Bioenergy, Biofuels, Lipids and Pigments—Research Trends in the Use of Microalgae Grown in Photobioreactors

Abstract

This scientometric review and bibliometric analysis aimed to characterize trends in scientific research related to algae, photobioreactors and astaxanthin. Scientific articles published between 1995 and 2020 in the Web of Science and Scopus bibliographic databases were analyzed. The article presents the number of scientific articles in particular years and according to the publication type (e.g., articles, reviews and books). The most productive authors were selected in terms of the number of publications, the number of citations, the impact factor, affiliated research units and individual countries. Based on the number of keyword occurrences and a content analysis of 367 publications, seven leading areas of scientific interest (clusters) were identified: (1) techno-economic profitability of biofuels, bioenergy and pigment production in microalgae biorefineries, (2) the impact of the construction of photobioreactors and process parameters on the efficiency of microalgae cultivation, (3) strategies for increasing the amount of obtained lipids and obtaining biodiesel in Chlorella microalgae cultivation, (4) the production of astaxanthin on an industrial scale using Haematococcus microalgae, (5) the productivity of biomass and the use of alternative carbon sources in microalgae culture, (6) the effect of light and carbon dioxide conversion on biomass yield and (7) heterotrophy. Analysis revealed that topics closely related to bioenergy production and biofuels played a dominant role in scientific research. This publication indicates the directions and topics for future scientific research that should be carried out to successfully implement economically viable technology based on microalgae on an industrial scale.

1. Introduction

Microalgae are single-celled and multicellular photosynthetic, prokaryotic or eukaryotic microorganisms that live in fresh and salt waters [1,2]. On the basis of their morphological features, microalgae are divided into Cyanophyta (e.g., cyanobacteria), Phaeophyta (e.g., brown algae), Rhodophyta (e.g., red algae) and Chlorophyta (e.g., green algae) [3]. From among approximately 200–800 thousand species of microalgae, only about 40 thousand species have been identified so far [4].

The advantage of microalgae is that they possess both the advantages characteristic of higher plants (efficient oxygen photosynthesis and simplicity of nutritional requirements) and positive biotechnological features of microbial cell culture (rapid growth in liquid culture and the ability to accumulate or secrete some metabolites) [5]. The efficiency of the photosynthesis process of microalgae, at a level of 3%, exceeds the biomass production efficiency of terrestrial plants (0.2–2%) [1]. Comparing the oil production capacity per hectare of plant crops (4–5 tons/year) and microalgae biomass (30 tons/year), it is evident that the microalgae culture efficiency is over five times higher than the best yields achieved in the cultivation of traditional plants. Easy access to light, CO₂ and nutrients in aerated liquid cultures make the cultivation of microalgae more attractive than terrestrial plants. Microalgae do not have non-productive (heterotrophic) organs, thanks to which all biomass is fully photosynthetically active. Microalgae show a flexible metabolism and a short biomass doubling time. For most species, it is a few hours in optimal growth conditions [2]. Microalgae farming does not require fertile land and can be grown in wastelands and deserted terrains using brackish water, wastewater or even seawater. As a result, they do not compete for resources with conventional food crops. It is also easier to select microalgae species for specific environmental conditions typical of the local climate than in the case of conventional crops [6].

Algae can use light and carbon dioxide to produce many valuable products, such as proteins, polysaccharides, lipids, vitamins, carotenoids and other biologically active compounds [7]. Scientific interest in laboratory algae cultivation dates back to 1919. The first mass-scale culture experiments were carried out in the 1950s in the USA and Japan. Commercial algae cultivation developed particularly rapidly in Southeast Asian factories [8]. Currently, the biomass of microalgae is used for the production of biofuels, for the production of food products, as fodder for fish and farm animals [9] and as high-value bioactive substances for the production of pharmaceuticals and cosmetics [10]. Microalgae are also used in environmental engineering, inter alia, for wastewater treatment and biomitigation of CO₂ in flue gases from coal-fired power plants [6], the integration of anaerobic digestion with microalgae cultivation [11] and biofixation [12]. The latest research shows that integrating wastewater treatment and CO₂ absorption processes with the production system of microalgae biomass is a feasible process [13].

Biofuels from microalgae are classified as third- and fourth-generation biofuels [4]. They are not made from edible crops, nor do they require large areas of land for food production [14]. This makes microalgae biofuels an alternative energy source to fossil fuels [15]. Microalgae biomass is used to produce biodiesel, biohydrogen, bioethanol, bio-oil and biogas [16,17]. It should also be remembered that the products of photosynthesis allow for the storage of energy, e.g., in the form of liquid fuels, which can be used in the future. Other renewable energy sources, such as hydro, wind or solar PV, do not possess this characteristic [2]. Microalgae with high lipid content in dry matter, such as Botryococcus braunii (25–75%), Nannachloropsis oculata (31–68%), Chlorella vulgaris (18–57%), Scenedesmus obliquus (up to 50%) and Dunaliella primolecta (up to 23%), are especially good for industrial production of biofuels [1,4]. In the food industry, microalgae enhance food’s nutritional and organoleptic properties (color, taste and texture) in products such as pasta, bread, mayonnaise and jelly desserts [5]. The most popular microalgae species in the production of high-value chemicals are the cyanobacteria Arthrospira platensis (formerly known as Spirulina) and the green microalgae Chlorella vulgaris, Dunaliella salina and Haematococcus pluvialis. They have long been used in large-scale industrial cultivation systems [2]. Carotenoid pigments such as astaxanthin, β-carotene, fucoxanthin and algal lutein are used in the production of nutraceuticals, pharmaceuticals, cosmetics, food and animal feed. The microalgae species Haematococcus pluvialis, Dunaliella salina, Chlorella spp., Scenedesmus spp., Spirulina platensis, Botryococcus braunii and diatoms are best known for their production of β-carotene, lutein, canthaxanthin, astaxanthin and fucoxanthin [3].

Carotenoids are one of the most popular natural products produced in microalgae cultivation. They are fat-soluble pigments known as tetraterpenoids, with a characteristic C40 carbon skeleton. They are divided into oxygenated carotenoids (xanthophylls) and hydrocarbon carotenoids, simply called carotenoids [3]. The most valuable of the carotenoids is the red ketocarotenoid with the chemical formula C₄₀H₅₂O₄ (red ketocarotenoid)—astaxanthin. It has strong pigmentation abilities, is an excellent antioxidant and has a broad, beneficial effect on human health [3,7].

The vast majority of produced astaxanthin is produced by chemical synthesis and is used as feed in aquaculture. Astaxanthin consumed by humans must come from natural sources. Natural astaxanthin can be obtained from the by-products of shellfish production, but it is economically unprofitable. Astaxanthin can also be produced by the red yeast Xanthophyllomyces dendrorhous (formerly referred to as Phaffia rhodozyma) on various carbon sources (glucose, xylose and molasses). Although the growth rate and the culture density are high, the produced astaxanthin content is only 0.16 to 1.1 mg·g⁻¹ dry weight, depending on the strain used. Of the higher plants, only the autumn love plant (Adonis annua) produces astaxanthin in the petals of its flowers. There are more and more reports of the possible accumulation of astaxanthin in transgenic plants such as tobacco, Arabidopsis, potato, carrot and even tomato [7]. In this way, an increase in the nutritional value of the edible parts of plants is achieved. The greatest potential for the production of natural astaxanthin (up to 4% of dry biomass) is shown by microalgae. Haematococcus pluvialis microalgae meal has been approved in many countries as a dietary supplement for human consumption. These algae grow relatively slowly with a low biomass yield. Additionally, they are susceptible to infections and require very strong light at the stress stage [18]. The green freshwater algae Chlorella zofingiensis can be an alternative due to the possibility of rapid growth in cultures with very high cell density, indoors and outdoors. In addition, these algae are characterized by low sensitivity to pollution and unfavorable environments and astaxanthin accumulation under heterotrophic conditions with glucose as the only source of carbon and energy. C. zofingiensis synthesizes and accumulates primary carotenoids (e.g., β-carotene, lutein and zeaxanthin) in chloroplasts. Under stressful conditions, these algae accumulate secondary carotenoids, such as astaxanthin, canthaxanthin and adonicixanthin, in lipid bodies (except chloroplasts) [7].

Natural astaxanthin obtained from microalgae is a trans-geometric isomer and occurs mainly in its esterified form. This form is more stable than artificial forms; therefore, a longer shelf life is achieved without oxidation. Due to its strong pigmentation, astaxanthin is used as a feed additive in aquaculture, e.g., in salmon and lobster farming, giving fish and crustaceans a red-orange color. Additionally, astaxanthin accelerates growth and increases the survival rate of fish and shrimp larvae [19]. The use of astaxanthin in the food, nutraceutical and cosmetic industries is determined by its strong antioxidant properties, which are beneficial to human health. Astaxanthin is more effective at scavenging free radicals than other carotenoids and vitamin E [7].

Astaxanthin production occurs under stressful conditions that differ significantly from optimal conditions for the normal development and growth of microalgae. The stress factor may be related to physical conditions (manipulation of environmental conditions—increase in light intensity, temperature, pH, salinity and electromagnetic field) and nutrition (manipulation of the composition of culture media (carbon source, nitrogen, phosphorus and iron deficiency) [5]. For this reason, properly selected and equipped cultivation systems used to produce astaxanthin play a significant role.

Two basic culture systems are used to conduct experiments and produce microalgae: open tanks and closed photobioreactors. Reports on the use of hybrid structures are also appearing more and more often.

Open ponds are the oldest, simplest and cheapest large-scale microalgae cultivation systems. These solutions were first used in the 1950s [15]. There are four basic open ponds: unmixed open ponds, raceway ponds, circular ponds and thin-layer ponds [6]. They are natural or artificially built shallow reservoirs of various sizes and shapes. The depth of open tanks ranges from 30 to 50 cm to ensure the best possible penetration of the culture medium by sunlight [20]. They are made of cheap materials and have a simple structure [21]. For recirculation of cultures in open tanks, solutions with mechanical agitation through a rotating arm or paddle wheel, consuming relatively small amounts of energy, are used [2,4]. The advantage of open tanks is that they can be built in areas that are not suitable for plant production. The most significant drawbacks of open tanks are the limited ability to control working conditions, e.g., maintaining temperature, pH, light intensity and dissolved oxygen [21,22]. Open tanks are susceptible to pollution from the air and soil; therefore, these systems cultivate species in selective environments (e.g., with high alkalinity and high salinity) [15,16]. Another disadvantage of open systems is their low productivity (<20 g·m⁻²·day⁻¹) resulting from poor gas exchange and the presence of dark zones. In turn, the low density of cell cultures necessitates the construction of systems over large areas, increasing biomass harvesting costs [2].

Closed photobioreactors (PBRs) are tanks made of transparent materials (glass or plastic) in the form of tubes, bags or plates [15]. Depending on the orientation of the elements used in the construction of photobioreactors, a horizontal, inclined or vertical configuration can be distinguished [6]. When designing photobioreactors, the basic principle is to obtain the highest possible ratio of the area on which the light falls to the volume of the tank [16]. Photobioreactors include lighting systems (natural or artificial), temperature and pH control systems, CO₂ supply and O₂ removal systems. Mixing is carried out employing mechanical or air pumps [4].

Among closed tanks, cylindrical, tubular and flat photobioreactors play dominant roles. Cylindrical photobioreactors are made in the form of bubble columns or airlift tanks. The columns are arranged vertically, aeration is provided from below, and light is provided through the transparent walls of the tanks [23]. Column photobioreactors provide the best control of culture conditions, efficient mixing and the highest volumetric gas transfer rates [15]. Tubular photobioreactors are systems in which the microalgae culture is pumped through long transparent tubes by mechanical pumps or air lifters, which also enables the exchange of CO₂ and O₂ between the liquid culture medium and the aerating gas [21]. Flat photobioreactors are built as flat transparent panels in which light is absorbed by a thin, several-millimeter-thick outer layer of the microalgae culture. Flat-panel systems provide higher culture densities and higher photosynthetic efficiency [15,22].

The cultivation in photobioreactors allows for precise control of the optimal environmental conditions for the development of microorganisms, including lighting intensity, nutrient content, proper pH and temperature, aeration rate, carbon dioxide concentration, and the appropriate size and phase of the inoculum [4]. Thanks to this, many different microalgae species can be cultivated in photobioreactors (which is not allowed using open systems), and the processes can be carried out in a repeatable manner [6,21]. Cultures in closed photobioreactor systems allow for higher productivity, at the level of 0.8–1.5 g·L⁻¹·day⁻¹, that is, ten times better than in open tanks [2]. The lack of direct gas exchange between the photobioreactor and the environment reduces the risk of contamination and chemical contamination [5,16]. There are limited carbon dioxide and substrate losses due to evaporation compared to open systems. The main disadvantages of photobioreactors are the high costs of their construction, operation and maintenance [4,15].

Hybrid production systems are systems in which scientists try to use the advantages of various types of photobioreactors [24]. Most often, they combine closed photobioreactors with open ponds [4]. Appropriate amounts of pollutant-free inocula are produced in photobioreactors and then transferred to open ponds to obtain maximum biomass efficiency [15].

These bibliometric, scientometric and content analyses aimed to identify the trends in scientific research related to algae, photobioreactors and astaxanthin production. A total of 367 scientific articles published between 1995 and 2020 in the Web of Science and Scopus bibliographic databases were analyzed. Based on the bibliometric analysis, the most influential journals, authors, research centers, countries and publications that were most cited were identified. Based on keywords analysis, seven significant research areas were identified, as well as the directions and topics for future scientific research that should be carried out to successfully implement economically viable technology based on microalgae on an industrial scale. Our article is the first one that combines bibliometric, scientometric and content analyses of articles published during the mentioned time.

2. Materials and Methods

The authors adopted the research protocol of bibliometric review described by Khan et al. [25] and Moher et al. [26]. In preparation for this work, the authors analyzed scientific articles from the Web of Science and Scopus as suggested by Teran-Yepez et al. [27]. The articles were published between 1995 and 2020, with the last one published on 15 December 2020. The research was conducted in the timespan between December 2020 and November 2021.

In the first step, the following keywords were used: ((algae OR algal) AND photobioreactor) AND (astaxanthin). The areas that were included in the search scope included: title, abstract and keywords. In the end, the authors identified 408 articles fitting the criteria. In the following step, the authors searched through peer-reviewed articles written in English. In the last step, the data were checked for duplicate records, which were removed. In addition, articles not containing an abstract, together with notes, editorial materials, books and book reviews, as well as an erratum and tutorials, were excluded. The final sample consisted of 367 articles. The authors prepared the pre-processed data as described by Wilson [28] and Sarkar and Maiti [29], using the Scopus database presentation as the foundation.

Once the search was complete, the data underwent a descriptive analysis and were organized according to the year of publication, the journals in which they were published, the contributions of different authors or different universities, and the contributions of different regions/countries.

With the descriptive analysis complete, a scientometric analysis was performed on a sample of 367 articles using the software VOSviewer (ver. 1.6.14.) (http://www.vosviewer.com/) to construct scientific maps. By analyzing this field’s most relevant studies, the authors were able to track the evolution of the research in this area over time. The methodology and assumptions of the analysis were based on Eck and Waltman [30,31]. In addition, the contents of the articles, together with co-citations, citations and co-authorships, underwent an analysis to identify the influences of said authors, articles, journals and keywords in the research area studied.

3. Results

3.1. Descriptive Analysis

The number of articles published from 1995 to 2020 (up to December) reveals that algae are gradually gaining the attention of researchers worldwide (Figure 1). From 1999 to 2014, there was only a very small number of publications. Then, an increasing trend was observed (December 2020).

In the analyzed sample, articles constituted 68%, and reviews accounted for 17% (Figure 2).

Only fourteen journals published five or more articles. These journals, presented in Figure 3, contributed 38% of the published articles within the studied domain. Bioresource Technology was the most productive journal, with 49 published papers. The subsequent places went to Algal Research (19), Journal of Applied Phycology (16) and Bioprocess and Biosystems Engineering (9). Many journals active in publishing articles on plant-based innovation suggest that different points of view have been presented to different audiences.

Lee C.-G. published 17 articles and was the most productive, with the highest contribution among individual authors. Sim S.J. published 11 and Kim Z.-H. published 10 papers within the studied area (Figure 4).

There were five universities affiliated with a minimum of four articles (Figure 5). The remaining 13 universities contributed only up to two publications. Two universities, Inha University from South Korea and the Chinese Academy of Sciences, showcase a strong representation of studies concerning algae.

In total, authors from China published 64 papers on algae (Figure 6). The USA came in second with 40 articles, followed by South Korea (33) and Spain (28 articles). As it turns out, there is a mismatch between the distribution of articles sorted by country/region and the authors’ region/country distribution. The total number is dependent on the frequency of their occurrence in papers, as pointed out by Sarkar and Maiti [25].

3.2. Scientometric Analysis

Firstly, a scientometric analysis was performed to analyze authors’ impact (VOSViewer); the minimum number of citations and the minimum number of authors were set at 5 and 1, respectively. A sample of 20 authors out of 1232 meeting the thresholds was obtained (

Table 1. Authors with high article impacts based on normalized citations (min. 5).

Author	Number of Articles	Citations	Total Link Strength	Normalized Citations	Average Year	Average Citation
Chang J.-S.	6	1317	0	14.33	2014.83	219.50
Wang J.	9	519	15	13.26	2015.00	56.67
Liu J.	8	414	11	11.89	2015.50	51.75
Sim S.J.	10	227	0	11.50	2015.10	22.70
Hu Q.	7	349	8	10.66	2014.71	49.86
Li Y.	9	240	6	10.42	2015.89	26.67
Lee C.-G.	17	613	22	9.79	2010.35	36.06
Zhang W.	5	456	12	9.05	2013.40	91.20
Liu T.	5	428	12	8.87	2013.80	85.60
Lee H.-S.	5	105	12	7.34	2010.00	21.00
Melkonian M.	6	123	0	7.34	2017.67	20.50
Morosinotto T.	5	422	0	7.27	2013.00	84.40
Kim Z.-H.	11	167	22	5.70	2012.91	15.18
Sun Z.	6	184	2	5.69	2014.83	30.67
Kumar A.	5	82	0	5.47	2017.60	16.40
Katsuda T.	5	167	0	3.82	2010.40	33.40
Guerrero M.G.	5	673	0	3.59	2007.40	134.60
Park H.	6	61	14	2.75	2016.33	10.17
Jeffryes C.	5	58	0	1.65	2015.40	11.60
Powtongsook S.	5	73	0	1.47	2014.40	14.04

). The number of authors in the largest connected author set was 7, which is shown in Figure 7.

Authors with the highest number of articles were not the most cited, as exemplified by Lee C.-G. and Kim Z.-H.

Table 2. Articles with high numbers of citations.

Authors	Year	Cited by	Normalized Citations	% of Total Sum of Citations (n = 26,015)
Chisti Y. [32]	2007	6137	472.08	23.59
Brennan L., Owende P. [33]	2010	2695	269.50	10.36
Chen C.-Y., Yeh K.-L., Aisyah R., Lee D.-J., Chang J.-S. [34]	2011	1088	120.89	4.18
Ugwu C.U., Aoyagi H., Uchiyama H. [35]	2008	660	55.00	2.54
Greenwell H.C., Laurens L.M.L., Shields R.J., Lovitt R.W., Flynn K.J. [36]	2010	527	52.70	2.03
Posten C. [37]	2009	412	37.45	1.58
Borowitzka, M.A. [38]	1999	743	35.38	2.86
Del Campo J.A., García-González M., Guerrero M.G. [39]	2007	434	33.38	1.67
Kumar K., Dasgupta C.N., Nayak B., Lindblad P., Das D. [40]	2011	287	31.89	1.10
Suali E., Sarbatly R. [41]	2012	239	29.88	0.92
Carvalho A.P., Silva S.O., Baptista J.M., Malcata F.X. [42]	2011	229	25.44	0.88
Lee Y.-K. [43]	2001	355	18.68	1.36
Janssen M., Tramper J., Mur L.R., Wijffels R.H. [44]	2003	310	18.24	1.19
Apt K.E., Behrens P.W. [45]	1999	268	12.76	1.03
Sum of citations of top 14 documents		11,426		43.92
Rest of the documents		14,589		56.08

lists highly cited articles as well as the most influential articles based on the normalized number of citations.

The top ten documents were cited by more than 51%. The most influential paper was authored by Chisti [32], which has a 23% share of citations of all of the documents in the analyzed database. However, the oldest document with a high citation number was by Borowitzka [38], with a 2.85% share of total citations. In turn, the newest article by Suali and Sarbatly [41] had a share of total citations equal to 0.92% (Table 2).

For scientometric analysis of the impact of countries, the fractional counting method was used. The threshold for the maximum number of authors per article was set at 25, the minimum number of articles from a country was set at 5, and the minimum number of citations for a country was set at 1. In the next step, 22 of the 57 countries were selected. A visual presentation of the impact of countries on the research domain is shown in Figure 8.

The countries displayed in Figure 8 are clustered in five groups based on their citation networks. The influence of countries’ research is presented in

Table 3. Impact of countries with high article impacts based on normalized citations.

Country	Documents	Total Citations	Normalized Citations	Average Year	Average Citation	Average Normalized Citations
China	64	2351	76.46	2015.34	36.73	1.19
United States	40	2566	40.61	2013.97	64.15	1.02
Australia	20	478	29.26	2015.25	23.90	1.46
Spain	28	1773	29.07	2012.46	63.32	1.04
South Korea	33	911	27.14	2012.79	27.61	0.82
Italy	22	830	25.82	2016.50	37.73	1.17
India	25	776	23.63	2016.76	31.04	0.95
Germany	18	787	17.83	2015.56	43.72	0.99
Japan	15	1195	17.31	2010.80	79.67	1.15
Portugal	12	614	15.29	2015.58	51.17	1.27
Taiwan	7	1387	15.07	2014.00	198.14	2.15
United Kingdom	11	728	13.89	2016.73	66.18	1.26
Netherlands	7	458	10.00	2016.29	65.43	1.43
France	12	589	9.72	2013.33	49.08	0.81
New Zealand	6	6517	9.23	2008.33	1086.17	1.54
Belgium	10	190	6.62	2015.20	19.00	0.66
Thailand	6	101	5.40	2015.17	16.83	0.90
Malaysia	10	481	4.83	2016.30	48.10	1.80
Iran	5	53	4.83	2018.20	10.60	0.97
Turkey	7	48	3.59	2016.43	6.86	0.51
Brazil	7	52	3.34	2017.00	7.43	0.48
Canada	6	52	2.72	2016.50	8.67	0.45

, where all of the countries are classified based on their “Normalized Citation” score. China, the United States, Australia, Spain and South Korea were the most influential countries with the highest contribution to knowledge within the studied research field.

Keywords denote the main contents of any study and narrate research themes in a particular domain. The authors’ keywords’ co-occurrence represents the closeness of the inter-relationships among them. Keywords having the same semantic meaning, e.g., “alga” and “algae”, were combined. Then, using VOSViewer with fractional counting, the minimum number of occurrences of a keyword was set at 5. This resulted in selecting as many as 44 keywords out of 889. The keywords displayed in Figure 9 are clustered into seven groups. Keywords in the same cluster have close inter-relationships; for example, “microalgae” and “photobioreactors” are co-studied in “productivity”.

The influence of obtained keywords is presented in

Table 4. Qualitative summary of the influence of authors’ keywords.

Authors’ Keyword	Occurren-ces	Total Link Strength	Average Year	Average Citation	Average Normalized Citation	Cluster
irradiance	7	6	2013.14	25.29	2.24	4
photosynthetic efficiency	6	5	2013.33	103.00	2.24	5
astaxanthin	52	51	2011.65	40.29	1.78	4
attached cultivation	5	4	2014.60	64.00	1.78	5
harvesting	5	5	2015.60	224.40	1.74	3
bioenergy	7	6	2013.86	402.86	1.73	1
lipids	18	18	2015.78	40.11	1.59	1
pigments	6	5	2013.00	58.00	1.57	1
stress	5	5	2012.80	63.60	1.45	6
heterotrophy	6	5	2014.67	73.17	1.44	7
Nannochloropsis	6	6	2013.00	79.17	1.34	1
algae	17	16	2015.00	87.41	1.29	2
photosynthesis	9	8	2016.00	52.78	1.28	1
biodiesel	30	29	2013.27	273.73	1.25	3
Haematococcus	11	10	2010.36	45.09	1.25	4
microalgae	145	118	2014.55	116.26	1.25	5
biomass	26	24	2014.35	71.50	1.23	1
biorefineries	9	9	2015.67	80.11	1.19	1
biofuels	35	35	2014.46	235.17	1.16	1
Haematococcus pluvialis	47	43	2012.87	34.32	1.15	4
photobioreactors	132	115	2013.35	131.33	1.15	5
flashing light	6	6	2013.83	29.33	1.12	1
continuous culture	5	5	2011.82	24.00	1.08	4
cyanobacteria	10	9	2014.90	52.10	1.08	5
light intensity	6	6	2015.67	46.17	1.08	2
microalgal cultivations	7	6	2013.71	55.71	1.05	3
scale up	11	10	2014.73	36.91	1.02	4
wastewater treatment	7	7	2018.00	20.57	1.02	5
Chlorella zofingiensis	10	8	2014.00	52.10	1.01	3
lumostatic operation	5	5	2009.40	26.20	0.95	4
productivity	7	7	2014.86	25.10	0.95	5
fatty acids	9	8	2014.78	30.67	0.82	6
outdoor culture	6	6	2008.83	71.17	0.81	3
carotenoids	21	20	2012.62	27.62	0.73	6
CO2	16	15	2015.44	24.56	0.69	6
tubular photobioreactors	7	6	2009.71	30.14	0.68	2
lutein	16	13	2015.12	36.94	0.62	2
Chlorella	16	15	2011.81	69.69	0.60	3
flue gases	5	5	2016.20	10.40	0.60	2
lipid content	5	4	2016.20	12.40	0.46	3
growth rate	6	6	2014.50	8.67	0.30	6
airlift reactor	5	5	2013.40	20.40	0.23	2
light	5	5	2017.40	63.60	0.19	6
Chlamydomonas	6	6	2013.17	18.00	0.15	2

, where the keywords are listed according to the “average normalized citation”.

The keywords “irradiance”, “photosynthetic efficiency”, “astaxanthin” and “attached cultivation” are at the top of the list according to the average normalized citation score. The “average publication year” reflects how recent the used keywords are in the research community. The results imply that these keywords are important in this research domain. It can be seen in Table 4 that the oldest keywords are, among others, “outdoor culture”, “lumostatic operation” and “tubular photobioreactors”. Recently, “flue gases”, “lipid content”, “light” and “wastewater treatment” have increased in frequency. According to the number of occurrences, keywords such as “microalgae”, “photobioreactors”, “astaxanthin”, “Haematococcus pluvialis”, “biofuels”, “biodiesel” and “biomass” have emerged at the top.

4. Content Analysis

On the basis of the content analysis, seven leading areas were identified, which were addressed in the research work:

• Techno-economic profitability of biofuel, bioenergy and pigment production in microalgae biorefineries.
• Influence of photobioreactor design and process parameters on the efficiency of microalgae cultivation.
• Strategies for increasing the amount of obtained lipids and obtaining biodiesel in the cultivation of Chlorella microalgae.
• Industrial production of astaxanthin using Haematococcus microalgae.
• Productivity of biomass and the use of alternative carbon sources in microalgae farming.
• Influence of light and carbon dioxide conversion on biomass efficiency.
• Heterotrophy.

The keywords in the clusters and the publication content served as a basis for each scientific area’s construction. The title of each area was proposed to reflect the specificity of the research work described. For example, the keywords: “harvesting”, “biodiesel”, “microalgal cultivations”, “Chlorella zofingiensis”, “outdoor culture”, “Chlorella” and “lipid content” were in Cluster 3 (Table 4). On this basis, the title was formulated: “Strategies for increasing the amount of obtained lipids and obtaining biodiesel in the cultivation of Chlorella microalgae”.

When analyzing the content of publications included in each cluster, the most significant emphasis was placed on the various strains of microalgae used in the research and the structures of photobioreactors used in the experiments. Topics related to bioenergy production and biofuels played a dominant role in the research. Of the seven identified leading areas, two clusters were closely related to the production of bioenergy and biofuels (Cluster 1—Techno-economic profitability of the production of biofuels, bioenergy and pigments in microalgae biorefineries; Cluster 3—Strategies for increasing the amount of obtained lipids and obtaining biodiesel in the cultivation of Chlorella microalgae). Additionally, the remaining leading areas related to research on new photobioreactor designs (Cluster 2), increasing the productivity of microalgae biomass (Cluster 5) or heterotrophy (Cluster 7) included many articles focused on the use of microalgae for energy purposes.

4.1. Techno-Economic Profitability of the Production of Biofuels, Bioenergy and Pigments in a Microalgae Biorefinery

Due to the excessive consumption and depletion of fossil fuels such as petroleum fuels, coal and natural gas, producing alternative fuels is becoming increasingly urgent, especially since the burning of fossil fuels is also one of the main factors contributing to global warming of the climate. Microalgae are considered an attractive source of bioenergy and biofuels due to their high lipid productivity (40–80% of dry weight), photosynthesis efficiency and efficient reduction of CO₂ [46]. They show a lower demand for land than plant crops and can grow in challenging conditions and in areas unsuitable for food plants [6]. The advantage of microalgae is their short cultivation cycles (1–10 days), low water quality requirements and smaller requirements for nutrients and fertilizers [47].

Strains capable of inducing large amounts of lipids and carbohydrates were used in research related to the use of microalgae for the production of biofuels and bioenergy. The lipid fraction of algae biomass is transformed into biodiesel by transesterification. Microalgae that can achieve high levels of polysaccharides as a component of the cell wall and inside the cells can be fermented to bioethanol [2]. The lipid productivity depends on both the biomass yield and the lipid content of the cells. Unfortunately, during microalgae growth under stressful conditions, the lipid content increases, but the biomass yield decreases [48]. The lipid content of microalgae usually ranges from 20–60% of dry biomass, depending on the species and growing conditions. Dunaliella tertiolecta ATCC 30929 shows the highest level of lipid content (60.6–67.8% of dry weight). The highest lipid productivity is achieved by Chlorella sp. (121–178 kg/m³/day with a fat content of 32–34%) [14]. The most popular types of microalgae used to produce biodiesel are microalgae of the genera Chlorella, Scenedesmus and Monoraphidium. The microalga Botryococcus braunii is also a rich source of lipids (up to 75% of dry weight), but its lipid productivity is very low due to its low growth rate [2]. When planning experiments, scientists first used these species. Song et al. investigated the requirements for micronutrients that increase biomass production and hydrocarbon recovery by the microalgae Botryococcus braunii UTEX 572 [49]. Kim et al. studied lipid accumulation under flashing light conditions using one of the most studied microalgae with a publicly available complete genome sequence, Chlamydomonas reinhardtii [50]. Dasan et al. [51], using aerated column photobioreactors with sequential atmospheric airflow (air exiting one photobioreactor flowed into the next), developed a strategy to induce stress for lipid accumulation and carbon dioxide binding in a culture of Chlorella vulgaris using a nutrient medium made of organic fertilizer [51]. Based on the same species of microalgae, Hossain et al. [52,53] based their research on techno-economic and environmental analyses. The microalgae C. vulgaris contains 55% carbohydrate and comprises two main components: cellulose in the cell wall and starch in plastids. Both cellulose and starch can act as carbon sources during hydrolysis and fermentation for bioethanol production [52,53].

For research on pigment production, scientists used the most popular and best-studied strain of microalgae, Haematococcus pluvialis [54,55]. At the same time, however, the results of experiments revealed new strains of microalgae that could be an alternative to H. pluvialis thanks to their unique physiological characteristics. Masojídek et al. [56] used Chlorococcum sp. with higher temperature tolerance and a faster growth rate in their research. The ability to produce secondary carotenoids under stress conditions (nitrogen deficiency, salinity and high light intensity) in an outdoor photobioreactor was experimentally assessed [56]. Additionally, Wannachod et al. [57] used the strain Chlorococcum humicola (TISTR 8641) in their study; apart from its high growth rate and tolerance to high doses of CO₂, it is distinguished by the possibility of culturing in wider pH and temperature ranges compared to H. pluvialis. Additionally, it is easier to harvest biomass using conventional methods such as filtration and sedimentation. This is due to the relatively large cell size and the tendency to self-flocculate. An interesting proposal also appeared in the research by Carbone et al. [58], in which the microalgae Galdieria sulphuraria strain 064 ACUF was used, which is a prospective alternative to Arthrospira platensis (Spirulina) for the production of phycobiliproteins. These thermophilic microorganisms thrive in geothermal volcanic areas at temperatures around 40 °C and high sulfuric acid concentrations. It allowed conducting experiments at 34 °C and pH = 1.5, with high biomass density and acceptable phycobiliprotein productivity [58].

Studies in larger volumes were carried out by, among others, Masojídek et al. [56], who optimized stress conditions for the synthesis of carotenoids by the microalgae Chlorococcum sp. The experiments were carried out in a tubular, horizontal photobioreactor built of 10 parallel glass pipes with a total volume of 50 L, placed outdoors [56]. In addition, Pérez-López et al. [59], in investigating the main factors responsible for the production of compounds with high added value for pharmaceutical, cosmetic and food applications by Tetraselmis suecica microalgae, used a column photobioreactor with aeration and a working volume of 80 L, working indoors [59]. The innovative solution of the photobioreactor structure proposed in the research by Moroni et al. [60], in which a sloping cascade photobioreactor with a wavy bottom was designed, where the microalgae culture flows by gravity, is also noteworthy. At the design stage, the scientists used the possibilities of computational fluid dynamics (CFD) modeling, i.e., simulating the dynamic behavior of fluids in complex physical systems with appropriate mathematical models [60].

In addition to issues related to technology, scientific publications dealt with the analysis of production costs, energy profitability and product life cycle. This is especially important for the production of biofuels, for which microalgae are regarded as the best alternative to fossil fuels [61]. In their research, Christiansen et al. [61] compared the costs and efficiency of microalgae production in open running ponds, tubular photobioreactors and mixed systems resulting from the combination of photobioreactors and running tracks. The smallest actual cost overrun in relation to the estimated design cost was noted for open running ponds and mixed systems. The highest production efficiency was recorded for the open running joints. Research results suggest that the economic viability of producing microalgae for biofuel purposes is highly uncertain and risky. The costs of starting the production of biofuels from microalgae may be 3 to 14 times higher than expected (

Table 5. Examples of techno-economical assessments of technologies using algae.

Description	Costs
Techno-economical assessment of bioethanol commercialization from Chlorella vulgaris in Brunei Darussalam. Two types of cultivation systems, namely, closed-system (photobioreactor—PBR) and open-pond approaches, were anticipated for an approximate total biomass of 220 t year−1 on 6 ha coastal areas. The biomass productivity was 56 t·ha−1 for the PBR and 28 t·ha−1 for the pond annually. The plant output was 58.90 m3·ha−1 for the PBR and 24.9 m3·ha−1 for the pond annually. The total bioethanol output of the plant was 57,087.58 gal·year−1, along with the value-added by-products (crude bioliquid and slurry cake).	The total production cost of this project was USD 2.22 million for bioethanol from microalgae, and the total bioethanol selling price was USD 2.22–2.87 million, along with a by-product sale price of USD 1.6 million [52,53].
Three production technologies were investigated: (1) open raceway ponds (ORPs), (2) tubular photobioreactors (PBRs) and (3) systems coupling photobioreactors to open raceway ponds.	The greatest cost growth (1.5–1.8) was estimated for PBR systems, while the lowest cost growth (1.2–1.4) was estimated for the ORP systems and coupled systems. Plant performance ranged from 13% to 40% of nameplate capacity [61].
Assumption: to leave scale out of the analysis to avoid disproportionate comparisons.	There was nearly a 25-fold difference in total cost per hectare between the ORP and PBR/PBR–ORP systems modeled, even with similar cost growth. If the algae were only 30% oil, the unit cost growth factor for algal oil would be somewhere between 10 and 46 times the proposed price [62].
Four production pathways, ranging from a base case with commercial technologies to an improved case with innovative technologies, were studied. All region-specific parameters were adapted to Belgian conditions.	The lowest carotenoid price for which the process had a positive NPV in the different scenarios, keeping all other parameters constant, was EUR 1059 per kg β-carotene (base scenario), EUR 657 per kg β-carotene (intermediate scenario), EUR 1379 per kg β-carotene (advanced scenario) and EUR 4725 per kg astaxanthin (alternative scenario). The most profitable scenario was the scenario that uses a specialized membrane for medium recycling and an open-pond algae cultivation [63].

). In the short to medium term, cheap algae biofuel is not expected [61]. A popular indicator of the profitability of a technological process is the net energy ratio, which is the ratio of the total energy produced (energy content of oil and residual biomass) to the energy needed to build the PBR and carry out all technological operations [6]. Razon and Tan [62] conducted a net energy analysis of two systems for producing biodiesel and biogas from microalgae grown in running ponds. The results leave no room for doubt. None of the studied systems would be cost-effective as a system designed solely for photosynthesis to produce energy for humans. The greatest energy demand is in oil recovery operations such as drying and cell disruption. A financially viable process is only possible if energy products are generated as by-products in a multifunctional biorefinery system, combined with, for example, carbon dioxide capture and wastewater treatment [62]. These conclusions are confirmed by the results of a study by Thomassen et al. [63]. They assessed the technological potential and economic viability of a microalgae-based biorefinery that produces two products: a high-value carotenoid and a fertilizer consisting of residual biomass. The scientists investigated four scenarios. The baseline scenario was farming Dunaliella salina in open paddlewheel ponds. A scenario with an added filtration step used an integrated permeate channel membrane (IPC), which thickens the biomass, allows bacteria and impurities to be filtered out, and allows water and salt to be recycled. The third scenario was the cultivation of D. salina in a closed photobioreactor, allowing for higher biomass yields, and an alternative scenario was the cultivation of Haematococcus pluvialis microalgae to produce astaxanthin and fertilizer. The scientists observed large differences between these scenarios’ technological and economic parameters, but the results confirmed the economic viability of the algae-based biorefinery [63]. Some details about techno-economical assessments are presented in Table 5.

The most effective method of environmental evaluation of sustainable microalgae biomass production is LCA (life cycle assessment), which can be performed in conjunction with economic analyses. Although the production costs of biofuels from microalgae are higher than those of fossil fuels, microalgae are a renewable and environmentally friendly source. For this reason, LCA for biofuel production becomes a more important factor than a standard cost analysis [6]. Hossain et al. [52,53] performed a techno-economic analysis, sensitivity analysis and LCA of microalgae as a raw material for commercial bioethanol production in the Brunei coastal zone. The scientists simulated a large-scale installation with technological details of the production system, CO₂ emissions, water and soil footprints and energy balance for a projected project duration of 20 years. Several life cycle assessments of different methods of cultivating microalgae and extracting value-added products such as vitamins (e.g., β-carotene), proteins, bio-oils and other biofuels (e.g., biodiesel, biohydrogen, biomethane and biochar) were carried out. The results of the analysis show a positive effect of microalgae-to-bioethanol conversion on energy and environmental aspects. The project was considered energy-efficient, environmentally friendly and economically beneficial, even when the sensitivity analysis covered variable ranges of all factors [52,53]. In the studies by Pérez-López et al. [59], life cycle assessment looked at the production of bioactive compounds using the microalgae Tetraselmis suecica and was based on the environmental performance of a pilot-scale installation. The use of microalgae residues after lipid extraction for biogas production and the production of substitutes for nitrogen-based mineral fertilizers was considered. The research took into account the impact of individual stages of the system (sterilization, inoculation and culturing, harvesting, extraction and biogas) on the environmental profile. The authors [59] identified the greatest demand for electricity at the stage of inoculation and cultivation and proposed alternative nitrogen sources, resulting in a significant reduction in the environmental profile. In turn, Monari et al. [64] analyzed greenhouse gas emissions and the energy balance of biodiesel production from microalgae grown in industrial-scale photobioreactors in Denmark. By combining different technologies at different production stages, the scientists tested twenty-four energy demand and greenhouse gas emission scenarios using LCA and compared them with fossil diesel fuel. The scenarios considered using fresh water, wastewater, synthetic and waste CO₂, flocculation, centrifugation, oil extraction with hexane and supercritical CO₂, and the management of glycerol, a by-product of biodiesel production. The research results showed that currently produced microalgae biodiesel has a higher energy requirement and higher greenhouse gas emissions than diesel extracted from the ground, especially if using wastewater and CO₂-rich flue gases from industrial power plants. Further development of biodiesel production technology is needed to obtain a positive energy balance [64].

4.2. The Influence of Photobioreactor Design and Process Parameters on the Efficiency of Microalgae Cultivation

4.2.1. Photobioreactors

Among the various designs of photobioreactors used to cultivate microalgae, the most popular solutions are presented in

Table 6. Examples of the most popular photobioreactors used for the cultivation of microalgae.

Type of Reactor	Description
Closed continuous-run tubular loop bioreactors	Connected with pipes with a diameter usually less than 0.08 m [65].
Vertical column reactors	Column diameter > 0.1 m [65].
Outdoor bubble columns and airlift bioreactors	0.19 m column diameter, 2 m tall, 0.06 m3 working volume; outdoor bubble column and airlift photobioreactors [65].
Panel photobioreactor	C-shaped flat vessel of various widths and heights (usually less than 1 m) and a thickness of 1–5 cm, made of transparent materials. PBR flat panels are positioned vertically or tilted towards the sun [44].
Hybrid reactors	Thin-film PBRs combined with bubble columns [66].
PBRs with internal lighting	Lighting in the form of submerged fluorescent lamps, LEDs, optical fibers or waveguides is a solution to the shading of the deeper layers of microalgae cultures [67].
The phosphorescent materials inside the PBR emit light in the dark phase, extending the microalgae culture’s illumination time.	The phosphorescent materials inside the PBR increased cells’ specific growth rate and dry mass by about 9% and 24%, respectively, due to the light emission of these materials in the dark phase [68].
Tubular PBR using dual Fresnel linear lenses	Lenses concentrated sunlight on the surface of glass culture tubes placed on a movable frame installed on a vertical wall and under a sloping roof window (42° angle). An automatic tracking system kept the glass cultivation tubes in the focus of the collector lenses as the sun changed its position. The use of collectors on vertical walls was beneficial in spring and autumn when the angle of inclination of the sun was small. In spring, the total radiation energy collected by both types of panels was similar. In summer, (sloping) roof collectors delivered, on average, 4–12 times higher radiation intensity on the pipe surfaces than vertical collectors [69].
Unusual geometric arrangements of 5-L X-shaped and H-shaped photobioreactors with two aeration bubblers and four serially arranged and connected columns with one bubbler in each column were tested	Increases in biomass and lipid production and high content of induced monounsaturated fatty acids were observed [70].
Transparent and gas-permeable micro-reactors made of poly(dimethylsiloxane)	There are special chambers and microchannels that allow obtaining the height of the culture substrate at a level of 3.5 mm [71].

.

Scientists hope for a strategy to cultivate microalgae in the form of biofilms, which are many times denser than plankton cells. Thanks to this, in addition to saving pumping costs, the dehydration of a kilogram of dry algae costs about 0.3% of that of open ponds [67].

Two thematic groups can be distinguished among scientific publications on photobioreactors (

Table 7. Mode of operation, parameters and efficiency of various types of photobioreactors—main achievements.

Description	Achievements
Biomass production in three types of vertical reactors with identical dimensions: bubble column, split-cylinder airlift device and a draft-tube airlift bioreactor.	The results proved that cells in all types of PBR were subjected to identical values of mean intensity of irradiation [65].
Compared light gradients and mixed light/dark (L/D) cycles and the performance of an aeration column, tubular reactor and flatbed reactor.	The light regimes and productivity in aerated and airlift column reactors were similar to each other [44].
Compared the results of H. pluvialis cultures in column and tube reactors in order to find the best reactor for outdoor production of astaxanthin.	Better biomass and astaxanthin productivity results were obtained in tubular photobioreactors [74].
Compared the mixing time, volumetric mass transfer coefficient kLa and the microalgae growth profile for an aerated column and airlift photobioreactor.	Better cell growth efficiency was observed in the airlift PBR, in which microalgae cells spent only 2 s in the riser (dark zone) and the remaining 10 s (or 84% of the time) in the downcomer (light zone) in a 12 s mixing cycle [75].
Studies of the influence of the geometric shape of photobioreactors on the cultivation of Cylindrotheca closterium diatoms. A bag photobioreactor made of polyethylene, a flat one made of plexiglass and a bubble made of glass were tested experimentally.	The highest values of cell and chlorophyll-a concentration, specific growth rate and doubling time were achieved in a bag photobioreactor [76].
Comparison of airlift photobioreactors and an agitated photobioreactor with the same working volume and operating conditions.	Due to the higher liquid height and different fluid flow patterns, culture in airlift photobioreactors resulted in a higher concentration of biomass and carotenoids at the end of the culture than when using an agitated photobioreactor with the same working volume and operating conditions [77].
Proposed a hybrid photobioreactor made of an opaque, columnar bubble reactor connected to a lighting platform built of eight open tubular structures arranged in a linear manner, with an inclination of 9°. The lighting platform was connected by a flow pump to the bubble column.	The hybrid PBR requires a much smaller area, ensuring the appropriate surface-to-volume ratio, which is extremely important, especially when scaling the solution to industrial conditions [66].
Study on the concentration of light in a PBR. They used waveguides illuminated by external light using three different specially designed and 3D-printed types of paraboloid mirrors to cultivate algal biofilm.	The productivity of algae biofilm biomass on the waveguide was 2.5 times higher when using mirrored concentrators [67].
Successfully tested a novel double-layered column photobioreactor consisting of two glass tubes, allowing the simultaneous growth of H. pluvialis microalgae cells and astaxanthin accumulation. An inner tube was destined for the vegetative growth of cells, which received light energy reduced by mutual shading by cells in the outer mantle.	An excessive amount of light reached the outer layer, which increased the accumulation of astaxanthin in the cells [78].
Study of a photobioreactor built from one tube inside the other but arranged horizontally, with aeration holes in the inner tube for both aeration and mixing of the culture.	The new photobioreactor assured the mitigation of changes in pH and dissolved oxygen along with the extension of the cultivation time, which resulted in obtaining high biomass productivity [79].
Proposed new construction of a flat airlift photobioreactor, the volume scaling of which consisted of increasing its length to deal with the problem of losing hydrodynamic properties with increasing culture scale.	The 90 L PBR performed just as well as the 17 L one, only with a slightly slower growth rate. The results of the economic analysis showed that although the operation of the 90 L PBR was associated with higher utility costs, the unit cost of production was lower due to the large number of cells grown in one batch. In smaller systems, it was necessary to repeat the process several times to obtain the same number of cells [80].

). These are publications in which scientists compare the mode of operation, parameters and efficiency of various types of photobioreactors [44,65] and articles presenting innovative solutions designed to achieve better results in the cultivation of microalgae [72,73].

Systems with lighting inside the photobioreactor are a potential way to solve the problem of uneven access of microalgae cells to light. Since 2015, there have been several scientific articles describing such solutions. Hincapie and Stuart [81] successfully tested a new airlift photobioreactor design with internal fiber optic lighting. The pipes were made of white polyvinyl chloride (PVC) to maximize light reflection and provide separation between the dark and light zones. Placing the optical fibers in the riser allowed them to be kept clean and prevented biofouling throughout the entire cultivation period [81]. Additionally, Jeffryes et al. [72] presented the concept of a flat photobioreactor with internal lighting, which, thanks to its construction, also acted as a barrier, forming an airlift photobioreactor. In this way, a very short light path of 2 cm in the riser and downcomer was obtained [72]. Rebolledo-Oyarce et al. [73] proposed an innovative version of the airlift photobioreactor with internal lighting. The light source was placed in the inner tube to ensure uniform light distribution, and baffles were added to the outer ring containing the microalgae to divide the riser and downcomer tanks. The scientists investigated the effect of different light colors and different radiation intensity models on biomass production [73].

Scientists are still faced with the problem of scaling the process to the level required for commercial production, as most of the research is carried out on a laboratory scale [82]. It is essential to understand the impact of critical scale-up parameters such as surface gas velocity, mixing time and circulation speed [83]. For the construction of photobioreactors, materials tested during the tests should be selected so that they will not interfere with the process (e.g., polyvinyl chloride (PVC) is suspected of supporting biofilm formation [81]). If possible, materials should also be used to reduce the cost of producing photobioreactors, such as cheap PVC films instead of thick polycarbonate plates [72]. When increasing the scale, it should also be remembered that the costs of materials and production of devices and systems for subsequent units, e.g., lighting systems, will decrease [44].

When producing microalgae on a large scale, particular attention should be paid to the production of appropriate amounts of inoculum, the monitoring of the culture to capture disturbances in the course of the process, the development of cultivation procedures in changing environmental (external) conditions and the prevention of loss of culture in the event of equipment failure. This task is difficult as it requires different procedures depending on the workplace of the cultivation system and the microalgae strain used [82].

Two-stage microalgae cultivation should be simplified, growth in the green phase should be increased, and better utilization of CO₂ should be ensured to increase the accumulation of bio-compounds [84]. High hopes are associated with the development of hybrid microalgae cultivation systems, which will allow the use of various types of photobioreactors at individual stages of cultivation, eliminating the problem of culture contamination and the use of one or more stress induction techniques [85]. In the case of the process of stressing the microalgae culture, there have been attempts to use electric and electromagnetic fields to increase the yield of microalgae [5]. The technology for the harvesting and filtration of biomass and lipid extraction also requires improvement. Extraction of CO₂ in the supercritical state seems to be of particular interest [14]. Research results also suggest using wastewater and CO₂-rich flue gases from industrial power plants in industrial-scale processes. Photobioreactor systems should be created near objects that are a source of CO₂. Large distances from such a source will significantly increase the costs of microalgae cultivation. The energy demand for mixing, pumping and other technological operations must also be significantly reduced [64].

4.2.2. Parameters of Microalgae Cultivation

The cultivation efficiency of microalgae depends on many factors, e.g., temperature, pH, light intensity, photoperiod, reactor design and hydrodynamic factors, such as the flow rate, mixing and mass transfer of CO₂ in the growth medium, and the nitrogen and phosphorus nutrient concentration and ratio. The combination of these factors can significantly affect biomass productivity, intracellular composition and chlorophyll content [84]. For this reason, scientists, in their research, have often addressed the issue of the influence of individual factors on the culture efficiency and the induction of valuable substances inside the cells of microalgae.

Of course, the primary environmental factors are temperature and pH. Microalgae cells react to their changes almost immediately by adjusting physiological changes, nutritional requirements and biomass composition [86]. Guedes et al. [86] investigated the influence of temperature and pH on the growth and antioxidant content of the microalga Scenedesmus obliquus. They aimed to find the optimal combination of temperature and pH for maximum biomass and antioxidant content. Temperature influenced the specific growth rate more intensely than pH. In the production of antioxidant compounds, both pH and, to a lesser extent, temperature play an essential role. The growth of biomass is favored by low pH = 6 and high temperature (30 °C), while high pH = 8 is conducive to producing antioxidant compounds [86].

One of the most critical components of the culture media is nitrogen, the main component of proteins, nucleic acids and chlorophylls, influencing biomass production and specific metabolites. The effects of culture parameters on the efficiency of biomass growth are presented in

Table 8. Effects of culture parameters on efficiency of microalgae biomass growth.

Description of the Culture Conditions or Parameters	Effect on Biomass Growth
The growth kinetics of microalgae and lipid synthesis for various temperatures and light colors were tested.	Red-orange light at a temperature of 24 °C obtained a 38% higher biomass productivity than blue light. In contrast, blue light at 32 °C was 13% more productive than red-orange light. The accumulation of lipids was favored by red-orange light and a temperature of 30–32 °C [87].
Tetradesmus acuminatus culture conducted in a flat-plate PBR was assessed. The increase in biomass and its relationship with the maximization of carotenoid production (astaxanthin and β-carotene) under the influence of light intensity, photoperiod, pH, NaCl and nitrogen concentration were studied.	The increase in light intensity and irradiation time favored the growth of microalgae biomass and resulted in the highest concentration of carotenoids. The maximum production of carotenoids in mg g−1 was observed at 85 μmol m−2 s−1 light intensity and a light/dark photoperiod of 15.8:8.2 h, and in mg L−1, it was at 595 μmol m−2 s−1 light intensity for 24 h [88].
The correlation between aeration and lighting was also experimentally verified in Scenedesmus obtusus microalgae culture conducted in an airlift photobioreactor with different inlet gas flow rates, different light intensities and different light/dark cycles.	Higher inlet gas flow rates (0.88 and 1.17 vvm) resulted in higher biomass productivity. The maximum biomass productivity of 0.07 g L−1 day−1 was achieved with an inlet gas flow rate of 3 Lpm. The maximum biomass efficiency of 0.103 g L−1 day−1 was achieved at an illuminance of 150 µmol m−2 s−1 in continuous light [83].
Investigated the effect and optimized the nitrogen availability in combination with high light intensity on antioxidant activity and carotenoid content in Nephroselmis sp. These microalgae produce sifonaxanthin—a rare pigment for biotechnology applications.	Under the conditions of unrestricted access to nitrogen, scientists found a 3-fold increase in the content and productivity of primary carotenoids and a 2.4-fold increase in antioxidant activity. Nitrogen availability had no effect on the content of lutein and β-carotene. The experiments were carried out at a constant temperature of 26.5 ± 0.3 °C, a regulated pH level of 7.75 ± 0.04 and a light intensity of 600 µmol m−2 s−1 [89].
The ability to produce lutein by thermotolerant strains of Desmodesmus sp. —the influence of substrate composition, nitrate concentration and light intensity.	The best cell growth and lutein production were achieved with a light intensity of 600 µmol m−2 s−1 and an initial nitrate concentration of 8.8 mM. The highest productivity (3.56 ± 0.10 mg L−1 d−1) and content (5.05 ± 0.20 mg g−1) of lutein were obtained in batch culture with a pulsatile feed of 2.2 mM nitrate when its content was almost exhausted [90].
Research with the arctic microalga Chlamydomonas malina RCC2488 at a temperature of 8 °C to produce carbohydrates, lipids and polyunsaturated fatty acids (PUFA). This strain tolerates a wide range of salinity and high light intensity with a high content of produced lipids.	The highest biomass (527 mg L−1 d−1), lipid (161.3 mg L−1 d−1) and polyunsaturated fatty acid (PUFA; 85.4 mg L−1 d−1) productivities were obtained at a salinity of 17.5, light intensity of 250 μmol m−2 s−1 and nitrogen-replete conditions. Nitrogen deprivation induced the accumulation of carbohydrates in cells (up to 49.5% w/w) at the expense of proteins, but without compromising lipid biosynthesis [91].
The effect of nutrients and light intensity on the growth and biochemical composition of the marine microalga Odontella auryta grown in columnar and flat photobioreactors.	The optimal composition of the medium for the photoautotrophic cultivation of O. aurite was found, and carbohydrates, consisting mainly of β-1,3-glucan, were found to be the main storage materials produced under stress conditions. The production of lipids and eicosapentaenoic acid (EPA) was low. The highest biomass increase was obtained at a light intensity of 300 μmol m−2 s−1) and a light path length of 3 cm. The biomass production of 3.8 g L−1 obtained in the 3 cm light path photobioreactor was 46% greater than that obtained with a 6 cm light path and 110% greater than with a 12 cm light path reactor [92].
Investigated the effects of different light intensities, forms and concentrations of nitrogen, phosphorus and salinity on the growth and production of docosahexaenoic acid (DHA) in a bubble column photobioreactor (PBR). Tested 19 natural Isochrysis strains.	A record high DHA productivity was achieved, amounting to 13.4 mg·L−1·d−1. The optimal light intensity for DHA production was 60–90 µmol m−2 s−1, and the optimal phosphorus concentration was 4.5 mg L−1. Of the three light paths of 1.9, 3.8 and 7.6 cm, the 3.8 cm PBR yielded the highest volumetric biomass productivity, 0.54 ± 0.05 (g L−1 d−1) [93].

.

4.2.3. Culture Strategies

Microalgae strains’ growth and biomass composition have been studied using various culture strategies, including continuous processes and two-step cultures. The experiments conducted by San Pedro et al. [94] on continuous cultures in tubular photobioreactors investigated the effect of the dilution rate and nutrient supply on the growth and biomass composition of Nannochloropsis gaditanastrain. The good results obtained in outdoor cultures and the ability to accumulate lipids under nitrate-limiting conditions make this solution a promising candidate for biodiesel production [94]. Van Wagenen et al. [95] compared mixotrophic growth with cyclic heterotrophic/autotrophic growth in a photobioreactor’s continuous culture of Chlorella sorokiniana. Cultures were carried out on a medium supplemented with sodium acetate at concentrations equivalent to those of volatile fatty acids in the effluent from an anaerobic fermentation chamber. The acetate was added during the light period for the mixotrophic strategy and the dark period for the autotrophic/heterotrophic cyclic strategy. Cyclic heterotrophy/autotrophy resulted in higher productivity than mixotrophic growth, using half the amount of sodium acetate [95]. Lakshmidevi et al. [96] assessed the mixotrophic cultivation of Scenedesmus sp. on a medium containing synthetic sewage supplemented with gradually added crude glycerol, which is the main by-product in the production of biodiesel. Scientists [96] found that the N/P ratio had the most significant impact on the productivity of microalgae biomass, followed by the concentration of crude glycerol as a carbon source and the concentration of inhibitors—methanol and KOH. The gradual addition of crude glycerol improved the yield of biomass and lutein [96].

Much less research has been carried out on the next steps in producing valuable substances with microalgae. The optimization of the carotenoid extraction method was undertaken by Schüler et al. [97]. The best extraction method from mechanically resistant microalgae Tetraselmis sp. CTP4 proved to be a combination of breaking by glass spheres when using tetrahydrofuran applied to wet biomass. Shattering by glass spheres using acetone applied to wet biomass was almost as effective and, additionally, less expensive and less time-consuming than using freeze-dried biomass or tetrahydrofuran as a solvent. These features predispose this extraction method to industrial use [97]. Mehariya et al. [84], in their research, looked at an integrated strategy for the production of Haematococcus pluvialis nutraceuticals from cultivation to extraction. They used the method of extracting astaxanthin, lutein and fatty acids using GRAS ethanol at 67 °C and a pressure of 10 MPa. Unfortunately, compared to other studies, the developed integrated strategy showed low production efficiency [84].

Scientists have also prepared a suitable system for growing microalgae in space. Zhang et al. [98] positively evaluated the possibility of efficiently growing mutagenized microalgae Chlamydomonas reinhardtii in aerated plastic tissue culture bags in the Veggie plant growth chamber, which is used at the International Space Station for the cultivation of terrestrial plants. The scientists identified a series of genes responsible for adaptation to batch culture in breathable plastic bags. Live cultures could be stored in bags at room temperature for at least 1 month [98].

4.2.4. Light

In their research, scientists dealing with various designs of photobioreactors have emphasized that the supply of light plays a key role in their design. The aim is to achieve the greatest possible surface-area-to-volume ratio. The remaining nutrients are cheap inorganic salts and can be supplied quickly and easily [44]. The construction of large transparent surfaces is difficult and expensive, and still, cells closer to the photobioreactor walls are at risk of experiencing photoinhibition, while too little light is supplied to the cells in the center of the tank. Both phenomena reduce the productivity of microalgae [81]. The effect of light on efficiency and biomass production is presented in

Table 9. The effect of light on microalgal growth in bioreactors.

Light Conditions	Achievement
Use of fiber optic spectrometer to determine the quantitative and qualitative light characteristics of the airlift photobioreactor downcomer.	The circular geometry allows for more efficient light penetration and illumination of a larger part of the PBR interior compared to the flat geometry. Limiting light availability depends on the applied wavelength, cell concentration, PBR geometry and the penetration distance of light [99].
The effects of light quality and the delivery strategy on the growth and production of carotenoids by Chlorococcum humicola.	For the one- and two-stage strategy, among white, red and blue light, the highest carotenoid productivity was obtained with two-stage lighting. During the growth period, white light at an intensity of 5000 lux was supplied, followed by a combination of white (100,000 lux) and blue (5000 lux) light in the stationary phase [77].
The effect of different light intensities under continuous lighting as well as alternating light and dark cycles at different frequencies.	Microalgae Nannochloropsis salina can use even very intense light efficiently, provided that dark cycles occur. If the alternation of light and dark is not optimal, the algae undergo radiation damage, and the photosynthetic efficiency drops significantly. This shows how important it is to optimize mixing in a photobioreactor to ensure efficient use of light energy by microalgae [100].
Experiments with a culture of Chlamydomonas reinhardtii with high light saturation with unlimited access to CO2 in turbidostatic photobioreactors.	Significant lipid accumulation can also occur under maximum growth conditions, without slowing down nitrogen starvation as a stress factor [101].
The effects of cultivating microalgae under fluorescent light and LED diodes in six different colors: blue, purple, orange, white, green and red.	Monochromatic blue light can substitute for polychromatic fluorescent light, and the colors generating the greatest amount of biomass are blue and red, as they are absorbed by chlorophyll a and b contained in Dunaliella tertiolecta cells [73].
Research on the growth rate, biomass yield and biosynthesis of carotenoids in the microalgae Dunaliella salina using red LEDs with a wavelength of 660 nm and blue LEDs with a wavelength of 470 nm and a narrow output spectrum (20 nm band at half the peak height).	The results showed that increasing the red light intensity to 170 μE/m2/s did not increase the accumulation of carotenoids. On the other hand, combining a red LED (75%) with a blue LED (25%) made it possible to increase carotenoids with the total photon flux. Additional blue light instead of red led to increased accumulation of β-carotene and lutein [102].
Three typical lighting systems were compared.	The influence of white light, blue-red LED light and blue-red LED light with far-red light on metabolic activity, chemical composition and yield of Chlorella vulgaris microalgae was analyzed. Classic fluorescent lamps turned out to be a much better source of light for the cultivation of C. vulgaris than LED sources emitting narrow bands of radiation in the blue, red and far-red ranges. Fluorescent light most effectively stimulated biomass growth and had the least impact on the physiological conditions of the culture. The fluorescent lighting was located between the reactor and the external coil, and the LED lighting only shone on the coil that covered the reactor [103].
Cultivation under blue, red or white light provided by light-emitting diodes and white light provided by fluorescent lamps to assess the growth characteristics of Dunaliella sp. in the green phase.	It was confirmed that for a batch culture run in PBRs with different LED lights, red and blue dichromatic lighting was more appropriate than monochrome, red or blue light. A stage one light delivery strategy was proposed for faster cell growth [104].
They used natural sunlight and wrapped the photobioreactors with filters of red, blue and red + blue light in the culture of Tetraselmis sp. in a bubble column photobioreactor outside.	The highest biomass and fatty acid productivity were obtained with a red light filter. Increased biodiesel production from algae can be achieved without artificial light sources, thanks to appropriate light filters transmitting selected wavelengths from solar radiation [105].
Maximizing the use of light and thus reducing the production costs of microalgae biomass.	They positioned mirrors around a column photobioreactor with internal lighting to increase the light intensity by reflecting the outgoing light rays back into the photobioreactor. The mirrors increased the light intensity by about 1.7 times without any additional energy consumption, and the specific growth rate and dry weight of C. vulgaris microalgae cells increased by about 25% and 91%, respectively, compared to the reference culture [68].
Investigated the influence of different colors of light-emitting diodes on the biomass composition of Arthrospira platensis (Spirulina) microalgae.	Different enzymatic steps are required to synthesize carbohydrates, lipids or proteins. Each of these stages uses a certain amount of energy. Hence, some colors that do not provide a sufficient number of photons (energy) allow for efficient synthesis of only less energy-consuming compounds [106].
Investigated high-density cultures.	Red light, which is heavily absorbed, is not necessarily the best wavelength for growing algae with monochrome lighting. Green light, which is less absorbed and can be scattered over a larger area of the photobioreactor, will be a better solution [107].
Experimenting with linear and exponential light intensity strategies in microalgae culture found that in the photoautotrophic mode, the light energy required by microalgae varies with the growth of the biomass.	In the early stages of growth, less light is needed to penetrate well through the algae culture, while moderately dense cultures require higher light intensities to counteract cell shading and enhance photosynthesis [108].

.

The quality of the spectrum (wavelengths of light) greatly impacts the photosynthesis process, for which the necessary energy is obtained from light by microalgae [99]. The ability to generate light with a narrow and specific wavelength is enabled by light-emitting diodes (LEDs) [105]. The use of a specific color lighting is closely related to artificial light sources of a specific wavelength (Table 8). An increase in light delivery efficiency may be achieved by installing sloping roof collectors, which may deliver, on average, 4–12 times (in summer) higher radiation intensity on the pipe surfaces as compared to vertical collectors [69].

One of the most important challenges is the optimization and scaling of light delivery and distribution systems to avoid photolimitation and photoinhibition during cultivation, reducing photosynthesis efficiency and overall productivity [109,110]. The use of artificial light sources is associated with enormous costs. High hopes are associated with the development of LED technology, which has resulted in lowering the cost of lighting systems, extending the service life and improving energy efficiency [110]. New solutions emerging from advances in electronics and the construction of new light sources, such as high-energy LEDs emitting UV radiation, should be constantly tested. Perhaps this will explain the phenomenon of the formation of photoprotectants in microalgae cells [9,110]. Further research on new lighting methods should also be conducted using optical fibers, waveguides and solar collectors [111]. It is also essential to study the influence of different spectral compositions of light on the production of biomass for different strains of microalgae [103] and to supplement natural light with artificial lighting, which would be used to intensify and extend the lighting time [67]. The potential of the flickering light effect has also not been fully used in large-scale processes. Progress in research related to this technology is still expected [109]. The challenge in the construction of photobioreactors is still the removal of excess O₂, as a concentration above a certain level inhibits photosynthesis [2].

4.3. Strategies for Increasing the Amount of Obtained Lipids and Obtaining Biodiesel in the Cultivation of Chlorella Microalgae

The selection of the appropriate strain is of fundamental importance in the production of biodiesel from algae, which should ensure rapid growth in a culture with high cell density, the accumulation of a large amount of lipids and suitable behavior in downstream purification processes [112]. In their research, scientists often used known microalgae cultures purchased from commercially available collections, e.g., Freshwater Algae Culture Collection of Hydrobiology, Chinese Academy of Sciences [113]; Algae Culture Collection of Wuhan Botanical Garden, Chinese Academy of Sciences [114]; The Culture Collection of Algae at UTEX (USA) [115]; or King’s College of London [116]. Involvement in the experiments of scientific centers from around the world resulted in the fact that research was also carried out with new strains of microalgae, isolated from water reservoirs located in various places on the Earth, e.g., from the Setúbal lagoon (Argentina) [117], from a local water reservoir in the Phoenix Metropolitan Area (USA) [118], Iraklion Bay (Greece) [116] and the wetlands of Nam Sang Wai (Hong Kong) [119].

The main difficulties in cultivating microalgae for biofuel outdoors are contamination by fungi, protozoa, bacteria and other algae. Farming is also not made easier by complex and changing weather conditions. The strains of microalgae used for biodiesel production, in addition to their high oil storage capacity, must have a very good ability to adapt to the external environment and survive. Chlorella microalgae have such features [120]. They can grow rapidly in mixotrophic and photoautotrophic conditions [121]. In the work of Sun et al., the photoautotrophic growth rate, lipid content and productivity, and the fatty acid profile of nine Chlorella strains from the three most oily species, Chlorella vulgaris, Chlorella zofingiensis and Chlorella protothecoides, were compared. The growth rate and oil content varied significantly between individual Chlorella strains in different culture conditions. The greatest potential for lipid production was demonstrated by the microalgae C. protothecoides CS-41, which accumulated lipids, triacylglycerol and oleate up to 55% [112]. The advantages of Chlorella vulgaris microalgae include its easy cultivation, fast growth rate, high lipid content and high CO₂ binding efficiency. Huang et al. attempted to enhance light transfer in microalgae culture by periodically pre-harvesting some cells from the medium. By pre-harvesting 30% of PBR microalgae cells, they achieved a daily increase in biomass production of 46.48% compared to the cultivation without compartments. The scientists attributed the performance improvement to optimized light distribution in the PBR [122]. Chlorella zofingiensis microalgae can accumulate lipids, canthaxanthin, zeaxanthin and astaxanthin under stressful conditions. Stress factors can be strong light intensity, nutrient deficiency, high salinity and low pH. Due to the low cell growth rate under stress, a two-phase culture process is used. A high growth rate and cell density can be obtained by heterotrophic growth [123]. However, it must be remembered that this growth requires an organic carbon source. With a low efficiency of the conversion of the carbon source to biomass, the production becomes costly and, therefore, less favorable for cheap products such as biofuels [112]. Imaizumi et al. [124], by experimenting with the microalgae of C. zofingiensis, tried to find the optimal light intensity in continuous culture. The minimum specific light intensity per cell to achieve sufficient growth was between 28–45 μE g-ds⁻¹s⁻¹. The high growth rate of 0.6–0.7 day⁻¹ was maintained over a wide range of light intensity per cell between 250 and 1000 μE g-ds⁻¹s⁻¹. Scientists found that the high adaptability to a wide range of strong light intensity predisposes C. zofingiensis to biomass production in tropical countries with strong light [124].

Scientists are constantly looking for optimal photobioreactor designs to support the cost-effective mass production of biodiesel using microalgae. Photobioreactors on a laboratory scale dominate, which may confirm the lack of effective solutions on an industrial scale (

Table 10. Typical photobioreactor designs for biomass production of microalgae.

Description of the Reactor	Achievement
Transparent and gas-permeable micro-reactors made of Poly(dimethylsiloxane), 3.5 mm in height.	The system provides high-throughput screening for growth and analysis of useful products such as lipids, carotenoids and polymers [71].
Small prototype reactors, acrylic tubes with a diameter of 1″, filled to 2 or 6 cm, which gave a total volume of 30 to 50 mL.	Verification of the possibility of exploiting mixotrophy (organic substrates as carbon source) in combination with excess CO2 in cultivation of Chlorella protothecoides and Nannochloropsis salina [107].
Multichannel photobioreactor made of 24 stands of 0.1 L bottles.	Pigment production from heterotrophically cultivated Chlorella sp. HS2 was optimized by regulating a suite of environmental conditions using the mcPBR system [125].
Laboratory scale—volume of 0.25 L to 0.5 L.	Impact of environmental conditions on biomass productivity and carbon dioxide fixation [126].
Photobioreactor with 350 mL cylindrical glass columns (ID = 4 cm), each with a 250 mL working volume.	Identified the optimum conditions and combined nutrient abatement from wastewater with CO2 fixation from flue gases [127].
Laboratory scale—volume of 0.25 L.	Characterization of secondary carotenoid production from Dactylococcus dissociatus MT1 isolated from the Sahara Desert of Algeria [128].
Laboratory scale—volume of 0.5 L.	High-dose CO2 significantly enhanced the light energy conversion and storage into lipids in the cultivation of Chlorella sorokiniana CS-1 [129].
Laboratory scale—volume of 0.5 L.	The effect of light quality on cell size and cell cycle, growth rate, productivity, PE and biomass composition of Tetraselmis suecica F&M-M33 [130].
Laboratory scale—volume of 0.25 L to 0.5 L.	Techno-economic feasibility of the cultivation process was based on measurements of long-term, sustained production at a demonstration scale [131].
Poly (ethylene terephthalate) cylindrical photobioreactors with an inner diameter of 80 mm and total volume of the PBR of 500 mL.	The effect of various colors of LED on the content of the main pigments of Arthrospira platensis, such as phycocyanin, chlorophyll and total carotenoids, and the contents of the proteins, carbohydrates and lipids in semi-continuous cultures [106].
Twin-layer biofilm photobioreactor with a volume of 800 mL.	The effect of light intensities and different concentrations of additional CO2 on biomass productivity and total biofilm dry weight of the green algae Halochlorella rubescens [132].
Bubble column photobioreactor with a volume of 1 L.	Mild-pressure-induced physical stress to promote rapid TAG accumulation in microalgae [133].
Column-type glass-fabricated PBRs (ϕ 6 cm × 80 cm high) with 1 L of working volume.	Growth and CO2 utilization efficiency of the Chlorella sp. AT1 with intermittent CO2 aeration in a semi-continuous long-term culture [134].
Bubble column photobioreactor with a volume of 2 L.	Flashing light as a light source in microalgae cultivation [135,136].
2 L flat-panel photobioreactors.	The effect of light quality on cell size and cell cycle, growth rate, productivity, PE and biomass composition of Tetraselmis suecica F&M-M33, with the objective of improving biomass quality and value for aquaculture and decrease production costs [137].
2 L airlift PBR.	Development of an optimal light-feeding strategy coupled with semi-continuous mode of reactor operation that resulted in greater lutein productivity, photosynthetic efficiency and CO2 fixation rate of Chlorella minutissima [108].
Reactors with a capacity of 3 L.	Optimization of a suite of environmental conditions for pigment production from heterotrophically cultivated Chlorella sp. HS2 [125].
Bubble column photobioreactors in batch operation mode 3 L.	Study of three key factors influencing cell growth of isolated microalga Scenedesmus obtusiusculus: CO2, aeration and light intensity and the accumulation of lipids assessed under nitrogen limitation [138].
Air-lift-type photobioreactor with a porous centric tube with a volume of 4 L and bubble column and centric-tube photobioreactors.	Comparison of culturing microalgae at high density [139].
Vertical tubular photobioreactors with a working volume of 4 L.	Research on the optimization of the Parietochloris incisa substrate for the accumulation of arachidonic acid [140].
Bubble column photobioreactor with a volume of 4.3 L.	Study of the medium’s composition in microalgae cultures of Chlorella sp. and Monodus subterraneus [116].
Airlift photobioreactor having a working volume of 5 L.	Cultivated a mixed culture of microalgae in an airlift photobioreactor in batch mode and studied the effect of varying CO2 concentrations and the combined CO2 and air flow rate, changes in pH with time at different CO2 concentrations and the effect of CO2 concentration on CO2 the biofixation rate [141].
Reactors with a capacity of 7 L.	Evaluation of the recycling of low-cost crude glycerol as a feedstock for heterotrophic cultivation of microalgae [142].
Prototype photobioreactors of various designs—volumes of 2.5, 5 and 20 L.	Effect of photobioreactor scale on cell growth and carbon dioxide fixation by Chlorella sorokiniana [143].
Double-walled polyethylene 20 L bags.	Control the tolerance of the unicellular green microalga Chlorella minutissima under extreme carbon dioxide concentrations, natural temperature and high-light conditions [144].
A 35 L bubble column photobioreactor	Experiments aimed at increasing the production of biomass and lipids by two autochthonous dinoflagellates (Alexandrium minutumand and Karlodinium veneficum) and one raphidophyte (Heterosigma akashiwo) [145].
A 50 L reactor cooperating with a 60 L open pool.	Developing a novel culture model for algal biomass and lipid production, namely, sequential heterotrophy–dilution–photoinduction [146].
Tanks with a volume of 60 L.	The tolerance to shear stress and the biochemical characteristics of the Phaeodactylum tricornutum microalgae in a column aerated photobioreactor and two versions of airlift photobioreactors with a split-cylinder airlift device and a concentric draft-tube airlift vessel [65].
Flat photobioreactors with a volume of 60 L.	The level of lipid accumulation and the growth characteristics of Chlorella zofingiensis microalgae were checked at different concentrations of nitrates and phosphates [147,148].
Pilot photobioreactors with a volume of 100 L.	Identification of the potential application of the biomass derived from microalgae cultivation with the use of industrial flue gas in the animal feed industry [149].
Ultrafiltration membrane fouling in a collection of algae from a 120 L tank was investigated.	Characterization of algal-related organic matter and evaluation of the impacts on ultrafiltration membrane fouling [150].
Photobioreactors with a volume of 150 L.	Pilot-scale outdoor cultivation of newly isolated strain Monoraphidium sp. CCALA 1094 with low requirements for cultivation temperature and a minimal requirement for light [151].
Pilot photobioreactors with a volume of 250 L.	The use of the waste CO2 recovered from a biogas upgrading process, in place of synthetic commercial CO2, in the cultivation of microalgae [152].
Photobioreactors with a much larger volume of 320 L or 540 L.	Culture performance of the dinoflagellate Amphidinium carterae in controlled conditions, in a closed system (PBR), in a large scale (>500 L), long-term culture (>8 months) grown in semi-continuous mode [153].
A pilot-scale photobioreactor, an open pond and a hybrid two-stage system.	The cultivation of microalgae for lipid targets [85].
The tubular photobioreactor was 1200 L, and the open raceway pond had a volume of 1000 L.	Comparison of microalgae cultivation systems, including open pond, closed PBR and hybrid cultivation [85].
Pilot photobioreactors with a volume of 1136 L.	Possibility of using waste heat from a combustion power plant to heat algae cultures in colder climates [154].

). The remaining studies were dominated by various designs of photobioreactors with a volume of a couple to several liters, laboratory bottles and flasks (Table 10).

Modeling biomass growth for photosynthetic organisms is much more complex than for bacteria, yeasts and fungi. Algae can store nutrients, and the pigments they contain suppress the light acting as an energy source. This strongly links biology (growth of microalgae) and physics (light penetration and hydrodynamics). For this reason, the vast majority of growth model tests were carried out in a practical way in the natural environment [155]. However, several scientific publications describing research results using mathematical modeling have appeared. These models are mainly related to light delivery to the microalgae culture. Kim et al. [115] proposed a one-dimensional model for interpreting batch growth kinetics under conditions of limited light and substrate saturation. The proposed model was derived from the combination of light attenuation phenomena and the light response curve based on the Beer–Lambert law and the Monod equation. The model was tested in flat photobioreactors with LED lighting. The model allowed them to determine the linear growth rate of the microalgae culture [115]. Heinrich and Irazoqui [117] developed a kinetic model of photoautotrophic growth of the microalgae Chlorella sp. They related the growth rate of microalgae to the availability of light in the culture medium. Two adjustable parameters were used in the model: biomass concentration and local volumetric photon absorption rate. The simulation of algae growth as a function of time showed good agreement with the experimental data for various operating conditions [117]. The mathematical model developed in the study by Chang et al. described the growth and biofixation of CO₂, taking into account the coupled effect of light intensity and dissolved inorganic carbon. Using the proposed model, it is possible to predict the growth of microalgae biomass and the rate of CO₂ biofixation at particular time points of the cultivation. The operation of the model was validated in experiments with the microalgae C. vulgaris FACHB-31 in flat-plate photobioreactors with a working volume of 1600 mL [156]. In turn, del Rio-Chanona et al. [157] developed a highly accurate, dynamic model suitable for use in real-time control systems. The operation of the model is based on chlorophyll fluorescence, which is a parameter that is immediately measurable. In order to maintain a high biomass concentration and high lipid productivity, it is very important to estimate nitrogen dosing requirements and implement advanced optimization strategies accurately. The results of simulations carried out based on mathematical models make it possible to identify the main limiting factors, e.g., production of biodiesel [157].

Scientists recognize the need to build mathematical models describing the processes of microalgae cultivation and the influence of individual environmental parameters on biomass efficiency and growth rate. This would allow for the improvement of experimental work by predicting the growth rate of microalgae as a raw material for producing, e.g., biofuels [6]. Mathematical models should consider the specificity of a particular microalgae species, type of farming, season or location [155]. In the future, these models should be incorporated into systems that control microalgae cultivation in real time. Predictive control should have a positive impact, in particular, on the optimization of the conditions for semi-continuous and continuous processes [157].

The most recent publications from 2018–2020 are dominated by the topic of using various types of waste as substrates for microalgae cultivation. In 2018, Katiyar et al. [158] used de-oiled algal biomass extract as a cheap medium to replace the commonly used BBM medium in both photobioreactors and open systems. The mixotrophic growth of algae cells caused a more than two-fold increase in algal biomass productivity and a more than four-fold increase in algae lipid productivity compared to BBM [158]. In turn, in 2019, Katiyar et al. [121] used the peel and pulp of the citrus fruit limetta (Citrus limetta) containing a suitable amount of carbohydrates and minerals as a nutrient medium in mixotrophic microalgae cultures for the production of biodiesel. In this way, they obtained two-fold higher lipid productivity as compared to BBM [121]. Zhao et al. [159] tried to increase the production of biomass and lipids in the cultivation of Monoraphidium sp. microalgae using walnut shell extract. This extract had a stimulating effect on lipid productivity by enhancing the level of lipogenic gene expression and nutrient removal efficiency [159]. Implementing the above ideas can contribute to lower costs and profitable large-scale production of biofuels.

4.4. Industrial Production of Astaxanthin Using Haematococcus Microalgae

Haematococcus pluvialis is the most commonly used microalgae strain in experiments focused on producing natural astaxanthin commercially. It owes its popularity to its efficiency of up to 5.9% astaxanthin in dry matter [160]. Astaxanthin biosynthesis usually takes place in a two-stage cultivation process [161]. First, there is a “green phase” aiming to multiply as many microalgae cells as possible. Later, the culture moves to the “red phase”, in which cells are stimulated to accumulate astaxanthin [160]. This transformation takes place under specific culture conditions known as the stress state. It can be caused, inter alia, by a deficiency of nutrients, lower or higher salinity, higher temperature, higher illuminance and their combinations [162]. The two phases differ significantly in terms of cultivation conditions and their end goal, making the production of natural astaxanthin a complicated process [161]. Only in six publications did researchers use algae that did not belong to the genus Haematococcus. However, only in the case of studies by Fujii et al. [163] was there an attempt to test the microalgae strain Monoraphidium sp. GK12, isolated from the activated sludge of a sewage treatment plant in Yamaguchi (Japan), for energy-efficient production of natural astaxanthin. In the remaining five cases, the research was related to the development of a new design of a thin-film photobioreactor [164] and a horizontal, floating photobioreactor without aeration or a mixing device [165], as well as work aimed at the application of a lumostatic lighting strategy in the cultivation of microalgae [166,167,168]. This confirms the dominant role of the H. pluvialis strain in experiments aimed at the production of astaxanthin using the natural method. Scientists know, however, that there is a significant physiological variation among Haematococcus strains, which, in the vegetative phase, amounts to as much as a three-fold difference in biomass production and growth rate. For this reason, from time to time, there are reports presenting the results of studies carried out with the use of new and less popular strains of this genus [161]. Since 2009, publications related to astaxanthin production by the Haematococcus lacustris strain have appeared systematically. Scientists have tried to optimize the cultivation conditions, such as CO₂ dosing, the best light absorption rate, nitrogen starvation and stress induction by high irradiance [169,170,171,172]. In 2019, Mazumdar et al. [161] published research results on the use of the new Haematococcus alpinus strain LCRCC-261f for higher biomass productivity. The optimal temperature range, CO₂ concentration, and the intensity and color of illumination needed to obtain a high-density culture were established [161].

The design of the photobioreactor has a decisive influence on access to light, mixing, the use of carbon dioxide and maintenance of the environmental parameters at an optimal level. All of these parameters translate into more efficient cultivation of microalgae [173]. When analyzing the scientific reports, it can be noticed that scientists used different versions of photobioreactors in their research (

Table 11. Typical photobioreactors used in astaxanthin production on an industrial scale.

Type of the Reactor	Description
Classic mechanical stirring reactors	The use of mechanical agitation photobioreactors to cultivate microalgae is not the best choice. High shear rates around the impellers of the agitators damage the cells of microorganisms, which are characterized by poor mechanical strength [162,174,175].
Various diameters of bubble columns	Higher volume-averaged light intensity, lower shear stress, higher astaxanthin content and higher mass concentration [168]. Higher biomass concentration and a longer light path will increase the extent of light attenuation [176].
Column aeration photobioreactors	The substrate circulates therein, rising in the central area and falling in the area close to the wall. In this way, the cells are ensured cycles of high light intensity, being close to the wall and lower illumination inside the column. At the same time, the absorption of carbon dioxide, which is a carbon source in photoautotrophic cultures, and the removal of photosynthetically produced oxygen, which inhibits photosynthesis, take place [171,177,178].
Classic airlift reactors	A very good solution for conducting photoautotrophic cultures [161,179,180,181,182].
Raceway pond with a gutter and oscillating partitions	It uses movable baffles to oscillate in the flowing substrate and fixed prismatic baffles to generate a vortex, which were developed to enhance the mixing efficiency of H. pluvialis cultures. The results showed that applying the new solutions could improve the mass transfer coefficient and shorten the mixing time, as well as the growth rate of microalgae, in the raceway pond [183].
Two-layer, slightly inclined (15°) photobioreactor with a porous substrate	Used to conduct immobilized microalgae cultures to obtain astaxanthin. The advantage of this solution is its simple structure, easy operation and technically simple harvesting of biomass, which translates into saving water, energy and culture time [160].
Horizontal, floating photobioreactor	Without aeration or a mixing device [165].

).

Photobioreactors were used for conducting experiments inside laboratories with artificial lighting [161,184], in laboratories with access to sunlight [164] and in experiments in which photobioreactors were placed outdoors [185,186].

The popularity of aeration columns is due to their simple structure, which facilitates their construction [175]. The circulation of the liquid in airlift reactors is even better due to the flow of the substrate from the riser into the annular space. This happens with even more regularity compared to conventional bubble columns. Regular light/dark cycles and the improved mixing of liquids in airlift photobioreactors may result in better biomass and/or secondary metabolite production [187]. In the case of popular types of photobioreactors, many design solutions have been experimentally tested to select the most efficient version possible (

Table 12. The experimentally tested popular types of photobioreactors and achieved effects.

Reactor Type	Achievement
V-shaped bottom slope photobioreactor, air volume flow (vvm), reactor height/diameter ratio and air bubble diameter.	They optimized the main factors related to substrate mixing in an aerated column reactor [173].
Tested the influence of the shape of the photobioreactor (flat panel, horizontal tubular and vertical tubular) on the efficiency of microalgae cultivation.	They tested various sparger shapes (ball, cube and cylinder) made of various materials (glass, plastic, steel and rubber) to determine mixing efficiency. Additionally, they investigated the effect of the light path length on the biomass efficiency by changing the photobioreactor diameter (5, 10, 15 and 20 cm) [164].
Effect of split-cylinder.	They used a specially placed partition forming an inner loop inside the cylinder [188].
To increase the illumination efficiency in split-column photobioreactors.	They used a photobioreactor, which was built of two interconnected bubble columns of different sizes with continuous circulation of the culture medium from one column to the other. Light was delivered to the smaller column only, allowing astaxanthin to be induced more efficiently by improving light distribution [189].
Comparison of bubble column photobioreactors with airlift.	The results clearly spoke in favor of the airlift. Higher cell density was obtained in the growth phase. Regular light/dark cycles and downstream laminar flow positively affected astaxanthin accumulation, increasing it by 16% compared to the aerated column [187].

).

The constant search for the optimal design is evident in many scientific works, in which new prototype versions of photobioreactors were used. An example is the specially designed raceway pond used in the studies by Yang et al. [183] or the two-layer, slightly inclined (15°) photobioreactor with a porous substrate used by Do et al. [160] (Table 11).

Scientists used transparent materials to build experimental photobioreactors (

Table 13. Typical transparent materials used for bioreactor construction.

Material	Ref.
Glass laboratory vessels and photobioreactors made of glass tubes.	[185,190,191]
Transparent plastics, such as a twin-layer photobioreactor made of a PMMA acrylic tube set on a polyvinyl chloride (PVC) base.	[192]
Plexiglass—a vertical panel-type 1.5 L photobioreactor.	[193]
Polypropylene film, which is transparent and has a very high durability (6 L column photobioreactors).	[174]
Polyethylene—angled twin-layer porous substrate photobioreactor (10 L bags).	[160]

).

While searching for alternative materials for the construction of photobioreactors (Table 12) that meet the criteria of transparency, low cost, high durability and ease of production, various types of films were found [164]. The advantage of this solution is the simple structure and cheap construction material. Tanks of this type can be easily produced in large numbers on a mass scale [173]. Many scientific articles omit information about the type of material that the photobioreactor was made from, but they inform about the geometric shape and dimensions (Table 9 and Table 10).

Studies have aimed to improve the design and functioning of the photobioreactors used to cultivate microalgae [194]. In the case of innovative prototype PBR constructions, further research is needed, among others, on the use of various materials, such as polyurethane or polycarbonate, for their construction and on the durability of these materials. Fluctuations in the way that PBRs work in relation to weather and seasonality should also be examined [195]. Further work on scaling cultivation systems is also necessary to verify the values of PBR technological parameters after rescaling, reducing energy consumption and system operation costs. All of these activities must be aimed at the possibility of commercializing the production on an industrial scale [111].

Scientists conducted experiments to determine the optimal values of the process parameters of microalgae cultivation. Giannelli et al. [196] confirmed the results of other scientists that the optimal temperature for the cultivation of H. pluvialis is 27 °C. Cells grown at a lower temperature (20 °C) achieved a smaller size and weight, which resulted in a lower dry matter content despite the higher number of cells. The economic analysis carried out additionally showed that cultivation at 20 °C is almost two times more expensive than at 27 °C while maintaining all other conditions at the same level [196]. Wang et al. [185] investigated the effect of the initial biomass content and nitrogen concentration on the astaxanthin production potential of H. pluvialis. The authors showed that the initial biomass content and the initial nitrogen concentration that led to the highest growth differed from that which resulted in the highest astaxanthin content in cells. The optimized initial biomass content and the limited nitrogen content allowed for the highest astaxanthin productivity [185].

The dominant method of cultivation of H. pluvialis is a two-stage strategy, i.e., the production of green biomass under favorable conditions and the induction of astaxanthin accumulation under stress [178]. Under photoautotrophic culture conditions, achieving high cell density in the first step is difficult. On the one hand, high cell density results in poor light penetration, and when the light intensity is increased, the cells are stressed and do not maintain their vegetative form [177]. For this reason, it is very common to use different culture systems for each stage. Vegetative growth is often carried out indoors using artificial lighting and sterile growing conditions. The induction of astaxanthin is usually performed outdoors using natural sunlight [192]. Although a comparison of the effectiveness of the one- and two-stage methods of astaxanthin production by H. pluvialis shows that the two-stage system fares better, scientists are still striving to simplify the process and develop an efficient one-stage production method [197]. Such an approach was presented in the research of Kiperstok et al. [192], who developed a technology for the single-phase production process of astaxanthin using high light intensity. The experiments were carried out in a two-layer photobioreactor, in which the culture medium circulated on the source layer, and the microalgae cells were immobilized on a layer made of microporous material [192]. On the other hand, other studies suggest increasing the number of production steps in order to better match the specificity of the astaxanthin production process. In Wan et al.’s [177] studies, a novel three-step cultivation strategy called “Sequential heterotrophy-dilution-photo-induction” was proposed. In the first stage, cultivation in heterotrophic conditions was responsible for achieving high cell density. Then, the cells were diluted to the appropriate concentration and acclimatized to the environment with the light source, restarting the photosynthetic apparatus of the cells. The third step, “photo-induction”, was responsible for producing high-value metabolites. Thanks to the application of the new strategy, high biomass productivity and large amounts of photosynthetic products were achieved, which positively impacted the economic effect [177]. Additional conclusions related to the linking of heterotrophy and autotrophy appeared in the studies by Zhang et al. [178]. They showed that light played a less important role than nitrogen in the acclimatization process, and that H. pluvialis cells can probably preserve their photosynthetic machinery in the dark [178]. An even more significant number of stages was proposed in the studies by Choi et al. [179], in which, due to the very high attenuation of light at particular stages of microalgae development, a multi-stage method of astaxanthin production was proposed. At individual stages, different sources of internal light would be switched on, e.g., one and two fluorescent lamps in the growth stage and four and seven fluorescent lamps in the astaxanthin induction stage [179].

Most publications are devoted to the subject of light among all environmental parameters of the astaxanthin production process with the use of H. pluvialis microalgae. Light energy is essential for the phototrophic growth of microalgae. Both the amount of light and its quality are essential. Excessive light often leads to a decrease in the growth rate of microalgae due to photoinhibition. On the other hand, at higher concentrations of the culture, the light supply is insufficient for the entire population because the effect of cell shading also results in a reduced biomass yield. By light quality, we refer to the wavelength and/or emission spectra of light, which also influence the cultivation efficiency of microalgae [198]. Scientists quickly noticed this problem and, in their research, paid a lot of attention to the issue of providing light to the cells of microalgae. Using the simplest constant light strategy throughout the entire culture often resulted in photoinhibition in the early stage of culture growth. The same light intensity after the phase of linear growth of the culture turns out to be insufficient for the efficient growth of the entire population of microalgae [168]. As early as 2003, Choi et al. [198] applied a lumostatic strategy to obtain a high-density green phase microalgae culture. As a control parameter, they proposed the light absorption rate, defined using the input and output light energy, photobioreactor column surface area, culture volume and cell concentration. This strategy made it possible to obtain high-density cultures in the green phase without inducing pigments compared to experiments with a constant supply of light energy [198]. A different approach to the lumostatic strategy was presented by Imaizumi et al. [168], who, instead of controlling the light intensity (in the case of natural lighting, this is impossible), controlled the intensity of light reaching the cells by changing the cell density. Maintaining the parameter of light intensity per unit cell biomass at an appropriate level allows obtaining the effect of a lumostatic strategy even in outdoor cultures [168]. Ifrim et al. [167] successfully designed and put into practice a lumostatic controller in experiments in a torus-shaped laboratory photobioreactor. For the construction of the feedback control system, they used a turbidity sensor, the operation of which was related to the concentration of biomass, and LED lighting, allowing for precise adjustment of the light intensity. Experimental data confirmed that the average intra-culture radiation intensity remained constant thanks to the implemented regulation system [167]. Scientists also developed mathematical models of how light propagates in cultured microalgae culture. These models can be an effective tool in designing and optimizing systems for microalgae cultivation. In their research, Gao et al. [199] developed a model of light distribution during biomass growth in the green phase of Haematococcus pluvialis microalgae cells cultivated with red LEDs with a wavelength of 655 nm. A similar task, but for the astaxanthin accumulation phase, was undertaken by Sheng et al. [176]. They examined the light distribution in a suspension of H. pluvialis in the red phase. To simulate the light distribution in the substrate, they used two light attenuation models—the Beer–Lambert model and the Cornet model [176].

4.5. Increasing the Productivity of Biomass and the Use of Alternative Carbon Sources in Microalgae Farming

In an effort to increase biomass productivity, scientists conducted research related to various strategies for microalgae cultivation. Jin et al. [200] optimized heterotrophic ultra-high cell density Scenedesmus acuminatus microalgae culture conditions and compared their lipid productivity with that of photoautotrophically grown cells. Larger-scale experiments have shown that heterotrophically cultured S. acuminatus cells are more productive for biomass and lipid accumulation than cells grown under photoautotrophic conditions. Additionally, based on experimental data, scientists conducted a techno-economic analysis, which showed the economic viability of ultra-high cell density cultures for the production of biofuels from microalgae. The cost of heterotrophic microalgae cultivation for biomass production is comparable to that of the pond system and much lower than that of the tubular PBR, with a biomass yield higher than 200 g·L⁻¹ [200]. In turn, Zhang and Lee [201] investigated the two-step process of ketocarotenoid production by Chlorococcum sp. Glucose was administered in the first stage, and light or chemical stress was responsible for inducing carotenogenesis in the second stage. The light-treated cultures produced three times more astaxanthin than those under chemical stress. The two-step strategy resulted in a significant increase in the yield of astaxanthin. The strain of Chlorococcum sp. MA-1 tolerated high temperatures and light intensity and provided a high biomass yield with glucose administration [201].

In addition to typical photobioreactor designs, scientists used innovative prototype solutions. It can be seen that the introduction of new PBR constructions was primarily aimed at improving the light diffusion efficiency in cultivation and achieving energy savings. Fu et al. [202] investigated the relationship between biomass productivity and light capture efficiency in the cultivation of Chlorella vulgaris in a photobioreactor with LED lighting with a wavelength of 660 nm. The experiments used flashing light at different work cycles but with the same frequency (10 kHz) and optimized the medium composition, gas velocity and CO₂ concentration. Based on the obtained results, the scientists estimated that LED-based PBRs could be economically viable in producing high-value compounds (price well over USD 20–30/kg dry weight). Unfortunately, in the case of biofuels, which have a lower unit value, LED lighting will not be an economical solution [202]. Instead of classic cylinder tubes, in the research of Wu et al. [203], there was a proposal to use spiral pipes. Computational fluid dynamics modeling results demonstrated the many advantages of spiral tubes, including strong swirl movements (no vortex was produced in the cylindrical tube), the occurrence of the light/dark cycle necessary for the growth of microalgae (such variability does not occur in cylindrical tubes) and greater illumination area than in cylindrical tubes for the same volume [203]. Liang et al. [204] investigated the effect of culture depth and cell density on the Chlorella sp. biomass growth rate and productivity in a shallow, circular articular photobioreactor. After performing experiments with a cultivation depth of 2 to 10 cm, the scientists found that the key to achieving the highest productivity is maintaining optimal biomass density. Biomass productivity obtained in shallow culture systems with high cell densities can be achieved in deep articular cultures with low cell density. However, the shallow cultivation system saves water and energy while maintaining a high density of microalgae cells [204]. A novel design of a thin-light-path photobioreactor for ethylene production using a genetically modified strain of Synechocystis sp. microalgae was presented by Jain et al. [111]. The photobioreactor was built of ten integrated plate waveguides that transported and distributed light evenly throughout the entire PBR volume. Its advantages are a short light path (~1 mm), preventing both photo-restriction and photoinhibition phenomena, reducing the need for energy-intensive and costly mixing, and a lower land footprint and the cultivation of high-density cultures. The optimal wavelength (red 630 nm, deep red 660 nm and blue 470 nm) and the incident light intensity regime were experimentally determined. Lighting optimization achieved a 4-fold increase in the ethylene production rate and even a 10-fold increase in the biomass density compared with the conventional flat PBR [111]. There was also the idea of harnessing the enormous potential of the oceans for the mass cultivation of microalgae due to their high specific heat, wave mixing energy and large area to be used. Kim et al. [195] presented a prototype of a floating photobioreactor made using a transparent low-density polyethylene film. Three types of internal baffles were tested for mixing performance. In cultures of Tetraselmis sp. microalgae, the productivity of biomass and fatty acids was 50% and 44% higher, respectively, compared to a PBR without partitions. Cultivation of microalgae in the ocean can reduce costs and lead to the economic profitability of production [195].

Scientists also investigated whether it is possible to profitably grow microalgae in countries with less sunshine. Fuentes-Grünewald et al. [205] conducted studies to compare the production of biomass and exopolysaccharides (EPS) in Porphyridium purpureum batch and fed-batch culture at higher latitudes (>50° N). The experiments were carried out in greenhouse conditions and on a pilot scale. The scientists assessed that it is possible to cultivate P. purpureum commercially in high latitudes and that the best strategy in terms of growth rate, biomass productivity and the production of EPS and phycoerythrin was semi-continuous culture [205].

Much scientific research has been related to using various raw materials as a potential carbon source in heterotrophic microalgae cultivation. Fujita et al. [194] investigated the effect of mixed organic substrate on the production of α-tocopherol by Euglena gracilis microalgae in photoheterotrophic culture. The organic carbon sources were glucose, galactose, fructose, ethanol, methanol, lactic acid, glycerol, sodium acetate and sodium propionate. Scientists using a mixture of glucose (suitable for cell growth) and ethanol (suitable for the synthesis of α-tocopherol) at a ratio of 6 g/L: 4 g/L as an organic carbon source achieved high efficiency in fed-batch culture in an internally lit photobioreactor airlift [194]. A way to reduce the cost of the medium was sought by Altunoz et al. [206]. They replaced the expensive commercial microalgae culture medium (BG-11 medium) with cheaper chicken manure. The experiments investigated the effect of the type of substrate and the different wavelengths, intensity and density of LED light (blue, blue combined with red, and white) on the growth of Neochloris oleoabundans as well as the content of pigments and lipids. When cultured on chicken manure medium with an appropriate combination of red and blue LED lighting, the growth of microalgae was comparable to that of strains fed with BG-11 medium. Blue or blue-red LEDs were more efficient with strains grown on chicken feces, while the BG-11 medium was more efficient with white light [206]. In their research, Sohrabi et al. [207] focused on minimizing nutrient costs and maximizing growth and productivity. They performed mixotrophic cultures of Dunaliella salina on crude glycerol (a by-product of the calcinated fatty acid production process) and on pure glycerin. The optimal concentration of crude glycerol was 2.5 g/L, and at concentrations above 5 g/L, there was a decrease in the maximum specific growth rate. In mixotrophic cultures of D. salina on glycerin, the content of carotenoids and proteins was more than twice as high as in autotrophic cultures [207].

Since 2018, the number of scientific articles related to the topic of wastewater management with the use of microalgae has significantly increased. Researchers’ attention has been drawn to the possibility of microalgae growth with minimal freshwater requirements and large amounts of nitrogen and phosphorus needed for their cultivation [208]. The ability to effectively use nutrients from wastewater of various origins predisposes microalgae to their inexpensive and environmentally friendly treatment [209]. The collected microalgae biomass can be used as a bioproduct and/or bioenergy, which is extremely interesting at the stage of transition to a circular economy [210]. An example of such an action is the study by Mendonç et al. [211], who dealt with the bioremediation and valorization of bovine wastewater. Scenedesmus obliquus microalgae were cultivated in vertical, 2.8 L flat-panel photobioreactors with aeration, operating in batch and continuous modes. Cattle wastewater was previously crushed in a hybrid anaerobic reactor. A higher CO₂ binding rate and biomass productivity were achieved in batch cultures where the maximum COD removal was 70%, while it was 61% in continuous cultures. Removal of greater than 96% NH₄⁺ and 70% PO₄⁻³ was also reported. The research results showed that this process can be considered promising for the bioremediation and valorization of wastewater produced by cattle breeding. The protein-rich microalgae biomass can be reused as cattle feed [211].

Larger-scale experiments were carried out by García et al. [210], who used three semi-closed horizontal tube photobioreactors with a volume of 11.7 m³ each to remove nutrients from a mixture of agricultural run-off (90%) and treated domestic wastewater (10%). During experiments carried out throughout the year, microalgae activity strongly depended on the insolation and temperature specific for a given season. There were large fluctuations in biomass production between seasons (over 6 times more in summer than in winter). The average efficiency of nitrogen removal ranged from 50% in winter to 90% in summer, and for phosphorus, in all cases, it exceeded 95%. Large-scale microalgae cultivation technology has proved to be an effective and reliable method of removing nutrients from wastewater [210]. In the same photobioreactor, García-Galán et al. [212] conducted experiments related to the removal of pesticides from agricultural run-offs with the use of microalgae. The scientists considered 51 pesticides of medium and high polarity identified, inter alia, in the EU list of priority substances (Directive 2013/39/EU). Of the sixteen pesticides present in agricultural runoff, ten were completely eliminated thanks to microalgae cultivation in a photobioreactor. The remaining six pesticides were still present, meaning either incomplete removal or formation during the treatment process (negative removal) [212]. Domestic wastewater was also used in the research by Aray-Andrade et al. [209]. From water samples collected from puddles and rain ditches around Guayaquil in Ecuador, they identified and isolated nine microalgae strains for cultivation in pre-treated domestic wastewater. The three strains with the best growth rates, i.e., Chlorella sp. M2, Chlorella sp. M6 and Scenedesmus sp. R3, were selected for outdoor cultivation in two types of photobioreactors: cylindrical and panel. The results showed that Chlorella sp. M2 farmed in domestic sewage was characterized by the highest growth rate, content of lipids and carbohydrates and total polyphenol content, regardless of the type of photobioreactor used [209].

A great contribution to the development of wastewater management was made by Arashiro et al. [213], who investigated the possibility of cultivating microalgae in wastewater in order to obtain valuable bioproducts—natural pigments (phycobiliproteins) and bioenergy in the form of biogas—from residual biomass. In experiments on municipal sewage and the liquid part of the digestate from a microalgae anaerobic digestion unit, they used the microalgae Nostoc, Phormidium and Geitlerinema. The combination of the extraction of pigments and the production of biogas from residual biomass allowed them to reduce costs and produce more energy (by approx. 5–10%) compared to the one-time recovery of biogas, which is in line with the idea of a circular bioeconomy [213]. Arashiro et al. [208] also investigated the possibility of using the cultivation of the microalgae Nostoc sp., Arthrospira platensis and Porphyridium purpureum for the production of natural pigments in industrial wastewater from the food industry. The removal of up to 98% of COD, up to 94% of inorganic nitrogen and 100% of PO₄³-P orthophosphates was achieved for the three tested species. Phycocyanin, allophycocyanin and phycoerythrin were extracted from the biomass. The results confirmed the possibility of effective industrial wastewater treatment while simultaneously obtaining high-value phycobiliproteins [208]. Cardoso et al. [214] dealt with the treatment of aquaculture wastewater. They noticed that the cultivation of the microalgae Spirulina sp. could be a biotechnological alternative. The best results were obtained by carrying out the cultures with 25% addition of Zarrouk culture medium to the wastewater. This allowed for high removal rates of harmful substances (sulfates 94.01%, phosphates 93.84%, bromine 96.77% and COD 90.00%). In addition, high production of proteins, phycocyanin, lipids and important fatty acids was achieved. The proposed solution will make it possible to reduce the costs of aquaculture production and minimize the environmental impact [214].

Scientists also cultivated microalgae immobilized on various types of supports and substrates. Rooke et al. [215] investigated whether it is possible to immobilize cyanobacteria of the genus Synechococcus in mesoporous silica matrices to use such a solution in new photobioreactor designs. Researchers were interested in comparing the performance of immobilized farming to classical tank farming. Liu et al. [216] assessed the productivity potential of the Scenedesmus obliquus biomass attached to microalgae cultivation in both internal and external conditions. Compared to traditional open ponds, 400–700% higher biomass efficiency was obtained with significantly reduced water consumption [216]. In turn, Ji et al. [217] investigated the growth and accumulation of lipids of the microalgae Aucutodesmus obliquus in a culture immobilized with different amounts of the nitrogen source and different volumes of the water medium, which constantly circulated inside the photobioreactor. Zhang et al. [218] investigated the effect of inoculum biomass density, light intensity and nitrogen supply on the growth and accumulation of astaxanthin in the immobilized culture of H. pluvialis in a biofilm. Piltz and Melkonian [219] investigated the possibility of recovering nutrients (nitrogen and phosphorus) from minimally diluted human urine using immobilized microalgae culture on porous substrate photobioreactors.

4.6. Influence of Light and Carbon Dioxide Conversion on Biomass Efficiency

A lot of scientific research has been devoted to various methods of carbon dioxide management and its influence on the course of microalgae cultivation. The interest in using carbon dioxide results from its growing concentration in the atmosphere, which contributes to global warming. Scientists are trying to fight this global environmental problem, and one of the ways is to use the biological method [139]. The method is based on the absorption of CO₂ and oxygen production through photosynthesis in plants or other organisms. The primary energy source for CO₂ sequestration is solar energy, which makes the cost lower compared to using physical and chemical methods. Additionally, CO₂ is converted into biomass that can be used in various ways [134]. Biological CO₂ fixation with microalgae is 10 times more efficient than that with terrestrial plants, and microalgae can grow about 10 to 50 times faster [34].

Microalgae of the genus Chlorella have been the most often used organisms in experiments. These were, inter alia, strains of C. minutissima [144,196], C. sorokiniana [129,220] and C. vulgaris [127]. Researchers also used microalgae of the genera Scenedesmus (S. obliquus [221,222], S. obtusiusculus [138], S. acutus [154]), Haematococcus [149,223] and Ostreococcus [224]. Chlorella is known for its relatively high lipid productivity. They grow in autotrophic, heterotrophic and mixotrophic conditions, which creates a lot of possibilities when it comes to designing the cultivation process [126]. They can also use a high dose of CO₂ (even up to 20%) without needing to adjust the pH of the culture [129]. Scientists are also starting to see and verify the possibilities of genetic modification. In the studies of Li et al., mutagenesis with ultraviolet radiation of cells was used to evolve an S. obliquus WUST4 mutant to be tolerant to high CO₂ concentration and have high CO₂ binding capacity [221]. In turn, in the studies by Kuo et al. [134], thanks to mutagenesis with NTG (N-methyl-N′-nitro-N-nitrosoguanidine), a mutant strain of Chlorella sp. AT1 resistant to high alkalinity in the environment was created. Very good results were achieved in both cases. S. obliquus WUST4 achieved a CO₂ removal rate of 67% in pilot-scale experiments and confirmed its potential suitability for CO₂ removal from real flue gas [221]. Chlorella sp. AT1 can grow well in a wide pH range from 6 to 11, with an optimal pH = 10, and has a stable growth efficiency, as well as a CO₂ utilization efficiency of 20.4% [134].

The key parameter influencing biomass productivity in microalgae cultivation is the photosynthetic efficiency of converting light energy into chemical energy. Although the maximum theoretical efficiency of solar energy conversion into biomass is about 12%, in practice, achieved efficiencies were between 1.5% for open ponds with a running track, 3.8–5% for tubular or flat photobioreactors in outdoor conditions and 6% in laboratory conditions. Higher efficiency of photosynthesis conversion should enable higher productivity and lower process costs. It should also be remembered that poor lighting conditions result in low biomass concentration and poor carbon dioxide fixation. In turn, excess light causes oxidative stress, damage to the protein involved in photosynthesis and, as a result, also reduced carbon dioxide binding [225].

The need for light in photosynthetic microalgae culture has been defined, and the conditions for efficient use of light in photobioreactors have been determined [143]. The role of the xanthophyll cycle in protecting microalgae against photoinhibition and the possibility of adapting microalgae to high irradiation was investigated [226]. Experimenting with microalgae, it was noticed that among artificial light sources, LEDs are a very promising solution. They have a long service life (over 50,000 h), are characterized by low heat generation and high conversion efficiency, and have a narrow spectrum and low costs [106].

It can be noticed that in the initial period, scientists tried to understand the mechanisms related to the influence of lighting on the cultivation of microalgae and the optimization of algae culture conditions, allowing for the management of as much carbon dioxide as possible. Recent publications not only contain the results of experiments carried out in larger volumes, but the experiments also focus on the practical application of microalgae cultivation. In the studies of Mohler et al. [154], microalgae cultivation focused on CO₂ recycling was carried out at Duke Energy’s East Bend Station coal-fired power plant in the USA, whose waste heat was used to heat algae cultures during the cold period. In a study by Hong et al. [149], the efficient conversion of waste CO₂ from industrial flue gases into high-value products, such as omega-3 fatty acids and astaxanthin, was achieved in photobioreactors installed near the LNG combustion stack at Korea District Heating Corporation (KDHC) in South Korea. Previously, in the work of Li et al. [221], a pilot installation of CO₂ bonding, built at the Wuhan Pingmei Wugang United Coking Chemical Co., Ltd., was used.

4.7. Heterotrophy

The constant search for new technologies using microalgae is evidenced by the results of research presented in scientific articles related to heterotrophy, the interest in which increased in 2017–2020. It was mainly based on species used on an industrial scale, e.g., Chlorella microalgae, including C. vulgaris, C. pyrenoidosa, C. ellipsoidea [146], Chlorella sp. MCC27 [142] and Chlorella sp. HS2 [125]. They make it possible to obtain high biomass productivity in both autotrophic and heterotrophic conditions [142]. The Scenedesmus sp. strain was also used and was able to accumulate lipids (up to 60%), proteins (up to 60%), polysaccharides (up to 50%) and high-value compounds. These features predispose Scenedesmus to be used in industrial applications related to the production of biodiesel, bioethanol, polymers and food additives [131].

Scientists considered the possibility of combining heterotrophic cultures, carried out in the absence of light, with a high-density culture, with its dilution and continuation of the process in autotrophic conditions [125,146]. A sequential heterotrophy–dilution–photoinduction strategy was proposed, in which the algae were first cultivated heterotrophically to achieve high cell density, after which the culture was diluted and transferred to an environment in which it was subjected to photoinduction [146]. A multichannel photobioreactor made of 24 stands was also used, which allowed for simultaneous cultivation in 100 mL bottles at different light intensities and temperature levels as well as with different aeration. This, in turn, allowed for a quick assessment of the influence of these environmental parameters on the accumulation of pigments [125].

To explore the possibility of using an alternative to glucose, cheap exogenous sources of organic carbon for heterotrophic farming, e.g., crude glycerol, which is the main by-product of biodiesel production [142] and wastewater from olive oil production [131], were also investigated.

5. Future Research

Among the many species of microalgae used in scientific experiments, the most often used strains are the most popular and best-known ones. There are no detailed studies with other species that have an attractive potential for the synthesis of bioproducts but have not been used commercially, e.g., Tetradesmus sp. microalgae, which shows the ability to accumulate lipids in photoautotrophic, heterotrophic and mixotrophic systems [88]. For new strains, the influence of basic parameters, such as pH or temperature, on increasing productivity and carotenoid content should be investigated [89]. An optimal cultivation strategy should be developed for promising strains, considering the greatest possible number of parameters [112]. Some microalgae species have been successfully tested on a laboratory scale, but there are still no large-scale or outdoor tank experiments utilizing them [90]. There is also a need for the selection of microalgae strains with high photosynthesis efficiency that can be obtained in continuous cultures with high cell density [168].

Scientists point to the growing role of genetic and metabolic engineering, which will be of key importance for the isolation of “smart strains” with metabolic pathways ensuring the traits desired in industrial production, such as increased tolerance to light and heat stress, increased resistance to pathogens and increased contents of desired products [2,15]. Using the effects of screening and molecular engineering, one should strive to select strains with a high growth rate, even in cultures with high cell density [4]. By identifying genes responsible for the growth rate, matching primary metabolism to mass culture conditions and detecting regulatory points of cellular pathways, it will be possible to manipulate and enhance key metabolic steps. By bypassing the biological limitations of the currently used strains, it will be possible to achieve increased efficiency and an economically viable production level [2,14].

Microalgae biomass production is still based on media whose content was developed 50 years ago [227]. The influence of the type and dosing strategy of the medium in heterotrophic processes included in hybrid systems should also be carefully investigated. Properly administered and used medium may result in an increase in the efficiency and growth rate in the photoautotrophic part of the culture system [200]. Future experiments should also investigate the possibility of using different types of waste as a raw material for cultivating microalgae [206].

Taking into account the scientific articles with the highest number of citations (Table 2), one can note that most of them belong to the first research area—techno-economic profitability of the production of biofuels, bioenergy and pigments in microalgae biorefineries—and to the third research area—strategies for increasing the amount of obtained lipids and obtaining biodiesel in the cultivation of Chlorella microalgae. The most frequently studied area by scientists was still biofuels and bioenergy production.

Future research should address the issues of economic viability and sustainable development [6]. Further cost reductions are needed to be able to start profitable production on an industrial scale. This will be possible by reducing the number of stages and increasing their efficiency, reducing the complexity and lowering the operating cost of the entire system [14]. It is also suggested to combine biomass production with wastewater treatment from the domestic and industrial sectors, which contain significant amounts of nutrients for microalgae [57]. Increasing the profitability of the process is also possible by using the fraction remaining after oil extraction from microalgae cells to produce other valuable products or energy [14]. In further research, great importance should be attached to the precise determination of the operating costs of photobioreactor systems, allowing the calculation of energy and economic savings of the implemented solutions [212]. It is necessary to conduct economic analyses to compare innovative solutions’ effectiveness with conventional culture systems [195]. Economic assessments are also needed to confirm that the increased efficiency of the developed cultivation systems will offset the additional capital costs required for their application. This will be especially important when implementing technologies related to the production of biofuels [228].

There have been more and more suggestions to carry out an LCA when implementing solutions on an industrial scale to precisely determine how individual technological changes affect the efficiency of the entire process, e.g., whether lower culture growth can be compensated by lower production costs (e.g., by using natural light, without temperature regulation) [153]. The most ecological effect of biofuel production is the simultaneous binding of CO₂ and climate change mitigation [6]. The type of land use, the consumption of fresh water and the possibility of using process water or groundwater, which require an additional treatment process to obtain the required quality for food applications, are also important. The relationship between environmental impact and economic costs should be the subject of additional research [63]. From a circular economy perspective, scientists should also investigate the benefits of water recovery [213], pesticide removal with microalgae [212] or alternative photobioreactor decontamination methods [229].

In future research, scientists should take a holistic approach to optimize microalgae culture by combining new strategies for metabolic engineering, strain selection, photobioreactor design and process optimization to produce low-cost, high-yield and environmentally friendly methods [10].

6. Conclusions

Scientists, in their research, focused mainly on problems related to the production of lipids and pigments by algae (especially Nannochloropsis) for use in biomass, biofuels, bioenergy and biorefineries. They touched upon the topics of tubular photobioreactors and airlift reactors used to cultivate microalgae, studying the effects of light intensity on combustion gas conversion and microalgae lutein production. They worked on developing microalgae cultivation strategies to increase the amounts of lipids and biodiesel production, carried out in external tanks using Chlorella microalgae. Particular attention was paid to the use of microalgae of the genus Haematococcus for the production of astaxanthin in a continuous, large-scale manner, with appropriately selected lighting, which is the most important environmental parameter. The possibilities of increasing productivity in microalgae and cyanobacterial cultures and linking the cultivation process to wastewater treatment were also analyzed. The influence of the most important environmental parameters—light, carbon dioxide and stress factors—on the biomass growth rate in microalgae cultures and the production of fatty acids and carotenoids were determined, and the phenomenon of heterotrophy was analyzed.

The main obstacles that make it challenging to increase the production scale in farms with the use of microalgae are too little scientific research with a precise cost analysis allowing for the calculation and comparison of energy and economic savings of implemented solutions. There is a lack of a holistic approach to research and development that takes into account the participation of specialists in experiments from various fields of science, including genetic engineering, strain selection, metabolic engineering, photobioreactor design and process optimization.

The successful implementation of an economically viable microalgae technology on an industrial scale means, as with any agricultural or industrial product, developing a technology that ensures high productivity and biomass quality while maintaining low production costs [109]. Achieving these goals in the case of complex biotechnological processes using living microorganisms with complex physiology requires integrating interdisciplinary knowledge, including biotechnology, molecular biology, genetic engineering, chemistry, bioprocess engineering, materials science, electronics, and economics and management [4]. Optimizing methods and solutions should really cover every aspect of the production process. Final success depends on making the right choice of the right strain of microalgae, the best-suited production system, the type and mode of operation of the lighting, the nutrient delivery strategy, the aeration and mixing method, the technique of collecting and processing biomass and the development of procedures to avoid infection and contamination of the substrate [2].