Application of microalgal-based technology for wastewater treatment and lipids production

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uef.fi

PUBLICATIONS OF

THE UNIVERSITY OF EASTERN FINLAND Dissertations in Forestry and Natural Sciences

ISBN 978-952-61-3246-4 ISSN 1798-5668

Dissertations in Forestry and Natural Sciences

DISSERTATIONS | EHSAN DANESHVAR | APPLICATION OF MICROALGAL-BASED TECHNOLOGY FOR... | No 357

EHSAN DANESHVAR

APPLICATION OF MICROALGAL-BASED TECHNOLOGY FOR WASTEWATER TREATMENT AND LIPIDS PRODUCTION

PUBLICATIONS OF

THE UNIVERSITY OF EASTERN FINLAND

Increasing population, industrialization, and urbanization have driven demand for water,

food, and energy. Overuse of fossil fuels, natural resource depletion, CO₂ emission, and

water pollution have created environmental crises that require urgent action. This thesis applies a biological system in which microalgae grown in wastewater consume CO₂ (climate action), remove pollutants (water treatment and resource recovery), and produce lipids which are used for biodiesel production

(clean energy).

EHSAN DANESHVAR

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Ehsan Daneshvar

APPLICATION OF MICROALGAL-BASED TECHNOLOGY FOR WASTEWATER TREATMENT AND LIPIDS PRODUCTION

Publications of the University of Eastern Finland Dissertations in Forestry and Natural Sciences

No 357

University of Eastern Finland Kuopio

2019

Academic dissertation

To be presented by permission of the Faculty of Science and Forestry for public examination in the Auditorium SN201 in the Snellmania Building at the University

of Eastern Finland, Kuopio, on December 16, 2019, at 12 o’clock noon

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Grano Oy Jyväskylä, 2019

Editors: Pertti Pasanen, Raine Kortet, Jukka Tuomela, Matti Tedre, Nina Hakulinen

www.uef.fi/kirjasto ISBN: 978-952-61-3246-4 (nid.) ISBN: 978-952-61-3247-1 (PDF)

ISSNL: 1798-5668 ISSN: 1798-5668 ISSN: 1798-5676 (PDF)

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Author’s address: Ehsan Daneshvar

University of Eastern Finland

Department of Environmental and Biological Sciences P.O. Box 1627

FI-70211 KUOPIO, FINLAND email: ehsan.daneshvar@uef.fi Supervisors: Professor Amit Bhatnagar, Ph.D.

FI-70211 KUOPIO, FINLAND email: amit.bhatnagar@uef.fi Professor Raine Kortet, Ph.D.

80101 JOENSUU, FINLAND email: raine.kortet@uef.fi

Reviewers: Associate Professor Yagut Allahverdiyeva, Ph.D University of Turku

Department of Biochemistry FI 20014 TURKU, FINLAND email: allahve@utu.fi

Professor Koenraad Muylaert, Ph.D KU Leuven University

Laboratory Aquatic Biology Etienne Sabbelaan 53 box 7659 8500 KORTRIJK, BELGIUM

email: Koenraad.Muylaert@kuleuven-kulak.be Opponent: Professor Maria J. Barbosa

Wageningen University

Department of Agrotechnology and Food Sciences P.O. Box 16

6700 AA WAGENINGEN, NETHERLANDS email: maria.barbosa@wur.nl

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7 Daneshvar, Ehsan

Application of microalgal-based technology for wastewater treatment and lipids production

Kuopio: University of Eastern Finland, 2019 Publications of the University of Eastern Finland Dissertations in Forestry and Natural Sciences 2019; 357 ISBN: 978-952-61-3246-4 (print)

ISSNL: 1798-5668 ISSN: 1798-5668

ISBN: 978-952-61-3247-1 (PDF) ISSN: 1798-5676 (PDF)

ABSTRACT

The rapid growth of the world’s population has accelerated the industrialization to supply human re-equipments. All the industries consume fresh water through production processes, and subsequently, a huge volume of industrial effluent is generated every day. The generated wastewater is contaminated by several types of organic and inorganic pollutants. These pollutants must be removed from wastewater before the discharging to the environment. In this work, microalgal- based technology and eco-friendly methods were used for the removal of pollutants from contaminated water. For this purpose, the growth of microalgae, water and wastewater treatment, and biochemical composition of microalgal biomasses were investigated under different cultivation conditions.

In the first study, nitrate removal efficiency from the synthetic medium by Chlorella vulgaris was investigated in response to different concentrations of macronutrients and micronutrients, nitrate concentrations, inoculum sizes, and pH. The highest nitrate removal efficiency was found at 94.81% in the cultivation medium, with a 25%

concentration of trace elements as compared with the standard Bold´s Basal Medium (BBM).

In the second study, pulp wastewater (PWW) and aquaculture wastewater (AWW) were mixed with different ratios to provide nutrients for the cultivation of C. vulgaris.

Among different mixture ratios, the highest dry mass of microalga was found at 80%

PWW:20% AWW and 60% PWW:40% AWW. In the experimental units without the addition of nitrate and phosphate, microalgal growth was improved by increasing the percentage of AWW from 20% to 40%. Results showed that higher concentrations of AWW are more appropriate for cultivation of microalga in PWW, as it could provide nutrients.

In the third study, one-time cultivation of Scenedesmus quadricauda (fresh water) and Tetraselmis suecica (marine water) microalgae in dairy wastewater (DWW) for versatile applications was investigated. Dry biomasses of fresh water and marine- water microalgae were observed as 0.47 g/L and 0.61 g/L, respectively, without the

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addition of extra nutrients to DWW. After 12 days’ cultivation of microalgae, 86.21%

and 44.92% of total nitrogen (TN) and 89.83% and 42.18% of phosphate were removed from DWW by S. quadricauda and T. suecica, respectively. After wastewater treatment, lipids were extracted from microalgal biomasses, which showed a variety of fatty acids from C14 to C18. Following lipid extraction, microalgal residues were used for the removal of tetracycline from water.

In the fourth study, the growth of microalgae, pollutant removal efficiency, and the percentages of pigments and fatty acids of S. quadricauda and T. suecica were examined in sequential cultivation cycles. Dry masses of S. quadricauda as 0.43 g/L and T. suecica as 0.58 g/L at the first cycle (12 days) increased to 0.79 g/L and 1.23 g/L, respectively, after two cycles of cultivation (24 days). Sequential cultivation of S.

quadricauda increased the removal efficiency of total nitrogen, phosphate, sulfate, and total organic carbon from 86.70%, 71.20%, 93.70%, and 69.10% at the first cycle to 92.15%, 100%, 100%, and 76.77% after two cultivation cycles. The lipids composition of microalgae after the first and second cycles of cultivation showed significant difference profiles.

In the fifth study, among different tested microalgal-based materials, microalgal biochar synthesized at 500°C showed the highest removal efficiency of Cr(VI) (1, 5, and 10 mg/L) from water up to 100%. Cr(VI) led to longer lag phase and negatively affected microalgal growth as compared with the control group (BBM without (CrVI)). The highest removal efficiency of Cr(VI) by living microalgal cells after 12 days’ cultivation was observed as 67%, which was remarkably lower than Cr(VI) removal efficiency by microalgal biochar (100% after 4 h).

The findings of this doctoral thesis showed that wastewater can be used as a source of macro- and micronutrients for the cultivation of microalgae, while at the same time microalgae remove pollutants from water and microalgal biomass is produced.

Further, the produced biomass can be used for the production of valuable products.

According to the findings of this doctoral thesis, the growth of microalgae, wastewater treatment, and the biochemical composition of algal biomass are affected by microalgae strains, wastewater types, and nutritional and environmental cultivation conditions.

Universal Decimal Classification: 544.723, 582.27, 628.316, 628.349, 628.35

CAB Thesaurus: wastewater; polluted water; effluents; wastewater treatment; algae;

Chlorella vulgaris; Scenedesmus quadricauda; Tetraselmis suecica; cultivation; chemical composition; nutrients; salinity; nitrate; phosphate; sulfate; organic carbon; chromium;

tetracycline; lipids; fatty acids; pigments; growth; biomass; efficiency; adsorption; desorption

Yleinen suomalainen ontologia: jätevesi; jäteveden käsittely; saasteet; poistaminen;

puhdistus; mikrolevät; viljely; kemiallinen koostumus; ravinteet; nitraatit; fosfaatit; sulfaatit;

kromi; lipidit; rasvahapot; pigmentti (biologia); kasvu; biomassa; tehokkuus; sorptio

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ACKNOWLEDGMENTS

First and foremost, thanks to God for his never-ending grace toward me.

I wish to express my deep appreciation and heartiest respect to my main supervisor, Professor Amit Bhatnagar, who encouraged and helped me at all stages of my research and thesis work with great patience and immense care. I am particularly indebted to Professor Raine Kortet, my co-supervisor, for his encouragement to participate in all spheres of academic activities and his useful comments on the earlier draft of my thesis. I would like to thank Maija-Riitta Hirvonen, head of the Department of Environmental and Biological Sciences, and Jukka Jurvelin, the dean of the Faculty of Science and Forestry, who provided such a friendly and peaceful atmosphere for research and learning.

I acknowledge the University of Eastern Finland, which gave me this opportunity to extend my experience. I learned how to solve problems; how to find relevant information; how to work independently and as a member of a team; how to communicate (by writing, by giving presentations, by speaking in public); how to meet deadlines; how to manage my time effectively; and how to prioritize my activities.

Special thanks to Timo Oksanen, Harri Kokko, and Pasi Yli-Pirilä for all their support and technical help with the laboratory set-up. I take this opportunity to thank Hanne Vainikainen, Jaana Rissanen, and Sirpa Martikainen for their help with the providing of chemicals and laboratory supplies as well as their concerns and guidelines about lab safety. Sincere thanks to Kaija Ahonen, human resources secretary, and Timo Kumlin, coordinator, for their helpful guidance and advice.

I am sincerely grateful to the reviewers, Professor Koenraad Muylaert and Associate Professor Yagut Allahverdiyeva in evaluating this thesis. The comments provided by them definitely improved the quality of the final version of my thesis.

As well, I am thankful to Professor Maria J. Barbosa who accepted the invitation to act as my opponent for the public defence.

I acknowledge the friendship and support of the members of the Water Chemistry group from 2016 to 2019. All the friends I made during this time in Kuopio and at the University of Eastern Finland are acknowledged for our time spent together. Last but not least, I am grateful to my family and friends in Iran for supporting me spiritually throughout my life.

Ehsan Daneshvar Kuopio, December 2019

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LIST OF ABBREVIATIONS

AWW aquaculture wastewater BOD biological oxygen demand

BBM Bold´s Basal Medium

CCU carbon capture and utilization

CN cetane number

COD chemical oxygen demand

CCAP Culture Collection of Algae and Protozoa

DWW dairy wastewater

ETC electron transport chain FAMEs fatty acid methyl esters

Glu glutamate

GHGs greenhouse gases Cr(VI) hexavalent chromium

IPCC Intergovernmental Panel on Climate Change MB-300 microalgal biochar synthesized at 300°C MB-500 microalgal biochar synthesized at 500°C MB-700 microalgal biochar synthesized at 700°C

MB microalgal biochar

MUFAs monounsaturated fatty acids

MTAB myristyltrimethylammonium bromide NADP⁺ nicotinamide adenine dinucleotide phosphate NADPH nicotinamide adenine dinucleotide phosphate

NH⁴⁺ ammonium

NO3- nitrate

NO^2- nitrite

OD optical density

PAWW mixture of pulp and aquaculture wastewater PAWW'N+P PAWW after the addition of nitrate and phosphate

PO43- phosphate

PUFAs polyunsaturated fatty acids

PAWW'BBM prepared BBM medium with PAWW instead of deionized water

PWW pulp wastewater

SFAs saturated fatty acids SCCO² Supercritical CO² TSS total dissolved solids

TN total nitrate

TOC total organic carbon (NH²)²CO urea

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LIST OF ORIGINAL PUBLICATIONS

This thesis is based on data presented in the following articles, referrred to by the Roman Numerals I-V.

I Daneshvar, E., Santhosh, C., Antikainen, E., & Bhatnagar, A. (2018).

Microalgal growth and nitrate removal efficiency in different cultivation conditions: Effect of macro and micronutrients and salinity. Journal of Environmental Chemical Engineering, 6(2): 1848-1854.

II Daneshvar, E., Antikainen, L., Koutra, E., Kornaros, M., & Bhatnagar, A.

(2018). Investigation on the feasibility of Chlorella vulgaris cultivation in a mixture of pulp and aquaculture effluents: treatment of wastewater and lipid extraction. Bioresource Technology, 255: 104-110.

III Daneshvar, E., Zarrinmehr, M. J., Hashtjin, A. M., Farhadian, O., &

Bhatnagar, A. (2018). Versatile applications of freshwater and marine water microalgae in dairy wastewater treatment, lipid extraction and tetracycline biosorption. Bioresource Technology, 268: 523-530.

IV Daneshvar, E., Zarrinmehr, M. J., Koutra, E., Kornaros, M., Farhadian, O., &

Bhatnagar, A. (2019). Sequential cultivation of microalgae in raw and recycled dairy wastewater: microalgal growth, wastewater treatment and biochemical composition. Bioresource Technology, 273: 556-564.

V Daneshvar, E., Zarrinmehr, M. J., Kousha, M., Hashtjin, A. M., Saratale, G. D., Maiti, A., Vithanage, M., & Bhatnagar, A. (2019). Hexavalent chromium removal from water by microalgal-based materials: adsorption, desorption and recovery studies. Bioresource Technology, 122064.

The above publications have been included in this thesis with their copyright holders’ permission.

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AUTHOR’S CONTRIBUTION

I) E.D. and A.B. designed the experiments. E.D. and C.S. took the samples and analyzed the growth of microalga and nitrate concentration. E.D.

wrote the manuscript. E.A. and A.B. revised and edited the manuscript.

All authors checked and approved the final version of manuscript before submission and proof of the paper after acceptance.

II) E.D. and A.B. designed the experiments. E.D. and L.A. took the samples and analyzed the growth of microalga and pollutant concentrations in wastewater. E.K. analyzed the lipid profile of microalga. E.D. wrote the manuscript. E.K., M.K., and A.B. revised and edited the manuscript. All authors checked and approved the final version of manuscript before submission and proof of the paper after acceptance.

III) E.D. and A.B. designed the experiments. E.D., M.J.Z., and A.M.H. took the samples and analyzed the growth of microalgae and pollutants concentrations in wastewater. E.D. and M.J.Z. analyzed the lipid profile of microalgae. E.D. wrote the manuscript. OF and A.B. revised and edited the manuscript. All authors checked and approved the final version of manuscript before submission and proof of the paper after acceptance.

IV) E.D. and A.B. designed the experiments. E.D. and M.J.Z. took the samples and analyzed the growth of microalgae and pollutant concentrations in wastewater. E.K. analyzed the lipid profile of microalgae. E.D. wrote the manuscript. E.K., O.F., M.K., and A.B. revised and edited the manuscript. All authors checked and approved the final version of manuscript before submission and proof of the paper after acceptance.

V) E.D. and A.B. designed the experiments. E.D., M.J.Z., and A.M.H. took the samples and analyzed the growth of microalgae and Cr(VI) concentration in the solution. E.D. and M.K. wrote the manuscript.

G.D.S., A.M., M.V., and A.B. revised and edited the manuscript. All authors checked and approved the final version of manuscript before submission and proof of the paper after acceptance.

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1 INTRODUCTION

Water, food, and energy are basic and primary requirements of human society.

There is a direct correlation between demands for these three essential resources and the growth rate of population. Figure 1 depicts and predicts the pattern of water, food, and energy consumption and world population for the years 1960 to 2050 (Kalair et al., 2019). The global demand for water will have risen 40% by 2030; food, 40%; and energy, 50% (Zhang et al., 2019). By 2050, those figures are estimated at 60%

for water and food and almost double for energy consumption (Lin et al., 2018).

Because of industrialization and overpopulation demands, the predicted consumption of water, food, and energy is beyond the carrying capacity of Earth.

Overuse of unequally distributed resources for the providing of those demands has resulted in the depletion of natural resources and widespread environmental issues such as freshwater shortages, food security, and fossil fuel exhaustion. The current situation may increase the inherent competition for accessing of these resources because of various conflicts among nations and manufacturers (Endo et al., 2017). For this reason, the challenges of providing water, food, and energy have attracted serious and universal attention from policy makers and scientists.

Figure 1. The pattern of water, food, and energy consumption and world population for 1960-2050 (Kalair et al., 2019) [Open access paper:

https://doi.org/10.1016/j.wen.2019.04.001].

Water is used a key operational element in most industries, and consequently, various industrial activities generate wastewater (Goh and Ismail, 2018). The effluents of various industries contain organic and inorganic pollutants, dyes, heavy metals, and nutrients (Mohammadzadeh Pakdel and Peighambardoust, 2018).

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19 Inadequate treatment of wastewater before discharging to natural water bodies, especially in developing countries, exposes freshwater resources (lakes, rivers and groundwater) to pollutants. On the other hand, worldwide freshwater resources are not distributed evenly, and reduced access to safe water in water-scarce zones results in limited development opportunities (Sato et al., 2013). Water scarcity, thus, is a global issue, and it is more pronounced in arid and semi-arid countries, where limited water resources have been consumed by misuse. Water scarcity as an environmental issue negatively affects the global economy as well as the sustainable development of human livelihoods and the environment (Elgallal et al., 2016).

Therefore, the efficient treatment of water and wastewater is necessary for the secure expansion of industrialization and consequently the development of societies.

Food security is another critical problem of the constant rise of world population.

The quantity of current food production will not be enough for the population in 2050 (estimated to reach nine billion). The numbers of undernourished and starving people are increasing. For example, the rate of malnourished population increased from 10.8% (equal to 794 million) to 11% (equal to 815 million) from 2015 to 2016 (Prosekov and Ivanova, 2018). Since agricultural products are the main sources of food, the development of agriculture for crop production might be the easiest way to solve the problem of insufficient food. However, a majority of farmable lands (more than 37% of Earth’s land) has already been used, and the rest is difficult to use because of unsuitable geoclimatic conditions (Tyczewska et al., 2018). Furthermore, food shortages will be further complicated by water shortages, as 70% of fresh water is used by agriculture (Winpenny et al., 2010). Vital action is necessary to increase food production. Otherwise, food insecurity because of lower productivity will increase the price of food and, consequently, affect the sustainable development of society.

Beside water and food, energy is another basic demand of each society. Almost 85% of energy in use is obtained from fossil fuels such as oil, gas, and coal, which are non-renewable. The resources of fossil fuels were formed during more than one million years, and restoring their original capacity would require a similarly long time (Abdalla et al., 2018). While the consumption of these resources is not comparable with their formation. It is estimated that fossil fuels, as the dominant source of energy, will be depleted significantly after 70 years (Weldekidan et al., 2018). The volatile, rising rate of the price of fossil fuels is another problem that negatively affects customers as well as the global market (Milano et al., 2016).

Furthermore, the excessive combustion of fossil fuel has emitted a considerable amount of greenhouse gases, the main cause of air pollution and ongoing global warming. The world faces an energy crisis because of the rise of its population, enhanced energy demand, the exhaustion of resources, and the environmental challenges resulting from the use of fossil fuels. An international effort is necessary to replace the current pattern of energy supply with clean, sustainable, and affordable sources to provide security for nations.

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Like water, food, and energy crises, climate change also is mostly an outcome of population growth and industrialization. After the Industrial Revolution and predominantly over the past 50 years, the concentration of greenhouse gases (GHGs) significantly increased in atmosphere. Currently, the atmospheric concentration of carbon dioxide (CO2) at 76% as the main GHG is 411 parts per million (ppm). It is estimated that this amount is highest over the past several thousand years (Tong and Ebi, 2019). Combustion of fossil fuels like oil, petroleum, coal, and gas is the main cause of CO2 emission. According to data collection through the world, the mean temperature of the planet has risen 0.75°C in last century, mainly since 1980. Climate change can result in environmental disasters such as ice melting, heat waves, tropical storms, severe droughts and floods, and rising sea levels (Ashrafuzzaman and Furini, 2019). At present, international parties and communities have focused on the anthropogenic emissions of CO2 as one of the main environmental problems. The Paris Agreement, which 194 countries signed in 2015, shows the universal importance of global warming as a public environmental challenge. The goal of this agreement is climate-controlled global warming at a maximum level of 2°C compared with pre-industrial times (Gao et al., 2017).

As water, food, and energy crisis and climate change caused primarily by CO² emission are universal issues, it is a main concern of all governments to suggest practical solutions. These issues have been reflected in such an important organization like United Nations (UN). To develop prosperity while protecting the Earth, this organization has defined 17 Sustainable Development Goals (SDGs), which need serious attention of all poor, middle-income and rich countries. SDGs are a blueprint that have been outlined to achieve a better future sustainably. Goals 2, 6, 7 and 13 have been specified to zero hunger, clean water and sanitation, affordable and clean energy, and climate action, respectively (SDGs, 2019). Many scientific communities and research institutes with different backgrounds and knowledge are trying to come up with new ideas to solve these problems. Numerous methods have been presented, all with their own advantages and disadvantages. Among them, the cultivation and use of microalgae has drawn increasing attention among scientists from different fields as a promising candidate for solving the environmental challenges and promoting sustainable development (Piloto-Rodríguez et al., 2017;

Wang et al., 2016; Udaiyappan et al., 2017). Microalgae are microscopic algae, which offer several noticeable advantages as compared with terrestrial crops and other microorganisms. Microalgae grow in fresh and marine water as well as the harsh conditions of wastewater. Microalgae can be cultivated year-round without competition with food for freshwater and arable lands (Chen et al., 2018). These microorganisms can successfully grow in wastewater and uptake nutrients. During photosynthesis, microalgae use sunlight and CO2 and produce microalgal biomass and oxygen (Suganya et al., 2016). Theoretically, 1.8 kilograms (kg) of CO² is used for the production of 1 kg of dried microalgal biomass (Chew et al., 2018). A high amount of valuable biochemical compounds of microalgal biomass, such as protein,

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21 carbohydrates, and lipids, can be used as a sustainable and appropriate source of food, feed, biomedicine, biopolymers, and natural pigments (de Carvalho et al., 2018). In addition, microalgal fatty acids can be converted to biodiesel by transesterification reaction.

Interestingly, microalgae can potentially solve water, food, and energy crises, and these organisms can capture CO2. However, despite many advantages of microalgae, their commercial application still is limited because of challenges in upstream and downstream processes including the selection of microalgae species, cultivation, harvesting, drying of biomass, and extraction. Accordingly, reducing the cost of microalgal biomass production and increasing the number of final products can improve the process. Therefore, providing affordable light, CO², water, and nutrients as the main elements for microalgae growth (Zhuang et al., 2018) is necessary to develop microalgae production sustainably.

2 LITERATURE REVIEW

2.1 CULTIVATION OF MICROALGAE IN WASTEWATER

Wastewater is a low-cost and available source of water and essential nutrients for the cultivation of microalgae. Wastewater often contains nitrogen and phosphorus compounds, pharmaceutical molecules, dye molecules, organic carbons, volatile fatty acids (VFAs), and heavy metals. Microalgae can consume these sources of environmental pollution as macronutrients and micronutrients (Ruiz et al., 2014).The integration of microalgae cultivation and wastewater treatment offers major benefits for reducing the cost of mass cultivation, a main challenge of microalgal-based biofuel production and biorefinery (Salama et al., 2017a). Oswald and Gotass (1957) have established large-scale cultivation of microalgae in municipal wastewater ponds for the first time. It has been reported that some microalgae can remarkably reduce the concentrations of nitrogen to 2.2 milligrams per liter (mg/L) and phosphorus to 0.15 mg/L in domestic wastewater (Boelee et al., 2011). Numerous studies have been conducted on the cultivation of different species of microalgae in various types of wastewater discharged from industries such as pulp and paper, food processing, textiles, dairy production, aquaculture, petroleum, sugar mills, piggery, pharmaceutical production, electroplating, and breweries (Wang et al., 2016). As a few examples, the cultivation of Scenedesmus obliquus in brewery wastewater, Botryococcus braunii and Chlorella saccharophila in carpet wastewater, Chlorella pyrenoidosa in dairy effluent,Chlorella vulgaris in soya whey, Chlamydomonas mexicana in piggery wastewater, Neochlorosis oleoabundans in livestock effluent and Scenedesmus sp. in fermented swine urine have been investigated for wastewater

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treatment and biomass production (Abinandan and Shanthakumar, 2015).

According to some reports, dry masses of species such as Scenedesmus sp. and Nannochloropsis sp. have a noticeable amount of 45% to 64% lipids (Moreno-Garcia et al., 2017). For instance, the lipid content of harvested microalgal biomass from various sewage and industrial wastewaters has been reported as follows: Chlorella ellipsoidea YJ-I (11.4 mg/L), Chlorella sp. (6.9 mg/L.d), Chlorella vulgaris C9-JN2010 (342 mg/g), Chlamydomonas mexicana (310 mg/L), Chlorella vulgaris YSW-04 (70 mg/L) and Chlorella pyrenoidosa (350-540 mg/L.d) (Abinandan and Shanthakumar, 2015). The transesterification of microalgal lipid as high-dense energy hydrocarbons to fatty acid methyl esters (FAMEs), which is the main component of biodiesel, has been extensively studied. The amount of biodiesel and FAMEs of Chlorella in sewage was 120 mg/L, and of Scenedesmus AMDD, 10–18 mg/L.d (McGinn et al., 2012; Li et al., 2011). The quantity of hydrocarbon in Botryococcus braunii biomass harvested from piggery was 950 mg/L, of that harvested from soybean wastewater, 696 mg/L (An et al., 2003; Yonezawa et al., 2012).

Although microalgae are renewable sources and potential feedstocks for a wide spectrum of industrial products from nutraceuticals to biofuels, large-scale production is economically unviable. Technical challenges in microalgae species selection, cultivation, harvesting, dewatering, fractionation, and extraction steps limit the industrial application of microalgae. In the next section, the recent literature on the main steps in upstream and downstream processes is reviewed. These steps affect the efficiency of microalgae system in wastewater treatment, biomass yield, and lipid production.

2.2 MICROALGAE SPECIES SELECTION

In recent years, researchers have studied the cultivation of numerous microalgae species in different types of wastewater. As noted in the published papers, these species have been supplied from commercial and research algae collections or isolated from local wastewater. The selection of suitable microalgae species for the cultivation in wastewater is an essential step for a successful plan. Osundeko et al.

(2013) state that the native microalgae species isolated from wastewater have naturally adopted to the condition of target wastewater and consequently, can grow better with higher bioremediation efficiency. On the other hand, as for their advantages, commercial microalgae are well-known species; much is known about their requirements for an optimal cultivation, and they can tolerate a wider range of physicochemical conditions. Overall, the selected microalgae should able to tolerate the harsh conditions of target wastewater. According to the literature review, high pollutants’ removal efficiency, high biomass productivity, and high lipid yield are other important features of microalgae selection for cultivation in wastewater (Sundar Rajan et al., 2019).

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2.3 OPTIMIZATION OF MICROALGAE CULTIVATION IN WASTEWATER

Many researchers have discussed the effects of light, nutrients, temperature, pH, CO2/O2 concentrations, salinity, grazers, and biological contamination (outside cultivation) as they pertain to the cultivation of microalgae in wastewater (Wang et al., 2013; Zhuang et al., 2018). In addition, several researchers have demonstrated microalgal growth, nutrient removal efficiencies, and lipid productivity under different cultivation modes (photoautotrophic, heterotrophic, and mixotrophic), cultivation systems (open ponds and photobioreactors), and cultivation methods (batch, semi-continuous and continuous). Herein, the effect of main parameters on microalgae growth are presented.

2.3.1 Light

Light properties including light intensity, light wavelength, and photoperiod play important roles in the growth of photosynthetic microalgae. When the light intensity in culture is low, microalgae consume high-carbon macromolecules faster than the synthesis rate. On the other hand, at too high a light intensity, photo-inhabitation and photo-oxidation damage the cell, and death can happen in severe cases (Lehmuskero et al., 2018). The energy of light arrives to microalgal cells in the form of photons with different wavelengths. Microalgae can use a fraction of light in the range of 400 to 700 nanometers (nm) (violet to red). Generally, the efficiency of light absorption and optimal wavelengths depends on the pigment content of microalgae. The maximum photon efficiency in microalgae containing chlorophyll a is in the range of 660 to 680 nm light wavelength (Schulze et al., 2014). In a study by Chang et al. (2011), the optimum growth of microalgae was observed at 674 nm wavelength (red light) among the 11 studied light sources.

In photoautotrophic and mixotrophic cultivation modes of microalgae, light penetration affects the efficiency of photosynthesis and consequently, microalgal growth. Wastewater is often very turbid due to total suspended solids (TSS).

Turbidity of wastewater limits the light penetration and interferes with microalgae photosynthesis. Microalgal growth significantly decreases in wastewater with turbidity higher than 1,000 mg/L (Acién Fernández et al. 2018). Kumar et al. (2019) reported that black color of kitchen wastewater and high turbidity was not favorable for microalgal growth. Appropriate mixing of a dark wastewater and other one with the low turbidity can solve the problem of light penetration and improve microalgal growth.

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2.3.2 Nutrients

Nutrients act as a building resource for microalgae and are essential requirements for the cultivation of microalgae. According to some references, providing nutrients imposes the predominant cost of microalgae cultivation (Liu et al., 2019). During photosynthesis, microalgal biomass is synthesized from nutrients as follows (Randrianarison and Ashraf, 2017):

16NO3-+124CO2+14H2O+HPO42- C106H263O110N16P+138O2+18HCO3-

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In the above equation, C¹⁰⁶H²⁶³O¹¹⁰N¹⁶P represents the general formula of microalgae. According to this formula, microalgal biomass is composed mainly of carbon (C), hydrogen (H), oxygen (O), nitrogen (N), and phosphorus (P) elements.

Except for elements that are essential for the growth of microalgae, other macronutrients and micronutrients are required for microalgal growth. Microalgae need higher amounts of macronutrients such as C, O, H, N, P, potassium (K), calcium (Ca), magnesium (Mg), sodium (Na), sulfur (S), and chloride (Cl) in their medium as compared with micronutrients. They also need several micronutrients or trace elements in very low concentrations (Procházková et al., 2014).

In photoautotrophic cultivation, three essential nutrients (C, H, and O) are provided from water and air. N and P, which are essential for the synthesis of biomolecules, must be supplied for the cultivation of microalgae. Industrial and municipal wastewater can be applied as available, as cheap source of N and P and other macronutrients and micronutrients. Simultaneously, the growth of microalgae decreases the concentration of nutrients and other contaminants in wastewater before it is discharged to the environment (Tan et al., 2018).

Nitrogen is an essential nutrient for the synthesis of compounds like proteins, enzymes, and nucleic acids. The concentration of nitrogen in a cultivation medium directly affects the lipid content of microalgal cells. Many researchers have reported an increase of lipid accumulation by microalgae because of nitrogen deprivation.

Different species of microalgae use inorganic and organic sources of nitrogen such as nitrite (NO^2-), nitrate (NO^3-), ammonium (NH⁴⁺), and urea ((NH²)²CO). Both NO^2- and NO3- should be converted to NH4+ before assimilation by microalgae. NO3- reduces to NO2- by nitrate reductase, and consequently, nitrite reductase reduces NO2- to NH4+. Finally, all the inorganic forms of nitrogen are reduced to NH^4+,and NH⁴⁺ismerged to amino acid glutamine by glutamate (Glu) and ATP (Salama et al., 2017a). Figure 2 shows the assimilation of nitrogen by microalgae.

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25 Figure 2. Mechanism of organic and inorganic nutrient assimilation by microalgae (Gupta et al., 2019) [Permission request granted Oct. 24, 2019, order number 4695450106241].

Phosphorus is one of the most important growth-limiting factors of microalgae in fresh water. The average amount of this essential element is 1% of dry microalgal biomass. The amount of phosphorus of microalgal biomass in waste stabilization ponds was found as 0.21-3.85% (equal to 25.9 to 476 kg biomass/1 kg phosphorus) (Powell et al., 2009). In another study, the ratio of kg biomass/kg phosphorus in Scenedesmus sp. LX1 cultivated in BG11 was 160 (Wu et al., 2012a). Different amounts of produced microalgal biomass per phosphorus might be related to microalgal species, cultivation media, and phosphorus concentration of media (Wu et al., 2013).

Phosphorus participates in vital metabolic processes like photosynthesis, synthesis of phospholipids, energy conversion, and signal transduction. Orthophosphates (- PO^43-) are the most appropriate forms of phosphorus for assimilation by microalgae during phosphorylation (Gupta et al., 2019).

The availability enough of sufficient amounts of trace elements or micronutrients like iron (Fe), nickel (Ni), molybdenum (Mo), boron (B), manganese (Mn), vanadium (V), zinc (Zn), cobalt (Co), cupper (Cu), and selenium (Se) guarantees a healthy culture of microalgae. These nutrients, depending on the microalgae species, are needed in very small amounts of µg, ng and in some cases even picogram (pg) per liter of culture medium. Thus, the boundary between their nutritional application as cell-growth promotion and their toxicity is narrow. Mainly, these micronutrients are recognized as metal constituents of enzymes, which participate in biochemistry reactions (Procházková et al., 2014). Several parameters, such as the availability of macronutrients and light intensity, affect the consumption of micronutrients by microalgal cells. For example, the form of nitrogen compounds determines the use of

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Ni, Mo, and Fe. Zn supply is needed for the assimilation of phosphorus and CO2

mitigation by microalgae (Sunda et al., 2005).

Moreover, there is a complex interaction among the chemical constituents (macronutrients and micronutrients), their availability and uptake of these constituents by microalgae in mass culture. In addition, the biological and physical variances enhance the complexity of process. Beyond the ability of microalgae to acclimation, the threshold concentrations of nutrients in cultivation medium can stop their growth. C/N/P elemental ratios in microalgal biomasses are approximately 106:16:1, which is known as Redfield ratio. The Redfield ratio is used to estimate the limitation effects of C, N and P as essential nutrients in microalgae cultivation (Grobbelaar, 2003). Many researchers have studied the effect of C, N and P ratios on microalgal growth. Nutrient ratios in wastewater often deviate from the Redfield ratio, which can negatively affect the performance of systems. A possible solution to this problem can be mixing of different types of wastewaters with different nutrient ratios. In addition to C, N and P as essential nutrient, this is necessary to evaluate the influence of availability and ratios of other macronutrients and micronutrients on microalgal growth rate as well.

2.3.3 Temperature

Different ranges of temperature, from 18 to 30°C (Vuppaladadiyam et al., 2018), 15 to 26°C (Seyed Hosseini et al., 2018), and 20 to 30°C (Enamala et al., 2018), have been suggested by researchers as the optimum temperatures of culture. That variety demonstrates that optimal temperature depends heavily on the microalgae species and strain. Very low or high temperatures disturb the activity of Ribulose-1,5- bisphosphate carboxylase/oxygenase (RuBisCO), which is an important enzyme in biofixation of CO². When the temperature of the medium is high, the solubility of CO2 in water is low and O2 is attached to RuBisCO instead of CO2. The activity of this enzyme is low in low temperature, which has led to decreased CO2 fixation and consequently, a lower growth rate (Seyed Hosseini et al, 2018).

Most microalgae can tolerate temperatures up to 15°C lower than optimal condition. However, increasing the temperature a few degrees higher than optimum can destroy microalgal cells and collapse the culture (Enamala et al., 2018). For example, Chlorella sp. and Scenedesmus sp. can grow in a wide range of temperatures from 5 to 35°C, though their optimum growth is observed at higher temperatures in the range of 25-30°C. For each species of microalgae, increasing the temperature raises the growth of microalgae up until the optimum zone, and after that increasing the temperature decreases the growth rate (Zhu, 2015).

2.3.4 pH and CO2

For each species of microalgae, there is a range of pH to gain the highest biomass and lipid yield (Chen and Durbin, 1994). For the most investigated microalgae,

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27 acceptable pH is in the range of 7 to 9; optimal, 8.2 to 8.7. However, other researchers believe that it is difficult to generalize a narrow range of pH to all microalgae, as optimum pH depends on the microalgae species and cultivation conditions. In spite of the wide range of acceptable pH for different microalgae strains, controlling the pH of culture improves the microalgal growth (Sakarika and Kornaros, 2016). The pH of culture strongly affects the abundance of carbonaceous species. CO2 is dissolved in water according to the following equation:

CO2+H2O H2CO3 HCO3-+H+ CO32-+2H⁺ (2) Figure 3 shows the fractions of inorganic carbons (CO², HCO^3-and CO^32-) versus pH. In an acidic medium with pH less than 5, CO2 is the dominant form of inorganic carbon because of the abundance of H⁺. The amounts of CO2 and HCO3- are equal at pH around 6.6. At pH 8.3, HCO^3- is almost the only form of inorganic carbon (Zhu et al., 2013). At pH higher than 7 up to 10, bicarbonate has the highest concentration as compared with the other two forms. Carbonate is the predominant form of inorganic carbon at pH higher than 10 in aquatic solutions. When CO2 is dissolved in water, carbonic acid (H²CO³) is formed, but it is not stable and dissociates to bicarbonate (HCO^3-) and H⁺ (Pedersen et al., 2013). The growth of microalgae and more consumption of CO² increase the pH of culture. That takes place because of the activity of carbonic anhydrase (CA) enzyme. Consequently, the availability of CO2 is limited at higher pH, and it negatively affects microalgal growth. Conventional methods such as CO² injection (CO² equilibrium) and buffer addition are used to control the pH of the medium. However, buffers are cost-effective especially in large- scale cultivation, and their side effect on microalgal metabolism is unknown (Qiu et al., 2017).

Figure 3. Relative speciation (%) of carbon dioxide (CO²), bicarbonate (HCO^3-), and carbonate (CO^32-) in water as a function of pH (Pedersen et al., 2013) [Open access paper: https://doi.org/10.3389/fpls.2013.00140].

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2.4 SCALING-UP OF MICROALGAE CULTIVATION

Large-scale cultivation of microalgae is generally performed in open ponds and closed photobioreactors. The first includes natural, raceway, and circular ponds and cascade (inclined) systems, which mainly are used for outdoor cultivation. Flat and tubular photobioreactors (PBRs) are common for both indoor and outdoor microalgae cultivation.

2.4.1 Open ponds

Raceway ponds, as the most cost-effective technique, are well known for the cultivation of microalgae and removal of nutrients from municipal wastewater (Tan et al., 2018). These ponds are constructed with concrete, compacting the earth and polyvinyl chloride (PVC). The foremost of them are concrete channels with a flat bottom and depth up to 30 cm. Raceway ponds are kept shallow to illuminate the sunlight in inner layers and maximizing the photosynthesis rate. Several facilities such as mixing rate, wastewater inlet (feeding) and outlet (harvesting), CO²injection, and cleaning should be considered in the designing of raceway ponds (Mathimani et al., 2019). A paddle wheel is used in front of a channel to introduce and circulate the mixture of microalgae inoculum and wastewater. The paddle system works continuously during the cultivation period to mix the medium properly and avoid sedimentation (Tan et al., 2018). The largest raceway pond, with an area of 440 hectares (ha), is in Calipatria, California, and is used for the production of Spirulina sp. as food (Spolaore et al., 2006). Raceway ponds are the first choice for large-scale cultivation of microalgae. This is because of the simple design and low capital investment of raceway ponds. However, some research has suggested that microalgal productivity in such systems is lower than in PBRs (Mathimani et al., 2019). In addition, several factors such as weather conditions, biological contamination by other microalgae species, and grazers affect the success of outdoor cultivation system in raceway ponds (Fazal et al., 2018).

2.4.2 Closed systems

PBRs as closed systems can be used for both indoor and outdoor microalgae cultivation. In contrast with open ponds that offer little if any control over the conditions of culture, almost all the biotechnologically factors can be controlled in PBRs. Because of a lower contamination risk, PBRs make single species cultivation possible for a longer time. PBRs are more efficient in use of CO² compared with open ponds, where the main portion of injected CO2 escapes into the atmosphere (Pulz, 2001).

Glass or transparent plastic tubes are used for the construction of tubular PBRs.

To provide appropriate light penetration, the diameter of tubes should be less than 10 cm. Horizontal, vertical, helical, and inclined are different types of tubular PBRs

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29 (Tan et al., 2018). In horizontal form, long tubes can be arranged alongside together on the ground (in panel form) or parallel up together (like a wall). One main drawback of horizontal PBRs is the accumulation of oxygen during microalgal photosynthesis, which inhibits microalgal growth. This problem needs to be solved by degassing the culture via adjusting the appropriate length of tubes (Chew et al., 2018).

Vertical PBRs, also known as column PBRs, are divided into bubble and airlift columns. In both bubble and airlift PBRs, an air sparger is connected to the end of a column for the mixing of culture. A bubble PBR is a simple column with no internal structure; air bubbles determine the pattern of medium flow. An airlift PBR is the improved form of the bubble PBR, with two interconnection parts, namely riser and downcomer zones. In an airlift system, air bubbles circulate the medium among dark (riser) and light (downcomer) zones. An external source of light is illuminated into vertical PBRs to provide the required energy of photosynthesis. These PBRs have a high surface-area-to-volume ratio, and the circulation of the medium by bubbles provides a suitable mass transfer. Oxygen, a limiting factor of microalgal growth, is discharged properly in vertical PBRs. The regulation of the gas flow rate in vertical PBRs is crucial because of its effects on light-and-dark cycles and the turbulence of the medium (69).

Flat-panel PBRs are the other type of closed system that has been used and developed in various sizes and forms for the cultivation of microalgae. Transparent and semi-transparent materials — such as glass, acrylic plastic (plexiglass), and polycarbonate — are used for the construction of rectangular flat-panel PBRs (Kiran et al., 2014). The aeration rate significantly affects the crucial aspects of flat PBRs such as mixing rate, heat and mass transfer, and CO2 and O2 gas exchange (Moreno-Garcia et al., 2017). As presented in the literature, the advantages of flat PBRs include high surface area for light absorption, low accumulation of O² in the system, flexibility in design and easy scale-up. However, as with open ponds and closed PBRs, there are some disadvantages for this system as well. For instance, there is potential of stress damage and hydrodynamic stress because of aeration. In addition, biofouling and temperature control are problems related to flat PBRs (Chew et al., 2018). Figure 4 shows different types of open ponds and closed PBRs for the cultivation of microalgae.

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Figure 4. (a) Vertical tube PBR, bubble column on left, airlift column on right; (b) horizontal tube PBR; (c) stirred tank mechanism; (d) flat-panel PBR; (e)racetrack- type pond (Chew et al., 2018) [Permission request granted Oct. 24, 2019, order numbers 4695460062377 and 4695460106012].

2.5 CULTIVATION MODES (PHOTOAUTOTROPHIC, HETEROTROPHIC AND MIXOTROPHIC)

Carbon capture and utilization (CCU) in microalgae has different metabolism pathways, including photoautotroph, heterotroph, and mixotroph modes. In photoautotroph mode, inorganic carbon, such as CO², is used as a carbon source, and energy is provided by light, which can be sunlight or artificial light. Heterotroph microalgae do not need light and use organic compounds as sources of carbon and energy as well. Mixotrophic microalgae are able to use light and organic carbon as sources of energy and consume both inorganic and organic carbon as sources of carbon (Chew et al., 2018).

The light dependency of photoautotrophic microalgae makes their cultivation process complicated. Light limits microalgal growth in high-density culture, because of the self-shading effect of cells and less photon absorption. On the other hand, exposing the cells to extensive irradiation can collapse the culture because of photo- oxidation and photo-inhabitation (Chang et al., 2011). The advantage of autotrophic microalgae is their contribution to the reduction of CO2 emissions as they use CO2 to grow. In addition, the risk of contamination of photoautotrophic cultivation is less than that of heterotroph and mixotroph cultivation (Chew et al., 2018).

The literature review indicates that cell density and lipid quantity in heterotrophic cultivation are significantly higher than in the photoautotrophic mode. Xu et al.

(2006) observed that the lipid content of Chlorella protothecoides increased 40% in heterotrophic cultivation as compared with cultivation in a photoautotroph system.

A light-independent and simple-design bioreactor decreases the production cost.

e

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31 Full-scale operation is easy, and the accumulation of specific products can be enhanced (Morales-Sánchez et al., 2015). Several wastewaters rich in organic carbon

— such as the hydrolysate of cassava starch, sorghum bagasse, corn powder, and sugarcane bagasse — have been tested for the economic heterotrophic cultivation of microalgae (Wang et al., 2016). The second main disadvantage of heterotrophic cultivation is a high risk of contamination, which can be improved by cultivation in a closed-indoor bioreactor.

Some researchers have stated that microalgae can grow better in mixotrophic modes as compared with photoautotrophic and heterotrophic ones. In mixotrophic cultivation mode, in the absence of light, organic carbon can be used as the source of energy, and light is no longer the only limiting-growth factor. In addition, it has been reported that the destructive effect of photo-inhibition is less in mixotrophic cultivation mode, and microalgae can grow better (Tan et al., 2018). Mixotrophic cultivation of microalgae has been conducted successfully in wastewaters contain various organic substrates, such as glucose, glycerol, fructose, and sodium acetate.

Among those, glucose is the preferred form of organic carbon for microalgae.

However, a high concentration of glucose decreases the lipid content, and the depletion of glucose decreases the growth of microalgae (Tan et al., 2018).

2.6 HARVESTING

Despite a great deal of attention on basic and applied research on microalgae, several barriers in cultivation, harvesting, and processing limit the commercialization of microalgal products. According to the literature, 20% to 30% of the total cost of biomass production is related to the microalgae harvesting from the culture (Wang et al., 2015b). The small size of microalgae cells and the low density of biomass (0.5-1.5 g/L) are the main challenges in microalgae harvesting (Vandamme et al., 2012). Centrifugation, flocculation, coagulation, electrophoresis, sedimentation, filtration, flotation, and magnetic separation are the commonly applied techniques for the harvesting of microalgae (Pahl et al., 2013).

2.6.1 Centrifugation

Cell size, cell weight (density), operation time, and centrifugal force are the parameters that affect the efficiency of microalgae separation by centrifuge. Molina Grima et al. (2003) report that increasing the centrifugal force from 1300×g to 13000×g increased the harvesting efficiency from 40% to more than 95%. Centrifugation as a chemical-contamination-free method is highly efficient and fast. However, microalgae separation by centrifugation is an energy-consuming and expensive method. I would suggest making this sentence read like this: Usually, centrifugation is appropriate for the separation of lower-quantity and higher-quality end products, where that high cost can be accommodated. It is not suitable for the harvesting of

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microalgae from a large volume when the end product does not have a high value.

For instance, the application of centrifugation for biodiesel production from microalgal biomass is not economically feasible (Bilad et al., 2014).

2.6.2 Membrane

Like centrifugation, separation of microalgae by membrane does not use chemicals. Compared with the other methods, it does not disrupt microalgal cells, and the quality of harvested biomass is good. Micro- (0.1-10 µm), macro- (>10 µm), ultra- (0.02-0.2 µm), vacuum, and pressure filtration are various types of membrane separation that researchers have introduced for microalgae harvesting (Milledge and Heaven, 2013). The size of microalgae typically has ranged from 3 to 30 µm, and a micromembrane is appropriate for most microalgae species, such as Chlorella sp., with 5 to 6 µm diameter (Milledge and Heaven, 2013; Molina Grima et al., 2003). Polyvinyl chloride, polyether sulfone, polytetrafluoroethylene, polyvinylidene fluoride, and polyacrylonitrile are among polymers that have been used as the materials of membranes (Bilad et al., 2014). Because of rapid fouling and the high cost of operating and maintenance, membranes have not been widely used for separation of microalgae.

2.6.3 Coagulation-flocculation

In coagulation, the negative surface charge of microalgae is neutralized by coagulants. Aluminum sulfate (Al2(SO4)3), sodium aluminate (NaAlO2), ferric chloride (FeCl3), ferric sulfate (Fe2(SO4)3), and ferrous sulfate (FeSO4) are the coagulants that have been used successfully for the harvesting of microalgae (Molina Grima et al., 2003). Electronegativity and solubility are two main features of these coagulants that affect the percentage of harvesting. In a study by Gerde et al. (Gerde et al., 2014) more than 90% of Scenedesmus sp. and Chlamydomonas reinhardtii biomasses were recovered at different concentrations of Al2(SO4)3. Following coagulation with flocculation increases the gravity sedimentation of microalgae and improves the harvesting efficiency. Long-chain polyelectrolytes are used in flocculation to form floc by the aggregation of neutralized particles.

Myristyltrimethylammonium bromide (MTAB) with the chemical formula of C¹⁷H³⁸NBr is a surfactant, which is used in the flocculation of microalgae. MTAB has a quaternary ammonium cation and a long alkyl chain. The cationic part neutralizes the un-neutralized functional groups that disturb the aggregation. The alkyl chain works as a bridge to link the particles together to enhance aggregation (Zhou et al., 2017).

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33 2.6.4 Auto- and bio-flocculation

Auto-flocculation could happen naturally because of a high rate of consumption of CO2 and increasing pH in the medium. The reason for this phenomenon at a high pH is the supersaturating of phosphate and calcium, which leads to the precipitation of calcium phosphate on the surface of microalgae cells (Uduman et al., 2010). In an outdoor cultivation experiment, the sedimentation of Scenedesmus dimorphus biomass was observed after the stopping of pond agitation and CO²injection (Sukenik and Shelef, 1984). Researchers who changed the pH of a medium artificially have also reported auto-flocculation. According to the observation of Wu et al. (2012b), more than 90% of freshwater and marine-water microalgae biomasses were harvested at pH 10.6. Because of the environmental changing of culture, auto-flocculation may not be economical at a commercial scale. In addition, auto-flocculation is slow and unreliable (Singh and Patidar, 2018).

The bio-flocculation process is defined as the flocculation of microalgae by excreted substances such as extracellular polymeric substances (EPS) from bacteria, fungi, and other microalgae. For instance, Solibacillus silvestris (one species of bacteria) could harvest 88.7% of Nannochloropsis oceanica from the medium (Wan et al., 2013). The bio-flocculation efficiency of 90% by another microalgae species, Pleurochrysis carterae, with microbes has been reported (Lee et al., 2009). The efficiency of bio-flocculation is different from one microalgae species to another via the same microorganism. The results of one study showed that the bio-flocculation of Botryococcus braunii, Selenastrum capricornutum and Scenedesmus quadricauda (green microalgae) was in the range of 91% to 95%, while it was 38% to 49% in the case of Anabaena flos-aquae and Microcystis aeruginosa (cyanobacteria) (Oh et al., 2001). Bio- flocculation followed by centrifugation can remarkably reduce energy demand for the harvesting of microalgae (Salim et al., 2012).

2.6.5 Flotation

Microalgae cells have a low density and can be carried by air or gas bubbles and float upward to the surface of a cultivation medium. The method, known as flotation, can be used for the separation of microalgae cells (Singh and Patidar, 2018). In foam flotation, bubbles capture the microalgae cells and move them to the surface of the medium. This technique has three main steps: the generation of bubbles, contact and adhesion between bubbles and microalgae cells, and the formation of a stable foam layer. The second step has a direct impact on the performance of foam flotation (Zhang and Zhang, 2019). To enhance the efficiency of foam flotation, the mass transfer between microalgae cells and bubbles is increased by the addition to the system of surfactants or coagulants. Cetyltrimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS), chitosan, and methylated egg ovalbumin are several synthesized and natural surfactants that are used in foam flotation. It has been

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reported that the percentage of microalgae harvesting was in the range of 70% to 99%

by the foam flotation method (Japar et al., 2017).

2.7 DOWNSTREAM PROCESSING

2.7.1 Drying

In the harvesting process, microalgae paste is concentrated to 11% to 23 wt%. An extremely high moisture content of microalgae biomass (90%) increases the costs of handling, storage, and transportation (Soto-Sierra et al., 2018). Therefore, drying is a necessary step before microalga cell disruption and oil extraction in the downstream process. Sunlight has been used as a conventional and cheap method for drying microalgae. However, this method is limited by inconsistent sunlight irradiance in different seasons, high demand for the drying surface, and loss of biomass (Tan et al., 2018). Fossil fuel-based methods are used to provide energy for the drying of microalgae. However, fuels such as natural gas can be expensive and can negatively affect the energy balance in producing final products, such as biodiesel (Lam and Lee, 2012). Spray-drying is another method and a recommended step before the extraction of high value-added compounds. However, high cost and the risk of destroying the pigment of microalgae are disadvantages of spray-drying. Freeze- drying has also been used to dry microalgae. Although expensive at a large scale, this method is efficient in disrupting microalgae cell and lipid extraction (Tan et al., 2018).

The temperature of the drying method significantly influences the biochemical composition of microalgae.

2.7.2 Disruption

The methods of cell disruption reported in the literature can be divided, generally, into mechanical and non-mechanical. Mechanical methods are subdivided into shear force (bead milling, high-pressure homogenization, and hydrodynamic cavitation), wave energy (ultrasonication and microwave), current (pulsed electric field), and heat (steam explosion, hydrothermal liquefaction, and freeze-drying). Non- mechanical techniques are classified as chemical (acid, ionic liquid, nanoparticle, oxidation, osmotic shock, and surfactant) and biological (enzymatic lysis and algicidal treatment) methods (Lee et al., 2017). Bead milling, ultrasonication, and high-pressure homogenization are appropriate methods for laboratory scales (Halim et al., 2012). The efficiency of cell disruption is high in the bead-milling method, and it is operable as single pass. In addition, the bead-milling method does not need high labor intensity. Because of these advantages, some researchers have suggested bead milling as a method with high potential for industrial use (Günerken et al., 2015). Al Hattab et al. (2015) have reviewed the economic efficiency of the current cell disruption methods on microalgae species such as Nannochloropsis sp., Chlorella sp.,

Application of microalgal-based technology for wastewater treatment and lipids production

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Dissertations in Forestry and Natural Sciences

EHSAN DANESHVAR

APPLICATION OF MICROALGAL-BASED TECHNOLOGY FOR WASTEWATER TREATMENT AND LIPIDS PRODUCTION

EHSAN DANESHVAR

Ehsan Daneshvar

APPLICATION OF MICROALGAL-BASED TECHNOLOGY FOR WASTEWATER TREATMENT AND LIPIDS PRODUCTION

ABSTRACT

ACKNOWLEDGMENTS

LIST OF ABBREVIATIONS

LIST OF ORIGINAL PUBLICATIONS

AUTHOR’S CONTRIBUTION

CONTENTS

1 INTRODUCTION

2 LITERATURE REVIEW

2.1 CULTIVATION OF MICROALGAE IN WASTEWATER

2.2 MICROALGAE SPECIES SELECTION

2.3 OPTIMIZATION OF MICROALGAE CULTIVATION IN WASTEWATER

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2.4 SCALING-UP OF MICROALGAE CULTIVATION

2.5 CULTIVATION MODES (PHOTOAUTOTROPHIC, HETEROTROPHIC AND MIXOTROPHIC)

2.6 HARVESTING

2.7 DOWNSTREAM PROCESSING