Biomass is the most relevant variable estimated in microalgae cultivation because almost all products obtained by microalgae cultivation are of intracellular nature and thus procured by biomass processing (Jacob-Lopes et al. 2020). Microalgal biomass is known to be a rich source of lipids, pigments, vitamins, amino acids, proteins, carbohydrates, e.g., polysaccharides and oligosaccharides, and essential minerals (Caporgno and Mathys 2018; Jacob-Lopes et al. 2020). Currently, the U.S. Food and Drug Administration (FDA) designates the microalgae Arthrospira platensis, Auxenochlorella protothecoides, Chlorella vulgaris, Chlamydomonas reinhardtii, Dunaliella bardawil, Euglena gracilis, Haematococcus pluvialis, Schizochytrium, Porphyridium cruentum, and Crypthecodinium cohnii with a Generally Recognized as Safe (GRAS) status (García et al. 2017; Diaz et al. 2023). It is nowadays common to find commercial presentations of microalgal biomass marketed as powders, pills and capsules.

The growth of microalgae cultures can be determined directly by measuring the abundance of the cells using cell counting by various methods and/or the increase in biomass, either by dry weight (DW), ash-free dry weight or chlorophyll a concentration. Methods using optical properties (e.g., turbidity or absorbance) are also used as a measure of algae abundance, however, precise correlations with direct methods (e.g., cell count, DW) should be considered for proper interpretation (Borowitzka and Moheimani 2013). The possibility of measuring cell count online together with statistics on cell size has been investigated by employing an in-situ, flow-through microscope installed in a microalgae cultivation bypass (Havlik et al. 2013b). Biomass measurement is not only important for its association with valuable compounds but also for estimating culture metabolic state variables (e.g., specific growth rate) necessary in fed-batch strategies (Wechselberger et al. 2013). Moreover, industrial processes using microalgae require the biomass as a variable for the estimation of process productivity (e.g., g DW/L, ton DW/ha), yields, and economics, e.g., €/kg DW (Norsker et al. 2011). Therefore, the use of devices to monitor this variable has been of interest to researchers, given its relevance in commercial applications.

The most common technique used in laboratory and industrial environments to determine microalgae growth involves the use of optical density (OD). Traditionally, the wavelengths at 750 nm (OD750) and 550 nm (OD550) have been employed for this purpose due to minimal interference with the cellular pigments that are present, e.g., chlorophyll and carotenoids (Borowitzka and Moheimani 2013; Wang et al. 2019).

On the other hand, authors have described the use of other wavelengths for cell growth estimation. In a short study, four different wavelengths comprised in a range between 677 and 688 nm (within the maximum chlorophyll absorption), using four different microalgae species, were employed for the purpose of correlating absorbance and cell concentration (Santos-Ballardo et al. 2015). In another study, the authors employed several wavelengths, 480, 510, 630, 647, 647, 650, 664 and 750 nm, to correlate them with cell concentration, but also to compare OD measurements and wavelengths employed. The authors used seven microalgae strains in this task. Their results showed consistency between OD measurements regardless of the wavelength used (Pearson coefficient = 0.92–0.97) which suggests that the wavelength selected is not a determining factor (Chirivella-Martorell et al. 2018).

Microalgae are an interesting source for different types of lipids. In general, the lipid profile of microalgae consists of neutral lipids (or non-polar lipids) such as triacylglycerols (TGA), free fatty acids (FFA) and carotenoids, i.e., lipidic pigments, and polar lipids mainly represented by glycolipids and phospholipids (Sarpal et al. 2015). The fatty acid profile in microalgae is characterized by a mixture of C16 and C18 saturated and unsaturated fatty acids, as well as long-chain (C20-C22) polyunsaturated fatty acids (PUFAS), e.g., eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and docosapentaeonoic acid (DPA) (Gouveia and Oliveira 2009; Barkia et al. 2019; Yang et al. 2020).

The type and amount of fatty acids present in microalgae can vary considerably among different phylogenetic groups, and even differ at the species level. Environmental conditions and growth conditions (e.g., pH, light intensity, nutrient limitation, and oxidative stress) are other factors that shape the lipid profile in microalgae (Andersen 2013; Borowitzka et al. 2016; Bi and He 2020; Morales et al. 2021). An extensive study of the long-chain fatty acid profile (C14-C24) in 2076 microalgae strains that investigate cyanobacteria and different phyla and classes of eukaryotic algae has been reported (Lang et al. 2011). Other studies on lipid characterization employing different microalgae can be found elsewhere (Yao et al. 2015; Shen et al. 2016).

Research with microalgae lipids started with the aim of biodiesel production (Barkia et al. 2019). However, this application has encountered several limitations in its step toward commercialization (Chisti 2013). The high costs of producing biofuels from microalgal biomass, both in open and closed systems, limit the economic success of the sector; however, scenarios that integrate biofuel production processes with "next generation cultivation systems and processes" could favor the profitability of this activity (Richardson et al. 2014; Bi and He 2020). For this reason, biotechnological applications have currently been directed towards lipid products not for biofuels but products with higher added value within the pharmaceutical and food sectors due to their beneficial properties for human health, e.g., PUFAs, phytosterols, food supplements and infant formulas (Barkia et al. 2019; Fabris et al. 2020; Fernandes and Cordeiro 2021).

As in plants, chlorophyll a (Chl a) constitutes the main pigment for photosynthesis in phytoplankton. However, these photosynthetic organisms also contain different accessory pigments (e.g., Chl b and c), carotenoids, and phycobiliproteins to carry out fundamental cellular processes, e.g., enhancement of light uptake and photoprotection (Andersen 2005). The pigments present in microalgae can vary between taxonomic groups and therefore be used to differentiate between them. For example, cyanobacteria, in general, produce Chl a, d and f, as well as the phycobiliproteins (e.g., phycocyanin, allophycocyanin, and phycoerythrin). An interesting exception within the cyanobacteria is the group of prochlorophytes, i.e., Prochloron, Prochlorothrix, and Prochlorococcus, which, in addition to Chl a, contains Chl b and lacks phycobiliproteins (Roche et al. 1996). Meanwhile, Chlorophyta, a group to which the genera Chlorella, Dunaliella, and Haematococcus belong, contains Chl a and b in addition to various types of carotenoids (e.g., β-carotene) and various xanthophylls, e.g., astaxanthin, canthaxanthin, lutein, and zeaxanthin (Barkia et al. 2019).

There are three main classes of pigments present in microalgae and whose commercialization is of great interest: chlorophyll, carotenoids and phycobiliproteins (Silva et al. 2020).

Chlorophylls are responsible for the photosynthesis process as well as light energy harvesting in oxygenic photosynthetic organisms, i.e., plants, algae, and cyanobacteria. Currently, five types of chlorophyll have been described: Chl a, b, c, d, and f, obtained from oxygenic photosynthetic organisms (Li and Chen 2015).

Chlorophylls exhibit variations in their side chains and/or reduced states. These structural variations in the rings and/or side chains give each chlorophyll distinctive characteristics in its absorption spectra. In general, chlorophylls present two main light absorption bands where their corresponding absorption maxima (λ max) are found; the short wavelength absorption band, i.e., the Soret band (in vitro and in vivo ∼400–470 nm) and the long wavelength absorption band, i.e., the Qy band (in vitro ∼620–710 nm and in vivo ∼640–710 nm) (Papageorgiou and Govindjee 2004; Chen 2014). Chl a in methanol exhibits a λ max of 436 and 665 nm. However, under in vivo conditions, Chl a, found in photosystem II, shows a λ max ∼680 nm and λ max ∼700 nm in photosystem I mainly attributed to the protein environment surrounding these molecules. On the other hand, Chl b under in vivo conditions presents values of λ max of ∼650 nm (Chen 2014). It should be noted that different types of Chl c, present in golden-brown eukaryotic algae but absent in plants, present an additional λ max at ∼580 nm under in vitro conditions (Zapata et al. 2006).

Interestingly, the Chl a is also almost the only chlorophyll performing fluorescence under in vivo conditions at ordinary temperatures. At room temperature, Chl a shows a heterogeneous behavior, however, a fluorescence band at 683–685 nm, which originates in PSII, and a small amount in the 710–760 nm region from the PSI antenna are observed (Papageorgiou and Govindjee 2004).

Despite being light- and oxygen-sensitive molecules, the biotechnological uses of chlorophylls are varied. For example, their use is common as dyes in the food, cosmetic and pharmaceutical sectors (Silva et al. 2020). In vivo evaluations of plant-derived chlorophyll extracts have demonstrated their protective capacity as antioxidants (Suparmi et al. 2016). A more recent work describes the process for stabilizing chlorophyll extracts with Cu(NO3)2 in paints (Sulaiman et al. 2019).

Carotenoids are lipid-soluble accessory pigments made up of isoprene units whose coloration mainly spans the visible light spectrum between yellow and red, i.e., 400–600 nm (Langi et al. 2018; Silva et al. 2020). In addition to their role in light harvesting, carotenoids present a photoprotective function against oxidative stress and adverse environmental conditions (Barkia et al. 2019). According to their function, there are two major groups of carotenoids: carotenes (e.g., α-carotene, β-carotene, lycopene) and xanthophylls (e.g., astaxanthin, zeaxanthin, lutein, violaxanthin, canthaxanthin). Based on their chemical structure, carotenes are considered oxygen-deprived hydrocarbon compounds (C40 polyenes), whereas xanthophylls present oxygenated groups, i.e., hydroxyl and keto groups, toward the end rings. This feature confers a relative hydrophilic character to xanthophylls (Langi et al. 2018).

The properties and functions of carotenoids depend on their molecular structure. For example, carotenoids can present different isomeric configurations (trans and cis), resulting in variations in the melting point, solubility and stability of the molecule. In addition, the conjugated polyene chromophore, present in carotenoids, defines the properties of light absorption and light harvesting (Langi et al. 2018).

Among their biological activities, carotenoids have been associated with antioxidant, anti-inflammatory, and anticarcinogenic properties. The effects of different types of carotenoids on human health (e.g., cardiovascular protection, prevention of liver fibrosis, prevention against different types of cancer) can be widely consulted in the literature (Park et al. 2010; Yoshida et al. 2010; Milani et al. 2017; Langi et al. 2018; Barkia et al. 2019). In addition, the carotenoids are used in the food sector as a food coloring additive and nutraceutical, in the animal feed industry, and in cosmetology (Silva et al. 2020).

Phycobiliproteins are photosynthetic light-harvesting protein pigments naturally found in cyanobacteria, red algae, cryptomonads, and glaucophytes (Silva et al. 2020). These proteins are hydrophilic and are found in superstructures called phycobilisomes in the chloroplast stroma. A classification of the phycobiliproteins includes three main groups: allophycocyanin, phycocyanins, and phycoerythrins (Stadnichuk et al. 2015). Covalently attached to their polypeptide structure through cysteine residues, chromophore molecules called phycobilins are found (Kovaleski et al. 2022). There are four types of phycobilins: phycocyanobilin (PCB, blue), phycoviolobilin (PVB, violet), phycoerythrobilin (PEB, red) and phycourobilin (PUB, yellow) (Dagnino-Leone et al. 2022). Other authors report only three types of phycobilins: PEB, PCB, and PVB (Kovaleski et al. 2022).

The amino acid sequence, the number of chromophores per monomer, and the type of chromophores present are criteria used to differentiate between phycobiliproteins. Based primarily on structural features and their absorption spectra, phycobiliproteins are divided into four groups: phycoerythrin (λmax = 490–570 nm), phycocyanin (λmax = 610–620 nm), phycoerythrocyanin (λmax = 560–600 nm), and allophycocyanin (λmax = 650–655 nm) (Kovaleski et al. 2022).

The online monitoring of phycocyanins using fluorescence spectrophotometry in the marine cyanobacterium Synechoccocus sp. has demonstrated the feasibility of this approach to obtain direct information from cell cultures (Sode et al. 1991). Moreover, the offline methods of extraction and purification of phycobiliproteins are relatively simple and widely known, a fundamental step in the training and validation of a software sensor (Kovaleski, 2022). Therefore, all these elements raise the possibility of using methods based on fluorescence spectrophotometry for online monitoring and software sensor development for estimating phycobiliproteins. Considering that the range of fluorescence spectra of phycobiliproteins is between 585 and 665 nm (Stadnichuk and Tropin 2017), selective online monitoring of different types of phycobiliproteins, e.g., phycocyanin or phycoerythrin, in microalgae cultivation systems poses interesting challenges.

Cyanobacteria of the genus Arthrospira sp. and Porphyridium sp. are the most relevant microalgae in the industrial production of phycobiliproteins, specifically phycocyanin (blue) and phycoerythrin (red), respectively (Silva et al. 2020). Commercial interest in phycobiliproteins is associated with their bioactive properties, such as antioxidant, anti-inflammatory, anti-metabolic diseases, anti-cancer, anti-neurodegenerative, and antibiotic (Dagnino-Leone et al. 2022). Other applications related to the use of these molecules are related to protein markers, cell sorting, and phycobiliprotein-derived conjugates that take advantage of their properties as fluorescent probes (Tounsi et al. 2023). Recent applications employ the fluorescent properties of phycoerythrin and phycocyanin to sense different analytes as part of nanoprobes or complexed with other molecules like DNA (You et al. 2020; Ghosh et al. 2020). Finally, the differential quenching effect of heavy metals such as silver and copper on phycocyanin fluorescence has demonstrated their potential use in selective monitoring for the presence of heavy metals (Bellamy-Carter et al. 2022).

Silva et al. (2020) determined, in a bibliometric study, that between 2009 and 2019, research was mainly focused on the study of the pigments phycocyanin, chlorophylls, β-carotene, and astaxanthin obtained from A. platensis, C. vulgaris, D. salina, and H. pluvialis, respectively. At present, there are reports where the use of software sensors has been employed for the estimation of chlorophyll and carotenoids (Sá et al. 2020b, a). However, the monitoring and estimation of phycobiliproteins in microalgae cultivation through the use of software sensors represents an interesting challenge to be explored.

Vitamins are organic compounds commonly required in low concentrations by organisms and indispensable in many vital cell processes. Vitamins are classified into two categories: fat-soluble molecules (e.g., vitamins A, D, E and K) and water-soluble molecules (e.g., B-complex vitamins and vitamin C) (Udayan et al. 2017).

Microalgae constitute a diverse source of different types of vitamins, e.g., vitamins A, D, E, K and several B vitamins, i.e., B1 (thiamine), B2 (riboflavin), B3 (niacin), B5 (pantothenic acid), B6 (pyridoxine), B7 (biotin), B9 (folic acid), and B12 (cyanocobalamin) (Del Mondo et al. 2020). For example, in A. platensis (formerly Spirulina platensis) (http://www.algaebase.org/) the presence of vitamins B1, B2, B3, B6, B9, B12, vitamin C, vitamin D and vitamin E has been reported (Jung et al. 2019). C. vulgaris constitutes another source of multiple vitamins, e.g., B2, B3, B9, and B12 (Edelmann et al. 2019). Dunaliella sp. is rich in fat-soluble vitamins and moreover, interesting concentrations of vitamin B2, vitamin B12, folic acid, vitamin C, vitamin B3 and vitamin E have been reported in D. tertiolecta (Udayan et al. 2017). More details on the production of different types of vitamins by some microalgae genera can be found elsewhere (Del Mondo et al. 2020).

There are some intrinsic characteristics in the fluorescence of some vitamins of the B complex that could be useful for monitoring microalgae cultures with software sensors using optical measurement methods. To make use of these characteristics, the knowledge of the excitation and emission spectra of these molecules is essential. For example, vitamin B1 in water presents a fluorescence region at λex/λem = 370/460 nm (Yang et al. 2016). Vitamin B6, B2 and B9, in aqueous solution, exhibit a fluorescence region at λex/λem = 330/380–390 nm, λex/λem = 445/520 nm, and λex/λem = 330/450 nm, respectively (Parri et al. 2020). Vitamin B2 also has a second fluorescence region that comprises λex/λem = 365/520 nm (Faassen and Hitzmann 2015).

Another component present in microalgal biomass is the protein fraction. Several microalgae species report high protein concentrations ranging from 42 to 70% in some cyanobacteria and up to 58% in C. vulgaris on a dry cell basis (Becker 2007; Barkia et al. 2019). Other species such as D. salina, H. pluvialis, Nannochloropsis sp., A. platensis and A. maxima show protein amounts of 49–57%, 29–45%, 50–55%, 46–63%, and 60–71% on a dry cell basis, respectively (Timira et al. 2022). A recent work shows a protein content of 51% and 64% (w/w) in two Galdieria sulphuraria extremophile strains (Canelli et al. 2023). The essential and non-essential amino acid profiles present are comparable with those of other protein sources, i.e., egg albumin, soybean and milk lactoglobulin (Williams and Laurens 2010).

Carbohydrates comprise between 12 and 64% of microalgal biomass (Becker 2007; Markou et al.