# Adding micronutrient of FeCl 2 and ZnCl 2 to culture medium in order to enhance high-value bioproduct extraction from microalgae

Zahra Khodabakshi, Bahman Nazari, Mohsen Taghavijeloudar

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Zahra Khodabakshi, Bahman Nazari, Mohsen Taghavijeloudar. Adding micronutrient of FeCl 2 and ZnCl 2 to culture medium in order to enhance high-value bioproduct extraction from microalgae. 13th international conference on new solutions in engineering, information science & technology of the century ahead, Nov 2022, Thessalokini, Greece. hal-04363449

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Distributed under a Creative Commons CC0 1.0 - Universal - International License
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13th international conference on

NEW SOLUTIONS IN
ENGINEERING, INFORMATION
SCIENCE & TECHNOLOGY OF
THE CENTURY A HEAD

<figure><img src="image_3.png" /></figure>

5 November 2022

www.13iciet.ir

Greece - Thessaloniki

# Adding micronutrient of FeCl₂ and ZnCl₂ to culture medium in order to enhance high-value bioproduct extraction from microalgae

Zahra Khodabakshi¹, Bahman Nazari, Mohsen Taghavijeloudar²*

1. MSc. Department of Civil and Environmental Engineering, Babol Noshirvani University of Technology, Babol, Iran. Email: mahsa999kh@gmail.com

2. MSc. Department of Chemical Engineering, Babol Noshirvani University of Technology, Babol, Iran. Email: bn.nazari1991@gmail.com

3. PhD. Department of Civil and Environmental Engineering, Seoul National University, Seoul, Korea. Email: mohsen@snu.ac.kr

**Abstract.**

High-value bioproducts extraction from microalge has received increasing attraction as a sustainable and promising alternative for traditional fuel sources. In this research, adding different trace elements (TEs) of FeCl₂ and ZnCl₂ to culture medium was evaluated to improve microalgae growth rate and enhance high-value bioproduct extraction from biomass. To do this, the effect of different dosage of TEs on biomass growth rate and bioproducts of protein, carbohydrate and lipid extraction were investigated. According to the results, maximum biomass concentrations of 3.24 and 3.84 g/L were achieved after adding 0.2 and 0.5 mg/L of FeCl₂ and ZnCl₂, respectively. While, in the case of microalgae without adding TEs, the maximum biomass concentration was 2.2 g/L during 12 days cultivation. The results revealed that treating microalge culture medium with micronutrients can significantly improve the high-value bioproducts of biomass. Maximum bioproducts values increased from 7 to 17 and 19 %MDW (carbohydrate) and from 21 to 45 and 52 %MDW (protein) and from 11 to 23 and 25 %MDW after adding 0.2 and 0.5 mg/L of FeCl₂ and ZnCl₂, respectively. The results of this research prove that treating microalgae culture medium with an appropriate level of TEs can significantly enhance both biomass growth rate and high value bioproduct extraction from biomass.

**Keywords:** Microalgae biomass, High-value bioproducts, lipid, protein, carbohydrate
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# 1. Introduction

The escalating worldwide need for energy, combined with the substantial environmental repercussions linked to conventional energy resources, has stimulated the exploration of sustainable and varied energy alternatives (Rostaghi Chalaki et al., 2020; Chalaki et al., 2016). Microalge have gained lots of attention as a sustainable and next-generation source of energy due to containing high-valuable bioproducts such as protein, polysaccharide and lipids (Calijuri et al., 2022). Microalgae protein can be used as a feed supplement for animals and aquatic organisms and in the manufacturing of nutritional supplements and cosmetics (Barros et al., 2015). Interestingly, it has been reported that microalgal proteins can be utilized for partial recovery of visual function in blind patients (Sahel et al., 2021). While, carbohydrates and lipids generated by microalgae can be utilized as feedstock for the production of biofuels (Gupta et al., 2018). Generally, the protein, carbohydrate and lipid content in microalgae can varies from 10 - 52%, 15 – 60% and 5 – 25% of microalge dry weight (MDW), respetively (Matos et al., 2016). However, these level of bioproducts can greatly be influenced by environmental factors, such as temperature, illumination, pH-value, growth phase physiological status and the season, even in the same species of microalgae (Shipin et al., 1999; Shirvani et al., 2020). Several reseracher have focused on increasing the level of bioproducts preduced from microalge by optimizing the environmental factors (Pereira et al., 2013; Lee et al., 2009; Vandamme et al., 2012). However, it should be noted that controlling environmental factors at large scale is very difficult or almost impossible. Therefore, some researchers attempt to increase biomass and high value products by adding chemical nutrient into culture medium (Gupta et al., 2016; Cheng et al., 2019; Wang et al., 2014).

In recent years, using trace elements (TEs) such as low dosage of heavy metals for improvement of microalgae growth has been noted by many investigators (chen et al., 2009; Ismail et al., 2002; Baumann et al., 2009). Hui-Ling et al. in 2012 investigated The impact of sub-lethal concentrations (0.05, 0.5, 5 µmol L⁻¹) of the heavy metals copper (Cu), chromium (Cr), zinc (Zn), cadmium (Cd) and lead (Pb) on the growth and photosynthesis of Chlorella vulgaris was studied during 96 h exposure experiments (Ouyang et al., 2012). Thier results showed that the effects of these five metals on the growth of *Chlorella vulgaris* were dependent on both concentration and exposure time. It was found that 5 µmol L⁻¹ treatments of Cu, Cr, Zn, Cd and Pb significantly inhibited the growth of *Chlorella vulgaris*, and the effect became weaker with an increase in exposure duration. The effect of FeCl₂ on growth and lipid accumulation in marine microalgae *Chlorella vulgaris* were investigated by Liu et al (2008) (Liu et al., 2008). Total lipid content in cultures supplemented with 1.2 ×10⁻⁵ mol/L FeCl₂ was up to 56.6% biomass by dry weight and was 3–7-fold that in other media supplemented with lower iron concentration. Manganese (Mn) is anonother essential trace element for plant growth which plays a key role in the light reactions of photosynthesis. According to Chen et al (2021) neutral lipid content in *P. tricornutum* was enhanced by Mn²⁺, likely as a result of increased production of enzymes in the C4 pathway (Chen et al., 2021). It should be noted that most of the previous studies regarding the impacts of heavy metals as TEs on algae were mainly focused on finding the optimum concentration of TEs for maximum biomass production. To the best of
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our knowledge limited studies have been conducted on the effect of TE treatment on bioproduct extraction from microalgal.

In this research, the effect of adding three well-known trace elements (TEs) of FeCl₂, ZnCl₂ and MnCl₂ on microalgae cultivation and high value bioproduct from microalgae growing in municipal wastewater were investigated. To do this, TEs with different concentration were added to the culture medium and then microalgae growth rate and the bioproducts protein carbohydrate and Lipid were measured for 12 days. *Chlorella Sorokiniana Pa.91* was selected as model microalgae in this study.

## 2. Materials and methods

### 2-1-Wastewater

The wastewater samples were obtained from real municipal wastewater (RMWW) influent after preliminary settling tank was collected from wastewater treatment plant (WWTP), Sari, Iran. After separation of solids, the wastewater was used as culture medium to evaluate the microalgae capacity for nutrient removal from real municipal wastewater. Before each experiment, wastewater medium was centrifuged at 11000 rpm for 15 min to remove solid particles. The properties of wastewater samples are given in Table 1.

All the chemicals including the heavy metals (e.g. FeCl₂ and ZnCl₂) were provided from Merck Company, Germany and were of analytical grade.

<table><caption>Table 1: Characteristics of the municipal wastewater sample in this study</caption><thead><tr><td>Parameter</td><td>Unit</td><td>Value</td></tr></thead><tbody><tr><td>pH</td><td>-</td><td>7.5</td></tr><tr><td>COD</td><td>mg/L</td><td>198</td></tr><tr><td>PO<sub>4</sub></td><td>mg/L</td><td>7.24</td></tr><tr><td>NO<sub>3</sub></td><td>mg/L</td><td>2.9</td></tr><tr><td>NH<sub>3</sub></td><td>mg/L</td><td>35.5</td></tr></tbody></table>

### 2-1-Microalgae and TE

A new strain of *Chlorella sorokiniana* pa.91 which was previously isolated from Gela dairy wastewater treatment plant (Amol, Iran) was selected and used in this study (Chang et al., 2016). This strain was recognized and analyzed phylogenetically based on 18S rRNA gene sequence (Chang et al., 2016). Because of the possibility of impurities and contamination, three purification phases were performed in plates containing agar and BG-11 culture medium. The microalgae cells were inoculated into several 500 ml Erlenmeyer flasks of BG-11 medium to prepare larger volumes of microalgae cultivation in RMWW. The trace
elements (TEs) of FeCl₂ and ZnCl₂ were purchased from Merck Company with laboratory grade.

## 2-3-Experimental procedure

Microalgae cultivation experiments were conducted in a pilot-scale flat-plate photobioreactor (FP-PBR) at the optimum condition to investigate the effect of TEs treatment on microalgae growth rate and byproduct extraction. An 8 L flat-plate photobioreactor with 34 cm (L) × 10 cm (W) × 40 cm (H) was used in this study. Liquid samples were also collected from a sample point at the side of the photobioreactor daily to determined temperature, biomass concentration, pH and nutrient concentrations. In addition, CO₂ gas (16%) was aerated by the sparger at the rate of 0.6 vvm into the photobioreactor to providing mixing. Various dosages of 0.2, 0.5, 1 and 2 mg/L of FeCl₂, ZnCL₂ and MnCL₂ were added into the reactor to investigate the effect of TEs on microalgae growth rate and nutrients removal. During the cultivation period, the concentration of main nutrients from the municipal wastewater influent were determined based on standard methods (Association et al., 1912). 50 mL sample of microalgae medium (RMWW) from the photobioreactor was collected and centrifuged at 3500 rpm for 15 min, then, the supernatant was used for measuring the nutrients concentration.

## 2-3-Analytical methods

The cell dry weight was measured by using the optical density at 680 nm in a UV spectrophotometer (2800 UV/VIS UNICO, China). The biomass concentration was determined by washing and drying the samples of microalgae cells in an oven at 70 °C for 24 h. The relation between OD₆₈₀ and biomass concentration was obtained according to Eq. 1 which was obtained from standard curve, as follows:

$$ \textit{Biomass concentration} = 1,824 \textit{OD}_{680}0,043 (R^2 = 0,9889) \qquad (1) $$

The concentration of protein, carbohydrate and lipid were measured in this study. First, the extracted products were filtrated throughout a 0.5 µm syringe membrane to remove residual microalge, then a 10 mL of the suspension was centrifuged at 5000 rpm for 10 min. The protein content was measured according to the Bradford assay method (Bradford, 1976). The absorbance of OD595 was recorded and the protein concentration was determined from BSA standard curve. The carbohydrate concentration was analyzed based on Anthrone method by phenol-sulfuric acid. Glucose (from 0.2 mg m/L to 1 mg m/L at an interval of 0.2 mg m/L) was used as a standard substance for the calibration (Lv et al., 2019). To determine the
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total lipid concentration, the method described by Abomohra et al. (2016) using chloroform:methanol (2:1) was applied in this study. The total yield of each bioproduct was presented based on percentage of microalge dry weight (%MDW).

**3. Results and Discussion**

**3-1-Effect of adding FeCl₂ on high-value bioproduct extraction**

Figure 1 shows the effect of FeCl₂ dosage on biomass growth rate (Fig. 1a) and final high-value bioproduct extraction of carbohydrate, protein and lipid (Fig. 1b). As can be seen from the Fig. 1a, maximum biomass concentration of 3.24 g/L (45 % improvement) was achieved by adding 0.2 mg/L FeCl₂ after 12 days cultivation. Also, it was observed that the final high-value bioproducts of carbohydrate, protein and lipid were increased from 7 to 17 % MDW, from 21 to 45 % MDW and from 11 to 23 % MDW, respectively, after adding 0.2 mg/L FeCl₂. The results revealed that adding TE of FeCl₂ positively effect on biopoduct content of microalgae. For example, in the case of adding 1 mg/L of FeCl₂, biomass concentration increased slightly from 2.2 to 2.26 g/L (3%), while final bioproducts values of carbohydrate, protein and lipid were significantly increased up to 98% (from 7 to 13 % MDW), 89% (from 21 to 40 % MDW) and 60% (from 11 to 23 % MDW) respectively.

<figure><img src="image_10.png" /><figcaption>Figure 1. Effect of FeCl₃ on (a) biomass concentration and (b) high value bioproducts extraction from microalgae</figcaption></figure>
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3-1-Effect of adding ZnCl₂ on high-value bioproduct extraction

Figure 2 shows the effect of ZnCl₂ dosage on biomass growth rate (Fig. 2a) and final high-value bioproduct extraction of carbohydrate, protein and lipid (Fig. 2b). As can be seen from the Fig. 2a, maximum biomass concentration of 3.87 g/L (76 % improvement) was achieved by adding 0.5 mg/L ZnCl₂ after 12 days cultivation. Also, it was observed that the final high-value bioproducts of carbohydrate, protein and lipid were increased from 7 to 19 % MDW, from 21 to 52 % MDW and from 11 to 25 % MDW, respectively, after adding 0.5 mg/L ZnCl₂. The results revealed that adding low dosage (<0.5 mg/L) of TE of ZnCl₂ positively effect on bioproduct content of microalgae. For example, in the case of adding 0.2 mg/L of ZnCl₂, biomass concentration increased slightly from 2.2 to 3.32 g/L (45%), while final bioproducts values of carbohydrate, protein and lipid were significantly increased up to 100% (from 7 to 14 % MDW), 81% (from 21 to 38 % MDW) and 73% (from 11 to 19 % MDW) respectively. However, at high dosage of ZnCl₂ (> 1mg/L) some negatively affect was observed in both biomass concentration and byproduct extraction.

<figure><img src="image_12.png" /><figcaption>Figure 1. Effect of FeCl₃ on (a) biomass concentration and (b) high value bioproducts extraction from microalgae</figcaption></figure>
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**4. Conclusion**

In this research, the effect of trace elements (TEs) of FeCl₂ and ZnCl₂ on microalgae growth rate and bioproduct extraction from biomass was investigated. According to the results, adding 0.2 and 0.5 mg/L of FeCl₂ and ZnCl₂, respectively could significantly improve biomass growth rate (Up to 50%). Beside the biomass growth rate, the results revealed that treating microalgae culture medium with micronutrients can significantly improve the high-value bioproducts of biomass. This research proves that treating microalgae culture medium with an appropriate level of TEs can significantly enhance both biomass growth rate and high value bioproduct extraction from biomass.

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13th international conference on

NEW SOLUTIONS IN
ENGINEERING, INFORMATION
SCIENCE & TECHNOLOGY OF
THE CENTURY A HEAD

5 November 2022

Greece - Thessaloniki

www.13iciet.ir

<figure><img src="image_16.png" /></figure>

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