1. Introduction

Water quality is a fundamental concern of the present century, considering the increasing scarcity of water resources. Degradation of water quality, either caused by anthropogenic activities (e.g., pollution; resources overexploitation) or natural phenomena (e.g., global warming; extreme climatic events) often leads to severe impacts on ecosystems, public health and economic growth, negatively affecting society and environment ^[1]. The improvement of water quality is a central issue included in the 2030 Agenda and on Sustainable Development Goals, recognizing the importance of good-quality water for a sustainable development of society and, therefore, the need to globally address this problem.

The discharge of untreated or inadequately treated effluents into rivers, lakes, aquifers and coastal waters supplies the aquatic environment with a myriad of chemical compounds that can impact aquatic organisms directly, by triggering hazardous effects, and indirectly, by changing some physicochemical features of the medium (e.g., oxygen concentration, pH, redox potential and nutrient concentration) ^[2],^[3].

Contaminants often result from domestic, agricultural and industrial effluents and their physical and chemical compositions differ according to their origins^[4]. The most frequently found are metals ^[5], pesticides^[6] and nutrients, such as nitrate ^[7] and phosphate^[8]. Moreover, the occurrence of emerging organic and inorganic pollutants, such as microplastics^[9], pharmaceuticals^[10], flame retardants^[11], personal care products^[12], hazardous and noxious substances^[13] has been increasing since conventional wastewater treatment plants (WWTP) are not yet equipped and suitable to remove these new contaminants.

The complexity of effluent composition is increasing as human activities intensify, so it is urgent to develop adequate wastewater treatment processes, which should be easily applicable, effective and eco-friendly, in order to prevent water quality degradation and to protect water resources.

Physicochemical processes alone are proven to be inefficient regarding the treatment of effluents with complex composition^[11]. A possible solution can be the combination of physicochemical and biological treatment technologies aiming at the development of sustainable treatment processes.

Biological treatments often involve the cultivation of microalgae. The capacity of microalgae to remove nutrients^[14], metals^[4], pharmaceuticals^[15][16], radioactive minerals^[17] or pathogenic organisms^[18] from the medium has been highlighted in literature. Using microalgae to remove pollutants from wastewaters or effluents before their discharge or reutilization is named phycoremediation^[6]. The basic mechanism behind this process is inherent to the algal metabolism, nevertheless, the removal of pollutants will be variable accordingly to different microalgae used and effluents’ characteristics.

Therefore, the design of the cultivation system is a critical parameter for microalgae production in order to achieve optimal growth rates and minimize costs^[19]. For microalgae production, several factors are important to take into account, namely, the biology of the microalgae, cultivation land area, light, nutrient supply, labor, energy, carbon dioxide, pH, temperature and the type of the final product^[20][21][22].

Several methodologies of microalgae production have been developed over time, from laboratorial systems under accurate controlled conditions, to open systems in the field under natural, uncontrolled and unpredictable conditions^[20]. Table 1 summarizes the main systems, methods and strategies used. Microalgae are typically cultivated in open or closed culture systems. Furthermore, diverse strategies, namely in batch, continuous and semi-continuous, are applied around the world and which differ mainly on nutrient supply and operation modes^[20].

Due to the high energy demand and operation costs of harvesting processes, solutions were investigated in order to simplify this process^[23]. Therefore, new methods of microalgae cultivation, like the immobilization method on alginate beads and the development of bioreactors that use microalgae biofilm, have emerged and have been coupled to the treatment of wastewater^[24][25]. Also, the biological interactions among microalgae and bacteria or fungi and microalgae’s ability to naturally flocculate under adverse conditions are under study as means to increase microalgae biomass productivity^[26][27] and to reduce harvesting^[28], respectively.

Phycoremediation shows great potential to complement traditional wastewater treatment processes. Microalgae cultivation is, thus, a promising approach for WWTP in municipalities or industries, promoting environmentally sustainable effluent treatment, and nutrient and carbon biosequestration; and yields economic benefits–cost reduction and revenue from innovative products originated from microalgae (e.g.,: pigments, enzymes, sugars, and lipids)^[6]. Furthermore, this approach addresses the need to reduce the costs associated with microalgae biomass production^[29], through the recycling of wastewater to obtain microalgal biomass instead of culture medium^[26]. Nowadays, there are already several cases of enterprises worldwide using wastewater sources for microalgae production: United States—Algae Systems and Sundine Enterprises, Inc.; United Kingdom—I-PHYC; Australia—Algae Enterprises; Israel—Aquonos Energy Ltd.^[4][30].

Microalgae have indeed gained attention from the scientific community and have been a growing subject in research and development (R&D) or research and technology development (R&TD) studies and projects over the last decades, mainly focused on their resilience to grow on different types of wastewaters, performing phycoremediation and promoting decarbonization^[31].

 Table 1. Summary of different systems, methods and strategies of microalgae cultivation commonly used.

Interest in this field of research is increasing related to the potential of microalgae cultivation for a circular economy, once the microalgal produced biomass can be used and valued in several industries (e.g., food, feed, textile, pharmaceutical, bioplastic, biofuel, biofertilizer and cosmetic ^[33][45][46]). Nevertheless, cost efficacy of commercialization is still an issue and essential to attract investors to up-scale production, so the technology is still being developed in terms of research and development^[46]. Current research lines focus on four strategic alignments: feedstock, process, products, and market uptake^[30][47].

This review aims to point out trends and hot topics concerning the use of microalgae in wastewater treatment and to identify potential paths for future research regarding microalgae-based bioremediation. In order to achieve this goal, available literature will be mapped using a bibliometric tool to assess and analyze the topics that attracted more attention among the science community and their evolution through time.

2. Methods

Publication Production

Over the last half a century, the progressive increment of publications in this research area showed the increasing interest in the study and development of microalgae applications for water bioremediation. Figure 1 clearly exhibits such interest: from 1981 to 1990, only the publication of 5 articles were registered within this research field, but a significant increment was observed in the time frame between 1991 and 2000, with 109 article publications. This trend continued in the subsequent analyzed time periods (2001–2010 and 2011–2018), corresponding to 250 and 2578 articles published, respectively. Therefore, it should be expected that this increasing trend will continue.

Figure 1. Number of articles published in each chosen period.

Within the period analyzed, several authors were quite prolific, producing a high number of publications and reaching high visibility and scientific impact. Table 2 summarizes the most productive authors according to the number of produced publications and h-index, which expresses the productivity of each author according to the impact of the publications.

Table 2. Most productive authors between 1981 and 2018.

Content Analysis

 The study regarding microalgae production and water bioremediation has focused on different themes over the years. The evolution maps resulting from the SciMAT analysis of 2942 published articles between 1981 and 2018 show the conceptual evolution and thematic areas throughout the periods analyzed through graphical interaction (Figure 2). Overall, analyzing the conceptual map, a growing interest in this research area seems quite clear, characterized by a progressive increment on the diversity of the investigated themes and the relationships between them. Here, the size of the spheres (Figure 2) represents the quantity of publications, while the thickness of the line represents the relationship between the themes^[50].

Figure 2. Evolution of thematic areas between 1981–2018 (SciMAT output).

Specifically, between the periods 1981–1990 and 1991–2000, investigation focused on “bioreactors” and “nutrient-removal”, these themes being strictly linked (Figure 2). In a closed system, the control of several parameters that affect microalgae growth and nutrient uptake is easier^[41]. Moreover, the growth of kinetic models applied to microalgae growth and developed during this period was essential for recognizing nutrients, light and temperature as growth-limiting factors^[52].

Between the periods 1991–2000 and 2000–2010, the research themes were related to biotic and abiotic factors that could impair the application of water bioremediation technologies. Parameters such as temperature, pH, oxygen, carbon and nutrient supply have a pivotal role in microalgae growth. Nevertheless, other factors can cause microalgae growth inhibition. Urban and industrial wastewaters generally present a complex composition with different types of inorganic and organic pollutants^[4]. Such compounds can be toxic to microalgae, at certain concentrations, and inhibit their culture development. For this reason, a characterization of wastewater is important for understanding the composition of the effluent and foreseeing possible effects on the microalgae community. For instance, the growth of the microalgae Chlorel

 (Chlorophyta) was inhibited in the presence of 400 mg/L of phenol^[54]. Nevertheless, at tolerable conditions, microalgae have also been shown to remediate pollutant-rich effluents^{[55][56][57][58]}. Green microalgae Chlorella spp., T. obliquuSys (Chlorophyta) and Litemnospira maxima (formerly 

Spirulina maxima) (Cyanobacteria) showed a capability to degrade organic compounds such as phenol^[55][56]. Moreover, Auxndine Entenochlorella pyrenoidosa (formerly Chlorprisella pyres

), 

, and Stichococcus bacillae Enterpris (Chlorophyta) were shown to have more affinity to metal compounds such as iron (Fe) and copper (Cu) due to their importance as micronutrients to microalgae metabolism. Uptake rates of 70% and 98% of Fe and Cu were obtained after exposure of the mentioned species at 4 mg/L metal concentration^[57]. Other microalgae, such as Tetraselmis mari

, Stigeocloniums tenuEne and Spirogyra spp. (Chlorophyta), have shown potential to metal pollution bioremediation^[59]. Apart from chemical toxicity, the removal of pollutants from effluents can be affected by several biotic factors, namely, the production of toxic compounds, enzyme induction, the composition of the microbial community and the number of microorganisms^{[11][26][27][28]}. Therefore, it was important to address such issues in order to developed efficient remediation systems.

• Quadrant Q1: considered motor themes, important for the development of the research field.
• Quadrant Q2: themes well developed.
• Quadrant Q3: represents emergent or declining themes.
• Quadrant Q4: characterized by basic and transversal themes, nevertheless not well developed.  

 Figure 3. Strategic diagrams. (a) Period 1981–1990. (b) Period 1991–2000. (c) Period 2001–2010. (d) Period 2011–2018. (SciMAT output).

 In the period 1981–1990 (Figure 3a), two research themes were identified—“effluent” and “bioreactor”—characterized by high centrality and density and, thus, considered important to the growth of the field of research. The first studies were developed in Thailand and focused on nutrient recycling, by promoting the growth of microalgae in Tilapia niloLtica aquaculture effluents. A preliminary characterization of the microalgae community in the ponds showed that cyanobacteria and Euglena sp. (Euglenozoa) were the microalgae with higher occurrence^[71]. Microalgae metabolized the nutrients present in the water and fish fed on microalgae, there being a recycling of nutrients. The optimal microalgae concentration in the ponds was, however, controlled to prevent negative impacts on fish cultivation^[72]. The application of aquaculture effluent bioremediation aimed at addressing the issue of water scarcity by promoting a sustainable use of water resources through the reuse of water for fish cultivation and, subsequently, utilization of water for irrigation^[73]. Studies focused on the presence of contaminants and pathogenic agents in recycled wastewater were reviewed by Emparan, Harun and Danquah (2019)^[6].

The Aquatic Species Program of research, funded by the United States Department of Energy’s Office of Fuels Development, between 1980 and 1996, was a key-driver for the development of investigation on microalgae cultivation in open and closed systems aiming at the production of alternative fuels^[74]. The main challenges addressed were the selection, isolation and maintenance of the microalgal strains, microalgae growth rate and operational conditions’ optimization in order to produce biomass at a big scale and in a cost-effective way^[44][75]. University of Berkeley applied an open raceway system in a deactivated wastewater treatment station, in Roswell, from 1988 to 1990, and obtained better results using native species collected from the wastewater and the surrounding environment. Furthermore, research and technological advances in the design of bioreactors, within this period, were crucial for the development of water bioremediation technologies coupled with microalgae biomass production. American researchers hold several patents of photobioreactors for microalgae mass culture^[76][77][78]. Photobioreactors allow a stricter control of biotic and abiotic parameters. However, for biofuel production, the application of this technology is not economically feasible due to the associated high energy demand and cost of technical operation^[74].

In the period 1991–2000 (Figure 3b), 10 themes were identified, in which “effluent” and “ponds” were characterized as motor themes. Within this time frame, socio-economic interests regarding the scale-up of microalgae production focused scientific research on open culture systems and on the reduction of the costs of microalgae biomass production using wastewater as a growth medium. Microalgae metabolize nutrients such as carbon, nitrogen and phosphorous and use metals as micronutrients^[79]. Carbon metabolization is needed to microalgae growth and cellular respiration^[80] and nitrogen and phosphorous are processed to synthesize essential amino acids and DNA^[49]. The literature produced also showed that microalgae have the potential to be used as a biosorbent agent for metal removal from wastewater. The polysaccharides present in microalgae’s cell wall are responsible for metal adsorption, nevertheless, this adsorption ability depends on the species and growth conditions^{[81][82][83][84][85]}. Furthermore, as previously mentioned, the success of bioremediation technologies using microalgae depends on their capacity to tolerate the pollutant concentration in the growth medium. Therefore, it is important to perform a preliminary characterization of the effluent and investigate, through toxicity bioassays the non-lethal concentrations of different pollutants for microalgae growth^[57].

Between 2001–2010, 21 research themes were observed (Figure 3c), and the motor themes were: “fatty-acids”, “effluent”, “wastewater”, “microalgae culture” and “environment”. Microalgal biotechnology investigation was enhanced within this period and, initially, research focused on microalgae as a source of biofuels due to their fast growth^[74]. On the other hand, nutrient pollution was a problem at a global scale and coupling biofuel production and microalgae growth in nutrient-rich effluents was pursued as a solution.

This problem should be handled through a multi-stakeholder cooperation^[47] given its worldwide dimension. Within this context, the European Union (EU)–Japan Centre for Industrial Cooperation, a non-profit association whereby its mission is to promote the collaboration between Japan and the EU in order to provide partnerships and guidance to prepare projects related to microalgae application on water bioremediation, promoting a circular economy^[30]. Furthermore, in 2003, the Japanese legal framework ‘’Basic Act for the Establishment of a Sound Material Cycle Society’’ formulated a five-year plan in which the main goal was to use properly the natural resources and develop eco-friendly procedures for waste management while minimizing the harmful impacts on the environment. In this context, governmental institutions such as the New Energy and Industrial Technology Development Organization (NEDO) and the Ministry of Agriculture, Forestry and Fisheries (MAFF) supported several companies and universities as partners or facilitated funding opportunities^[30]. This framework also launched the environmental goal of developing alternative WWT in detriment to the traditional WWT, aiming at nutrient recovery and recycling^[84].

Furthermore, in the European Union, the Water Framework Directive, published in 2000, addresses and encourages the improvement of urban wastewater treatment with biological methods. More recently (2016), the Government of the Republic of Lithuania has, inclusively, approved a management plan in order to reduce the nutrient input in the Baltic Sea^[47].

Microalgal biomass is a valuable resource with potential applications in several industries and is a key factor in the blue economy because of the value of its commercial applications and for being an alternative to land-based products or non-renewable resources. Hence, microalgae have gained increasing attention by the academia, industry and policy makers, especially since 2001.

In the last analyzed decade (2011–2018) (Figure 3d), the main motor-themes were: “cultivation”, “aqueous-solution”, “diatoms”, “fatty-acids composition”, “high rate algal-ponds” and “pharmaceuticals”. Advances of more sensitive analytical equipment and methods led to the determination and identification of emerging water pollutants in aquatic ecosystems^[85] resulting from human activities, such as pharmaceuticals, personal hygiene products, chemical cleaning agents and natural or synthetic hormones^[86]. Under the scope of the European framework of the Water Directive and Wastewater Treatment Directive, numerous projects have been funded to overcome the challenge of water-emerging contaminants. Networks among reference laboratories, research centers and related organizations were created to monitor emerging environmental substances (NORMAN) and develop a database of geo-referenced monitoring and bio-monitoring data on emergent substances in water^[87]. Studies regarding the use of microalgae as bioremediators of contaminated effluents were, as previously mentioned, conducted. Furthermore, ecotoxicological studies have been carried out with diatoms or other microalgae, which are primary producers and present higher sensitivity in the marine environment in comparison to other organisms^[17]. The toxicological effects of fossil fuels^[88], organophosphorus compounds^[89] and polycyclic aromatic hydrocarbons^[90], easily found in water, on microalgae were also studied. Nevertheless, some strains of microalgae were shown to be resilient with acclimatization capability through biochemical and physiological modifications^[6].

High-rate algal ponds (HRAPs) have demonstrated feasibility at an operational and economical level for bioremediation of a secondary effluent from a wastewater treatment plant. Gentili and Fick (2017) obtained a 60%–80% and 60%–70% nitrogen (N) and phosphorous (P) removal, respectively, in a HRAP system^[16]. Furthermore, a mixed freshwater population, in which Tetradesmus dimorphus (Chlorophyta) dominated, was shown to successfully grow in a HRAP system and to deplete different types of pharmaceuticals. Removal rates higher than 90% on pharmaceuticals, namely atenolol, atracurium, bisoprolol, bupropion, citalopram, diltiazem and metoprolol were reported 

The production of microalgal biomass into bioproducts is also a current theme of research and will promote the manufacture of innovative biological products as well as the creation of employment^[47]. Photosynthetic microorganisms are efficient producers of high-value compounds, like polyunsaturated fatty acids (PUFAs)^[92]. Studies showed that n-3 PUFAs have therapeutic properties, namely in cardiovascular and mental diseases^[93]. Furthermore, it is important to characterize microalgae lipidic and protein content in order to understand the range of bio-products that can be developed. The protein and fatty-acids composition is distinctive among microalgae species, and factors like effluent physic and chemical characterization, aeration rate, temperature, photoperiod and light type and intensity, can influence the biochemical profiles of microalgae^[94].Within this scope, several funding opportunities have been created. For instance, the European Union has directed funds to support investigation within the field of bioeconomy given its social, economic and environmental impacts in the communities. The international project “Algae Service for LIFE” supported by LIFE Environment and Resource Efficiency sub-program aims to enhance good practices on ecosystem services and promote circular economy. Also, in Japan, the Agency for Natural Resources and Energy presented, in 2015, a report about the valorization of microalgal biomass from wastewater treatment processes^[30]. Over the last decade, the United States Department of Agriculture (USDA) has supported several projects regarding wastewater bioremediation using microalgae aiming at water reutilization for agriculture purposes and as a measure against water scarcity^[95].