolation and identification of Chlorella sorokiniana HDMA-16 and its

cultivation potential in flax retting wastewater

Meiqi Wang1, Jing Xu1, Jialin Zou1, Hongzhi Ling1, Jingping Ge1, Yimeng Lin1,2, * Wenxiang

Ping1, *

1
Engineering Research Center of Agricultural Microbiology Technology, Ministry of Education

& Heilongjiang Provincial Key Laboratory of Ecological Restoration and Resource Utilization

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for Cold Region & Key Laboratory of Microbiology, College of Heilongjiang Province & School

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of Life Sciences, Heilongjiang University, Harbin 150080, China

2
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Hebei University of Environmental Engineering, Hebei Key Laboratory of Agroecological
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Safety, Qinhuangdao 066102, China
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Abstract
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The utilization of microalgae for wastewater treatment presents a promising and cost-
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effective solution due to their ability to remove pollutants while simultaneously synthesizing

biomass from wastewater nutrients. This research identified a novel strain of Chlorella

sorokiniana and investigated its growth ability in flax retting wastewater (FRW), as well as its

potential for FRW treatment. Chlorella sorokiniana HDMA-16 was inoculated into FRW, both

with and without additional nutrient supplementation. The biomass, lipid content, and fatty acid

profile of Chlorella sorokiniana HDMA-16 were determined, along with the changes in total

nitrogen (TN), ammonia nitrogen (NH4+-N), total phosphorus (TP), and chemical oxygen
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demand (COD) in FRW. The results revealed that Chlorella sorokiniana HDMA-16 exhibited

moderate growth in FRW without the need for additional nutrients. The strain achieved

maximum biomass and lipid productivity levels of 785.7 mg L-1 and 151.5 RFU L-1 d-1. The lipid

produced by Chlorella sorokiniana HDMA-16 displayed a favorable fatty acid profile, making

the strain a potential candidate for the synthesis of by-products such as biodiesel. The removal

rates of NH4+-N, TP, and COD in FRW reached a maximum of 26.86%, 99.59%, and 29.39%.

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This study is significant in that it shows that the newly discovered Chlorella sorokiniana

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HDMA-16 can thrive in FRW and effectively remove pollutants. These findings provide a novel
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approach for FRW treatment and the large-scale cultivation of microalgae, offering promising
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prospects for sustainable wastewater treatment and microalgae-based industries.
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Abbreviations
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FRW Flax retting wastewater

TN Total nitrogen
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TP Total phosphorus
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COD Chemical oxygen demand

SFA Saturated fatty acid

MUFA Monounsaturated fatty acid

PUFA Polyunsaturated fatty acid

DU Degree of unsaturation

Keywords

Microalgae; Flax retting wastewater; Lipid; Biomass

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1. Introduction

Flax is a plant with a high economic value due to its wide use, particularly as an important

raw material in high-end textiles and clothing. The flax industry is highly polluting, generating a

large amount of pollutant-rich, low-pH wastewater that is high in nitrogen, phosphorus, and

chemical oxygen demand (COD) during processing and production[1,2]. Due to the physical and

chemical characteristics of this flax retting wastewater (FRW), there are no reports on the use of

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FRW for microalgae cultivation within the literature to date.

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Microalgae are widely known as a third-generation bioenergy alternative to traditional
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biofuels. Microalgae biomass can be used to produce lipids, proteins, polyunsaturated fatty acids,
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pigments, and other high-value products, making it a product with great economic worth[3]. They
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not only absorb and utilize carbon dioxide in the atmosphere but also have higher lipid
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production per unit area than traditional lipid crops. The need for large amounts of water and
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nutrients in microalgae biomass lipid production results in high culture costs. The cost of culture
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medium has been estimated to account for approximately 50% of the total cost of large-scale

microalgal cultivation[4]. Utilizing FRW containing nutrients such as nitrogen and phosphorus as

a culture medium may be an effective way to reduce the cost associated with the mass cultivation

of microalgae.

Cultivating microalgae in wastewater has many advantages, including improving lipid

production, removing pollutants, and reducing wastewater treatment costs[5]. Combining

microalgae cultivation and wastewater treatment is an environmentally friendly strategy to

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reduce the culture cost for microalgae and harvest high-value biomass[6]. Using wastewater for

cyanobacterial culture, Shaheed et al. were able to harvest up to 26–36% carbohydrates, 15–28%

proteins, 38–43% lipids, and 6.3–9.5% phycobilins of total biomass[7]. The use of microalgae to

treat palm oil mill effluent has been shown to significantly remove organic nutrients and

pollutants[8,9]. It has been suggested that microalgae have the potential to effectively remove

pollutants such as nitrogen, phosphorus, and COD from wastewater. Most wastewater requires

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additional fresh water dilution before it can be used for microalgae culture, which increases the

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process cost of wastewater treatment. Guangpu studied the effect of different dilution ratios on
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the growth of Scenedesmus, finding that microalgae accumulated a large amount of biomass in a
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20-fold dilution of swine wastewater[10]. Wastewater diluted 5-fold was suitable for the growth of
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Chlorella emersonii ACOI516[11]. Identifying algal strains that can tolerate highly concentrated
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flax retting wastewater will improve the economics of the wastewater treatment process.
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Although there have been many studies on the treatment of wastewater with microalgae, no
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attempt has been made to cultivate microalgae from FRW due to its high COD. This study aims

to explore the potential of a newly discovered strain of native Chlorella sorokiniana HDMA-16

to treat flax retting wastewater. Chlorella sorokiniana HDMA-16 was cultured using both flax

retting wastewater and flax retting wastewater with added nutrients to determine the lipid and

biomass content of Chlorella sorokiniana HDMA-16 and its effect on nitrogen and phosphorus

content and COD in FRW. The results of this study demonstrate that the use of Chlorella is a

feasible and cost-effective means of treatment for flax retting wastewater.

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2. Materials and methods

Chlorella sorokiniana HDMA-16 was cultivated utilizing flax retting wastewater, both with

and without additional nutrients. The growth of the microalgae was evaluated by quantifying the

number of algal cells, biomass, and lipid content. Analysis of water quality indicators to assess

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the potential of Chlorella sorokiniana HDMA-16 in treating flax retting wastewater.

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2.1 Isolation and purification of algal strains
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Seven layers of gauze filtration were used to remove larger protozoa from water samples
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collected from Guowu Lake in Daqing, China. The microalgae were enriched with Blue-green
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(BG-11) medium, after which the samples were diluted in a gradient and spread on sterilized
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solid plates of BG-11 medium. The plates were incubated in an HPG-400H artificial climatic
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chamber (China Donglian Electronic Technology Co.) at 25 °C with a light intensity of 3000 lux
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and a 12h/12h light/dark cycle. After approximately 20 days, yellowish-green algal colonies

grew on the plates, and the single algal colonies were inoculated into fresh BG-11 autotrophic

medium and incubated using the same parameters as the first incubation in an artificial climatic

chamber. After a further 7 days of incubation, the colorless medium gradually became light

yellow or green, at which time samples were taken for microscopic observation.

2.2 Algal strains and cultivation conditions

The algae were cultivated in a 250 mL Erlenmeyer flask with 100 mL BG-11 medium
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containing Na2CO3 (0.02 g L-1); NaNO3 (1.5 g L-1); MgSO4·7H2O (0.075 g L-1); K2HPO4·3H2O

(0.04 g L-1); CaCl2·2H2O (0.036 g L-1); EDTANa2 (0.01 g L-1); Ferric ammonium citrate (0.006 g

L-1); Citric Acid (0.006 g L-1); Na2MoO4·2H2O (0.00039 g L-1); Co(NO3)2·6H2O (0.00005 g L-1);

MnCl2·4H2O (0.00181 g L-1); ZnSO4·7H2O (0.00022 g L-1); CuSO4·5H2O (0.000079 g L-1);

H3BO3 (0.00286 g L-1). Algal cells were cultured at 25 °C with a light intensity of 3000 lux and a

12h/12h light/dark cycle in light shaker equipment (China Formosa Co.) at 100 rpm.

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2.3 FRW and pretreatment

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The FRW used in this study was obtained by retting flax in fresh groundwater at 30°C for
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seven days. After retting, the FRW was dark yellow-brown with an unpleasant odor. The FRW
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was first centrifuged at 4500 g for 15 min to remove solid contaminants. Next, the supernatant
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was added to TAP medium, i.e., extra nutrients, and for control samples, the supernatant was
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used without the addition of TAP medium, sterilized at 121 °C for 15 min, and used to culture
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microalgae. Microalgal strains were cultured in 500 mL flasks containing 200 mL of either FRW
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or FRW with added nutrients, under the same growth conditions as described in Section 2.2.

The components of TAP medium used in the dilution process were: NH4Cl (0.375 g L-1);

MgSO4·7H2O (0.1 g L-1); CaCl2·2H2O (0.05 g L-1); K2HPO4·3H2O (0.288 g L-1); KH2PO4 (0.144

g L-1); EDTANa2 (0.05 g L-1); ZnSO4·7H2O (0.022 g L-1); H3BO3 (0.0114 g L-1); MnCl2·4H2O

(0.005 g L-1); CuSO4·5H2O (0.0016 g L-1); FeSO4·7H2O (0.005 g L-1); CoCl2·6H2O (0.0016 g L-

1
); (NH4)6Mo7O24·4H2O (0.0011 g L-1).

2.4 Phylogenetic analysis

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The total genomic DNA of Chlorella sorokiniana HDMA-16 was extracted using the

Fungal Genomic DNA Extraction Kit (China Bori Technology Co.). Two primers (ITS-up:

5'-GGAAGTAAAAGTCGTAACAAGG-3'; ITS-down: 5'-TCCTCCGCTTATTGATATGC-3')

were used for PCR amplification of partial ITS genes. Each PCR reaction mixture (50 μL) was

composed of 5 μL of 10× Pfu Buffer; 4 μL of dNTP Mixture; 10 μL of primers; 1 μL of template

DNA; 0.2μL Pfu DNA polymerase and 29.8 μL of sterile water. The PCR program was as

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follows: pre-denaturation for 6 min at 94 °C, 30 cycles at 94 °C, 55 °C and 72 °C for 30 s each,

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and a final extension step at 72 °C for 10 min.
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The ITS gene was amplified, sequenced, and aligned with selected sequences obtained from
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NCBI. Subsequently, the NJ model bootstrap within Mega X was set to 1000, and
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Monoraphidium sp. KT-2021 (MZ852873.1) was selected as the outgroup to construct the
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phylogenetic tree.
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2.5 Growth of Chlorella sorokiniana HDMA-16

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Algal cell density was calculated using a hemocytometer and the sample optical density was

determined with a V-500 UV spectrophotometer (China Yuan Analytical Instrument Co.) at 680

nm using fresh water as a blank to further verify the data of algal cells [12].

2.6 Determination of biomass and lipid content

The dry cell weight of Chlorella sorokiniana HDMA-16 in the different FRW

concentrations was measured by gravimetric analysis. The algal precipitate was obtained by

centrifuging 70 mL of algal culture at 3800 g for 20 min. The precipitate was then dried to
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constant weight. The biomass was calculated as the mass of algae powder per unit volume [13].

Neutral lipid was analyzed by the Nile Red fluorescence method as described by Xiao et

al.[14] 50 μL of DMSO was added to 948 μL of algal solution with an OD680 density of 1.4. To

break up the algal cells, the sample was shaken vigorously and left to stand for 1 min, after

which 2 μL of Nile Red was added. Samples were then shaken and incubated in a constant

temperature incubator at 37℃ under darkness for 30 min. The relative fluorescence intensity was

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measured with SpectraMax iD3 Multi-Mode Microplate Reader (USA Molecular devices). The

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fixed excitation wavelength was set at 430 nm and the generation wavelength at 685 nm. The
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relative fluorescence intensity is representative of the relative lipid content
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2.7 Analysis of fatty acid methyl esters
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To analyze the fatty acid composition of the lipid derived from Chlorella sorokiniana
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[15]
HDMA-16, the following procedure was used . First, 0.3 g of dried Chlorella sorokiniana
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HDMA-16 sample was ground thoroughly and added to 7.5 mL of a sodium hydroxide methanol
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solution (0.5 mol/L). The mixture was then heated in a 70 ºC water bath and refluxed for 5

minutes. After the initial reflux, 10 mL of boron trifluoride methanol solution was added to the

flask and the sample was allowed to reflux for another 5 minutes. Following the addition of 4 mL

of n-hexane, the sample refluxed for 8 minutes. An appropriate amount of saturated saline

solution was added to the flask and the mixture was left to stand for 5 minutes. Next, 1 mL of the

upper layer of oil was collected for analysis of the fatty acid methyl esters using gas

chromatography–mass spectrometry (GC–MS), according to the parameters determined in the

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experiments conducted by Lin et al. [16].

2.8 Measurement of nutrients in FRW

An appropriate amount of microalgal culture was sampled every 24 h and centrifuged at

4000 g for 15 min to obtain the supernatant for subsequent sample dilution and nutrient

determination. The contents of COD, TN, TP, and NH4+-N were analyzed according to the

Chinese national environmental water and wastewater monitoring and analysis method [17].

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2.8.1 Ammonia nitrogen (NH4+-N)

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Diluted samples were placed in colorimetric tubes, ddH2O was added to the cuvette scale
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line and 1.0 mL of 500 g L-1 potassium sodium tartrate solution and 1.0 mL of Nessler's reagent
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were added to the tube. After 10 min, the absorbance was measured with SpectraMax iD3 Multi-
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Mode Microplate Reader (USA Molecular devices) at a wavelength of 420 nm, using water as a
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reference.
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2.8.2 Total nitrogen (TN)

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Each diluted sample was added to 10.00 mL of water in a 25 mL glass colorimetric tube,

followed by 5.00 mL of 40 g L-1 alkaline potassium persulfate solution. The colorimetric tube

was placed in a high-pressure steam sterilizer for 30 min with a temperature maintained between

120–124 °C. After cooling to room temperature, 1.0 mL of 0.119 g mL-1 hydrochloric acid

solution was added to each colorimetric tube, which were then diluted with water to 25 mL. The

sample was transferred to a 10 mm quartz cuvette and the absorbance was measured at 220nm

and 275nm on a V-500 UV spectrophotometer (China Yuan Analytical Instrument Co.)

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2.8.3 Total phosphorus (TP)

The sample and 4 mL of 0.05 g mL-1 potassium persulfate solution were added to the

cuvette which was tightly capped and placed in an autoclave. Heating was stopped 30 min after

the pressure reached 1.1 kg cm-2 and the corresponding temperature was 120 °C. Then, 1 mL of

100 g mL-1 ascorbic acid solution and 2 mL of 0.13 g mL-1 molybdate solution were added. After

15 min at room temperature, the absorbance was measured at a wavelength of 700 nm using a

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cuvette with an optical path of 30 mm and water as a reference.

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2.8.4 Chemical oxygen demand (COD)
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In an Erlenmeyer flask, 10.0 mL of the water sample, 5.0 mL of 100 g L-1 mercury sulfate
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solution, and 5.0 mL of 0.025 mol L-1 potassium dichromate solution were combined. The flask
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was then connected to the lower end of the condenser tube of the reflux device, and 15 mL of
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10.0 g L-1 silver sulfate-sulfuric acid solution was slowly added from the upper end of the
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condenser tube to prevent the escape of low-boiling organic matter. The micro-boiling reflux was
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maintained for 2 h after the solution began to boil. After reflux cooling, 45 mL of water was

added to the upper end of the condenser tube to flush it, resulting in a total solution volume was

approximately 70 mL, and the flask was removed. After the solution cooled to room temperature,

3 drops of ferrous iron indicator solution were added and titrated with 0.005 mol L-1 ammonium

ferrous sulfate solution, until the color of the solution changed from yellow to blue-green to an

endpoint of red-brown, after which the consumption volume of the titrant (V1) was recorded.

A blank test was carried out using 10.0 mL of reagent water instead of an FRW sample
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following the same steps outlined above, and the volume of titrant consumed for the blank

titration (V0) was recorded.

Mass concentration of chemical oxygen demand in the sample, ρ(mg L-1), was determined

using the following equation：

𝐶 × (𝑉0 − 𝑉1) × 8000

𝜌= ×𝑓
𝑉2
Where：

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C is the concentration of ammonium ferrous sulfate solution, in mol L-1; V0 is the volume

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of ammonium ferrous sulfate solution consumed in the blank titration, in mL; V1 is the volume
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of ammonium ferrous sulfate solution consumed in FRW sample titration, in mL; V2 is the
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volume of the FRW sample, in mL; f is the time of dilution of the sample; and 8000 is a
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conversion of the molar mass of 4 O2 in mg L-1.
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2.9 Statistical analysis

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All experimental groups were set up in three parallels, and the data were analyzed and
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plotted using Origin 2021 software. SPSS Statistics 22.0 software was used for variance analysis

and a p-value of 0.05 was used to determine statistical significance.

3. Results and discussion

Microalgae are autotrophic organisms capable of absorbing nitrogen and phosphorus from

wastewater to support their growth. They utilize photosynthesis to biocatalytically eliminate

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organic matter, thereby purifying the water. Throughout the water purification process,

microalgae can also generate biomass and lipids, which, in turn, can be harnessed for by-

products like biodiesel, maximizing resource utilization. In this study, we assessed the biomass

and lipid content of Chlorella sorokiniana HDMA-16 and closely monitored the levels of COD,

TN, TP, and NH4+-N in FRW, aiming to investigate the potential of using Chlorella sorokiniana

HDMA-16 as a means of flax retting wastewater treatment.

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3.1 Identification of microalgal strain

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The preliminary observation of the isolates’morphology under the light microscope showed
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that the algal cells are green-colored, spherical, solitary cells without any mucilaginous envelope,
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[18]
bristles, and spines outside the cell wall (Fig. S1) . Microscopic cells with a diameter of 3–8
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μm. Preliminary morphological identification indicated that the strain belonged to the genus
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Chlorella.
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Fig. 1. Phylogenetic tree of Chlorella sorokiniana HDMA-16 based on partial ITS sequences.

In order to further identify the algal species based on morphological species identification,

the ITS region of the strain was amplified by PCR. The sequences were compared to related

sequences in NCBI. Results showed that the ITS sequence of the microalgae (HDMA-16) used

in this study shared 100% identity with Chlorella sorokiniana (Figure 1).

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3.2 Growth of Chlorella sorokiniana HDMA-16 in FRW

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Chlorella sorokiniana HDMA-16 was cultured in FRW and FRW with added nutrients for 7
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days (Figure 2a), showing a similar growth trend in different FRW. In the raw FRW, Chlorella
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sorokiniana HDMA-16 entered a stagnant phase during days 0–2 and a logarithmic growth phase
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on days 2–5. The cell count reached a maximum of 6.37 × 107 mL-1 on day 5. In FRW with
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added nutrients, Chlorella sorokiniana HDMA-16 grew rapidly in 2–5 days and the cell count
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also reached a maximum on day 5, this time at 5.46 × 107 mL-1.

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The trend of the optical density curve (Figure 2b) was consistent with the growth curve,

further confirming the growth state of microalgae. The initial OD680 of the FRW was much

higher than that of the FRW with added nutrients, which may be due to the influence of the color

of the FRW itself. Generally, the growth of Chlorella sorokiniana HDMA-16 in FRW alone was

significantly better than in FRW with added nutrients when using the same number of inoculated

cells, indicating that the nutrient and trace element content of raw FRW are more suitable for the

growth of the microalgal strain.

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The current treatment methods for FRW primarily use traditional filtration and membrane

treatment processes. Most of these processes require the use of large quantities of fresh water,

which increases the cost of wastewater treatment. Wastewater used for microalgae culture

usually needs to be diluted as well, which increases the cost of microalgae cultivation. Elystia et

al. found that tofu wastewater was most suitable for culturing Chlorella sp. when diluted to a 40%

concentration, resulting in the highest cell density and highest COD and NH4+-N removal

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efficiency [19]. In the present study, Chlorella sorokiniana HDMA-16 grew better in raw FRW

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than in FRW with extra nutrients, indicating the potential of Chlorella sorokiniana HDMA-16 to

be cultured in highly concentrated organic wastewater.

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(a) 7 (b)
2.0 FRW with added nutrients
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FRW with added nutrients

FRW FRW
6 1.8
Optical density(680nm)

5 1.6
Cell density(107/mL)

1.4
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4
1.2
3
1.0

2 0.8
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0.6
1
0.4
0
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0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7
Time(days) Time(days)

Fig. 2. The growth of Chlorella sorokiniana HDMA-16 in FRW (a): The growth curve based on

cells number of Chlorella sorokiniana HDMA-16 in FRW or FRW with added nutrients within 7

days. (b): The growth curve based on optical density of Chlorella sorokiniana HDMA-16 in

FRW or FRW with added nutrients within 7 days. Each value is mean ± SD.

Biomass and lipid production based on microalgae wastewater treatment systems has
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become increasingly popular over the past decade because it minimizes production costs

compared to synthetic media[20]. The biomass and lipid production of Chlorella sorokiniana

HDMA-16 are summarized in Table 1. After 7 days of culture, the biomass of Chlorella

sorokiniana HDMA-16 was 785.7 mg L-1 in raw FRW and 442.9 mg L-1 in FRW with added

nutrients. The increase in biomass was consistent with changes in cell number and oxygen

demand (OD) (Figure 2).

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Chlorella sorokiniana HDMA-16 also achieved a high biomass productivity of 112.2 mg L-

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1
d-1 in FRW. The lipid productivity of Chlorella sorokiniana HDMA-16 in raw FRW and FRW
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with added nutrients was 151.5 RFU L-1 d-1 and 46.19 RFU L-1 d, meaning the lipid content in
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raw FRW is approximately 227.9% higher than in FRW with added nutrients. The fact that
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Chlorella sorokiniana HDMA-16 can produce lipid directly in FRW with significantly higher
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lipid productivity than FRW with added nutrients will not only save fresh water but also
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simplifies the process of using wastewater for microalgae culture.

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Table 1. The biomass and lipid productivity of Chlorella sorokiniana HDMA-16 after

wastewater culture
biomass
biomass Relative lipid Lipid productivity
productivity
(mg/L) productivity (RFU/L/d)
(mg/L/d)

FRW with 442.9 63.3 0.73×107 46.19

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added nutrients

FRW 785.7 112.2 1.35×107 151.5

..

3.3 Fatty acid analysis by GC–MS

In recent years, the production of lipids from microalgae has garnered significant attention

worldwide. Microalgae thrive under easily attainable cultivation conditions and produce high-

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quality biodiesel, which has the potential to gradually replace the use of fossil fuels. In order to

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explore the lipid quality of Chlorella sorokiniana HDMA-16 cultured in flax retting wastewater,

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the fatty acid composition of the strain was determined.
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The lipid extract of Chlorella sorokiniana HDMA-16 was subjected to GC-MS analysis,
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with the resulting algal fatty acid profile presented in Table 2. The analysis revealed that
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Chlorella sorokiniana HDMA-16 contains 16 fatty acids, with a dominance of long-chain fatty

acids ranging from C12 to C24. Notably, the saturated fatty acid (SFA) content in these
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microalgae was found to be 43.78%, while the total unsaturated fatty acid content exceeded 50%.
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Within the unsaturated fatty acids, 36.05% were monounsaturated and 20.1% were

polyunsaturated. It is worth highlighting that the monounsaturated fatty acid content was

significantly higher compared to the values of 27.58% and 25.17% observed in Chlorella

vulgaris cultures using Pyropia-processing wastewater and BG-11, as reported by Zheng et al.

[21]
. Prior research has demonstrated that the presence of C16:1 and C18:1 monounsaturated fatty

acid structures in oils and fats plays a pivotal role in enhancing the chemical stability of biodiesel
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[22]
. The lipid derived from Chlorella sorokiniana HDMA-16 revealed a degree of unsaturation

(DU) value of 76.17, which meets the requirement set by the European Committee for

Standardization (less than 137). Chlorella sorokiniana HDMA-16 is an excellent candidate for

biodiesel production due to its favorable fatty acid profile.

Table 2. Fatty acid composition of Chlorella sorokiniana HDMA-16 after culture in flax retting

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wastewater

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Fatty acid Name Yield (%, w/w)

C6:0 -p
Hexanoic acid,methyl ester acid,methyl ester 0.01
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C12:0 Undecanoic acid, 10-methyl-, methyl ester 3.08
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C15:0 Methyl 13-methyltetradecanoate 0.17

C16:0 Hexadecanoic acid,methyl ester 15.41

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C16:1 9-Hexadecenoic acid,methyl ester 13.03

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C16:2 7,10-Hexadecadienoic acid,methyl ester 2.63

C16:4 Methyl 4,7,10,13-hexadecatetraenoate 3.30

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C18:0 Methyl stearate 16.79

C18:1 8-Octadecenoic acid,methyl ester 18.53

C18:1 6-Octadecenoic acid,methyl ester 4.49

C18:2 9,12-Octadecadienoic acid,methyl ester 7.00

C18:3 Methyl 9 cis,11 trans 13 trans octadecatrienoate 4.50

C18:3 9,12,15-Octadecatrienoic acid, methyl ester, (Z, Z, Z) 2.63

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C20:0 Methyl 18-methylnonadecanoate 0.36

C22:0 Docosanoic acid, methyl ester 2.95

C24:0 Tetracosanoic acid, methyl ester 5.10

SFA 43.87

MUFA 36.05

PUFA 20.06

DU 76.17

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..

3.4 Nutrient removal efficiency of Chlorella sorokiniana HDMA-16 in FRW

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The excess nitrogen and phosphorus in wastewater diminishes the dissolved oxygen levels
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in the water, undermining the stability and biodiversity of aquatic ecosystems. The elevated
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COD in FRW indicates the significant presence of reducing agents, primarily in the form of
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organic pollutants. Failure to promptly address this issue can lead to the long-term deposition of
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these organic pollutants, resulting in persistent toxic effects on aquatic organisms for many years.
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Nitrogen, phosphorus, and COD all serve as crucial parameters for assessing the quality of

wastewater.

3.4.1 Characteristics of FRW

Due to the decomposition of the outer layer of flax during the retting process, FRW

contains a large amount of organic matter and inorganic nutrients. The concentration of organic

matter in FRW far exceeds the Chinese national wastewater discharge standards. The high

concentration of COD can lead to increased turbidity in the water, which affects the light
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absorption of microalgae, thereby inhibiting the growth of microalgae and the efficiency of

nutrient removal [23]. If such wastewater is discharged without treatment, it will cause great harm

to natural water resources. The physical and chemical properties of FRW are shown in Table 2.

The concentrations of NH4+-N, TN, and TP were 65.19 mg L-1, 993.45 mg L-1, and 2.52 mg L-1.

The content of COD in the FRW was particularly high, approximately 3480 mg L-1, which can

be attributed to the large amount of organic matter produced in the process of flax retting. The

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concentrations of NH4+-N, TN, TP and COD were 69.50 mg L-1, 1098.08 mg L-1, 3.16 mg L-1

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and 3538. mg L-1. Results showed that the levels of NH4+-N, TN, TP, and COD in the obtained
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FRW are higher than acceptable limits. Many wastewaters currently require dilution treatment
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before being used for microalgae culture. If Chlorella sorokiniana HDMA-16 can grow directly
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in raw FRW and achieve the desired degree of water purification, it would greatly reduce the
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cost of macerated wastewater treatment.

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The pH of FRW is low, while the optimal pH for most freshwater microalgae is 7–9.
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Preliminary experiments identified that the cultivation of Chlorella sorokiniana HDMA-16 in

raw FRW with an unadjusted pH was suboptimal. The pH of the raw FRW was fine-tuned at the

beginning of this experiment to facilitate better growth of Chlorella sorokiniana HDMA-16.

Compared with the commonly used medium, the content of nitrogen and phosphorus in FRW

meets growth requirements, confirming the possibility of directly using FRW to cultivate

Chlorella sorokiniana HDMA-16.

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Table 3. Physicochemical properties of flax retting wastewater and flax retting wastewater with

added nutrients
parameter flax retting wastewater content nutrient-added flax retting wastewater

pH 5.6 ± 0.20 6.4± 0.40

NH4+-N (mg/L) 65.2 ± 1.30 69.5± 1.39

TN (mg/L) 993.5 ± 19.70 1098.08± 21.96

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TP (mg/L) 2.5 ± 0.0 3.1± 0.06

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COD (mg/L) 3480.0 ± 70.7 3538± 70.76

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3.4.2 Ammonia nitrogen and total nitrogen removal from FRW by Chlorella sorokiniana
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HDMA-16
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Nitrogen is one of the most common pollutants in water bodies. Excessive ammonia

nitrogen can lead to eutrophication, red tide, algal blooms, and the poisoning and death of
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aquatic organisms. It is difficult for wastewater with high ammonia nitrogen content to meet the
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requirements for treated effluent due to its complex composition and poor biochemical properties.
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It is valuable to explore an economical and sustainable method of ammonia nitrogen removal.

As shown in Figures 3a and b, the ammonia nitrogen in FRW was continuously removed by

Chlorella sorokiniana HDMA-16 over the 7 days of culture, decreasing from 76.32 mg mL-1 to

55.82 mg mL-1 and reaching a removal rate of 26.86%. The removal rate of ammonia nitrogen

from FRW with added nutrients was slower than in raw FRW. The removal was relatively

efficient in the first two days of FRW with added nutrients treatment, then decreased
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significantly on the third day. The ammonia nitrogen concentration in FRW with added nutrients

decreased from 80.10 mg mL-1 to 59.62 mg mL-1 and the removal rate was 25.56%.

In the RW and FRW with added nutrients, the total nitrogen content gradually decreased

during the culture time, and the removal rate of total nitrogen by Chlorella sorokiniana HDMA-

16 increased significantly (Figures 3c and d). After 7 days of cultivation, Chlorella sorokiniana

HDMA-16 reduced the total nitrogen concentration in raw FRW and FRW with added nutrients

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to 79.54 mg mL-1 and 85.89 mg mL-1. The removal rate of total nitrogen in raw FRW was 16.84%

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and the removal rate of total nitrogen in FRW with added nutrients was 17.76%.
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It is worth noting that microalgae generally exhibit a limited capacity to remove organic
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nitrogen and the total nitrogen present in wastewater comprises both inorganic and organic
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nitrogen components[24]. In the present study, the observed removal rate of total nitrogen from
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FRW by Chlorella sorokiniana HDMA-16 was not particularly high, which may suggest that the
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FRW used in this study contains a relatively high concentration of organic nitrogen. This
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observation highlights the importance of further investigating the characteristics and composition

of the organic nitrogen present in flax retting wastewater to better understand its impact on

microalgae-based treatment processes.

Overall, Chlorella sorokiniana HDMA-16 showed potential for the removal of ammonia

nitrogen and total nitrogen from FRW. Chlorella sorokiniana HDMA-16 cultured in FRW was

more effective in removing ammonia nitrogen and TN than in FRW with added nutrients. Since

the number of algal cells inoculated in different FRW treatments was approximately the same in
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this study, the experimental group with better algal cell growth also exhibited a higher removal

rate of ammonia nitrogen and TN in combination with the growth status of algal cells (Figure 2a).

It is speculated that the removal rate of TN and NH4+-N may be related to algal cell growth. Zhai

et al. previously found that algal density was positively correlated with the effectiveness of

nutrient removal when using Chlorella sp. HQ to treat starch wastewater [25].
85 30
(a) FRW with added nutrients
(b) FRW with added nutrients

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FRW FRW
80
NH4+-N concentration (mg/L)

Removal rate of NH4+-N(%)

25

75

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20
70

65

60
15

10
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55
1 2 3 4 5 6 7 1 2 3 4 5 6 7
Time(days) Time(days)
110 20
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(c) FRW (d)

FRW
105 FRW with added nutrients FRW with added nutrients
TN concentration (mg/L)

Removal rate of TN(%)

100
15
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95

90

85 10
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80

75
5
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70
1 2 3 4 5 6 7 1 2 3 4 5 6 7
Time(days) Time(days)

Fig. 3. Treatment effect of Chlorella sorokiniana HDMA-16 on ammonia nitrogen and total

nitrogen. ( a )The concentration change of ammonia nitrogen in FRW or FRW with added

nutrients; ( b ) The change of ammonia nitrogen removal rate in FRW or FRW with added

nutrients; ( c )The change of total nitrogen concentration in FRW or FRW with added nutrients;

( d )The change of total nitrogen removal rate in FRW or FRW with added nutrients.
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3.4.3 Total phosphorus

High phosphorus levels in water bodies can cause algae bloom and damage to the water

ecosystem, so phosphorus must be removed from wastewater before discharge. Phosphorus is an

essential element in the fertilizer and feed industries, so recovering phosphorus from wastewater

for reuse could bring significant social and economic benefits. Traditional wastewater treatment

methods are less efficient in removing phosphorus[26]. Using microalgae for phosphorus removal

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from wastewater has shown great potential in recent studies[27].

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The total phosphorus content in the FRW showed a downward trend (Figure 4a). In the raw
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FRW, the TP decreased rapidly on the first day, after which the removal rate decreased
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significantly. The TP concentration fell to 0.0079 mg L-1, representing a removal rate of 99.59%.
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In the FRW with added nutrients, the removal of TP was steady over the 7 days of treatment,
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decreasing from 2.49 mg L-1 to 0.59 mg L-1, with a final removal rate of 76.25%.
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The TP concentration in the two media decreased to a very low level on days 5–7.
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Combined with the growth results of Chlorella sorokiniana HDMA-16 (Figure 2a), the low

concentration of phosphorus may be an explanation for the significant decrease in the number of

Chlorella sorokiniana HDMA-16 during the same time period, limiting the growth of possibly

leading to microalgal death in the later logarithmic growth.

In this study, Chlorella sorokiniana HDMA-16 showed a very high removal efficiency of

TP of more than 99.00% from raw FRW, but only 76.25% in FRW with added nutrients (Figure

4b). The concentration of ammonia nitrogen in a water body has been shown to be an important
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factor affecting the removal rate of TP. Beuckels et al. found that the nitrogen concentration in

wastewater is closely related to the ability of microalgae to absorb phosphorus[28]. When the

ammonia nitrogen content in wastewater is high, Chlorella and Scenedesmus may exhibit a better

removal effect on phosphorus, and even further affect the content of microalgae biomass

components. The high efficiency of phosphorus removal in this study may be for this reason.

Future work should continue to investigate how the nitrogen content affects phosphorus removal

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and how they influence the biochemical composition of the biomass, which is important for the

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valorization of microalgae biomass.
(a) 3.0
FRW with added nutrients
(b)
100
-p FRW with added nutrients
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FRW FRW
2.5
90
TP concentration (mg/L)

Removal rate of TP(%)

2.0 80
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70
1.5

60
1.0
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50
0.5
40

0.0 30
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1 2 3 4 5 6 7 2 3 4 5 6 7
Time(days) Time(days)
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Fig. 4. Treatment effect of Chlorella sorokiniana HDMA-16 on total phosphorus. ( a )The

concentration change of total phosphorus in FRW and FRW with added nutrients; ( b )The

removal rate of total phosphorus in FRW and FRW with added nutrients.

3.4.4 Chemical oxygen demand

The composition of high organic wastewater is complex and diverse. The COD of highly

organic wastewater is usually above 2000 mg L-1. The malodor, foam, and coloration occurring
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in high-concentration organic wastewater reduce the use values of water bodies. High-

concentration organic wastewater may also contain a large amount of toxic organic substances,

which can accumulate and be stored in the natural environment, entering the human body

through the food chain, and endangering human health. It is crucial to reduce the COD of FRW

before discharge.

In this study, Chlorella sorokiniana HDMA-16 was incubated in FRW and FRW with

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added nutrients for 7 days, and changes in COD were observed (Figure 5). Within 7 days of

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treatment, Chlorella sorokiniana HDMA-16 reduced the COD in raw FRW from 2620.0 mg L-1
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to 1850.0 mg L-1, indicating a removal rate of 29.39%. In FRW with added nutrients, the
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reduction effect of algae on COD was poor. COD only fell from 2628.0 mg L-1 to 2360.0 mg L-1,
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and the removal rate was 11.94% (Figures 5). The COD reduction rate in FRW was better than
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that in FRW with added nutrients. A higher number of algal cells was seen to lead to a higher
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COD removal rate, and vice versa (Figure 2a), which also confirms the findings of Brar et al.[29].
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The removal rate of COD in raw FRW by Chlorella sorokiniana HDMA-16 was not as high

as in some previous studies[30]. This may be due to the large amount of macromolecular organic

matter, such as tannin, lignin, and cellulose, in the FRW. These organic matters cannot be

effectively utilized by Chlorella sorokiniana HDMA-16. Nagarajan et al. previously reported

that Scenedesmus sp. cannot utilize most of the organic nutrients in wastewater and other steps

are required to improve the reduction of COD[31]. It is important to mention that this present

study presents only preliminary results using FRW for microalgal culture. Additional nutrients
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and CO2 aeration were not used. Future studies should adjust the composition of FRW by adding

additional phosphorus, trace elements, and carbon dioxide to further improve the reduction of

COD in FRW.

3000
First day
Seventh day
Remove rate 30
2500

of
COD concentration (mg/L)

2000 25

Remove rate
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1500
20

1000 -p
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15
500
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0 10
FRW with added nutrients FRW
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Fig. 5. Treatment effect of Chlorella sorokiniana HDMA-16 on COD in FRW and FRW with
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added nutrients.
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4. Conclusion

In this work, higher removal rates of NH4+-N (26.86%), TP (99.59%), and COD (29.39%)

were observed in untreated flax retting wastewater cultured with Chlorella sorokiniana HDMA-

16 compared to nutrient-added flax retting wastewater. Good biomass and lipid production were
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demonstrated by the microalgal strain, and a suitable fatty acid profile was observed, suggesting

the potential of FRW treatment by Chlorella sorokiniana HDMA-16 for various applications,

such as the production of fertilizers and biodiesel. This method has the potential to reduce costs

associated with microalgae cultivation and FRW treatment, while also contributing to the

improvement of the ecological environment. In future investigations, factors such as CO2 supply

and culture temperature will be further examined to enhance the treatment potential of Chlorella

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sorokiniana HDMA-16 on flax retting wastewater. The co-cultivation of Chlorella sorokiniana

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HDMA-16 with algae strains possessing superior lipid production capabilities, or even the
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exploration of co-cultivation with multiple algal species, is also of interest. These approaches
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aim to enhance the efficiency of FRW purification and maximize the utilization of its nutrient
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content for higher lipid yields. The feasibility of large-scale microalgae cultivation derived from
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flax retting wastewater must be established to contribute to a sustainable treatment approach.

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Funding
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This work was supported by Heilongjiang Provincial Natural Science Foundation of China

(LH2020C089), the Heilongjiang Province University Basic Research Foundation (2021-

KYYWF-0014, 2022-KYYWF-1080) and the National Natural Science Foundation of China

(32071519).

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Author contributions:

Meiqi Wang (First Author): Conceptualization, Methodology, Software, Investigation, Formal

Analysis, Experiment, Writing - Original Draft;

Jing Xu: performed the experiment;

Jialin Zou: contributed to data analysis and manuscript preparation;

Hongzhi Ling: helped perform the analysis with constructive discussions.

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Yimeng Lin (Corresponding Author): Conceptualization, Funding Acquisition, Resources,

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Supervision, Writing - Review & Editing.
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Jingping Ge: Conceptualization, Funding Acquisition, Resources, Supervision, Writing - Review
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& Editing.
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Wenxiang Ping (Corresponding Author): Conceptualization, Funding Acquisition, Resources,

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Supervision, Writing - Review & Editing.

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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.

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☐ The authors declare the following financial interests/personal relationships which may be

considered as potential competing interests:

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Highlights:

l. An algal strain isolated from a freshwater lake was identified as Chlorella sorokiniana

HDMA-16.

2. HDMA-16 can grow in flax retting wastewater.

3. HDMA-16 has a good potential in the removal of NH4+-N, TN, TP and COD in flax retting

wastewater.

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4. HDMA-16 has high biomass and oil productivity while culturing in flax retting wastewater.

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