Microalgae: Cultivation, Recovery of Compounds and Applications

By Charis M. Galanakis

Microalgae: Cultivation, Recovery of Compounds and Applications supports the scientific community, professionals and enterprises that aspire to develop industrial and commercialized applications of microalgae cultivation. Topics covered include conventional and emerging cultivation and harvesting techniques of microalgae, design, transport phenomena models of microalgae growth in photobioreactors, and the catalytic conversion of microalgae. A significant focus of the book illustrates how marine algae can increase sustainability in industries like food, agriculture, biofuel and bioprocessing, among others.

This book is a complete reference for food scientists, technologists and engineers working in the bioresource technology field. It will be of particular interest to academics and professionals working in the food industry, food processing, chemical engineering and biotechnology.

• Explores emerging technologies for the clean recovery of antioxidants from microalgae
• Includes edible oil and biofuels production, functional food, cosmetics and animal feed applications
• Discusses microalgae use in sustainable agriculture and wastewater treatment
• Considers the techno-economic aspects of microalgae processing for biofuel, chemicals, pharmaceuticals and bioplastics

• Language: English
• Publisher: Academic Press
• Release date: Oct 5, 2020
• ISBN: 9780128232149

Book preview

Brazil

Chapter 1: Cultivation techniques

Luisa Fernanda Rios Pintoa; Gabriela Filipini Ferreirab; Marija Tasicc     a Chemical Engineering, Research Fellow Position at the Faculty of Chemical Engineering at the University of Campinas (UNICAMP), Campinas, Brazil

b Chemical Engineering, Student at the Faculty of Chemical Engineering at the University of Campinas (UNICAMP), Campinas, Brazil

c Chemical Engineering, Associate Professor at the Faculty of Technology at the University of Nis, Leskovac, Serbia

Abstract

Production of microalgal biomass usually takes place in open ponds or closed photobioreactors. Open systems (raceway and circular ponds) are cheaper and easier to construct and operate; thus they are chosen mainly for cultivation at industrial scales. However, they often depend on weather conditions, have poor culture parameters control (temperature, pH, dissolved O2 and CO2), and are prone to contamination with other microorganisms. As photobioreactors (tubular and flat-plate) achieve higher biomass productivity and to improve final product quality, hybrid systems could be a promising alternative. Therefore, a robust axenic inoculum would be obtained by first cultivating microalgae on a smaller scale followed by open ponds where stress conditions could be applied to increase a metabolite production, e.g., lipids. However, most studies that involve microalgae cultivation to attain different bioproducts are performed on a laboratory scale. Scaling up should then consider hydrodynamic limitations as well as its economic and environmental impacts.

Keywords

Microalgae; Cultivation; Biomass; Open system; Closed system; Photobioreactor; Bioproducts; Nutrient removal; Wastewater treatment

Statement

We declare that all figures and tables presented in this chapter were made by the authors and have not been published elsewhere.

1: Introduction

Microalgae have considerable potential as a feedstock for different bioproducts (Borowitzka & Moheimani, 2013; Klok, Lamers, Martens, Draaisma, & Wijffels, 2014; Moheimani, McHenry, de Boer, & Bahri, 2015; Raheem, Prinsen, Vuppaladadiyam, Zhao, & Luque, 2018), from biofuels to edible oils and proteins, and can be cultivated in different environmental conditions and systems. As the human population increases, it is required the production of more food, energy, and water, thus producing more carbon dioxide (CO2). For this reason, microalgae cultivation brings a new opportunity and solution for all these problems toward a sustainable bioeconomy (Bussa, Eisen, Zollfrank, & Röder, 2019; Ferreira et al., 2018; Rizwan, Mujtaba, Memon, Lee, & Rashid, 2018).

The growth characteristics and metabolites accumulation strongly depend on the type of cultivation: phototrophic (using light and CO2), heterotrophic (without light and organic carbon), and mixotrophic (using light, CO2, and organic carbon). Phototrophic cultivation is the most common type used in large-scale microalgae production (Suparmaniam et al., 2019).

One of the essential parameters in algae cultivation is the type of system used. Several companies in some countries have developed the commercial production of microalgae biomass and/or bioproducts, also coupled with CO2 fixation (Zhou et al., 2017).

Cultivation systems can be divided into two broad groups: open systems and closed systems (Okoro, Azimov, Munoz, Hernandez, & Phan, 2019). The most commonly used open systems are ponds (cylindrical, rectangular, and elliptical-bottomed) or largely used raceway ponds. It is called open systems because the microalgae culture is in contact with atmospheric air. These systems are not sophisticated; the cultures are usually grown under natural conditions and have lower control on variables (e.g. temperature, light, and pH). Several challenges are confronting with the use of open ponds: water evaporation, climate variations, contamination, among others. These systems are generally used to obtain low added-value products. On the other hand, in closed systems, generally called photobioreactors (PBRs), the cultivation is developed to achieve higher yields. These systems can be achieved by the high luminosity surface and the control of growth and contamination variables within the system. In these systems, the atmospheric air has no direct contact with the microalgae culture. Several PBRs have been developed so far, among them, vertical cylindrical, vertical or horizontal flat panel, tubular panels, and vertical and horizontal serpentines. The material may be of glass, plastic, or polycarbonate.

Microalgae cultivation, in particular growing systems, were developed thinking in terms of volume production, so at first, it was quite rustic and simple. With the development of different products from microalgae biomass (high-value products), these systems have been advancing technologically, using engineering concepts in the construction of improved systems, always aiming to achieve high productivity and cost optimization (Narala et al., 2016). Both of actually systems for microalgae cultivation, in particular growing systems, have advantages and disadvantages. Notably, open systems have minimal capital and operating costs and lower energy but require large areas and come with contamination problems. By contrast, the closed systems are more expensive; they do not require large areas, and the contamination problems decrease.

Nowadays, microalgae cultivation is well stabilized for food, feed, and pigments. However, in some areas, it is still not feasible. Microalgae biorefinery is a possibility for becoming microalgae cultivation a viable economic process, producing high-value products for all the cell fractions (Chew et al., 2017). Currently, many companies work in microalgae large-scale production. Following is a few facilities that produce microalgae in large scale: (a) Earthrise Nutritionals is used to produce cyanobacterial biomass for food (Chisti, 2007), (b) Phycom is an industry that produces Chlorella vulgaris and Chlorella sorokiniana for feed and food, and (c) Algawise that is a part from Corbion, that produce algae oils, among others. More industrial facilities are listed in Table 3.

This chapter deals with conventional and emerging cultivation techniques for microalgae cultivation.

1.1: The history of microalgae cultivation system

The large-scale systems of microalgae cultivation were established during the first decades of the 20th century (Hamed, 2016). Before this, algae culturing was restricted to laboratory-scale operations. Notwithstanding, simple systems for food chain enhancements commanding to algal production and following growth of comestible organisms exist for centuries, and natural colonies of Spirulina have been initially collected for food in Africa and Mexico. The outdoor microalgae production began in around 1948. Fig. 1 depicted when a few cultivation systems were created to enhance production over time. The first large cultivation was performed by growing microalgae in an open pond for the food chain. These systems were initially used in Germany and Japan during 1940–50. The construction of German ponds was in a plastic-lined, with approximately 20 cm depth and using air to improve mixing and obtain a homogeneous culture. The Japan design was a single flat-bottomed of 22 m² surface area and bubbling CO2 enriched air thought aeration pipes and 15–20 cm deep (Terry & Raymond, 1985). In the early 1950s, the Research Corporation of New York contacted the Stanford Research Institute (SRI) in Palo Alto, California, to conduct the study for the continuous culture of Chlorella, but the results were not conclusive enough, one of the reasons was the contamination culture. The SRI was one of the first workers to recognize the need for a closed and sterilized system to maintain an uncontaminated algal culture (Lee, 1986).

Fig. 1 Timeline for microalgae cultivation system.

Following the United States concentrated on microalgal-bacterial systems for wastewater treatment, even though during 1950–60, the application of microalgae for the conversion of CO2 to oxygen in spacecraft and submarines was also explored. The more significant international effort in algal biomass culture with strongly mixed systems started in Eastern Europe in the 1960s, headed by researchers at the Czechoslovak Academy of Sciences and based at least in part on earlier Soviet work (Terry & Raymond, 1985).

Between the 1960s and 1970s, a deep channel system patterned after with Oswald's design, ponds of up to 100 m², was developed in California for the treatment (to remove nitrogenous) of runoff waters from agricultural tiling is in the San Joaquin Valley. Another model developed in the Czech Republic in the 1970s was a cascade of descending film.

The Commission of the European Communities began the algal biomass research for bioenergy in 1978. The project entitled Mariculture on Land (MCL), includes researchers in Germany, France, Italy, and Brazil. It proposed the utilization of arid coastal lands and seawater for the culture of micro and macroalgae for methane production. Later in 1979, the Solar Energy Research Institute (SERI), began a research program on the biomass production of aquatic plants for energy, which included the use of microalgae as a mechanism for the fixation of solar energy (by photosynthesis) as liquid biofuels (Terry & Raymond, 1985).

As already mentioned, the cultivation system will be selected among existing or new designs depending on the final product. This chapter provides a short review of the present systems and their applications. The cultivation system is a principal criterion after the selection of a microalgae species for optimal cultivation in order to be a cost-effective process.

2: Laboratory cultivation techniques

Generally, laboratory cultivations are used to study and optimize parameters, variables, and growth conditions. Lab cultivation is a mandatory step when the objective is a scale-up. Many of the research published in the literature are results of laboratory studies, through the propose of studying growth conditions for a specific microalga (Liu, Chen, Tao, & Wang, 2020; Zhou et al., 2019). Microalgae cultivation offers benefits in terms of climate change, but still has many challenges that need to overcome. The first factor that needs to be considered is the strain selection; the best strain is that one grows highly and has the metabolic accumulation necessary for the objective and can survive in severe conditions. The next challenge is energy consumption, as many steps in the microalgae cultivation process need power to work, including upstream and downstream. Closed PBRs need energy for mixing the culture, water pumping, gas bubbling (CO2), harvesting/dewatering the culture, among others, that depend on the final product. Another challenge that needs to overcome is the availability of the nutrients and water, large amounts of water and nutrients are used for cultivation, but nowadays microalgae cultivation are overcoming these obstacles by using wastewater from industries, which have abundant nutrients and water often undervalued. The final challenge is the distribution of CO2; the systems for the CO2 distribution are problematic, as they demand high energy and require long pipelines to transport the gas.

The factors that affect the microalgae cultivation for biomass production are light intensity, photoperiod, temperature, nutrients, mixing, aeration, pH, and CO2 absorption. Microalgae photosynthesize, which means that they assimilate inorganic carbon for conversion into organic matter. Light is a common source of energy, which drives this reaction. The light intensity is one of the most important variables, once the microalgae cell metabolism uses light as a source of energy for synthesizing the cell protoplasm. For lab cultivation, artificial illumination is necessary by incandescent lamps or without light (heterotrophic cultivation). Fluorescent tubes in the blue or red-light spectrum are ideal as these are the most active portions of the light spectrum for photosynthesis (Koc, Anderson, & Kommareddy, 2013). The depth and the density of the microalgal culture influence at the light intensity: at high depths and cell concentrations, the light intensity needs to be increased to access through the culture. The optimum light intensity depends on the microalgae strain; in the literature, it is possible to find that for Desmodesmus sp., a 98 μ mol m− 2 s− 1 (Ji et al., 2013) source is the optimum, on the other hand, for Dunaliella viridis the optimum was 700 μ mol m− 2 s− 1 (Gordillo, Goutx, Figueroa, & Niell, 1998). Some strains suffer photoinhibition when is increased the light intensity. It occurs with disruption of the chloroplast lamellae growth and inactivation of the enzymes involved in the carbon dioxide fixation. However, some strains can survive without light in heterotrophic cultivation.

The light intensity and duration affect photosynthesis of microalgae, and its biochemical composition, very low or very high luminescence, is not efficient for growth (Khan, Shin, & Kim, 2018). The range of lightness used in a lab-scale is 20–1000 μ mol m− 2 s− 1 (ResearchGate, n.d.); being the most common, 150 μ mol m− 2 s− 1. The lamps are placed, usually, at 25–30 cm of distance from the flasks.

Light cycles (dark:light) are important for the performance of the microalgae (Jacob-Lopes, Scoparo, Lacerda, & Franco, 2009). The most used photoperiod is 12:12 h (dark:light). Experimental studies report that the minimum photoperiod is (8:16) and the maximum (24:0). Many studies reported that 24 h of light exposed increased biomass productivity (Rai, Gautom, & Sharma, 2015). Biomass productivity reached for lab-scale is around 0.02–0.7 g L− 1 d− 1 (Ferreira, Ríos Pinto, Maciel Filho, & Fregolente, 2019; Ishika, Moheimani, Laird, & Bahri, 2019; Rashid, Ryu, Jeong, Lee, & Chang, 2019).

The temperature influences the growth velocity, cell dimension, metabolites composition and accumulation, and nutrients requirements. The best temperature for cultivation also highly depends on the strain. The cultivation in a lab-scale is prepared in a cultivation room (indoor) with a stable temperature in a range of 16–27 °C. Some studies report that the optimum temperature is 18–24 °C but strongly depends on the microalgae strain.

In terms of pH, in a lab-scale is common uses a range of 6–9 (Qiu, Gao, Lopez, & Ogden, 2017; Song et al., 2020), but it is reported that the optimum is 8.2–8.7 for most microalgae strain. In order to control the pH value, it is strongly used the addition of carbon dioxide to reach acceptable levels (Valdés, Hernández, Catalá, & Marcilla, 2012; Vonshak & Coombs, 1985).

Another factor that influences in microalgae productivity is mixing. This factor is necessary to prevent cell sedimentation and the homogenization of all the cells. To good mixing granter, all the cells are exposed to the light and nutrients. In practice, among different ways to mix microalgae culturing, the most used are the mechanical and air bubbles. High velocities and turbulence can damage the cells; the optimum levels depend on the microalgae strain (Eriksen, 2008).

Finally, nutrient concentration affects the microalgae growth. Some nutrients are essential for the cultivation, principally macronutrients (phosphorus and nitrogen) (Zhuang et al., 2018). Many culture media are used for laboratory cultivation (BG 11 (Rippka, Deruelles, Waterbury, Herdman, & Stanier, 1979), f/2 Guillard (Guillard, 1975; Guillard & Ryther, 1962), CHU 13 (Chu, 1942), among others) that strongly depends on the microalgae strain, Chew et al. (2018) list the nutrients that are needed for different types of microalgae.

Lab cultivation generally is operating in a batch culture that consists in the inoculation of cells into a flask, following for a growth period of several days (7–40) and finally harvested. The scale-up in the laboratory is usually carried out by successive transfers of cultivation systems from small to larger (Pérez et al., 2017). The inoculum (2%–10%) is transferring to a bigger flask adding more cultural media (Cruz et al., 2018).

A few of lab systems are shown in Fig. 2, (a) Petri dishes that are used to isolate a microalga species, once microalgae usually grow in a colony that is easy to identify in a solid media, but the sample to be inoculated need to be in a deficient concentration of microalga cells. (b) Tubes are used for inoculum conservation, usually is the purest microalgae colonies that have in a lab, generally, from this sample, the experiments start. (c) Flasks are primarily used for lab cultivations; usually, the experiments start with a 250-mL flask. Various sizes and capacities are used for this purpose (100, 250, 500, 1000, and 2000–10,000 mL). Commonly is used 80% of the maximum volume, with 10% of inoculum, the remaining is cultivation medium. (d) Plastic bottles: there are several plastic bottles from water bottles to a gallon of water.

Fig. 2 Systems cultivation (A–D) Lab-scale systems, (E–I) closed systems, and (J and K) open systems.

In laboratories, microalgae can also be cultivated in different systems; for example, PBRs, bags, and other technological systems. The cultivation technique is strongly influenced by the microalgae strain (Chew et al., 2018).

3: Pilot cultivation techniques

The cultivation parameters (Bux & Chisti, 2016; Gim et al., 2014; Najafabadi, Malekzadeh, Jalilian, Vossoughi, & Pazuki, 2015; Ruangsomboon, 2012) that are possible to manipulate at a pilot-scale are the same as laboratory-scale systems: nutrients concentration, nutrients source (wastewater and synthetic medium), carbon source (organic and inorganic), salinity, temperature, pH, light source (artificial and natural), light intensity, light spectra (Vadiveloo, Moheimani, Cosgrove, Parlevliet, & Bahri, 2017), photoperiod, and agitation. Among these parameters, the temperature ensures that the selected microorganism grows appropriately. The pH is also essential, as it interferes with cell growth. The agitation, on the other hand, has the role of ensuring a good transfer of mass and heat, keeping the medium homogeneous throughout the cultivation process as well as improving the access of algae cells to gases in the case air or CO2 are supplemented. Considering that each parameter will be adjusted according to the need and characteristics of the process, stress conditions can be applied by manipulating these parameters to produce a specific metabolite. Nevertheless, better control is possible by using closed systems compared when scaling up PBRs.

Regardless of choosing a specific cultivation system to produce microalgae at a larger scale, it is desired to maintain the biomass productivity and its composition when scaling up the production from laboratory to pilot to industrial scales. To achieve this goal, a few aspects must be taken into consideration, such as hydrodynamic and environmental conditions (Cuello, Hoshino, Kuwahara, & Brown, 2016). Additionally, it is essential to arrange a large pond or multiple tubes in a way that all culture volume is roughly under the same conditions and close to a complete mixture (homogeneous), with minimum dead zones, light incidence variation, among others. Thus larger-scale production of biomass must consider economic as well as technological limitations. In summary, the selection of a cultivation system will depend on the microalgal species selected, the cost of land, environment conditions, desired bioproduct (Lee, 2013) and process economic viability.

The microalgae cultivation technique is divided into two categories: open and closed systems (Klinthong, Yang, Huang, & Tan, 2015). Open systems (mostly open ponds) are commonly chosen to be used in larger scales due to lower construction and maintenance costs, thus resulting in a lower estimated cost of production (Zakariah, Abd. Rahman, Hamzah, & Md Jahi, 2015), direct exposure to outdoor sunlight and other technical advantages (Acién et al., 2017). However, these are more susceptible to contamination (fungi, bacteria, and other microalgae) and dependent on climate conditions. Therefore, robust cultures with high tolerance to adverse ambient conditions, such as pH and temperature variations, and microalgae prone to resist harmful invaders should be selected for this type of cultivation. To that purpose, it is essential to invest in upstream processes, such as isolation and selection of microalgae strains, as well as optimizing downstream processes (harvesting, dewatering, drying, cell disruption, and lipid extraction).

On the other hand, numerous PBRs with different shapes and sizes have been developed to achieve high productivity, which could be competitive to open systems if production costs are reduced, and revenue is also increased by targeting high-value bioproducts. Moreover, they allow axenic cultivation of monocultures, less water evaporation, lower CO2 loss, and better control of conditions (El-Baz & Baky, 2018).

3.1: Photobioreactors

Closed algal cultures exhibit several benefits disregarding the economic barrier. They reduce contamination risks and limit CO2 losses (Moheimani et al., 2015) in addition to the previously mentioned increase in biomass productivity. Although many economic studies show a higher price of construction and operation compared to open ponds, there are still process steps that could be explored to reduce costs. For example, expanses with nutrients and carbon sources could be significantly reduced if wastewater (Ayre, Moheimani, & Borowitzka, 2017; Chinnasamy, Bhatnagar, Hunt, & Das, 2010; Guldhe et al., 2017) and flue gases (Duarte, Fanka, & Costa, 2016; Hanifzadeh, Sarrafzadeh, & Tavakoli, 2012; Ji et al., 2017) were used during the cultivation step. Closed PBRs are usually divided by their geometrical configuration, such as tubular (cylindrical) and flat-plate (rectangular prism) types.

3.1.1: Tubular photobioreactors

These systems consist in glass or plastic tubes usually assembled vertically with upward flow obtained by using pumps and/or gas bubbling (air-lift), area-to-volume ratios of 80–100 m− 1, diameters up to 10 cm to improve photosynthetic efficiency (Acién et al., 2017) and liquid velocities of 0.5 m s− 1 (El-Baz & Baky, 2018). They are usually applied to obtain concentrated cultures and produce high-quality biomass for high-value applications such as pharmaceutical, food, and cosmetic industries. However, excess O2 accumulation, above air saturation, is a disadvantage of this configuration as it can inhibit photosynthesis.

Another common tubular PBR is the air-lift or CO2-lift type, which can achieve high-density cultures (Chiu, Tsai, Kao, Ong, & Lin, 2009). They consist of larger diameter tubes with two internal zones and gas bubbling that allows recirculation. The central zone, called a riser, is where gas is dispersed. Then the liquid is moved to the outer zone, called downcomer. This configuration differs from other tubular PBRs mainly due to the mixing mechanism that promotes better homogeneity and facilitates mass transfer. Also, they can have high photosynthetic efficiency due to the use of a LED light jacket covering most of the bioreactor's surface.

3.1.2: Flat-plate photobioreactors

The rectangular-shape PBRs were conceptualized to obtain optimal light absorbance as the rectangular panels, made of glass or a polymer, are transparent, have a high area-to-volume ratio (400 m− 1) and a light path around 7 cm (Płaczek, Patyna, & Witczak, 2017). Consequently, cultures can achieve high volumetric biomass productivities (Acién et al., 2017). They show less O2 accumulation than tubular PBRs but present a temperature variation challenge, overheating. Thus, as in some cases of tubular, flat-plate PBRs may require cooling. Similar mixing mechanisms are applied (pumps and gas bubbling).

3.1.3: Different designs of photobioreactors

Other PBR systems are (1) the oldest widely used serpentine and (2) manifold or the unusual (3) helical configurations (Acién et al., 2017; El-Baz & Baky, 2018). The first refers to several tubes usually displayed horizontally, connected by U-bends. They have been adapted and upgraded over the last decades to the recent model with improved mass transfer and mixing. The second is similar to serpentine as it consists of a series of straight, long, and parallel tubes, which are connected by two manifolds for distribution and collecting culture suspension. Finally, helical PBRs are smaller diameter circularly bent tubes coiled around an upright supporting structure with a separate or connected degasser. They may be advantageous in a space-limiting situation.

3.1.4: Comparison of photobioreactors

Photobioreactor selection must consider the best design to achieve high biomass productivity with a specific composition (target bioproduct), as well as the economic viability of the proposed system. Therefore, enclosed PBRs are often chosen for high-value metabolites production (Suparmaniam et al., 2019). Table 1 shows a collection of different microalgae cultivation using PBRs at a laboratory or pilot scale. Studies developed in recent years were chosen to compare biomass productivity using different systems. It can be seen that flat-plate configurations usually yield higher biomass production probably due to superior photosynthetic efficiency. On the other hand, tubular PBRs proved to be a suitable system for high-lipid production as well as wastewater treatment.

Table 1

a Measured from days 4–6 of cultivation.

Another important parameter to be considered when scaling up PBRs is the cultivation regime, similar to chemical reactors, batch, semi-batch, and continuous. Batch reactors are the most used in industry when involving microorganisms due to easier control of the process variables and final product quality. However, a continuous process is desired to increase production and obtain constant growth throughout the year. Once defined the cultivation regime, a kinetic study of cell growth if often conducted to optimize production.

3.2: Open ponds

Open ponds are often divided into the raceway and circular configurations (Suparmaniam et al., 2019). They consist of cheap systems where the cultivation is usually conducted in outdoor conditions. Consequently, it depends on weather conditions being favorable such as abundant light incidence throughout the year; it has poor control of parameters such as temperature and pH; it is highly exposed to contamination (grazers, bacteria, fungi, viruses, and other microalgae) (Bux & Chisti, 2016). Although they usually achieve lower biomass productivity than closed PBRs due to these factors, and others, economic studies show that they still are the most viable option. For example, cost-efficient carbon supplies could improve biomass production and lipid accumulation by bubbling CO2 from inexpensive flue gases.

3.2.1: Raceway ponds

One of the oldest developed open systems is raceway ponds, widely used for large-scale cultivation of microalgae (El-Baz & Baky, 2018). They have large areas (100–5000 m²) and are divided into 2 or 4 channels for recirculation, the liquid velocity of 0.2 m s− 1 to avoid settling of cells, short height to ensure light reaches to bottom (0.2–0.4 m) and low surface-area-to-volume (S/V) ratios (5–10 m− 1) (Acién et al., 2017). Culture circulation is usually obtained by using a paddle wheel or a propeller and a centered baffle. Deflector baffles may also be used to improve mixing by promoting a turbulent flow.

Besides fluid dynamics, mass transfer limitations are an issue of raceway ponds, as CO2 biofixation and O2 removal are also determining for system performance.

3.2.2: Circular pond

A rounded pond with a rotating arm usually has 20–30 cm depth and 40–50 m diameter. It is often employed in wastewater or other effluent treatments, especially in Asia (El-Baz & Baky, 2018), and is associated with a cheaper cost. However, due to high exposure to the surroundings, it can easily contaminate with other microorganisms.

3.2.3: Different designs of open systems

Inclined (cascade) systems allow the algae culture to flow down an angled surface of a few 100 m², be collected in a larger-volume recipient, and pumped back to the top (Borowitzka & Moheimani, 2013). During the day, the culture is continuously recirculated to increase light exposure and promote photosynthesis. They can achieve high production once the shallowest pond stores concentrating biomass cultures.

Thin-layer systems are employed to obtain high microalgal biomass concentrations by using a low-depth culture (< 50 mm) and maximizing light-efficiency (Acién et al., 2017). They consist of inclined platforms, sloping cascades, or near-horizontal raceways with a high S/V ratio (25–50 m− 1), that enables the optimal sun incidence to achieve high productivity, in contrast with other open ponds or raceways. Thin-layer cascades can have more than 100 m− 1 S/V, with biomass productivity and density higher than 30 g m− 2 d− 1 and 10 g L− 1, respectively (Grivalský et al., 2019).

Algal turf scrubbers consist of a similar system with a substrate that supports attached growth on a sloped surface, where algae absorb nutrients from the flowing wastewater (Hoffman, Pate, Drennen, & Quinn, 2017). They are simple in design and allow a more natural biomass harvest compared to other systems. Furthermore, they exhibit stable and promising biomass productivity, being one drawback of the high content of ash (Hess et al., 2019).

3.2.4: Comparison of open systems

Open ponds are the cheapest system to cultivate microalgae on a large scale; thus, they are mainly found in industrial production. The most common are raceway and circular ponds, but attention has been recently given to thin layer and microalgal turf scrubber to increase biomass productivity. Moreover, new designs have been developed, such as a flat-plate continuous open PBR (Luo et al., 2019), which was able to achieve 0.47 g L− 1 d− 1 biomass productivity while treating a piggery biogas slurry. Table 2 compares different open systems used for microalgae cultivation at a laboratory or pilot scale. Studies developed in recent years were chosen to compare biomass productivity using different systems. Biomass productivities are overall lower compared to closed systems (Table 1), but promising results were obtained using different designs of unconventional open ponds with wastewater or another effluent as nutrient and carbon sources. Moreover, the use of flue gases instead of pure CO2 can be a sustainable and economical option to improve biomass productivity, with improved mixing and mass transfer (Cheng, Yang, Ye, Zhou, & Cen, 2016).

Table 2

a Obtained during summer.

Table 3

Glossary: eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), arachidonic acid (ARA), research and development (R&D).

3.3: Hybrid system

A hybrid system for microalgae cultivation involves the integration of different phases into a two-stage system (Płaczek et al., 2017). Usually, the initial growth takes place in a closed system to obtain high concentration cultures using a closed PBR. Then following larger-scale cultivations are performed using open ponds. This strategy is employed to benefit from the advantages of both systems (Rawat, Ranjith Kumar, Mutanda, & Bux, 2013), closed and open, by achieving a strong inoculum with minimum contamination and applying nutrient stress conditions toward the desired metabolites, respectively. These steps are not only applied to improve biomass production based on the advantages and avoiding the drawbacks of each system individually but also aim to understand and then manipulate the stages of cell growth. The first step usually implies in growing microalgae to reach a late exponential growth phase, and the second, where lipid accumulation occurs more expressively, for example, is conducted during the stationary phase, even though the culture will naturally have an adaptation and exponential phase when scaling up.

Liu, Chen, Wang, and Liu (2019) cultivated Scenedesmus dimorphus using an open pond–PBR hybrid system. They achieved a 46.3%–74.3% increase in biomass productivity using the hybrid system compared with the open pond and a 12.5% increase compared with the PBR. Similarly, Narala et al. (2016) indicated that a hybrid system could lead to significantly higher lipid production by combining exponential growth in an air-lift PBR followed by growth under nutrient depletion in open raceway ponds. Consequently, these systems cannot only be a solution to open the pond's lower biomass and lipid productions compared to closed systems, but also improve PBR's performance in some cases.

4: Industrial cultivation techniques

Increasing the scale of microalgae production from laboratory to industrial scales poses even more difficulties than pilot systems. Among the discussed cultivation techniques, open ponds lower costs and become more attractive. However, control of the environmental conditions and contamination is a greater challenge.

Although many research papers found in the literature are focused on the production of biodiesel from microalgal biomass, the existing sites for microalgae cultivation at an industrial scale aim the development of aquaculture and production of carotenoids, essential fatty acids and/or animal feed. Table 3 gathers details of specific commercial products from different species, being Haematococcus pluvialis, Spirulina, Chlorella, or marine microalgae cultivated in open ponds the most common. The data were collected from the company's websites, and the year is referred to as the date they were founded, not exclusively for algae products.

In general, any facility with enough sunlight incidence, cheap nutrient sources, and water availability are adequate for microalgae cultivation. However, the selection between open pods and PBRs should be based on the microalgae and target products as well as parameters other than solar radiation, temperature, and day length, such as wind and rainfall onsite (Schade & Meier, 2019). Also, CO2 bubbling on growth media that enables carbon credit and improves both biomass production and lipid accumulation is another important factor. Typical locations are coastal areas, and the majority of existing sites for microalgae cultivation are found in the United States.

Both new companies and existing ones aiming to expand their product catalog are investing in the production of microalgae. The recent alarming news of climate change effects encourages the market to provide clean energy sources with minimum impact on the environment. In that sense, microalgae are promising biomass regarding land use (Georgianna & Mayfield, 2012), CO2 biofixation, biomass productivity, water consumption, and many other aspects compared to terrestrial plants (Nakamura & Li-Beisson, 2016).

5: Dark fermentation—Fermenters

Dark fermentation is the process cultivation of some microalgae strains in the dark, heterotrophic environment.

5.1: Heterotrophic microalgae strains

The dinoflagellates, the green algae, and the thraustochytrids strains can grow under heterotrophic conditions. The easiest way to determine if microalgae are heterotrophy is to use the test with Biolog, Inc. to sell microtiter plates. In this test, there are 96-well plates with different organic carbon compounds that can be used to determine if and on which carbon source microalgae grow.

The genera suitable to heterotrophically grown are Amphora, Ankistrodesmus, Arthrospira, Chlamydomonas, Chlorella, Chlorococcum, Crypthecodinium, Cyclotella, Dunaliella, Euglena, Nannochloropsis, Nitzschia, Ochromonas, and Tetraselmis (Behrens, 2005). However, the heterotrophic Dunaliella strains or mutants were not commercially developed (Ben-Amotz, 2007).

Most microalgae culture collection strains cannot grow under heterotrophic conditions since their maintenance is in the phototrophic regime. Therefore, the isolation of strains from organic material sources is a better alternative.

5.2: Heterotrophic cultivation

The custom heterotrophic cultivation conditions are media with glucose or acetate, nitrogen and phosphorus compounds, no light, agitation (200–480 rpm), pH (6.1–6.5), with or without oxygen (Chew et al., 2018). Besides glucose and acetate, fructose, sucrose, galactose, glycerol, and mannose can be a carbon source for microalgae fermentation, too (Liang, Sarkany, & Cui, 2009). As a source of nitrogen, ammonia and nitrate are most common. The agitation of heterotrophic cultures is necessary for uniform nutrient and oxygen distribution. Oxygen is involved in the reproduction and growth of heterotrophic algae. The limiting factor for most microalgal heterotrophic cultures growth is oxygen, which makes increasing operating cost. It is due to the low solubility of oxygen in aqueous media. After a strong algae colony is formed, heterotrophic algae may ferment under anaerobic conditions.

Recently, scientists investigate cheap organic carbon sources. The agricultural waste, cattle slurry, food waste, whey permeate are some of the studied cultivation media. The solid and liquid wastes from the treatment plants and industry are very common nowadays (Table 4). These substrates are rich in carbon, nitrogen, and phosphorus, so they supplement the wastewaters for better growth of microalgae (Gladue & Maxey, 1994; Guldhe et al., 2017). However, these waste substrates are rich in various contaminants and need purifying pretreatment. Fungi and bacteria can contaminate media, too. Thus, sterilization is essential. Marine microalgae must be capable of growth in low-salinity medium. Namely, a combination of high chloride levels and the high temperatures during cultivation and sterilization respectively will result in corrosion of stainless-steel vessels (Harel & Place, 2007).

Table 4

a Synthetic medium supplemented with glucose.

High-energy density in the organic carbon source provides high cell densities (> 100 g L− 1) in heterotrophic cultures of microalgae (Barclay, Kirk, & Dong, 2013). However, the maximum specific growth of heterotrophic cultures is generally lower (i.e., from 0.008 to 0.098 h− 1 for Spirulina platensis and Chlorella vulgaris, respectively) than that in phototrophic cultivating regime (Lee, 2007). These lower growth values are the consequence of low affinity for organic components. Excess organic compounds concentrations inhibit cell growth instead of increasing maximum specific growth (Chew et al., 2018). The only exception is Chlorella vulgaris, whose photosynthetic and heterotrophic maximum specific growth rates are comparable.

Algae can successfully synthesize several products. The lipids (lipids in general and docosahexaenoic—DHA), pigments, and carotenoids are the most common heterotrophic microalgae aerobic products, whereas hydrogen, alcohols, acetate, lactate, and succinate are linked to anaerobic heterotrophic microalgae production.

Owing to the absence of light, the pigmentation on the algae is eliminated. Thus, lipid productivities increase and light-induced metabolites decrease (Chew et al., 2018). Even higher lipid productivities rates are associated with low levels of nitrogen or silicate (in diatoms) (Perez-Garcia, Escalante, de Bashan, & Bashan, 2011). A type of sugar determines the accumulation of specific types of lipids. Low temperatures favor the synthesis of polyunsaturated fatty acids. The carbon-limited but nitrogen-sufficient heterotrophic cultures of Galdieria sulphurariam and Spirulina platensis (Sloth, Wiebe, & Eriksen, 2006) accumulate a high concentration of pigments (C-phycocyanin). On the other hand, Chlorella pyrenoidosa, Chlorella protothecoides, Chlorella zofingiensis, Haematococcus pluvialis, and Dunaliella sp. under nitrogen-limited and at very high C/N ratios synthesize carotenoid—astaxanthin, while with glucose and urea in medium synthesize carotenoid—lutein.

Under anaerobic conditions in the dark, Chlorella, Scenedesmus, and Chlamydomonas produce formate, acetate, ethanol, glycerol with small amounts of H2, and CO2, from carbohydrates (mainly starch) from cells biomass Ethanol and hydrogen, are most common anaerobic products of microalgae heterotrophic cultivation. Microalgae such as Chlamydomonas reinhardtii synthesize ethanol (66.7 mg g− 1 biomass) due to the high-starch content via the dark fermentation process (Hirano, Ueda, Hirayama, & Ogushi, 1997). While in sulfur-deprived medium, the same strain can produce sustained quantities of H2 (Kosourov, Seibert, & Ghirardi, 2003). In, acid-rich effluents, such as food wastes and wastewater, Chlorella sp. showed the dominant H2 producing comparing to consortia of Scenedesmus sp. and Diatoms under starvation (Chandra & Venkata, 2011).

5.3: Fermenters

Fermenters or stirred-tank bioreactors are process vessels used to cultivate heterotrophic microalgae. Strictly speaking, eukaryotic microalgae grow in a bioreactor under lower mixing intensity, and prokaryotic microalgae grow in a fermenter. However, manufacturers and scientists still refer to both as fermenters. Laboratory-scale fermenters are mainly batch operation mode. This mode reduced growth and productivities, and so feed-batch or continuous mode is more implied. Where product formation is strictly associated with rapid biomass growth, the pulsed fed-batch strategy is applicable (Venkata Mohan, Rohit, Chiranjeevi, Chandra, & Navaneeth, 2015). The continuous fermenter modes are chemostat (Fig. 3) and perfusion culture systems (Fig. 4).

Fig. 3 Chemostat.

Fig. 4 (A) Perfusion and (B) perfusion-bleeding culture systems.

Chemostat is a static fermenter, which could maintain high-density algal cells. While fermenter is stocked with cells, tubes continually add nutrients and oxygen and remove the spend medium and CO2. Therefore, intracellular molecules and ions concentrations, as well as cell populations, are maintained constant over an indefinite period. Chemostat can provide 100 times higher productivity comparing to the batch mode. For example, the highest obtained cell concentration of C. reinhardtii was 1.5 g L− 1 using heterotrophic chemostat culture (Chen & Johns, 1996a).

Perfusion culture systems (Chen & Johns, 1995) are applied when inhibitory metabolites need to be removed. Perfusion retains cells inside the fermenter while fresh media and waste and spend media are continuously provided and removed, respectively. Hollow fiber filtration or gravity settler can be used to achieve perfusion. However, clogging of the membrane is frequent and reduces cultivation efficiency. Therefore, the strategy of bleeding the cells, where the spend medium was removed while algae cells were continuously harvested, allowed higher biomass and product productivity than the single perfusion culture. For example, the highest productivity (175 mg L− 1 d− 1) of eicosapentaenoic acid (EPA) in microalgal cultures was reported for diatom Nitzschia laevis cultivated in perfusion–cell bleeding culture system (Wen & Chen, 2001).

Fermenters maintain a controlled environment to maximize productivity in strictly aseptic conditions. Owing to repeated sterilization, noncorrosive and nontoxic material must be used in fermenters design. Laboratory (research or bench-top, 1–50 L) fermenters can be of borosilicate glass while pilot-plant vessels (50–1000 L) are of stainless steel. Regardless of fermenter type, (1) the culture vessel, (2) input–output systems ports for nutrient media, gas, and product, and (3) control systems are common to all. Mixing is crucial for fermentation since it causes oxygen dispersion and homogenization of media. Also, during heterotrophic cultivation, high biomass levels can be expected, so proper mixing is critical (Barclay et al., 2013). Airlift, bladed turbine, and impellers are generally used mixing systems for laboratory, pilot plant- and industrial scale, respectively. For the design and construction of fermenters, sterilization must be included too.

Finally, fermenters are geometrically designed as packed-bed fermenters, bubble-column type, air-lift fermenter, and tank fermenters (Figs. 5–8). The fed-batch, chemostat, and membrane cell recycle systems were specially studied to overcome growth inhibition of low organic substrates concentrations (Chen, 1996; Hochfeld, 2006; Liu, Sun, & Chen, 2014). Very few processes for heterotrophic microalgal cultivation use membrane cell recycle systems to overcome the inhibitory effect products (Chen & Johns, 1995, 1996b).

Fig. 5 Packed-bed fermenter.

Fig. 6 (A) Bubble column, (B) stirred bubble column, and (C) bubble column with external loop.

Fig. 7 Air-lift fermenter.

Fig. 8 (A) Tank fermenter pressure cycle with external loop, (B) stirred tank with internal loop, and (C) tank fermenters pressure cycle with internal loop.

Packed-bed fermenters are systems where microalgae are immobilized in beds (usually gel beds) to increase the productivity of biomass and products. Frequently immobilized genera of microalgae were Chlorella and Scenedesmus (Lebeau, Robert, & Subba Rao, 2006), mainly for wastewater treatment. A co-immobilized cell system can also improve the productivity of microalgae. For example, heterotrophic cultivation of Chlorella sp. co-immobilized with bacteria Azospirillum brasilense provides higher biomass productivity and higher total starch content per culture comparing to packed-bed fermenters with Chlorella sp. immobilized alone (Choix, de Bashan, & Bashan, 2012). The bacterial presence significantly prolonged the production of starch in this immobilized heterotrophic system.

Bubble-column type is aerobically fermenters because bubbles come from oxygen. The fermenter is a long tube with a sparging device at the bottom, which creates rising bubbles to mix algae content. During cultivation, cells grow and increase weight, settle out, and therefore, can be easily separated from the fermenter. However, bubbles cannot provide enough mixing across the whole length of the tube. Only lower tube parts maintain high densities algal cells since bubbles cannot pass faraway. Fermenting is fast and cells do not have enough time to produce desirable products. To overcome this problem, agitators or external pumps can be used in bubble column (Fig. 6B and C). Bioremediation with heterotrophic Phormidium sp. was performed in a bubble column in the secondary treatment of cattle-slaughterhouse wastewater (Kalk & Langlykke, 1986).

Air-lift fermenter (Fig. 7) has a similar mechanism to a bubble column. Air from the bottom push the media and creates circulation differences in cell density over the unit. As well as the bubble column, the air-lift fermenter is not suitable for algae with both a high and low specific gravity. Algae with a high and low specific density tend to settle down and foam, respectively.

The tank fermenter pressure-cycle fermenter with an external loop is an air-lift unit (Fig. 8A). It is equipped with an external loop that provides heat removal for thermosensitive algal strains. Air is pumped from the bottom, and mixing can be enhanced by installing a mechanical mixer (Fig. 8B) or an external pump (Fig. 8C). Among these three modes of tank fermenter designs, stirred tank with the internal loop are most frequently utilized, since most of the commercially available fermenters are this construction type. The high-lipid content (55.2%) was reached from a microalga C. protothecoids (15 g L− 1) through this type of cultivation technology (Xu et al., 2006). The highest phycocyanin production rate of 861 mg L− 1 d− 1 was obtained in the continuous stirred tank with the internal loop flow culture of Galdieria sulphuraria 074G-G1 (110 g L− 1) with glucose as substrate (Graverholt & Eriksen,