Full Length ArticleThe effects of indole-3-acetic acid and hydrogen peroxide on Chlorella zofingiensis CCALA 944 for bio-butanol production
Graphical abstract
Introduction
Cultivation of microalgae can be performed with artificial media or wastewaters. Microalgae have many benefits in wastewater treatment such as removal of pollutants and biofuel production, but their practice needs to be developed to obtain high biomass for industry [1]. Interference of wastewater treatment causes accumulation of pollutants as nitrogen and phosphorus in wastewater and it results in downstream eutrophication and environmental problems. Excess deposit of these pollutants leads to death of living systems and cyanotoxin production. To discard these toxins, biological, chemical and physical treatment technologies must be used. These technologies load extra cost [2]. Therefore, proper treatment of wastewater is important. Microalgae can help removal of these nutrients. Recently, depletion of fossil derivative fuel has caused the investigation of novel energy sources. To produce high amounts of bio-fuel, biomass and microlagal growth can be tightly regulated with pH, temperature, salinity, light, macro, micro elements and hormones affecting metabolic components of microalgae [3]. Wastewaters can be classified to three main groups. They can be artificial, domestic, and agricultural. Artificial wastewater used instead of municipal wastewater did not give deserved results at previous studies for biomass production [1]. Microalgae include many various genera. One of them, Chlorella has been used for protein source, animal feed and its active compounds. Nowadays, it has been studied for biofuel production and environmental problems [4]. Chlorella zofingiensis belongs to class Trebouxiophyceae and order Chlorellales. C. zofingiensis has high kinetic parameters under autotrophic, heterotrophic and mixotrophic conditions. It includes natural astaxanthin and lutein. C. zofingiensis has been studied for biodiesel production in detail [5]. However, there still have not been enough studies related with bio-butanol production from microalgae in the literature.
Indole-3-acetic acid (IAA) is one of the auxin members and has many properties of auxins [6]. IAA declines reactive oxygen species (ROS) under stress conditions [7]. To our knowledge, the effect of IAA on microalgae grown in wastewater has not been investigated for bio-butanol in detail yet.
Hydrogen peroxide (H2O2) which was used in this study for the pre-treatment procedure is a highly reactive substance with great oxidizing power. It functions as catalyst because of its capability to form various hydroxyl radicals. It can be easily converted to oxygen and water. So, it does not remain as contaminant for the experiment. Ansari and co-workers [8] studied different disruption techniques for microalgae pellets in wastewater and indicated that the microalgae obtained a biomass concentration of 1.64 g/L.
Photosynthesis is important for green microalgae. Microalgae can be followed with accumulation of pigments. Photosynthesis is biochemical process and it uses sun energy and converts to chemical energy to accumulate organic molecules such as carbohydrate, protein and lipid. At the any stress conditions, photosynthetic capacity can be suppressed and amount of biomass decreases [6]. Photosynthesis capacity can be determined with chl a, chl b and carotenoid content. Amounts of carotenoids increase in stress conditions of environment [9]. Growth of microalgae can be followed with chl a, chl b and carotenoid content.
Formation of free radical in microalgae generally occurs in metabolic pathway. Free radicals are formed by metabolic activities of organisms and decomposition of macromolecules. These reactions lead to the formation of ROS via superoxide, hydroxyl radicals and hydrogen peroxide [10]. Superoxide dismutase (SOD) and catalase (CAT) are the linked enzymes that scavenging ROS and neutralize their negative effects [11]. Low levels of H2O2 affect metabolic cascade and results in growth of microorganisms [12]. In addition, glutathione (GSH) is a non-protein tripeptide compound. GSH is also considered as the most potent natural antioxidant molecule, functions scavenging of free radicals (ROS) and maintains detoxification of hydrogen peroxide [13]. Malondialdehyde (MDA) is an endogenous source of oxidative stress. Lifestyle and environmental exposure can lead to an increase in MDA. High amounts of antioxidant enzymes are connected with stress conditions [14].
Bio-butanol (C4H9OH) consists of four carbons involving colourless alcohol. Bio-butanol can be used as new renewable energy source compared to traditional fuels. It has some advantages. Bio-butanol is more useful than bio-methanol and bioethanol due to higher energy density. It can be formed with the acetone-butanol-ethanol (ABE) fermentation. Microalgae are substrate for this fermentation. C. acetobutylicum is usually used for anaerobic digestion. Reaction generates butyric and acetic acids. Then, at the second reaction, butanol, acetone and ethanol are produced [15]. In current study, we examined the role of Chlorella zofingiensis grown in municipal wastewaters and IAA via its productivity, carbohydrate concentration and bio-butanol content in the flat-photobioreactor (FPBR). We carried out activities of antioxidant enzymes and molecules (SOD, CAT, GSH and MDA) under stress conditions. Also, we tried H2O2 as pre-treatment method to apply hydrolysis procedure.
The purpose of this study is to enhance bio-butanol production with Chlorella zofingiensis cultivated in various concentrations of wastewater, IAA and H2O2 as pre-treatment hydrolysis agent in FPBR. Also, we explained how activities of antioxidant enzymes changed with these agents in the current study.
The present study is the first report demonstrating bio-butanol production from C. zofingiensis CCALA 944 grown at various concentrations of municipal wastewater and IAA after H2O2 pretreatment in a flat-photobioreactor and the change of antioxidant enzymes and molecules.
Section snippets
The microalgal strain and set up of flat-photobioreactor
Chlorella zofingiensis CCALA 944 was purchased from CCALA in Czech Republic. Microalgae firstly were cultivated in agar plates. Then, Chlorella zofingiensis was maintained in modified BG-11 medium during the experiments. Municipal wastewater was prepared synthetically. The total organic carbon (TOC), total nitrogen (TN) and, and total phosphorous (TP) in the municipal wastewater were nearly 85 mg/L, 12 mg/L and 3.5 mg/L, respectively. Value of pH was 7.6 in municipal wastewater. The all
The effect of wastewater on C. Zofingiensis
Propagation of microalgae depends on various genetic and environmental factors such as macro and micro nutrients, pH, light, salinity index and temperature. Stress factors affect microalgae related with their growth rates and percentages of macromolecules. Absorbance values can indicate pre-specific growth rate of microalgae. In this study, we firstly carried out the effects of various concentrations of municipal wastewater and plotted them according to absorbance versus time (days) for the
Conclusion
This study demonstrated that Chlorella zofingiensis had maximum biomass content and bio-butanol yield at 25% wastewater, IAA 80 µM and 4% H2O2 in a flat-photobioreactor. At these conditions, carbohydrate concentration increased from 23.9 to 34.2% compare to control. Maximum biomass concentration and bio-butanol content were 2.77 g/L and 0.084 g of bio-butanol/g of microalgal biomass, respectively. It corresponds to an increase of 35%. IAA and wastewater caused the formation of stress conditions
CRediT authorship contribution statement
Melih Onay: Conceptualization, Methodology, Software, Data curation, Writing - original draft, Visualization, Investigation, Supervision, Validation, Writing - review & editing.
Declaration of Competing Interest
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.
Acknowledgements
Author would like to thank Van-YYU-Department of Environmental Engineering for technical support.
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