Introduction

In applied biology, the term ‘microalgae’ usually refers to prokaryotic cyanobacteria and eukaryotic microalgae [1]. These organisms are widespread and can be found in almost all ecosystems, from extremely cold polar regions to dry deserts [2]. Photosynthetic microalgae provided the Earth with the initial oxygen supply, creating an environment conducive for the evolution of various forms of aerobic life over time. Furthermore, microalgae are important CO2 consumers and major producers because of which they have attracted attention in recent decades as one of the most effective converters of solar energy into biomass.

From natural resources to artificial culture

Microalgal biomass has been used since antiquity (Fig. 1). Initially, it was used to cope with food shortage. For centuries, natural biomass of the blue–green alga, Arthrospira, was harvested in certain environments of alkaline soda lakes in countries, such as Chad or Mexico, and was used as a food supplement [2]. During the rule of the ** Dynasty in China (about 1500 years ago), another cyanobacterium, Nostoc sphaeroides (known as Ge-** functional food material in the 1960s [10]. Some progresses have also been made in industrial-scale processes for heterotrophic cultivation of microalgae. For example, in the late 1970s, Chlorella producers in Japan and Taiwan attempted to supplement acetate or glucose as carbon and energy sources to heterotrophically cultivate Chlorella in stainless steel tanks [11, 12]. Notably, in the 1970s, another microalga, Arthrospira, was used in large-scale outdoor cultivation near the alkaline soda lakes in Mexico, and production reached 1000 kg day−1 in 1974 [13]. In the 1980s, large-scale cultivation of Dunaliella was established in the USA and Australia to produce β-carotene [14]. By the end of the twentieth century, thanks to the progress in large-scale cultivation processes, several microalgal species were in commercial production or were at the pilot stage, and microalgae cultivation became popular worldwide.

Biotechnological contributions to biomass production

In the twenty-first century, the global demand for microalgae is dominated by food, health products and feed [15,16,17,18]. However, these demands require further increase in biomass production and strict control of product quality, as well as the development of production strains. Various techniques have been investigated to achieve the above objectives. For example, the ultrahigh‐cell‐density heterotrophic cultivation technique was developed for two Chlorella species and Scenedesmus acuminatus, which provided an important technical foundation for promoting its industrial application as an alternative high-quality protein source of food and feed [19,20,8]. Currently, the photoautotrophic growth mode is still the most commonly used technique used in microalgal industries, which contributes to the vast majority of global biomass. The advantages and disadvantages of different microalgal cultivation systems are summarized in Table 1.

Table 1 Comprehensive comparison of common microalgal biomass production systems
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Open ponds are the oldest microalgal cultivation systems. Typically, natural or artificial ponds, raceways, and circular ponds represent open pond systems for microalgae, where algae are cultivated under conditions identical to the external environment [2, 11]. Natural or artificial ponds represent extensive open systems and usually comprise a large pond without special modifications, i.e., stirring and CO2 addition. This system has minimal construction and operation costs, although maintaining of monocultures and controlling of environmental parameters is difficult. In addition, lower cell density means lower productivity and increase in the cost of harvesting. For example, artificial shallow ponds (2000–5000 m2) used for Dunaliella cultivation in Western Australia can only produce 1 g dry weight m−2 day−1 [2]. Intensive open pond systems on a commercial scale mainly contain raceways and circular ponds. An intensive open pond is smaller than natural or artificial ponds. In this system, some facilities for improving growth conditions of microalgae, such as stirring and CO2 supplement devices, are installed [11]. Therefore, sufficient CO2 supplement can be provided for microalgal photosynthesis, and stirring enhances the light utilization efficiency of cells. The world's earliest specially modified raceway pond was designed by workers in Germany in the 1950s for evaluating the possibility of biological utilization of CO2 from waste gases [9]. This open-air plant consisted of four culture trenches with a fall of 6 mm m−1, each 9 m long and 70 cm wide [9]. These trenches were rammed down in loam and were lined with plastic. In addition, devices including pump, centrifuge, collecting vessel, and gas pipeline were installed to control the growth parameters and harvesting. Today’s commercially available raceways largely follow or are improvements upon this design. The circular pond, the most common open system for mass production, was first developed and used in Japan in the 1960s. This system mainly included a rotating arm for mixing and a circular pond with a maximum diameter of 50 m. The design of the open circular pond limits the size to about 10,000 m2, because relatively even mixing by the rotating arm is no longer possible in larger ponds. Notably, most of the culture ponds for Chlorella cultivation used in Japan are circular in shape and up to 50 m in diameter. However, the cultures in these intensive open systems are usually grown at biomass concentrations in the range of 0.5 to 1 g L−1, and the light utilization efficiency of cells is the main limiting factor, which depended on the culture depth and stirring.

Circulation cascades

Circulation cascades (i.e., inclined-surface systems) were considered as high cell-density open culture systems for microalgae. The first experimental circulation cascade was designed by Dr. Ivan Šetlík and was built at the Botanical Garden of the Slovak Academy of Sciences in the late 1950s [33]. The system was constructed as stepped arrangement shallow troughs made of reinforced polyester resin. Circulation cascades have several advantages; in particular, microalgal cultures can flow over slo** planes arranged in inclined surface, which allows the culture depth to be controlled at a low level (usually below 1 cm), while the turbulence generated by the device also prevents self-shading of cells. Therefore, high productivities can be achieved easily in this open system [34]. Recent studies using this system for culturing Chlorella sp. MUR 268 and Scenedesmus obliquus have achieved productivity in excess of 20 g dry weight m−2 day−1 [35, 36]. However, although circulation cascades are transportable with long working life, higher construction costs limit complete scale up of this system.

Challenges of open pond systems

A major challenge of open ponds is the sensitivity to pollutants. Thus, only few species can be cultivated in these ponds for biomass production on a commercial scale, such as Arthrospira, Chlorella, and Dunaliella. The main characteristic of these species is that they can only grow in specific environments, which is hostile to most other competitors. For example, D. salina grows in salty water (NaCl concentrations > 20% w/v) and A. platensis grows in highly alkaline environments (pH > 9.2). Other microalgae that can be grown in open ponds are rapidly growing dominant species, such as Chlorella and Scenedesmus. Some other contaminants, including heavy metals and microplastics, are also unacceptable for microalgae cultivation for food and food supplement purpose. Furthermore, massive water loss due to evaporation and low cell concentration and biomass productivity are also the intrinsic disadvantages of open ponds. Therefore, the future techniques for open ponds should address these bottlenecks while maintaining lower production costs. Notably, using open ponds for the production of valuable microalgal products is unlikely to be sustainable or economic, thus, attempts have been made to overcome some of their limitations using closed pond or enclosed photobioreactor systems [11].

Enclosed photosynthetic mass cultivation–photobioreactor systems

Typical photobioreactors

Photobioreactors represent sophisticated and flexible systems working either outdoors or indoors, in which a single species is inoculated to keep a clean culture operation. A photobioreactor is usually equipped with lighting, stirring, CO2 addition, and cooling facilities, and it can be better optimized according to the biological characteristics of the microalgal species cultivated. Compared to open systems, enclosed photobioreactors have several advantages, mainly including (i) larger surface-to-volume ratio, (ii) low CO2 losses, (iii) reduced risk of contamination, (iv) smaller area requirements, (v) ability to prevent evaporation, and (vi) higher cell productivities. So far, various photobioreactors consisting of glass or transparent plastic tubes, and columns or panels, have been designed using either natural or artificial lighting.

The most commonly used photobioreactors are vertical-column and tubular. The former is a relatively simple system, in which stirring is achieved by air or high concentration of CO2 bubbling up from the bottom. Vertical-column reactors described by Cook in 1950 were the first real enclosed systems for microalgae culture [37]. In the 1980s, algae workers evaluated two vertical-column reactors and found that maximum productivity of 20–26 g dry weight m−2 day−1 for Chlorella and Nannochloropsis could be achieved in a vertical air-lift photobioreactors [38], while 23 g m−2 day−1 could be obtained in a vertical glass tube for Monoraphidium [39]. Despite the very gentle stirring and good light penetration of this reactor, its potential for scale-up appeared difficult. In fact, vertical-column reactors are commonly used in a seed culture in microalgal factories. The reactors used commercially for biomass production are tubular. Since the pioneering work of Tamiya et al. [40], several tubular reactors have been studied and developed. In general, the tubes are made of glass, plastic, or acrylic as the solar receptor and are arranged as a serpentine loop or as manifold rows. Recirculation of the culture suspension and removal of O2 are achieved using a pump (mainly using centrifugal or peristaltic pumps) or an air-lift (injecting a stream of compressed air into an upward-pointing tube) [40]. The cell growth temperature is regulated by a heat exchanger, or by spraying water onto the surface of the photobioreactor. In addition, tubular photobioreactors for commercial production are usually of modular design, which allows easy installation in any open space; for example, the culture systems developed at Batelle in 1980s for the production of polysaccharides from Porphyridium cruentum [41]. The two‐plane tubular photobioreactor is another type of tubular reactor first developed by Torzillo et al. [42] in Florence (Italy) for outdoor culture of Arthrospira, which led to a high biomass productivity of 30 g dry weight m−2 day−1. In particular, the largest known microalgae plant using tubular reactors has been established in Pataias (Portugal), with a total-volume of 1300 m3 and occupying one hectare of land, operated by A4F-AlgaFuel at the Secil Cement Company, for producing food-grade C. vulgaris and Nannochloropsis [43].

New photobioreactor designs

More recently, several new photobioreactor designs have been reported. For example, Carone et al. [44] designed an alveolar flat panel photobioreactor; this reactor enhanced the CO2 bio-fixation rates using 1.3 cm thick alveolar flat-panels as light receptor. Gifuni et al. [45] developed an ultra-thin (3 mm) flat photobioreactor for increasing both biomass concentration and productivity, and maximum biomass productivity of 1.34 g m−2 h−1 was obtained with C. sorokiniana. Furthermore, a hybrid photobioreactor consisting of a bubble column reactor coupled to an illumination platform has recently been reported [46]. The reactor presented higher hydrodynamic performance (mixing time of 98 s), and biomass yield of 2.8 g L−1 was achieved in the reactor with S. obliquus CPCC05 [46]. However, scaling up of these systems may be difficult because of their complexity and potentially high cost.

Challenges of enclosed photobioreactors

Although higher biomass density can be maintained, the construction and maintenance costs of photobioreactors are ten times higher than those of open ponds, making them uncompetitive for the industrial production of microalgal biomass. Thus, photobioreactors can be used commercially to produce high-value bioactive substances, such as obtaining astaxanthin from H. pluvialis; it is foreseeable that these enclosed bioreactors will be continuously used to produce high-value products from microalgae in the future under aseptic conditions. Furthermore, there are several other problems, such as the accumulation of high concentration of O2 in the cultures and difficulties in cleaning. Until these problems are solved, the commercial application of enclosed reactors for microalgae will be challenging.

High cell-density heterotrophic cultivation-fermenters

Characteristics of heterotrophic cultivation

Some microalgal species can grow in the dark or under light limitation, using organic carbon (e.g., acetate or glucose) as their sole carbon and energy source, a process known as heterotrophy. Heterotrophic cultivation in fermenters may result in higher cell productivity than that in open ponds and photobioreactors, as this growth mode eliminates the requirement for light. Therefore, this process may provide a cost-effective and large-scale alternative strategy for microalgal biomass production. Fermenters and photobioreactors represent enclosed systems that share many common features, such as pH and temperature control, and the progress in stirring and harvesting. The main differences between fermenters and photobioreactors include their energy source, oxygen supply, and sterility, which may lead to differences in the final biomass production of these two systems. Notably, high cell concentrations also mean the lower downstream process costs. Hence, the focus is now on heterotrophic cultivation of microalgae.

Development of heterotrophic cultivation

The key to heterotrophic production is that microalgal cultures must be axenic. This issue can be well-solved by drawing on proven techniques in microbial fermentation, for example, sterilization of fermenters and medium can be achieved using steam. Early attempts have been made to develop industrial production processes for microalgal heterotrophic cultivation. For example, studies on heterotrophic production of microalgae began in Japan in the late 1970s for Chlorella, and this process was applied to the industrial production of Chlorella in the mid-1990s. Subsequently, Cell Systems (Cambridge, UK) developed a process for the heterotrophic cultivation of T. suecica in 5000 L scale fermenters [47]. In addition to these heterotrophic batch processes, an industrial heterotrophic cultivation process for docosahexaenoic acid (DHA) production by Cryptheconidium cohnii was set up at Martek Biosciences in 1990s (Columbia, USA) [11].

During the last two decades, heterotrophic cultivation of microalgae has attracted attention. On one hand, high productivity of heterotrophic production has attracted the interest of microalgae enterprises. This process has been used to produce high-value products. For example, DSM (Heerlen, Netherlands) has commercialized the production of DHA and microalgal oil rich in DHA from two heterotrophic microalga, Schizochytrium sp. and C. cohnii, respectively, using a two-stage fed-batch process [48]. Duplaco (Oldenzaal, Netherlands) produces Chlorella for human nutrition using a proprietary process in which the microalgae are ‘fed’ with carbon source and grown in sterile fermenters; this process is expected to be expanded in the future to produce 1500 tons year−1 of Chlorella biomass. On the other hand, the heterotrophic cultivation processes are also being optimized constantly. Studies have reported increase in the yields of biomass and their by-products in E. gracilis and by C. vulgaris by optimizing complex medium composition and culture conditions, respectively [49, Flocculation

Flocculation represents a low-cost harvesting method; this process increases the particle size, reducing the energy requirement in dewatering process [61, 62]. Flocculation was first used in wastewater treatment and was investigated for microalgae harvesting using chitosan as the flocculant in the 1980s [63, 64]. It has been recognized as an excellent technique for harvesting microalgae, as it can be used on a large scale for various microalgal species [65]. Currently, three main processes have been extensively studied: chemical flocculation, physical flocculation (electro-flocculation) and bio-flocculation. Chemical flocculation, the most common method, usually uses cationic flocculants, such as metal salts (Al3+, Fe3+, Ca2+, and Mg2+) and macromolecule polymers (chitosan, polyacrylamide, and polyethyleneimine). The process causes aggregation of algal cells due to neutralization or reduction of the negative charge on the surface of the microalgae and/or due to the formation of bridging bonds. A study has shown that cationic polyelectrolytes are more effective than metal salts, with the ability to induce up to 35 times biomass concentrations [66]. Although economical, the chemicals used for flocculation can be hazardous and may contaminate the algal biomass. In particular, metal salts remain in the biomass residue after the lipids or carotenoids have been extracted. These metals may interfere with the use of the protein fraction of this residue as animal feed. Electro-flocculation is also used for algae harvesting. The technique uses an applied electric field to disrupt the electrostatic balance of individual microalgae, causing the algal cells to aggregate. Although the removal efficiency is high (80–95%), the process requires electrode replacement and maintenance, and metal residues may be present in the recovered biomass. Bio-flocculation is a promising method, as it does not require chemicals or specific culture conditions [59]. Bio-flocculation is assumed to be caused by extracellular polymeric substances (EPS) in the medium [67]. EPS can be produced by bacteria, microalgae, and fungi [59]. Therefore, flocculating species can be supplied to the algal growth medium to harvest microalgae. However, the mechanism underlying bio-flocculation is poorly understood. Some studies have suggested that bio-flocculation may be triggered by info-chemicals [68, 69]. The use of bacteria or fungi as flocculants is the principal drawback of bio-flocculation, as it leads to microbial contamination. This may also limit the use of microalgal biomass for food or feed applications. Thus, this technique is often used for wastewater treatment [70, 71].

Other techniques

Other techniques, such as flotation and gravity sedimentation, have also been developed for microalgae harvesting. Flotation involves introduction of air bubbles to transport the suspended matter to the top of the liquid surface, where it can be collected using a skimming process [59]. The technique is more effective than gravity sedimentation, especially for cultures with low density and self-floating properties. The main advantages are short operation time, low space requirement, and low initial equipment cost. The process is usually used after the flocculation process. However, the surfactants used for flotation may be toxic. Gravity sedimentation is a simple and inexpensive process, but disadvantages such as low efficiency and time-consuming limit its use in microalgae harvesting.

Harvesting strategies for bulk biomass production

Harvesting strategies based on the final application

Currently, good harvesting methods are lacking, as the major drawbacks of each harvesting technique prevent them from being applied on a large scale in a non-toxic, cost-effective, or energy-efficient manner at the same time. Similarly, no single method or combination of harvesting methods appears to be suitable for all species. Nevertheless, thorough comparative analysis is required to determine the most appropriate harvesting method. These analyses should be based on some of the most critical factors in harvesting technique, such as recovery efficiency, concentration factor, biomass quantity and quality, cost, processing time, toxicity, and suitability for large-scale application [72, 73]. Considering that microalgae can be used in various applications and that different applications focus on different key criteria, the application of each microalgae should be analyzed specifically, i.e., the described parameters should be prioritized in a different order depending on the final application of the microalgal biomass [73].

A possible approach that may be followed to determine the most appropriate method for each microalga should include the following: (i) the final application of the biomass recovered from microalgae should be clarified; (ii) the most important criteria should be considered for each application; (iii) the most satisfactory harvesting method for each criterion should be considered [59, 73]. Several studies have conducted similar analyses [59, 72, 73]. In these studies, six important criteria, including biomass quantity, biomass quality, cost, processing time, toxicity, and suitability for large-scale application, were used to assess the applicability of harvesting techniques to the main or potential applications of microalgal biomass. These applications include the production of human food, animal feed, high-value products, water quality restoration and biofuels. The most appropriate harvesting technology for each biomass application is selected by establishing a prioritized list of criteria for that application. Figure 3 summarizes the evaluation for harvesting techniques considering each criterion based on the main advantages and disadvantages of each harvesting method described in this study. The current demand for microalgal biomass is mainly for health food. In this regard, biomass quality, toxicity, and suitability for large-scale application are considered the key factors. This is also applicable for the production of animal feed. For high-value products, toxicity, biomass quality, and quantity are even more important. Algal biomass can be potentially used for making biofuels, and considering the current demand for low-cost biofuels, biomass quantity, cost and processing time are considered to be the most important criteria for biofuel production. Briefly, for industrial-scale production of microalgal biomass, flocculation, filtration, and centrifugation are the main options for harvesting. Centrifugation is the most suitable option for the production of high-value compounds due to its advantages in terms of biological quality, processing time, and suitability for large scale applications. In terms of biomass quality, filtration is considered to be the most suitable method for harvesting for human food and animal feed. Considering the cost requirements, flocculation appears to be the best option for wastewater treatment and biofuel production.

Fig. 3
figure 3

Comprehensive evaluation of optimal harvesting techniques for different applications. a Order of suitability of harvesting techniques for various criterions; b order of the most important criterions should be considered for various applications; c order for suitability of harvesting techniques for various applications

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Two-step harvesting process

In many cases, the use of a combination of two or more harvesting methods can lead to further improvements in harvesting efficiency, production cost, and processing time [74]. A typical combination of harvesting techniques includes a pre-concentration/concentration step, followed by dewatering. In a two-step process, the microalgal suspension from the culture system is first concentrated to an algal slurry with 2–7% total suspended solids; then, in a second step, the slurry is dewatered to 15–25% TSS [59, 75]. The processes used for the first step of concentration include flocculation, sedimentation, flotation and electro-assisted technique. Centrifugation and filtration are usually used for the final dewatering process. This step is more expensive as it requires a higher energy input than the thickening process. Several studies have reported the advantages of using different combinations of harvesting methods. For example, Hapońska et al. [76] evaluated the application of pH-induced sedimentation combined with dynamic filtration for microalgal dewatering at a pilot scale. High concentration factors of 207.4 for D. tertiolecta and 245.3 for C. sorokiana were achieved using a combination of these two techniques. More recently, Min et al. [75] used the resonance vibration submerged membrane system as a pre-treatment process prior to centrifugation for concentrating C. vulgaris; the system was evaluated and was found to be less energy intensive than conventional systems. Thus, these studies proved the potential benefits of using multiple methods for microalgae harvesting in terms of recovery efficiency, processing time, and process economics.

Microalgae drying: balancing cost and quality

Drying techniques of commercial microalgae

Drying is usually as the last harvesting step. This process requires the removal of moisture to ≤ 12% to obtain dry microalgal biomass for downstream product production. The dried microalgae are easy to store and transport, as well as to use in bio-refinery and in the food and feed industry. Drying also represents a significant fraction of the total production costs. Since the mass cultivation of microalgae, several drying techniques have been developed. The commercial techniques mainly include (i) solar drying, (ii) convective drying, (iii) spray drying, and (iv) freeze drying. The different processes have their own distinctive features. The selection of drying method is critical for the subsequent processing and quality of the final products.

Solar drying

Solar drying, the most traditional and cost-effective method for microalgal powder production, has been used for hundreds of years to stabilize the moist algal biomass. In some open processes, the heat for water evaporation is provided by solar radiation and moisture removal by natural air currents. This may be time-consuming, and a large drying surface and the efficiency of the process is directly dependent on the weather conditions. Moreover, longer processing times and exposure to open environments may increase the risk of spoilage or development of off-flavors. Several strategies and facilities have been developed to address these issues. Some closed solar dryers can reduce the moisture content of the final product to less than 10% within 5 h of drying and remain low energy and exergy efficient [77, 78]. These dryers usually consist of a solar heater, a drying chamber, and an airflow system. Although the process can further improve the quality of the algal powder, research on this is negligibhas focused on solar dryers for microalgae.

Convective drying

Convective drying is popularly used for drying microalgae. It is performed in a type of convective hot air dryer and is commonly used in small-scale production. It usually includes draft oven drying, convective tray drying, microwave oven drying, convective tunnel drying, and continuous conveyor belt drying [79,80,81,82,83]. Several studies have evaluated the potential for large-scale application of these processes. For example, Chen et al. [84] assessed the effect of heating rate on the pyrolysis of C. vulgaris and measured energy consumption using microwave drying. A study indicated the strong influence of process temperature on chlorophyll a content and hue angle (relative to sample color) under the same conditions of convective drying, with a sharp reduction in chlorophyll concentration at drying temperatures up to 40 °C [85]. This fact was also confirmed by Oliveira et al. [86] who evaluated the effect of drying temperature on the functional components of Arthrospira; results showed that convection drying temperatures above 45 °C could cause phycocyanin degradation. Therefore, the optimization of convective drying conditions is important for pilot-scale applications. Moreover, studies are required to minimize energy consumption.

Spray drying

On a commercial scale, spray drying is the most commonly applied method. This technique was first proposed in the early 1950s for the production of microalgal powder [8]. Spray drying involves the atomization of the algal slurry to produce droplets, which are dried into individual particles while moving through the hot air. Although the algal slurry is exposed to higher temperatures in a shorter period of time, the drying of single droplets provides a large surface area per unit volume of liquid, which facilitates rapid drying and reduces degradation of product quality. Therefore, this process is the preferred method for drying high value microalgae products [129]. Therefore, for complete removal of macrobiotic contaminants, the microalgal solution should be filtered continuously for 3–4 days. In addition, the resistance of different algal strains to zooplanktons varies. Some algal populations have successfully resisted grazing pressure, such as Chlorella spp. and Tetreselmis spp. [140]. A potential strategy involves using high resistance strains for open cultivation wherever possible. In contrast, algal strains with weak or no resistance to grazing are cultivated using enclosed systems or alternative species. Considering that the higher cost of filtration only allows its use on a small scale, the above strategy appears to be more feasible.

Chemical control

Chemical control is a potentially viable method for eliminating biological contaminants. Many studies have reported chemical treatments for controlling biological contamination in open microalgal cultures. For example, Moreno-Garrido and Cañavate [141] reported that 10 mg L−1 quinine was effective in killing ciliates, with less damage to D. salina cells. In addition, the use of ammonium bicarbonate in culture can control rotifers and cladocerans, and can also provide an additional source of nitrogen and carbon at low cost [142]. However, the effectiveness of ammonium bicarbonate in controlling zooplankton contamination is significantly reduced at high temperatures as the ammonia evaporates [143]. An effective, safe, and low-cost method of controlling biological contamination is required. Botanical pesticides have been proposed as a potential control agent for zooplanktons in microalgal mass cultivation [144]. Various botanical pesticides, such as celangulin, matrine, azadirachtin, and toosendanin, are all being considered for use as biological control agents in microalgal mass cultivation [144]. For example, toosendanin has been shown to be effective for rotifer and ciliate contamination control [143, 144]. Considering its relative safety toward microalgal cells and low cost, the use of toosendanin for zooplankton control in microalgal mass culture appears promising. The use of chemical agents must be considered in terms of their impact on the final application of microalgal biomass, especially when it is used as animal and human food. In summary, the development of biopharmaceuticals that do not produce chemical residues without damaging the target microalgae may be more promising.

Changes in environmental conditions

Changes in certain environmental conditions, such as temperature and pH, can also be used to control biological contamination. This strategy depends on the survival conditions of the target microalgae and/or contaminants. Several studies have confirmed the effectiveness of this approach. For example, Hallegraeff et al. [145] confirmed that Gymnodinium catenatum and Alexandrium catenella can be easily killed using temperatures as low as 35 °C and 38 °C, respectively. This has potential application in the treatment of input water for algal cultivation. Adjustment of the pH of cultures is commonly used for killing or removing of biological contaminants [129, 146]. Becher [147] recommends lowering the pH to 3.0 for 1–2 h to control rotifers. Moreover, the amounts of contaminants in the biomass can also be limited by controlling the nutrient composition of the medium to maintain the target microalgal population. For example, harmful algal growth increases when diatom populations are starved by low silica levels; thus, managing silica levels is key to ensure that diatoms grow faster than other species [148].

Research needs for control of biological contamination

The control of biological contaminants is essential for the production of sufficient and high-quality microalgal biomass. However, a number of key issues still need to be addressed. For example, the application of control strategies also requires comprehensive understanding of the range of adaptation of target microalgae and biological contaminants to ecological factors. Bacterial contamination in open ponds is inevitable; hence, methods of reducing harmful bacteria and increasing beneficial bacteria (e.g., nitrogen fixing bacteria) is the focus of future research. Moreover, research on algal lysing viruses should be intensified, as current knowledge regarding algal viruses is limited. The other caveats regarding the control of algal toxins in biomass production include the lack of standards for acceptable levels of toxicity in algal biomass or compound feeds. In addition, the development of sensors for monitoring various biological contaminants is also necessary.

Biotechnologically gifted strains of microalgae

In microalgal biotechnology, suitable species can be grown as production strains in aquaculture. Although tens of thousands of microalgae exist in nature, only a few gifted strains are used for commercial biomass production. In particular, over the past decade, the bulk of annual biomass production is dominated by six species, namely, the cyanobacteria, Arthrospira and Nostoc (cultivated only in China), the green microalgae, Chlorella, Dunaliella, and Haematococcus, and the flagellate, Euglena. Table 5 lists the gifts, bottlenecks, and technologies for further improving biomass production of these species.

Table 5 Gifts, bottlenecks, and key technologies in biomass production of some important commercial microalgae
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Arthrospira

Arthrospira (Cyanophyta) is a multicellular filamentous cyanobacterium that grows naturally in subtropical alkaline lakes with an optimum temperature of approximately 35 °C. It represents the most successful commercially available microalga. In productive cultures, two species, A. platensis and A. maxima, were widely cultivated in open raceways or tubular photobioreactors. The first commercial production started in the 1970s in Mexico. Currently, Arthrospira is produced in over twenty countries. The mass cultivation of this cyanobacterium contributes to over 50% of the global microalgal production, with total annual production estimated at about 12,000 metric tons [25]. China, in particular, produces more than 60% of the world’s Arthrospira biomass, thanks to improvements in raceway ponds (Fig. 5a, b) and the breeding of low-temperature tolerant species. Recent statistics showed that 8327 metric tons of Arthrospira were produced in China in 2021, and 8828 metric tons are expected to be produced in 2022.

Fig. 5
figure 5

Examples of various biomass production systems for cultivation of commercial microalgae. a, b cultivation facilities (open raceways in greenhouse) of Arthrospira in Erdos (China); c circular ponds for Chlorella cultivation (Sun Chlorella, Japan); d heterotrophic culture facilities for Chlorella; e cultivation of D. salina at Cargill lakes in San Francisco Bay (USA); f D. salina cultivation using open raceway ponds by NBT Co., Ltd. (Eilat, Israel); g open raceways in greenhouse for H. pluvialis cultivation by Green-A (Yunnan, China); h tubular photobioreactors used for H. pluvialis cultivation by Algatech (Israel); i E. gracilis cultivation using circular ponds by Euglena Co., Ltd. (Japan); j a demonstration site for the indoor cultivation of N. sphaeroides in Plateau algal Research Center (China)

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Arthrospira biomass is mainly used as human food, animal feed, and source of certain chemicals. In addition, mass cultivation of this alga has also been attempted for sewage treatment. The challenges for the Arthrospira industry are monotonous application markets and unclear market positioning. In addition to the food and nutraceutical sectors, the Arthrospira industry needs to develop other new application markets to increase the resilience of the industry. Much remains to be elucidated about the pharmacological activities and mechanisms of action of Arthrospira, particularly in the areas of antioxidant and antitumor activities; therefore, the key to future medical research is to develop the technology for isolation and purification of antioxidant and antitumor active substances. The cost of biomass production and the quality of the product still do not meet the demands of the market, which needs to be addressed through technological innovation and improvement of standards. Reducing the loss of nutrients during processing or obtaining fresh Arthrospira as dietary supplement by combining with the rapidly develo** Internet of Things is another direction for the development and extension of this industry chain. Briefly, the various processes in the value chain of industrial production of Arthrospira all pose significant challenges.

Chlorella

Chlorella (Chlorophyta), a genus of unicellular green microalgae living in freshwater, seawater, and terrestrial habitats [2], was the first species to be used commercially for biomass production. Commercial species mainly include C. vulgaris and C. pyrenoidosa. Currently, it is usually cultivated phototrophically in open ponds, cascades, and enclosed tubulars, as its high growth rate prevents contamination by other microalgae. A circular pond is the most common device for commercial biomass production of Chlorella (Fig. 5c). It can also grow under mixotrophic and heterotrophic conditions with the addition of acetic acid and glucose (Fig. 5d). Countries and regions where commercial production has been achieved include Japan (companies, such as Sun Chlorella and Yaeyama), mainland China (King Dnarmsa and C.B.N Microalgae, etc.), China Taiwan (Chlorella Manufacturing and Far East Bio-Tech, etc.), Korea (Daesang), Germany (Algomed), and Portugal, with total annual biomass production of about 5000 metric tons [26].

The success of mass cultivation of this microalga photoautotrophically, heterotrophically, and mixotrophically has led to a stable Chlorella industry for human nutrition and animal feed due to its high nutrient content. In recent years, the mass cultivation of Chlorella has also shown its potential for applications, such as bioremediation, biofuel production, and as a raw material for biofertilizers. However, the current production systems and processes of Chlorella are neither cost-effective nor energy-efficient, rendering these potential applications impractical. In particular, the cell processing requires both effective and efficient harvesting and mechanical disruption of cellulose cell walls. Breakthroughs and innovations in the next generation of production technology are, therefore, urgently required.

Dunaliella

D. salina (Chlorophyta), a unicellular biflagellate green microalga, represents the most salt-tolerant eukaryotic organism. It is one of the most industrially important species of microalgae because of its extremely high β-carotene content, which accounts for up to 16% of the dry matter. The first outdoor pilot of this microalga was attempted in the USSR in 1966; however, mass cultivation on a commercial scale was first achieved in the USA and Australia in the 1980s. Currently, the large production plants are mainly established in Australia (companies, such as Western Biotechnology and Betatene) and Israel (Nature Beta Technologies). The biomass production systems mainly include open natural/artificial ponds (Fig. 5e) and raceway ponds (Fig. 5f). The mass culture of D. salina is mainly used for natural β-carotene production. A two-stage process has been developed in which the alga is first grown in nutrient-rich media for rapid biomass production and then transferred to nitrogen deficient media to stimulate β-carotene production. This process has been used in open raceway ponds, but is difficult to use in natural or artificial culture ponds. Today, the total annual production is estimated at 1200 metric tons of dry biomass [27].

Commercial D. salina is used in various forms. For example, the algal powder can be used for food and feed coloration, and β-carotene can be used for health care. Optimization of biomass production is the main bottleneck of the D. salina industry. In particular, existing outdoor cultivation techniques allow the density of this microalga in open ponds to reach only 8 × 105 cells mL−1. This should be addressed by develo** high-density cultivation techniques or by breeding high-yielding strains. Moreover, large-scale outdoor cultivation also means higher harvesting costs.

Haematococcus

H. pluvialis (Chlorophyta) is a freshwater unicellular green microalga. The high content of astaxanthin (up to 4% of the dry weight) makes H. pluvialis attractive to biotechnologists for large-scale production in raceway ponds (Fig. 5g) or enclosed photobioreactors (Fig. 5h) at around 25–28 °C. However, the cells in open ponds systems are susceptible to contamination by other microorganisms, such as algae, fungal parasites, and zooplankton predators. Thus, a two-stage process has been developed and used for biomass production. For the first stage, green zoospores are usually cultivated in enclosed tubulars to maximize cell density. Then, the cultures are exposed to high irradiance in open ponds under nutrient stress to induce astaxanthin synthesis. Currently, H. pluvialis is produced in only few countries: USA (companies, such as Cyanotech), Japan (Yamaha and Biogenic, etc.), Israel (Algatech), China (Alphy and Green-A, etc.), and India (Bioprex). The total annual worldwide commercial production is estimated to be at 800 metric tons [28].

H. pluvialis is the main producer of natural astaxanthin. This pigment can be used as an anti-oxidant for human nutrition or as a natural colorant for the aquaculture of salmonoid fish. However, the production of astaxanthin is still restricted to that of a few hundred kilos. In fact, the H. pluvialis industry is still in the early stages of industrialization. This is reflected in the small number of companies capable of large-scale production and the lack of derivative products. Further expansion of biomass production will depend on the development of superior strains and a significant increase in the cells’ resistance to environmental stresses, especially fungal diseases.

Euglena

Euglena (Euglenophyta) is the protist genus consisting of unicellular freshwater flagellates. These species can be grown photoautotrophically, heterotrophically, or photoheterotrophically, and have been studied extensively. In particular, E. gracilis has long been used as a model organism. This microalga has attracted the attention of cultivators as it is able to accumulate more than 50% of the dry weight as polysaccharides. The first outdoor pilot of E. gracilis was attempted in Japan in 2005, and the commercial cultivation was started in 2007 by Euglena Co., Ltd. (Japan) [24]. Circular ponds and raceway ponds are most common culture systems (Fig. 5i). A high cell density cultivation process, SHDP, has also been used for the mass production of biomass. Currently, only Japan and China produce E. gracilis commercially, with an annual production of about 70 metric tons [29, 30]. The biomass is mainly used for human nutrition. A variety of foods, drinks, and supplements containing Euglena have been developed as commercial products. However, the widespread cultivation and commercial application require further optimization of biomass production systems and methods.

Nostoc

N. sphaeroides (Cyanophyta) is an edible cyanobacterium with high nutritional value. Wild N. sphaeroides can grow naturally both in terrestrial and aquatic environments. It has been consumed as food in China (Ge-** algal industry. Therefore, the establishment and improvement of microalgae-related standards is essential. According to the current status of standardization in the microalgae industry, the construction of the standardization system should focus on the following aspects: (i) research and development of rapid detection technologies for algal active compounds should be intensified to provide microalgae producers with simple and reliable testing methods; (ii) Timely revision of relevant industry standards to meet the development needs of new species and products of microalgae. (iii) Continuous strengthening of the function of industry associations, highlighting the importance of group standards, and stimulating the vitality of market players; (iv) International cooperation should be strengthened to promote the development of international common standards.

Future directions and perspectives of microalgal biotechnology

  • Microalgal biotechnology is limited by a few algal strains available, which indirectly reduces the diversity of commercial products. Therefore, breeding techniques should be developed to screen for high-quality algal strains that can be used for mass production.

  • High cell-density culture techniques are key for reducing costs, although there is scarcity of available production facilities. Similarly, culture conditions also have to be optimized to further increase productivity.

  • Quality control is an important direction for the future development of biomass production technology, in particular, the need to avoid the loss of active ingredients from microalgae under inappropriate conditions.

  • The main obstacle impeding the control of biological contaminants is the lack of information regarding the biochemical pathways of contamination. The prevention of biological contaminants and development of control technologies that are both economically efficient and environmentally friendly are top priorities. The development of techniques for the detection of contaminants or toxicity factors in cultures and standards for determining the acceptable levels of toxicity in algal biomass are also necessary.

  • The functional and molecular mechanisms of action of the microalgal active ingredients are not completely understood, which to some extent, affects the market positioning of microalgae and development of new applications.

  • Innovative strategies are obligatory, which would help realize some potential applications of microalgae, such as genetic modification (directed evolution and rational design), to increase oil content and render microalgal biofuels commercially viable.

  • The untapped bioeconomic potential of microalgae should guide the exploration of the vast undiscovered possibilities of these biotechnologically important species, such as microalgal power generation.

Conclusions

This review systematically summarizes current biomass production technologies for commercial microalgae. We concluded that high cell-density cultivation process is important for producing biomass on commercial scale in the future, and that cost-effective processes and strategies are required for the development of microalgal harvesting. Moreover, microalgal drying should not only be cost-effective, but should also consider the quality of the product, while basic research into the control of biological contaminants should be strengthened. The article then briefly reviews the current status of commercial production of some biotechnologically important microalgae and highlights the importance of improving microalgal industry standards. In summary, it is clear that before the wider application of microalgal biomass can be achieved, significant investments in technology development and technical expertise will be required.