Microalgae: A Promising Source of Valuable Bioproducts

1 Institute of Living Systems, Immanuel Kant Baltic Federal University, A. Nevskogo Street 14, 236016 Kaliningrad, Russia; ur.liam@fv_kunaglod (V.D.); ur.liam@airad-aninotna (D.B.); ur.liam@34.hcilo (O.B.); moc.liamg@93eztakd (D.K.); ur.liam@98_80_devs (N.P.); ur.liam@psa-sats (S.S.)

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Daria Belova

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Olga Babich

2 Laboratory of Biocatalysis, Kemerovo State University, Krasnaya Street 6, 650043 Kemerovo, Russia; ur.xobni@vokesorp.a

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Alexander Prosekov

2 Laboratory of Biocatalysis, Kemerovo State University, Krasnaya Street 6, 650043 Kemerovo, Russia; ur.xobni@vokesorp.a

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Svetlana Ivanova

3 Natural Nutraceutical Biotesting Laboratory, Kemerovo State University, Krasnaya Street 6, 650043 Kemerovo, Russia

4 Department of General Mathematics and Informatics, Kemerovo State University, Krasnaya Street 6, 650043 Kemerovo, Russia

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Dmitry Katserov

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Nikolai Patyukov

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Stanislav Sukhikh

2 Laboratory of Biocatalysis, Kemerovo State University, Krasnaya Street 6, 650043 Kemerovo, Russia; ur.xobni@vokesorp.a

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2 Laboratory of Biocatalysis, Kemerovo State University, Krasnaya Street 6, 650043 Kemerovo, Russia; ur.xobni@vokesorp.a

3 Natural Nutraceutical Biotesting Laboratory, Kemerovo State University, Krasnaya Street 6, 650043 Kemerovo, Russia

4 Department of General Mathematics and Informatics, Kemerovo State University, Krasnaya Street 6, 650043 Kemerovo, Russia

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

Abstract

Microalgae are a group of autotrophic microorganisms that live in marine, freshwater and soil ecosystems and produce organic substances in the process of photosynthesis. Due to their high metabolic flexibility, adaptation to various cultivation conditions as well as the possibility of rapid growth, the number of studies on their use as a source of biologically valuable products is growing rapidly. Currently, integrated technologies for the cultivation of microalgae aiming to isolate various biologically active substances from biomass to increase the profitability of algae production are being sought. To implement this kind of development, the high productivity of industrial cultivation systems must be accompanied by the ability to control the biosynthesis of biologically valuable compounds in conditions of intensive culture growth. The review considers the main factors (temperature, pH, component composition, etc.) that affect the biomass growth process and the biologically active substance synthesis in microalgae. The advantages and disadvantages of existing cultivation methods are outlined. An analysis of various methods for the isolation and overproduction of the main biologically active substances of microalgae (proteins, lipids, polysaccharides, pigments and vitamins) is presented and new technologies and approaches aimed at using microalgae as promising ingredients in value-added products are considered.

Keywords: microalgae, biologically active substances, proteins, lipids, polysaccharides, vitamins, pigments

1. Introduction

Recently, the issues of microalgae cultivation have been of increasing interest among researchers due to their ability to synthesize various biologically active substances, the rapid growth of biomass and the ability to adjust their biochemical composition depending on cultivation conditions. Microalgae are marine or freshwater microorganisms consisting of a single eukaryotic cell. These are unicellular flora representatives with huge potential for application in various branches of science and technology [1]. Previously, cyanobacteria, which later became considered bacteria, were also classified as blue-green algae. Currently, there are many types of eukaryotic unicellular microorganisms. Their diversity can be compared with the diversity of insects [1,2]. Unlike heterotrophic microorganisms, which require various organic compounds for growth, unicellular photosynthetic organisms produce biomass from completely oxidized inorganic substances and mineral elements due to the light energy converted during photosynthesis. Furthermore, microalgae biomass production technologies do not pollute the environment, use carbon dioxide while generating oxygen, consume a relatively small amount of water and may occupy land unsuitable for cultivation of agricultural crops [2].

At present, two main areas of use of microalgae can be distinguished: the production of biomass as a biologically active additive, and the cultivation of microalgae for the subsequent isolation of biologically active substances from biomass.

Microalgae are rich in nutrients and biologically active substances, such as proteins, polysaccharides, lipids, polyunsaturated fatty acids, vitamins, pigments, phycobiliproteins, enzymes, etc. Biologically active substances from microalgae are capable of exhibiting antioxidant, antibacterial, antiviral, antitumor, regenerative, antihypertensive, neuroprotective and immunostimulating effects [3]. These compounds are in demand in pharmacology, medicine, cosmetology, the chemical industry, fish farming, the energy industry, agriculture in the production of feed and functional foods [4].

Microalgae are less studied than seaweeds, but their advantages are associated with rapid growth, high photosynthetic efficiency and the possibility of cultivation under production conditions. In addition, the biodiversity of microalgae will increase the number of different sources of biologically active substances, such as polysaccharides, lipids, proteins and pigments.

Currently, integrated technologies for the cultivation of microalgae aiming to isolate various biologically active substances from biomass to increase the profitability of algae production are being sought. The purpose of this review is to analyze recent works (less than 10 years) aimed at isolating biologically valuable substances from microalgae and assessing their biological activity. This review aims to assess the potential of microalgae as a raw-material basis for biologically valuable substances of various activity spectra.

2. Factors Affecting Biomass Production

The level of biomass accumulation and productivity of biologically active substances is an important indicator of the effectiveness of the microalgae strain. These parameters are influenced by many conditions, including the composition of the culture medium, temperature, pH, growth phase, method of harvesting and illumination [5].

The optimum growth temperature for the most commonly used microalgae, such as Chlorella, Chlamydomonas, Botryococcus, Scenedesmus, Neochloris, Haematococcus and Nannochloropsis, is in the range of 15–35 °C depending on the strain [6]. It has been shown that some microalgae strains have high stress resistance at high temperatures. For example, strains of Asterarcys quadricellulare and Chlorella sorokiniana, isolated from soil near a steel mill, not only grow at 43 °C, but are also resistant to high concentrations of CO₂ and NO [7]. It is known that the use of thermotolerant microorganisms can significantly reduce the growth of incidental microflora. In addition, the use of such strains allows cultivation under natural conditions. The effect of technological parameters of the microalgae cultivation process (temperature, duration, stirring) on biomass production is presented in Table 1 , Table 2 and Table 3 .

Table 1

The influence of temperature on the growth of various microalgae (table reconstructed using data from Varshney et al. [7]).

Microalgae	Biomass Yield, g/L
Microalgae	27 °C	30 °C	35 °C
Chlorella vulgaris	0.77	0.83	0.36
Chlamydomonas reinhardtii	0.33	0.75	0.18
Botryococcus braunii	0.79	0.81	0.64
Scenedesmus obliquus	0.65	0.67	0.23
Neochloris cohaerens	0.58	0.77	0.25
Haematococcus pluvialis	0.68	0.72	0.55
Nannochloropsis gaditana	0.45	0.59	0.13

Table 2

The influence of cultivation time on the growth of various microalgae (table reconstructed using data from Qiu et al. [8]).

Microalgae	Biomass Yield, g/L
Microalgae	7 days	14 days	21 days
Chlorella vulgaris	0.68	0.72	0.47
Chlamydomonas reinhardtii	0.78	0.79	0.65
Botryococcus braunii	0.64	0.69	0.37
Scenedesmus obliquus	0.75	0.80	0.29
Neochloris cohaerens	0.76	0.79	0.52
Haematococcus pluvialis	0.73	0.75	0.67
Nannochloropsis gaditana	0.68	0.73	0.53

Table 3

The influence of culture stirring on the growth of various microalgae (table reconstructed using data from Varshney et al. [7]).

Microalgae	Biomass Yield, g/L
Microalgae	0 rpm	50 rpm	100 rpm
Chlorella vulgaris	0.24	0.66	0.32
Chlamydomonas reinhardtii	0.39	0.62	0.42
Botryococcus braunii	0.26	0.59	0.33
Scenedesmus obliquus	0.43	0.69	0.52
Neochloris cohaerens	0.41	0.71	0.59
Haematococcus pluvialis	0.23	0.63	0.67
Nannochloropsis gaditana	0.36	0.70	0.55

The recommended cultivation temperature for maximum biomass yield of various types of microalgae is a temperature ranging from 27 °C to 30 °C. With increasing cultivation temperature to 35 °C, the biomass yield decreases sharply.

A significant accumulation of biomass was observed by the 14th day of cultivation of all the studied microalgae.

The growth of microalgae biomass under stationary conditions is slowed down compared to cultivation with stirring (50–100 rpm).

The hydrogen potential (pH) is of great importance in the cultivation of microalgae, as it determines the solubility of minerals and carbon dioxide in the medium, in addition to the direct effect on the microalgae themselves [8]. The pH of the culture medium can be influenced by such factors as composition and buffer capacity, the amount of dissolved carbon dioxide, temperature and metabolic activity of the cells. Different types of microalgae have different levels of tolerance to the pH of the culture medium, which may affect their growth rate. For some microalgae, the optimal pH ranges from 6 to 8. In a number of cases, a steady increase in the microalgae biomass is observed at extremely low pH values, as, for example, for Chlorella protothecoides var. acidicola isolated from the surface of the microbial mat and having a growth optimum at pH 2.5 and 30 °C [9]. The use of buffer solutions reduces pH fluctuations in cultures, however, for large-scale cultivation systems, the use of buffers increases the cost of production, making it impossible. One way to control pH changes is to aerate cultures by pumping atmospheric air (0.03% CO₂) or CO₂-enriched air through the medium, since carbon dioxide, when dissolved, reduces the pH of the medium [10]. The effect of pH on the biomass yield of various types of microalgae is presented in Table 4 . The optimal pH indicator is in a range from 6 to 8, however, when the biomass of the microalgae Chlorella vulgaris is produced, a decrease in pH to 4 leads to the greatest yield.

Table 4

The influence of pH on the growth of various microalgae (table reconstructed using data from Varshney et al. [7]).

Microalgae	Biomass Yield, g/L
Microalgae	pH = 4	pH = 6	pH = 8
Chlorella vulgaris	0.75	0.67	0.34
Chlamydomonas reinhardtii	0.17	0.72	0.68
Botryococcus braunii	0.16	0.74	0.78
Scenedesmus obliquus	0.18	0.65	0.69
Neochloris cohaerens	0.21	0.73	0.68
Haematococcus pluvialis	0.16	0.69	0.67
Nannochloropsis gaditana	0.18	0.78	0.76

Combining the given data, the recommended values of technological parameters for growing microalgae are presented in Table 5 .

Table 5

Optimal conditions for growing microalgae biomass (table reconstructed using data from Nancucheo and Johnson [9]).

Microalgae	Cultivation Temperature, °C	Cultivation Duration, Days	Culture Stirring, rpm	pH of Culture Medium
Chlorella vulgaris	29	13	60	4.1
Chlamydomonas reinhardtii	30	13	75	6.3
Botryococcus braunii	27	11	70	6.8
Scenedesmus obliquus	29	12	90	7.1
Neochloris cohaerens	27	12	85	6.9
Haematococcus pluvialis	28	14	90	7.3
Nannochloropsis gaditana	30	11	70	7.6

The salinity of the solution is another factor affecting the growth and development of microalgae. Some types of microalgae are very limited in terms of salinity, especially those found in freshwater. In general, microalgae can be divided according to salinity resistance into oligogaline when they can develop only in water with low salinity (maximum salinity 0.5–5.0 g/L), mesogaline—develop in media with moderately saltwater with a salinity of 5–18 g/L and polyhaline—develop in saltwater with a salinity of 18–30 g/L [11].

An ideal culture medium for microalgae should contain inorganic elements such as nitrogen (N) and phosphorus (P), which can vary depending on the cultivated species. After carbon, which is approximately 50% of the fraction of elements in the biomass of microalgae, nitrogen takes second place, with a concentration of 1% to 14% in the dry mass. It can be absorbed in inorganic forms of NO₃, NO₂, NO and NH₄ and in some cases as N₂ or in an organic form via urea or amino acids. The decrease in nitrogen concentration during cultivation leads to the predominant synthesis of lipids and polysaccharides. The phosphorus concentration in the dry biomass of microalgae can be from 0.05% to 3.3% [12]. In natural environments, as well as in wastewater, phosphorus is present in various forms, such as orthophosphate, polyphosphate, pyrophosphate and metaphosphate. In addition, there are various types of agricultural fertilizers that can be used to saturate the microalgae cultivation environment with phosphorus, such as phosphates and superphosphates derived from phosphorites. For adequate growth of microalgae, the medium must contain other nutrients—trace elements. The main trace elements are Mg, S, Ca, Na, Cl, Fe, Zn, Cu, Mo, Mn, B and Co, with an emphasis on Mg, S and Fe [13].

Nutrient limitation has a direct impact on the synthesis of biologically active substances, biomass growth and photosynthesis processes in microalgae [14]. High illumination and limitation of nutrients (nitrogen or phosphorus) lead to an increase in the size of the lipid fraction and stimulate the accumulation of triacylglycerols. In low light conditions, mainly polar lipids (phospholipids and glycolipids) accumulate, which are structurally and functionally associated with cell membranes. Cell growth is reduced under such conditions. However, there are exceptions, for example, diatoms, in which the lipid content in the long phases does not respond to nitrogen starvation [15].

Stress caused by nutrient deficiency also leads to the formation of free radicals in the cell and a change in antioxidant content. Primary carotenoids (chlorophylls, β-carotenoids, violaxanthin and voheriaxanthin) are synthesized under normal favorable microalgae growth conditions, especially in Eustigmatophyceae strains. However, secondary carotenoids are produced under stress caused by nitrogen deficiency, synthesized from primary carotenoids and accumulating outside the chloroplast after the cell growth phase [16]. Under intense exposure to light, microalgae are photo-damaged, and some become pale green and decrease in size, which indicates a decrease in the density of chlorophyll and affects cell development [17]. Furthermore, with excess light, violaxanthin is converted to zeaxanthin by removing epoxides using the anthraxanthin monoepoxycarotinoid. Zeaxanthin is epoxidized in low light conditions, and nitrogen deficiency contributes to the accumulation of astaxanthin pigment in some microalgae, such as Haemotococcus pluvialis [15].

During autotrophic growth, microalgae carry out oxygen photosynthesis and fix carbon dioxide. One part of fixed carbon is used to maintain cells and growth, while the other part is stored in several forms, depending on different types of microalgae. Microalgae require 1.8 to 2.0 kg of CO₂ to produce 1 kg of biomass. Given this stoichiometric ratio, the amount of CO₂ present in the air (0.03%) is not enough for high culture productivity. Thus, in order to increase the efficiency of photosynthesis, media with microalgae cultures should be supplemented with carbon, either in the form of salts, such as bicarbonate, or by introducing CO₂-enriched air. A study by Dúran et al. [18] demonstrated that when the air was supplied (600 mL/min) to the photobioreactor, microalgae showed optimal growth with a CO₂ content of up to 20% in the supplied air. This makes it possible to use CO₂ from industrial combustion, generating on average 5.0% of CO₂, in microalgae cultivation. This approach combines an inexpensive carbon source for microalgae and reduces CO₂ emissions. The supply of CO₂ to microalgae cultures can increase biomass productivity, however, a decrease in pH due to an increase in the availability of CO₂ in the aqueous phase may impede the growth of certain types of microalgae [19].

The optimal conditions for the growth of microalgae depend on the light intensity, wavelength and photoperiod to which the cells are exposed during cultivation [20].

Light intensity is directly related to the photochemical phase of photosynthesis, when light is absorbed through chlorophyll molecules, the synthesis of adenosine triphosphate (ATP) and the photolysis of water. In general, photosynthesis can be defined as a process in which light energy synthesizes polysaccharides and oxygen from carbon dioxide and water [21]. Light intensity is one of the important aspects of growing microalgae and requires special attention. The amount of light received by cultured cells is directly related to carbon, which will be fixed, affecting the growth rate of cultures [22]. The light source can be either artificial or natural (sunlight), the latter being the most cost-effective due to its availability. Photosynthesis in microalgae increases with increasing light intensity until it reaches a maximum speed at the saturation point [23]. Above the saturation point, excess light leads to a phenomenon called photoinhibition, which is determined by the change and possible inactivation of photosystem II (PSII), affecting electron transport in the chain of reduction reactions from NADP + to NADPH. Photoinhibition and photosynthesis can be classified as moderate or intense, which determines whether this inhibition is dynamic or chronic [24]. The light intensity does not affect its accessibility in the depths of the plant environment. In other words, even if the light intensity is high enough to cause inhibition of photosynthesis, the light may not reach the shaded cells, which affects the efficiency of biomass production. Therefore, to supplement natural light or even for cultures under artificial lighting, it is recommended that LEDs be placed inside the medium to improve photon delivery and distribution.

Photosynthetically active radiation, useful for microalgae, is in the range from 400 to 700 nm from light radiation, which corresponds to 50% of solar radiation and intensity from 800 to 1000 W/m 2 [25]. This is confirmed by the results of Fu et al. [26], who demonstrated that Dunaliella salina shows the highest biomass and carotenoid productivity (β-carotene and lutein) when 75% of red light (wavelength about 700 nm) and 25% of blue light (wavelength about 400 nm) are used together, compared with the only red light.

There are light/dark (L/D) cycles in the microalgae growth tanks provided by the medium stirring system. These cycles affect the efficiency of photosynthesis conversion and biomass productivity in microalgae. It was also found that the efficiency of photosynthesis in Chlamydomonas reinhardtii improved when L/D cycles of less than 20 s were applied, with an increase in the growth rate of up to 40% [27].

Using the example of various types of marine and freshwater microalgae, it was shown that the duration of the photoperiod affects the intensity of photosynthesis, productivity, cell division rate and carbon dioxide consumption. Almost always, the listed indicators for various light and dark modes were higher than for continuous illumination. However, at present, there is no consensus on the mechanisms that explain the increase in productivity under the influence of the photoperiod [28].

There are several hypotheses in the literature that explain the phenomenon of an increase in the growth rate of lower phototrophs in the presence of a photoperiod. For example, under continuous illumination, some products of photosynthesis that inhibit it may accumulate in the cells, the outflow of which occurs in the dark period. Furthermore, in the presence of a photoperiod, the activity of ribulose-1,5-bisphosphate carboxylase may increase, which may be due to the regulation of carbon consumption mechanisms. It can be assumed that when limiting the growth of microalgae culture by CO₂ concentration, the increase in productivity is due to an increase in carbon concentration during the dark period due to the dark respiration of the cells. Additionally, if there is a dark period in the daily cycle, a change in the biochemical composition of algae occurs, in particular, the respiratory consumption of reserve compounds, which, in turn, can lead to a decrease in respiration rate [29].

An effective stirring of the culture medium is important for obtaining high cell concentrations. Stirring maintains cells in suspension, eliminates thermal separation, distributes nutrients and increases gas exchange efficiency. Stirring can reduce the degree of self-darkening and the likelihood of photoinhibition by uniformly distributing light among all microalgae cells [30]. In addition, stirring is also responsible for the possibility of CO₂ capture from the atmosphere and facilitates the transfer of biosynthesized O₂ from the liquid phase to the gaseous one, which stimulates photosynthesis in microalgae culture [31]. The connection between stirring and illumination becomes more obvious when the culture has a high concentration of cells, since under this condition the light is blocked by cells of the surface region and the light intensity decreases sharply with the depth of the culture [32]. In a study by Sánchez et al. [33], it can be noted that in cultivation systems with stirring blades, the daily growth of the microalgae culture Isochrysis galbana was two times higher compared to a system without stirring (8.8 × 10 5 and 4.0 × 10 5 cells/mL).

The growth rate of photosynthetic microorganisms increases with increasing turbulence caused by stirring, but above the optimum level of turbulence, this growth decreases sharply due to damage to the cells. Many photosynthetic microorganisms have a fragile cell wall; some are mobile or threadlike and may be subject to physical exertion. Thus, it is desirable that the stirring is carried out with the lowest possible hydrodynamic stress.

3. Methods of Microalgae Cultivation

The conditions for the cultivation of microalgae can be divided into three main methods: photoautotrophic, heterotrophic and mixotrophic. Photoautotrophic is the most commonly used cultivation method. Microalgae use light (usually solar) as an energy source and inorganic carbon (e.g., carbon dioxide) as a carbon source to generate chemical energy. With heterotrophic cultivation, microalgae can grow not only under photoautotrophic conditions, but also use organic carbon in the absence of light. Owing to the ability to grow in the dark, microalgae growing during heterotrophic cultivation are much less demanding on the ratio of surface area to volume than autotrophic cultivation [34].

Mixotrophic cultivation is a generalized two-stage mode in which microalgae have a high initial content of organic carbon, but are induced for assotrophic assimilation of CO₂ due to depletion of organic substances and oxygen production through photosynthesis [35,36]. During mixotrophic cultivation, microalgae grow under optimal autotrophic and heterotrophic conditions, combining the advantages of both cultivation modes [37]. Under such conditions, acetyl-CoA is produced and maintained both by fixing CO₂ and extracellular organic carbon, which indicates a decrease in photoinhibition. Mixotrophic cultures are often observed in ecological water bodies, where they are supported by both chemical, physical and organic activity of biota. Venkata Mohan et al. [38] presents in detail metabolic schemes for each cultivation mode.

The design and operation of an algae cultivation system play a decisive role in both the productivity of photosynthesis and economic efficiency. The design of the cultivation system should have a short path of light, the optimal volume of liquid for appropriate stirring and scattering of light. Working with large volumes of fluid requires high hydrostatic pressure and higher energy consumption for proper stirring [39]. Various large-scale cultivation systems have been developed for the large-scale production of microalgae. They usually work under photoautotrophic conditions and can be divided into two main groups: open or closed cultivation systems. Ninety percent of microalgae biomass production worldwide is through open cultivation systems. Open systems usually use open shallow ponds made of aligned raceways (2–10 m wide and 15–30 cm in depth) in the form of simple loops or meandering systems where turbulence is provided by rotating impellers [40]. These reactors are easily expandable, and investment and operating costs are relatively small. The main disadvantages of open cultivation systems are the complexity of process control, as well as the inability to organize a continuous cultivation process. High susceptibility to contamination by other microalgae and microorganisms, dependence on weather conditions (light, temperature) and daily temperature fluctuations make the open pond system unsuitable for large-scale culture [35,41,42].

Closed photobioreactors were designed to overcome the aforementioned disadvantages of open pond systems, resulting in a series of reactor designs that increased surface-to-volume ratio and system reliability [32,43]. Closed systems allow continuous operation and offer higher biomass productivity and quality, as well as higher photosynthesis efficiency. Liquid evaporation is minimized through the use of a closed system. However, such systems are difficult to scale due to their complex organization and reduction of light penetration with increasing cell concentration and the removal of oxygen generated during photorespiration is a serious problem. Closed photobioreactors are certainly more expensive than open systems [44].

There are bubble-airlift, bubble-column, tubular and panel photobioreactors. Mostly in pilot tubular photobioreactors, microalgae grow at a depth of illumination of 3–6 cm, where light penetration is less than 50%. Panel photobioreactors are the simplest solution for laboratory-scale microalgae cultivation. Of all these types of photobioreactors, they have a maximum illuminated surface and allow achieving high cell densities. However, during their operation, it is necessary to maintain a balance of illumination to prevent photoinhibition. The main achievements in the field of closed photobioreactors have been considered by Wang et al [45].

Currently, several basic technologies for the production of microalgae biomass are being developed in Russia and abroad, which include cultivation: (a) in open waters; (b) in greenhouse ponds; (c) in closed photobioreactors. Genomic and biochemical technologies are used for microalgae cultivation. In the framework of this work, the effect of all three types of the described methods for the cultivation of microalgae was studied [46]. The characteristics of the possible methods of microalgae cultivation under various conditions are presented in Table 6 .

Table 6

Characteristics of possible methods for microalgae cultivation.

Cultivation Method	Energy Source	Carbon Source	Cell Biomass Accumulation Rate	Reactor Type	Price	Features
Phototrophic	Light	Inorganic	Low	Photobioreactor/open waters	Low	The cell density of the culture is low; water evaporation
Heterotrophic	Organic matter	Organic	High	Bioreactor	Medium	The high price of the nutrient medium components; possibility of microbial contamination
Mixotrophic	Light, organic matter	Organic and inorganic	Medium	Closed photobioreactor	High	The high price of the nutrient medium components; possibility of microbial contamination