Microalgae as Multiproduct Cell Factories

Abstract

Microalgae are a highly diverse group of microorganisms that are currently produced at industrial scale at comparably high specific costs for various applications (e.g., food supplements and cosmetics). Although a multitude of high-value, market-relevant products, such as fucoxanthin and eicosapentaenoic acid (EPA), are present in the biomass, currently single-value chain products are produced and marketed, limiting profitability of microalgal biotechnology, as well as potential application. The chapter provides an overview on microalgae-based lead compounds and their bioactivities providing the basis for the multiproduct cell factory concept. Furthermore, a general overview of current downstream processing (DSP) methods is given that are currently used for microalgal biorefineries at industrial scale. The latest advancements in the research and development of multi-products are showcased, highlighting its role in facilitating the microalgal bioeconomy.

1. Introduction

Microalgae are predominantly photosynthetic and mainly single-celled microorganisms. Their metabolic complexity enables them to produce a diverse range of algae-specific compounds; thus, microalgae have emerged as promising multiproduct cell factories. Moreover, the composition, as well as the proportion of their biochemical composition, is highly variable and controllable by adjusting cultivation conditions. Therefore, microalgae are subject of increasing interest of various industrial sectors seeking solutions to prevail future global sustainability challenges. Among these, microalgae can contribute to food, feed, and high-value chemicals supply [1, 2, 3]. However, the establishment of a microalgae-based bioeconomy faces substantial challenges, first and foremost economic hurdles in both upstream (USP) and downstream processing (DSP) [4], as well as market entry barriers and consumer acceptance.

DSP plays a crucial role in the production process, involving several unit operations such as solid-liquid separation, milling, extraction, and chromatographic separation of the desired compound. In its complexity, it contributes substantially to the production costs and represents a major economic bottleneck for new microalgae-based products [5]. This limitation reasons that commercial exploitation of microalgae is currently restricted to a few single-value chained products (e.g., astaxanthin from H. pluvialis [6] and phycocyanin (PC) from A. platensis [7]) [8, 9, 10]. However, using microalgae as multiple product cell factories to produce several high- and medium-value components and utilizing the residual biomass for the production of low-value bulk commodities (e.g., food and feed additives, fertilizers) seems to be a promising way to generate sufficient value to render the overall bioprocessing economically viable. To achieve this goal, straightforward, optimized, and most importantly scalable unit operations have to be combined for DSP allowing microalgal biomass to be fractionated into their most valuable components, including n-3 and n-6 very-long-chain polyunsaturated fatty acids (VLC PUFAs), carotenoids, (phycobili) proteins and functional polysaccharides (e.g., β-glucans). While mechanically based solid liquid separations such as centrifugation for cell harvest and bead milling for disintegration do not represent big contributors to energy requirements and specific costs, extraction and adsorption-based chromatographic techniques are costly in terms of specific energy input and operational expenditures (OPEX). If cheaper solutions, for example, aqueous two-phase extractions (ATPE) and liquid-liquid chromatography for fractionation can be established and their robustness and efficiency can be shown; as a consequence, an increased number of microalgae-producing companies and products will enter the market, which, in turn, will enhance product recognition and acceptance [5, 9].

2. Current status of microalgal products

Microalgae present a unique diversity in the biosphere in terms of their phylogenetic characteristics, which results in an array of metabolic pathways. This metabolic flexibility leads to the synthesis of a wide range of chemical constituents with diverse biological activities (e.g., anti-inflammatory, anti-aging, and anti-proliferative), making microalgae a potential candidate for sustainable multiproduct cell factories [11, 12]. Driven by this unique metabolomic versatility, microalgae have recently gained significant interest for bioprospecting from industries such as Nestlé [13] and L’Oréal [14] owing to their potential as sustainable sources of bioactive and functional compounds.

Attempts to develop microalgae-based products, including food, cosmetics, and pharmaceuticals, are not new [11]. Harder and Von Witsch (1942) and Kathrein (1964) suggested using microalgae as a sustainable source of lipids and carotenoids, and substantial research was conducted on using microalgae as a source of single-cell protein in the 1970s [15]. However, owing to unresolved economic hurdles, to date, only a few species of microalgae have been produced commercially and are currently exploited as single-value chain products by the industry (see Table 1) [4].

Microalgae	Single-value component and its content [w/w% dry biomass]	Primary product form	Biomass producers (examples)
Arthrospira platensis (Spirulina)	phycocyanin (≤20%) [16]	Extract	Cyanotech Corp.
	Spirularin®	extract	n.s.
Haematococcus pluvialis	astaxanthin (≤5%)	Biomass/extract	Algatech LTD, Algalif Iceland ehf
Dunaliella salina	β-carotene^e31 (≤10%) [17]	Biomass/extract	BASF SE, NBT Ltd.
	glycerol (≤10%) [17]	Extract	n.s.
	Pepha®CTIVE	Extract	n.s.
Chlorella vulgaris	protein (≤60%) [8]	Biomass	Roquette Frères, Algaspring B.V.
Tetraselmis spp.	EPA, C20:5 (≤2%)	biomass/extract	Reed Mariculture Inc.
Euglena gracilis	paramylon (>50%) [18]	Biomass	Euglena Co., Ltd.
Porphyridium cruentum	ARA, C20:4 (≤2.2%) [19]	Extract	Necton S.A.
	phycoerythrin ($\le$8%) [20]	Extract
	SILIDINE®OH & SILIDINE®	Extract	n.s.
Cyanidium caldarium	TEGO® Stemlastin	Extract	n.s.
Phaeodactylum tricornutum	EPA (≤4.5%) [21]	Extract	Simris Group AB
	fucoxanthin (≤1.9%) [21]	Biomass/extract
	MEGASSANE®	Extract	n.s.
Skeletonema costatum	Grevilline™PF	Extract	n.s.
Nannochloropsis spp.	EPA (≤5%) [22]	Biomass/extract	Xiaozao Tech
	Pepha®TIGHT	Extract	n.s.
Isochrysis galbana	DHA (≤ 1.8) [23]	Biomass/extract	Reed Mariculture Inc.
	fucoxanthin (≤1.45) [23]	Biomass/extract
	Lipout™	extract	n.s.
Schizochytrium (protist)	DHA (≤20%)	Biomass/extract	Xiaozao Tech

Table 1.

List of commercially produced microalgae and their extracts.

n.s.: Not specified.

For instance, the dried biomasses of Spirulina and Chlorella spp. have been utilized as dietary supplements for essential amino acids and vitamins in various forms (e.g., tablets, capsules, and powders) or have been incorporated into foods (e.g., smoothies and pasta) to enhance the nutritional value of these products [24, 25, 26]. H. pluvialis and D. salina, the leading natural sources of commercial astaxanthin and β-carotene, respectively. These carotenoids are high-value antioxidants that can accumulate in the cells in high quantities (astaxanthin up to 5% and β-carotene up to 10% DW, respectively); thus, their biomass and processed extracts are commonly used as natural colorants and increase the nutritional value of feed and food applications [27, 28, 29]. E. gracilis is a widely commercialized microalgal species in East Asia since it produces paramylon in large quantities (> 50% of DW). Due to its immunomodulatory functions, the β-1,3- glucan is used for the preparation of high value-added nutritional supplements and medicinal products [30, 31].

Some microalgal species, such as Nannochloropsis spp., Tetraselmis spp., and I. galbana, and the protist Schizochytrium are known to contain considerable amounts of n-3 and n-6 VLC PUFAS such as EPA and DHA (Table 1), which are products of both high market relevance and commercial value. Microalgal species with high PUFA content are currently utilized as feed in aquaculture and animal farming as important sources of these fatty acids [32, 33, 34, 35]. Biomass is used as rotifer feed or is incorporated into formulations for larval and juvenile feed, which is essential for breeding crustaceans and fish [36, 37].

Other single-value chained products from microalgae are lipophilic astaxanthin extract (5, …, 10%) from H. pluvialis [6], algal oil from P. tricornutum [38]), the hydrophilic extracts Pepha®TIGHT from Nannochloropsis oculata [39], and SILIDINE® from Porphyridium cruentum (Table 1). These extracts have found industrial applications owing to their anti-oxidative, anti-inflammatory, anti-aging, and skin regeneration properties in nutraceuticals and cosmeceutical industries [40, 41]. However, beside containing the main valuable and effect causing component(s) of interest alone, also a myriad of other extracted cellular products are present in the raw extracts. Further fractionation of these extracts to obtain a high level of purity for one valuable compound is currently limited to niche products with high market value but very low market volumes (e.g., analytical grade phycoerythrin (PE) from P. purpureum [42]), where the special application justifies the effort and price of the required purification steps [5]. Hence, the generation of economically feasible microalgal products can either be accomplished due to the hyperaccumulation of a specific high-value product or in the co-accumulation of multiple high and low-value products.

3. High-value lead compounds from microalgae

Lead compounds are biomolecules with sufficient potential (measured by potency, bioavailability, therapeutic efficacy, etc.) to progress into a full product development program in the cosmeceutical, nutraceutical, and pharmaceutical industries [43]. However, these lead compounds are only commercially exploitable from microalgae if there is no alternative source, the respective microalga contains the lead compound in higher contents, or the compound possesses specific activities compared to other sources (e.g., galactolipid (GL) esterified EPA compared to EPA from triacylglycerol (TAG)). Such microalgal metabolites that have been recognized for their potential as next-generation lead compounds are special lipids such as carotenoids (e.g., fucoxanthin and astaxanthin), n-3 and n-6 VLC PUFAs, phycobiliproteins, phenols, and polysaccharides. Thus, this section provides a general overview of these high-value lead compounds that are currently commercialized with high market prices and considerable volume [44, 45, 46, 47].

3.1 Lipids

Lipid molecules are a broad class of structurally diverse compounds that are insoluble in water but soluble in organic solvents such as chloroform [48]. Among these, considerable interest of the microalgal-produced lipids are glycolipids, phospholipids, carotenoids, triglycerides, and sterols [49] (Figure 1). Due to their special structure and functionalities, these are used for diverse applications, for example, in nutraceuticals, aquaculture, and cosmeceutical formulations [50].

Figure 1.

Schematic figure of the three different isoforms (triradiate, ovoid, and fusiform, from left to the right) of the oleaginous microalgae P. tricornutum representing the localization of the major lipid constituents of the microalgal lipidome such as glycolipids (purple), phospholipids (brown), special ether bond-based betaine lipids (red), chlorophylls (green), carotenoids (orange), and neutral lipid such as triacylglycerols (yellow) (R represents fatty acid acyl chains with different length (12–22 carbon atoms, for example, EPA. Created with BioRender.com.

3.1.1 Very-long-chain polyunsaturated fatty acids

In addition to being building blocks of cellular membranes, n-3 and n-6 VLC PUFAs have been shown to play a significant role as mediator molecules [51], in pre- and postnatal brain development [52], regulation of cytokine storms [53], antidepressant mechanisms [54], and prevention of cardiovascular diseases [55]. As the de novo synthesis of n-3 and n-6 VLC-PUFAs is restricted to microorganisms, among which marine microalgae, such as the EPA containing oleaginous marine algae P. tricornutum, are considered rich and sustainable sources of these PUFAs (arachidonic acid (ARA, C20:4, n-6), EPA and docosahexaenoic acid (DHA, C22:6n-3)) [1, 3]. Principally, FAs are present in the glycerolipidome of cells, which refers to the complete set of glycerolipids in which two fatty acid acyl chains (R₁ and R₂) are esterified at the sn-1 and sn-2 positions of the glycerol backbone, whereas the third hydroxyl group of the glycerol molecule is esterified with headgroups of different polarities or a third fatty acid acyl chain (R₃) [56, 57].

Microalgae synthesize several types of glycerolipids, which are based on the chemical structure of the headgroup, subclassified into glycolipids, phospholipids, special ether bond-based betaine lipids, and the neutral lipid fractions, such as triacylglycerols. Figure 1 shows the lipidome and its distribution in the commercially exploited diatom P. tricornutum. Among these lipid classes, n-3 and n-6 VLC PUFAs in microalgae are predominantly reported to be esterified into medium-polar thylakoid localized lipid classes [58, 59]. Incorporating these PUFAs into membranes enables algae to maintain optimal membrane fluidity. This characteristic is particularly vital in cold environments as it ensures the efficient light-driven electron transfer through the thylakoid membranes, thereby sustaining the functionality of the photosynthetic apparatus [56]. Interestingly, n-3 and n-6 VLC PUFAs, which are esterified to the GL fractions, have demonstrated enhanced bioavailability and bioactive properties (e.g., antitumor-, antiproliferative-, and anti-inflammatory activities). These bioactive effects surpass those observed with the triacylglycerol-esterified form derived from fish oil, however, the underlying mechanism behind this phenomenon is still not fully understood [60, 61, 62].

3.1.2 Carotenoids

Microalgae have long been recognized for their ability to synthesize and accumulate carotenoids, which have been shown to exhibit several remarkable health-promoting properties. They are well-known for their neutralizing activity against harmful free radicals in the body, thus acting as protective agents against oxidative damage. In addition, carotenoids exhibit further biological activities, such as anti-inflammatory, immune-enhancing, and anticancer properties, making them high-value compounds [63, 64]. In microalgae, these pigments accumulate to higher contents than in alternative sources, such as heterotrophic microorganisms or plants, making them attractive sources for extraction. Astaxanthin (6.000, …, 7.150 US$ kg⁻¹ [65]) β-carotene (300, …, 3.000 US$ kg⁻¹ [66]), and fucoxanthin (40.000, …, 80.000 US$ kg⁻¹ [67]) are the most valuable commercially sold carotenoids [68, 69, 70] at scale.

Astaxanthin is a red secondary ketocarotenoid produced predominantly by green microalgae species. H. pluvialis accumulates astaxanthin contents of up to 5% being natures richest source of this dye. The molecule is known for its superior antioxidant activity, making it highly effective in neutralizing free radicals and suppressing inflammatory processes [6].

β-Carotene, in turn, is a common orange-colored hydrocarbon (Figure 1) and acts as provitamin A, thus contributes to healthy vision and supports immune functions [69]. β-Carotene is found in all microalgae species as primary carotenoid. However, in D. salina under unfavorable cultivation conditions, it can accumulate up to 10%, which made D. salina an attractive choice for production purposes [71].

Fucoxanthin has a unique chemical structure characterized by an allenic bond and an epoxy group (Figure 1). This structural characteristic contributes to its additional health benefits such as anti-obesity and antidiabetic properties. Fucoxanthin serves as primary carotenoid and is mainly distributed in the chloroplast of diatoms. In P. tricornutum fucoxanthin content can reach up to 1.9% [21, 72]. This 5, …, 60-fold higher fucoxanthin content compared to brown seaweeds [73] makes P. tricornutum a suitable alternative production source.

Although frequently being investigated and purified from Chlorophyceae, lutein and zeaxanthin from microalgae can product wise currently not compete with the industrial scale extraction from Tagetes erecta petals, which moreover lack chlorophyll [74]. This may change in the course of successfully applied multiproduct biorefinery approaches, where a separation will also yield these purified carotenoids.

3.1.3 Phytosterols

Microalgae are also sources of phytosterol compounds (e.g., Pavlova lutheri, 0.8, …, 2.6%, P. tricornutum 0.1, …, 0.3% [75]. Phytosterols are plant and algae-derived cholesterol analogs, which are vital for the structural integrity and proper functioning of cell membranes and serve as precursors for the synthesis of hormones, vitamins, and bile acids [47]. Two microalgal phytosterols, β-sitosterol and stigmasterol, have been extensively studied for their ability to reduce low-density lipoprotein (LDL) cholesterol levels, exhibit anti-inflammatory and neuroprotective activities, and have been shown to suppress tumor growth [76]. Therefore, they are potential candidates for the development of novel pharmaceuticals targeting inflammatory diseases such as arthritis and oxidative stress-related disorders. The utilization of microalgal sterols in the formulation of drug delivery systems, including liposomes and nanoparticles, is gaining momentum since these microalgal-based sterol systems improve drug solubility, stability, and bioavailability and enhance therapeutic efficacy. Moreover, they have been shown to be promising in reducing side effects [77, 78].

3.2 Phenols

Microalgae-derived phenols are a diverse group of secondary metabolites that make up a small percentage of microalgal biomass (0.1, …, 0.9%). For example, in the case of N. oculata, the phenolic content can range between 0.1, …, 0.34%, while in I. galbana 0.2, …, 0.8%) have been reported [79]. These phenolic compounds have been found to possess various beneficial properties. Microalgal phenolic compound enriched extracts were shown to exhibit potent antioxidant- and anti-inflammatory properties and considerable antibacterial- and antifungal activity against pathogenic strains [80]. Furthermore, phenol-rich fractions have also demonstrated cytotoxicity and induced apoptosis in various malignant cancer cell lines. Therefore, these substances are promising chemopreventive agents and are currently being investigated to examine their potential in cancer treatment [81, 82]. In addition, several phenolic compounds derived from microalgae have been shown to exert neuroprotective effects as well. They can cross the blood-brain barrier and exert nervous system-localized neurotrophic activities, making them potential candidates for the treatment and prevention of neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases [83, 84, 85].

3.3 Proteins and peptides

Microalgae-derived protein hydrolysates, containing typically oligopeptides (10, …, 20 amino acids), often possess radical scavenging activities and exhibit inflammatory pathway modulatory functions, thus have potential applications in targeting oxidative stress and inflammation-related conditions (e.g., arthritis and inflammatory bowel diseases) [86]. Furthermore, certain microalgae-derived oligopeptides also exhibit antihypertensive effects by inhibiting enzymes involved in blood pressure regulation such as angiotensin-converting enzyme [87], while the inhibition of the growth and proliferation of cancer cells was also proven [88].

PC and PE are two types of phycobiliproteins (PBP) found in certain microalgae [89] (e.g., Rhodophyta, P. purpureum (8%) [20]) and Cyanobacteria (A. platensis, (≤20%) [16]). PC is a blue PBP that has been approved safe for consumption by regulatory agencies and is used primarily as a natural food colorant [90]. Furthermore, it exhibits anti-oxidative and immunomodulatory properties, and research suggests its potential in various therapeutic applications, including as a neuroprotective and anticancer agent [16, 91, 92]. PE is a red-colored PBP primarily found in red microalgae (Rodophyta), which exhibits fluorescence properties and has been utilized as a fluorescent probe in various scientific applications, including flow cytometry and microscopy. Like PC, PE has also shown potential therapeutic activities (e.g., anti-oxidative and anti-cancer); however, its industrial applications are primarily focused on the use of natural fluorescent dyes [90, 93, 94].

3.4 Polysaccharides

Polysaccharides are classified into two main groups: high-molecular-weight (HMW) polysaccharides and low-molecular-weight (LMW) polysaccharides or also referred sometimes as oligosaccharides as the boundary is not precisely defined [95]. Among the HMW polysaccharides, the extracellular ones are of major interest because of their remarkable versatility and wide-ranging applications [96]. For instance, certain species of C. vulgaris produce polysaccharides that exhibit immunomodulatory and anti-oxidative activities [97], and thus have potential in immune health and functional food products. Additionally, some microalgae, such as Porphyridium spp. and Tetraselmis spp., produces sulfated polysaccharides that have been shown to possess antimicrobial- and anticoagulant activities [19, 98]. These sulfated polysaccharides, along with chrysolaminarin (β-glucan) from P. tricornutum (contains up to 32%) [99]), also showed immunomodulatory, anti-oxidative, and antitumor activities, thus holding potential for biomedical research [72, 100, 101]. Additionally, these HMW polysaccharides possess exceptional water-binding capacity and viscosity [100]. A recently exploited sulfated polysaccharide is sacran, derived from the freshwater cyanobacterium Aphanothece sacrum. The chain length exceeds 30 μm and the extremely high molecular weight of 10 to 22 MDa accounts for its elasticity and water-binding capacity. The manifold beneficial characteristics make the biopolymers excellent matrices, emulsifiers, stabilizers, and thickening agents in formulations for the food, pharmaceutical, and cosmetic industries [102].

4. Downstream processing

Exploiting the full potential of microalgae requires an effective DSP strategy to extract, purify, and concentrate the desired products from the complex biomass or their culture broth. However, the DSP of microalgae presents unique challenges, owing to the intrinsic, highly variable characteristics of these microorganisms [103]. In addition to physical characteristics (e.g., cell size, cell wall composition, thickness, and rigidity), which primarily affect harvesting and cell disruption processes. The diverse range of biochemical characteristics of microalgae plays also a significant role in tailoring the overall DSP. Therefore, these differences in the localization (e.g., intra- or extracellular polysaccharides) and content of the compounds of interest necessitate the development of specialized processes for different genera [104]. Thus, the following section aims to discuss the main steps involved in DSP applied for microalgae.

4.1 Harvesting

In general, harvesting techniques aim to concentrate the microalgal suspension for the next DSP unit operation as the diluted algal suspension (0.03, …, 0.5% DM) cannot be used directly from the effluent of the photobioreactor for efficient cell disruption or extraction. However, the small size of the single-celled microalgae (usually below 10 μm), the low density difference between cell and medium, and the low solid volume fraction connected to a large volume of the algal suspension result in a long processing time. Therefore, harvesting is a substantial contributor to the high production expenses. Numerous harvesting methods, such as centrifugation, filtration, and flotation, have been examined to optimize the productivity and economic viability of this DSP unit operation [105, 106, 107].

Disk stack centrifuges (separators) are currently predominantly employed for concentrating microalgal suspensions, while hydrocyclones present an additional option to remove extraneous solid matter when harvesting from open ponds. Depending on the physicochemical properties of the algal suspension, a concentration factor of 50 to 150 for closed photobioreactors and up to 400 for open ponds can be achieved, resulting in slurries with dry matter contents of up to 25%. The feed capacity for the separator can reach up to 90 m³ h⁻¹. However, to achieve the required separation efficiency, which is equivalent to minimal discharge of centrifuged cells with the clear run of the separator, the physiochemical properties of the suspension have to be considered. Here, the particle size (e.g., H. pluvialis 15, …, 50 μm (cyst state) vs. Nannochloropsis 2, …, 4 μm), the viscosity of the algae suspension (e.g., Porphyridium sordidum of 4.2 mPas at 5.0 g L⁻¹ [108] vs. Spirulina of 1.25 mPas at 3.40 g L⁻¹ and 25°C [109]), as well as the solid volume fraction, influence the feed flow rate at a given harvest efficiency. Both achievable harvest cell biomass concentration and the daily harvest volume is dependent upon the photobioreactor design. Particularly for raceway ponds pre-concentration steps, such as pH-induced flocculation and precipitation, have been investigated. These methods do not possess industrial relevance as the multivalent cations (Al²⁺, Al³⁺) or the base (20 mM NaOH) cannot be efficiently removed from the clear phase, preventing re-usage of the medium. Therefore, flotation or vibration sieves are applied, wherever it is feasible with respect to cell and suspension characteristics [106], due to the low volume fractions of the cells in the microalgal suspension (1, …, 2% (v/v) corresponding to 0.3, …, 0.5 g L⁻¹ dry matter). In the pre-concentration step, the suspension is concentrated to, and this pre-thickened slurry is further processed to obtain a final slurry of a dry biomass content of 10 to 25%. Due to acceleration of up to 20,000 g the energy consumption of disk stack centrifuges is high, but the biomass-specific energy consumption is, depending on the type of algae, between 0.4 and 0.8 kWh kg⁻¹ acceptable and moreover cost effective [105, 107, 110].

Among the filtration techniques of small and compressible algal cells, dead-end filtration suffers from the high cake resistance and resulting low filtrate flow rates. Under extreme conditions (e.g., 3 M NaCl), depth filtration using silane-treated glass wool can be applied [111]. With the development of suitable polymer and ceramic membrane materials, crossflow filtration has become an option for harvesting microalgal biomass. In this technique, a tangential flow is applied across the membrane surface, which prevents a rapid decline in flux, setting it apart from dead-end filtration. Thus, with crossflow filtration (CF), a concentration factor of approximately 300 in a single step has been reported for instance for C. minutissima [112]. The energy demand of crossflow filtration techniques, ranging from 0.64 to 3 kWh kg⁻¹ [112, 113, 114], is comparable to that of centrifugal harvesting techniques. However, it is important to consider that CF relies on specific membrane suppliers, necessitating filter replacements with uncertain standing times, as well as process interruptions due to backflushing cycles. Consequently, disk stack centrifuges are currently the preferred and most cost-effective harvesting method for microalgae, especially for small and single-cell morphologies.

4.2 Cell disruption

To facilitate the release of intracellular molecules and enhance the recovery of special membrane-bound high-value components (e.g., carotenoids and GL- esterified n-3 and n-6 VLC PUFAs), cell disruption is a pre-requisite [115]. As key performance indicator, the disintegration degree is defined and targeted at values of greater than 90%, preferably above 95%. The thickness and structure of the cell wall predestinate the required power demand and exposure time of the cell disintegration method applied for this purpose [104]. For instance, a study conducted by Tan et al. demonstrated that an effective disruption of the cell wall of A. platensis can be reached by one freeze-thaw cycle. Its cell membrane, consisting of four 15 nm peptidoglycan layers [116], was easily disrupted using a freezing/thawing cycle of 2/24 hours being sufficient to maximize the yields of PC. This approach is used at scale for industrial PC production if slurry is used as raw material for extraction.

In contrast, the cell wall of certain microalgae, such as Nannochloropsis spp., presents a unique composition consisting of algaenan, cellulose, and hemicellulose layers. This structure results in a cell wall thickness of approximately 110 nm [117], which provides exceptional rigidity for the cells. Consequently, a significantly higher energy input is required to disrupt these cells. Among the mechanical disintegration techniques, stirred bead milling (SBM) and high-pressure homogenization (HPH) are the preferred methods for industrial-scale applications [118]. While industrial-scale bead mills with rotating drums do not provide sufficient specific energy input, the SBM is established as an efficient and favorable disintegration method owing to its high throughput, easy scalability, and high disruption efficiency. In SBM, cells are damaged and ground into smaller particles by applying a force through collisions between the cells and beads, pressure differences, and shearing between the spheres. The effectiveness of microalgal cell lysis was found to be primarily dependent on the sphere diameter (0.35, …, 1.9 mm) to cell diameter ratio (60, …, 230), the material of the beads (e.g., glass, zirconium, steel), the filling degree (0.8, …, 0.85), the circumferential speed (8, …, 14 m s⁻¹), the geometry of the stirring elements, as well as the dry matter content (up to 25%) [119]) and the temperature of the microalgal slurry [119, 120, 121, 122].

HPH is also a mechanical technique used to rupture the cells by suitable conduction of flow of the biomass suspension through a homogenizing valve, converting high hydrostatic into hydrodynamic energy. The efficiency of cellular disintegration relies predominantly on the pressure applied (up to 2500 bar), the number of cycles, the geometry of the valve, and the solid content (up to 25%) [123]) of the slurry. Although this technique has shown effective cellular disintegration, an excessive amount of heat is generated that may influence the product composition. As the valve is not cooled directly, the process operation requires additional intermediate cooling, while the SBMs typically employ a cooling double jacket [123, 124, 125].

The importance of species-specific tailoring for achieving optimal cellular disintegration degrees (>90%) was demonstrated by several studies. Montalescot et al. revealed that specific processing parameters in an SBM were required for N. oculata and P. cruentum [120]. The optimal conditions proved to be 0.6 mm zirconia beads with a filling ratio between 80 and 85% and an agitator speed ranging from 8 to 10 m s⁻¹, for N. oculata whereas for P. cruentum, satisfactory disintegration degrees were achieved at 45% filling ratio with 1.3 mm glass beads. However, it should be noted that these optimized conditions were reported to have an exceptional high biomass-specific energy consumption due to the 7 to 200 times lower dry matter content, compared to other studies. In parallel, studies have focused on systematically optimization of SBM operating conditions, resulting in a considerable reduction in biomass-specific energy demands. For instance, it was shown by Postma et al. that the minimum specific energy consumption was obtained for C. vulgaris, Neochloris oleoabundans and T. sueccica with 1.42 kWh kg⁻¹, 1.78 kWh kg^−1, and 0.47 kWh kg⁻¹ respectively, while still achieving >95% cellular disintegration degree [121]. Furthermore, several studies have optimized HPH process parameters and found that it is also possible to achieve cellular disintegration degrees higher than 95% for microalgae species possessing a rigid cell wall, such as N. gaditana, with a specific energy input of less than 0.5 kWh kg⁻¹ [126]. Based on energy considerations, there is currently no universally preferred method for cell disruption. Further DSP influencing phenomena such as stable emulsion formation hampering extractability need to be considered in each individual case.

4.3 Extraction

In microalgal biorefinery, one of the crucial steps is to effectively extract the valuable components of interest. This extraction process commonly uses both dry and wet biomass [127, 128]. Currently, water-based extraction techniques are the most frequently applied extraction methods for the generation microalgae-based extracts. They offer several advantages that contribute to their widespread use such as safety, environmental friendliness, and mild extraction conditions (e.g., ambient pressure) [129]. For the generation of hydrophilic extracts (containing e.g., carbohydrates and polyphenols), hot water extraction by maceration is frequently used (e.g., for Pepha®TIGHT from N. oculata). In the process, the harvested biomass slurry is diluted and under continuous stirring at elevated temperatures (up to 100°C) for several hours extracted. Following extraction, the raw extract is centrifuged for the removal of cells and debris and subsequently ultrafiltrated to remove macromolecules, which may precipitate or have allergic potential. Finally, the clarified extracts are formulated into products (e.g., topically applied cosmetics) and preserved according to their application [39].

For the extraction of the lipophilic compounds, many methodologies have been investigated; however, solvents, such as chloroform, are considered highly toxic. In agreement with the 2009/32/EC directive, which regulates the use of extraction solvents for foods [130], only a small number of solvents can be used without any restrictions (e.g., ethanol, ethyl acetate). Currently, ethanolic maceration is a widely used technique for the extraction of several lipophilic compounds (e.g., fucoxanthin, n-3, and n-6 VLC- PUFAs). Many studies have evaluated the feasibility of ethanol-based maceration for lipid extraction using multiple species of marine microalgae (e.g., P. tricornutum). Thus, ethanolic extraction of lipophilic molecules seems to be a feasible extraction method; however, to efficiently extract valuable compounds of interest with ethanol, large biomass-to-solvent ratios (usually 1:10 w/v) are required. Furthermore, to increase the extraction yield, elevated temperatures and prolonged extraction times are used. Due to the flammability and explosiveness of ethanol, additional safety precautions, such as explosion protection measures, must be implemented. Additionally, the large volume of extracts produced using ethanolic extraction needs to be evaporated before transport or further processing for solvent recycling.

Therefore, as an alternative to conventional organic solvent extraction, supercritical CO₂ (ScCO₂)-based extraction has gained attention and is currently in use as green alternative for the extraction of lipophilic molecules. Specialized processing companies are producing algal extracts such as astaxanthin oleoresins from H. pluvialis [131], as well as fucoxanthin and EPA containing oleoresins from P. tricornutum [132, 133]. However, only a few companies have experience with ScCO₂ at industrial scale, and as this process requires long processing hours and a high technical demand this technique is the most expensive method available. Furthermore, because of the low solubility of, for example, carotenoids in ScCO₂, high extraction pressures (up to 1000 bar) are required for efficient recovery of the highly apolar astaxanthin fatty acid esters from H. pluvialis. Therefore, in industrial processes the use of ethanol as an entrainer recovers carotenoids, in this case astaxanthin, at reduced pressures [6] and process costs.

4.4 Multiproduct refinery

While the number of bioactive compounds of potential commercial interest from microalgae is continuously increasing, the launch of new microalgal-based products remains challenging. Microalgae typically contain low amounts of target compounds in their biomass. This low content makes the DSP even more crucial for extracting and concentrating as many valuable components as possible. Therefore, instead of commercializing single-value chain products, such as algal biomass and raw extracts (Table 1), it is important to establish a biochemical class-based production platform for multiple products to fully capitalize the components produced by microalgae [8, 134]. A possible multiproduct purification scheme is presented in Figure 2, generating nine valuable fractions, an additional residual, and insoluble biomass fraction.

Figure 2.

Multiproduct generation platform from microalgal biomass. * represents the single-value chained products and residual biomass that is currently discarded. Created with BioRender.com.

Prior to extraction, the harvested algal biomass is either processed directly in the form of slurry (frozen and thawed). Alternatively, the biomass is disrupted and dried, or solely dried. Then, the concept of cascade extraction is implemented to microalgal bioprocessing. The biomass is consecutively extracted using solvents of different polarities (e.g., water and ethyl acetate) to obtain two fractions: one enriched in hydrophilic (e.g., proteins and carbohydrates) and one in lipophilic compounds (e.g., n-3 VLC-PUFAs and carotenoids). The generated extracts are then separated using different fractionation techniques based on the nature of the molecules found in the extract (e.g., based on the molecular size; HMW sulfated polysaccharides from LMW polysaccharides). The residual biomass from the cascade extraction may undergo additional hydrolysis, resulting in polyalcohols (e.g., inositol, glycerol, sorbitol), bioactive peptide-rich fractions, or leave the bioprocessing chain being sold as low-value bulk commodities (e.g., fertilizer). Therefore, this approach can result in a spectrum of high- and low-value products, which balances the production costs of both bulk and specialty microalgae-based products. Subsequently, the total revenue from a given algal biomass can increase, and the production of sustainable algae-based bulk commodities in an economically viable way becomes possible. However, it is important to note that the focus should not be solely on optimizing individual unit operations, but rather on enhancing the efficiency of the overall process. Improving a single-unit operation based on a benchmark may lead to a more intricate and expensive overall process. Therefore, increasing the efficiency of utilization by implementing a zero-waste multiproduct biorefinery has become a prominent area of research.

In a study conducted by Gallego et al., a sequential accelerated solvent extraction-based platform was developed to recover PE, sulfated polysaccharides, and carotenoids (β-carotene and zeaxanthin) from P. cruentum [135]. Similarly, Derwenskus et al. applied this pressurized liquid extraction (PLE) technique for the co-production of EPA and fucoxanthin from the diatom P. tricornutum (Figure 1) [21]. They obtained EPA- and fucoxanthin-rich fractions through a single extraction step, applying ethanol, which contained approximately 90% of the initial fucoxanthin and EPA contents. The obtained fraction was further purified by precipitation and filtration, resulting in product purities exceeding 90%. Furthermore, the use of subcritical n-hexane at 100°C led to direct precipitation of the carotenoid at a temperature drop to ambient conditions. Similarly, Zhang et al. investigated the concept of an integrated biorefinery process to produce three valuable compounds, fucoxanthin, EPA, and chrysolaminarin, via cascade extraction [136]. In their study, freeze-dried P. tricornutum was processed using microwave-assisted ethanolic extraction. The obtained extract and residual biomass were subsequently subjected to n-hexane extraction via maceration to obtain fucoxanthin- and EPA-rich fractions. After n-hexane extraction, the residual biomass was further processed using sulfuric-acid-catalyzed hot water extraction to obtain chrysolaminarin. Although the compounds of interest were isolated, their yields were relatively low (34 and 23%, respectively) [136].

While these studies represent progress in multiproduct generation, they also underscore the importance of scalable DSP unit operations because the scalability of microwave-assisted extractions and PLE processes from the laboratory to the industrial scale remains a general cost issue and currently exceeds the technical state of the art at scale. Here, solvent extraction is applied at ambient or low overpressure, which results in lower extraction yields compared to PLE. To ease scale-up classical maceration and mixing techniques using various solvents have been studied. For instance, Chaiklahan et al. evaluated different scenarios of sequential product recovery, such as PC, FAs, and polysaccharides from A. platensis, using conventional maceration techniques to extract dried biomass [137]. PC was the main product of the extraction process, and approximately 67% of the original PC content from the raw materials could be recovered. Crude fatty acid and polysaccharide-rich extracts were also obtained, which have shown to increase the overall revenue. Similarly, Li et al. applied cascade extraction under atmospheric conditions using cold water, 95% ethanol, and hot water to recover PE, lipids, and polysaccharides from P. purpureum [138]. The cascade extraction process achieved considerable recovery rates for the target compounds (63.3, 74.3, and 75.2%, respectively).

However, when high-purity fractions have to be generated, it is necessary to separate the compound of interest from other molecules, which primarily reduce purity and value of the lead component. For this purpose, different separation techniques, depending on the nature of the molecule, can be used such as solid-liquid phase chromatographic techniques (e.g., adsorption or molecular sieve-based). Several studies have demonstrated the successful use of silica gel for purifying microalgal oil, including the separation of specific compound-rich fractions, such as γ-linolenic acid from Spirulina [139], EPA and ARA from Porphyridium spp. [140], and carotenoids, such as fucoxanthin [141]. These purifications are currently restricted to the laboratory scale [119]. Despite these efforts, microalgae extracts are rarely purified to single components owing to the high cost of purification with respect to the “ceutical” application and a lacking market demand for highly pure compounds. Thus, simple, robust, and scalable isolation methods will aid broader availability of valuable microalgae-based fractions at reasonable market prices. For this purpose, liquid-liquid separation techniques, such as centrifugal partition chromatography (CPC), high-speed countercurrent chromatography (HSCCC), and aqueous two-phase systems (ATPS), have been increasingly investigated in natural product recovery.

ATPSs are formed when two nonmiscible aqueous solutions are mixed (e.g., two polymers, polymer-salt, ethanol-salt, or ionic liquids (IL)-salt or polymer) [142]. ATPSs have been investigated in the study of Suarez Ruiz et al. using different systems, including PEG400-Cholinium dihydrogen phosphate, PEG400-citrate, and Iolilyte 221PG-citrate, for the development of a single-step fractionation method for microalgal carbohydrates yielding a high recovery for glucose [143]. In a different study, 98% of pigments were recovered from the polymer-rich phase, separated from a protein-rich cholinium dihydrogen phosphate phase using a disintegrated N. oleoabundans suspension at a PBR-typical concentration (2.5 g L⁻¹). In the IL phase, a recovery of 82 and 93% for proteins and soluble sugars, respectively, was obtained. In addition, 79% of the insoluble polysaccharides and 71% of the lipid molecules were recovered from the interface [144] underlining the suitability of the multistep fractionation approach. Sarkar et al. further confirmed the efficacy of the ATPS approach [145]. Using an aqueous ammonium sulfate solution (40%) combined with ethanol at a ratio of 0.25:1 (v/v), it was possible to separate proteins, carbohydrates, and chlorophylls simultaneously in a single step from the biomass of C. thermophila. The recovery yields were 71, 40, and 22%, respectively [145]. Thus, the use of ATPS for extracting multiple products from microalgae has recently gained significant interest. Although the scalability of ATPE was demonstrated up to 1000 L systems (e.g., Human insulin-like growth factor [142]), its industrial application in microalgae processing has not been examined, yet. Technically scalability of ATPE is comparably simple as it combines a stirred tank with a centrifuge. Thus, studies should aim to investigate the still existing gap to explore the possibility of using this technique in the microalgal biorefinery processing [142, 143, 144].

CPC and HSCCC are separation techniques that use two immiscible liquid phases, composed mainly of organic solvents. One phase is held stationary in sequentially joint separation chambers (CPC) or in a multilayer coil (HSCCC) by centrifugal force, whereas the other phase is forced to flow through the stationary phase. Dissolved components are separated on the basis of their distribution coefficient between the two phases. Unlike other chromatographic techniques, a solid support is not required; therefore, irreversible sample adsorption is prevented. In addition to a recovery rate of over 95% and a purity rate of over 99% [146], CPC and HSCCC also provide high injection capacities, making this technique an efficient DSP unit operation in the field of natural product isolation [147, 148]. Recently, the applicability of these techniques in algal biorefineries has been investigated. For instance, in a study by Bárcenas-Pérez (2021), HSCCC was employed to isolate fucoxanthin from an extract of P. tricornutum using a multiple-sequential-injection HSCCC method. With ten consecutive sample injections, 38 mg of fucoxanthin with 97% purity and 98% recovery was obtained. The process throughput was 0.19 g extract h⁻¹, while the fucoxanthin throughput was 3 mg h⁻¹ employing a 135 mL rotor [149]. In another study, Bárcenas-Pérez et al. al (2021) focused on the separation of DHA ethyl ester and docosapentaenoic acid (DPA, C22:5, n-3) ethyl ester from algal oil using HSCCC. Similarly, consecutive sample injections of 1 g of algal oil were performed, resulting in the isolation of 1.8 mg of DHA ethyl ester with 99% purity and 99% recovery. In addition, 2.16 mg of DPA n-6 ethyl ester with 97% purity and 92% recovery was obtained. The process throughput for algal oil was 1.15 g h⁻¹, and the throughput of the two target compounds was about 0.11 g h⁻¹ [150]. Additionally, a novel two-step HSCCC method was developed, resulting in the isolation of 980 mg EPA ethyl ester-rich fraction with a purity of 71%. This fraction was further purified, yielding 700 mg of EPA ethyl ester with a purity of 99% and a recovery of 98% with an EPA ethyl ester throughput of 0.05 g h⁻¹ [151]. Furthermore, Fábryová et al. developed a new isolation procedure for the purification of lutein from C. vulgaris. They utilized a multi-injection HSCCC method, processing a total of 250 mg of extract per hour, which resulted in a total recovery of 60 mg lutein within a processing time of 8 h at a purity of 92%. Fábryová et al. also demonstrated that HSCCC separation can be used for the pre-fractionation of astaxanthin fatty acid esters from H. pluvialis [152, 153].

The abovementioned studies collectively emphasize the potential of liquid-liquid chromatographic techniques as versatile tools for the recovery and purification of valuable natural compounds derived from microalgae since these techniques offer efficient separation, high purity, and high recovery rates. Therefore, these separation techniques may represent a useful and moreover scalable tool for the efficient production of purified microalgae-based compounds such as fucoxanthin and VLC PUFAs. With respect to scalability CPC rotors of a volume of 50 L are currently available [154], which represents a scale-up factor of 200 to 500 for CPC methods developed. Processing capacities of crude extracts lie, assuming optimized methods, in industrial relevant dimensions of several kg of extract per day.

Summarizing the currently applied and possible DSP unit operations, Table 2 presents an overview as a showcase of the state of the art and the potential multiproduct biorefinery for P. tricornutum. At industrial level, the applied DSP process currently involves four-unit operations, including solid liquid separation, cellular disintegration, drying, and SC-CO₂ extraction. At the end of this production process, one fucoxanthin and EPA-enriched fraction are commercialized, while the residual biomass is not used.

Number of DSP unit operations	Gained fractions	Valuable fractions	Discarded fractions	References
4	2	1	1	Industrial process
5	3	2	1	[21]
5	10	10	0	Potential

Table 2.

Current and possible DSP unit operations for P. tricornutum.

As shown by Derwenskus et al., by implementing one additional DSP unit operation, the number of valuable fractions also increases. In this study, an EPA and fucoxanthin-rich fraction was obtained by PLE subsequent to harvesting and disintegration [21]. Further crystallization and filtration of precipitating fucoxanthin yielded two purified fractions. However, if after harvesting and cellular disintegration, adequate bi-phasic extraction techniques are utilized (e.g., ethyl acetate, water) the process would already result in the hydrophilic and lipophilic enriched extracts (see Figure 3). Subjecting these extracts to liquid-liquid separation techniques, such as CPC and ATPE, up to 10 different products could be generated. For P. tricornutum, from the lipophilic fraction fucoxanthin, VLC PUFAs, polyphenols, and sterols could be purified, while from hydrophilic extracts the more polar polyphenols, proteins, and chrysolaminarin could be obtained. These hydrophilic compounds then could be further fractioned by ATPS into proteins and carbohydrates and separated by filtration based on their different molecular size. The residual cell debris from the extraction could consequently undergo additional hydrolysis to generate polyalcohols, peptides, and amino acids. The residual solids would leave the value chain and potentially utilized as fertilizers. This concept then would result in a close to zero-waste biomass utilization of P. tricornutum without a significantly higher number of different unit operations.

Figure 3.

Indicative flow diagram containing unit operations and products of the multiproduct DSP concept for P. tricornutum.

In summary, previous studies have presented advancements and a step forward in the extraction and purification of multiple high-value compounds. However, the financial implications of the multiproduct biorefinery concept, resulting from the implementation of additional DSP unit operations at the industrial scale, still need to be explored. Additionally, the choice of process units, design characteristics, and process conditions for each step of the algal biorefinery chain affects the performance of the subsequent processing steps. Thus, owing to the lack of information on the total specific energy demands, processing times and yields of the overall process, further investigations on the subject are required.

5. Conclusion

In summary, this chapter highlights the large potential of microalgae as multiproduct cell factories. Despite the economic and legislative hurdles faced in establishing a microalgae-based bioeconomy, the unique metabolic versatility of microalgae makes them promising candidates as sustainable bioresources. The current status of microalgal products is presented and it is evident that the commercial exploitation of microalgae is currently limited to a few single-value chained products. However, identifying market-relevant products derived from algae of high commercial interest, such as fucoxanthin and EPA, and the utilization of the currently discarded residual biomass seems to be a promising way to enhance the credibility of a microalgae-based bioeconomy.

It is also evident that there have been notable achievements in the research and development of multiproduct biorefinery approaches; however, these are still limited to laboratory-scale experiments. Additionally, research activities have been focusing on the microalgae-specific optimization of single DSP unit operations, thus neglecting the overall performance of the whole multiproduct DSP platform. Furthermore, there is a need to develop suitable purification methods for multiple products with a high throughput, yield, and recovery. For this approach, liquid-liquid separation techniques, such as CPC and ATPE, appear to be promising. However, further economic feasibility investigation (e.g., product-specific energy input, solvent reusability, yield) for the generation of multiple products with consecutive liquid-liquid chromatographic techniques has to be carried out in order to provide further insights into microalgae-based bioprocessing.

Acknowledgments

We thank the University of Applied Sciences for research funding and the Open Access Publishing Fund.