UV radiation is mainly used to generate random mutations in microalgal cells. Depending on the wavelength, UV radiation is classified as UV-A (315–380 nm), UV-B (280–315 nm) and UV-C (200–280 nm). As presented in Figure 12, UV exposition induces several types of DNA alterations; however, it has to be taken into account that phototrophic cells might be resistant to certain physical mutagens due to their photon-capturing and quenching properties. For instance, Zygmena circumcarinatum and Chlorella protothecoides revealed a high resistance to ionizing radiation, while Nostoc sp., Stylidium javanicum and some extremophiles showed UV protective properties ^{[1][2][3][4][5][6]}[45,46,47,48,49,50].

Further, 80% of mutation events caused by UV, especially UV-C radiation, are related to the formation of pyrimidine dimers within the DNA. 5-methylcytosine is frequently involved in this type of mutation as it deaminates spontaneously to thymine; hence, the energy absorption shifts to higher wavelengths compared to non-methylated cytosine. Additionally, pyrimidine(6-4)pyrimidone photoproducts can be formed by UV radiation with neighboring pyrimidines between positions 6 and 4 ^[7][8][51,52]. Radiation at 260 nm (UV-C) leads to the most efficient formation of cyclobutene pyrimidine dimers and 6-4-photoreaction products, as DNA absorption reaches its maximum level at this spectral range. Therefore, UV-C irradiation has been recommended for random mutagenesis approaches, including microalgae ^[9][53]. A comprehensive overview on UV-radiation-induced mutagenesis approaches is presented in Table 1.

Ionizing Radiation, such as gamma irradiation, X-rays or ion beams, can also act as physical mutagens ^[10][54]. Due to the higher energy density compared to UV radiation, ionizing radiation causes serious genetic damages ^[11][55], such as the ionization of molecules, the alteration of bases, the breaking of phosphodiester bonds and the production of chromosomal aberrations, such as deletions, translocations and chromosomal fragmentation ^[12][56].

In view of the lack of knowledge on interactions between gamma radiation and microalgae, Gomes et al. ^[13][57], investigated the effects of various gamma ray intensities on the green alga Chlamydomonas reinhardtii, revealing modifications to the PSII energy transfer and a decrease in photosynthetic activity due to the induced formation of reactive oxygen species (ROS) by gamma radiation. Senthamilselvi and Kalaiselvi ^[14][58], analyzed the effects of gamma radiation on the microalgae Chlorella sp. in a range of 100 Gy to 1100 Gy, showing a 1.4-fold increase in the intracellular neutral lipid content compared to the wild type. Even the biomass production increased in 10 out of 12 mutants compared to the wild type by up to 27.16%.

New physical mutagenesis approaches have been recently presented using atmospheric and room temperature plasma (ARTP) for several bacterial and microalgal strains ^[15][59]. ARTP approaches involve the exposition of cells to charged particles ^[16][60], electromagnetic fields ^[17][61], neutral reactive species ^[18][62] and heat ^[19][63]. Due to low, controllable gas temperatures, the rapid performance, the high diversity of mutants and the tool’s environmentally friendly operation, ARTP mutagenesis shows high potential; however ^[20][64], comprehensive datasets, including survival rates of cells or the mutation rate, are not available yet ^[15][59].

The use of laser radiation in the near infrared and visible spectrum has already been reported for fungi and bacteria ^[21][65]. In recent years, it has also been adapted for microalgae. Due to natural heat dissipation and fluorescence quenching, many microalgae show a higher tolerance to radiation in the visible light spectrum. For a significant mutagenesis effect, higher intensity has been realized by using lasers, including semiconductor lasers (632.8 nm), (He-Ne) lasers (808 nm) or Nd:YAG lasers (1064 nm). This mutagenesis approach provides short-term exposure of microalgae in the minute range. Due to the ease of application and the good results obtained in initial studies, e.g., for the improvement in lipid production, there still seems to be potential ^[22][23][66,67]. Table 1 provides an overview on physical mutagens applied to microalgae.

Alkylating agents (AAs) are commonly used in random mutagenesis to induce nucleotide substitutions within the DNA. AAs transfer alkyl residues, predominantly methyl and ethyl groups, yielding a change in base pairing, followed by typical point mutations after replication of the DNA. It was observed that chloroethylating drugs can also cause sister chromatid exchange or DNA breaks ^[38][77], even though AAs cannot induce the direct scission of the DNA backbone ^[39][78]. Alkylation leads to the formation of adducts on either O- or N-atoms of nucleotides or O-atoms in phosphodiesters. O-alkylations are particularly potent mutagens, while N-alkylations act predominantly cytotoxic rather than mutagenic ^[38][40][77,79].

One widely used chemical mutagen is ethyl methanesulfonate (EMS), which induces point mutations, in particular, by guanine alkylation, yielding an A·T→G·C transition. Other AAs (shown in Table 2) applied to induce random mutations include methylnitronitrosoguanidine (MNNG) ^[41][80], diethyl sulfate (DES) ^[42][81], N-methyl-N-nitrosourea (NMU) ^[43][82] or N-methyl-N′-nitro-nitrosoguanidine (MNNG) ^[44][45][83,84], which can methylate almost all O- and N-atoms, up to several hundred times more effectively than similar concentrations of other monofunctional AAs ^[39][78].

AAs have also been used in combination with other mutation approaches, such as exposure to UV radiation (MNNG and EMS) ^[46][47][85,86] or base analogs (MNNG) ^[39][78], in order to achieve a higher mutation rate.

Chemicals that are capable of replacing DNA bases during the replication process are called base analogs (BA). If the BA is chemically bound to deoxyribose, there is a possibility that it will change shape and, thus, pair with an incorrect base during replication. Depending on the BA used, different types of changes in DNA pairing can be induced ^[48][49][26,87].

5-bromodeoxyuridine (5BrdU) is a uridine/thymidine analog. If 5BrdU is bound to deoxyribose, it is capable of a tautomeric shift to its enol form, leading to a guanine–cytosine-base pairing after DNA replication (A·T → G·C) ^[50][33]. Since it changes the structure by tautomeric probability, it can also cause a mutation in the opposite way, pairing with thymine instead of cytosine (G·C → A·T) ^[51][88].

2-aminopurine (2AP) is an adenine analog that causes similar changes in DNA pairing to 5BrdU ^[52][89]. 5-azacytidine (5AZ) is one of the most commonly used cytidine analogs due to its unique mutagenic specificity, changing only from cytidine BA to a guanine BA (C·G → G·C) ^[53][90].

When combined, some BAs have been detected to show a higher mutagenic effect than they could normally accomplish on their own. Combining 2AP and zebularine (ZEB) resulted in a 35-fold increase in mutation frequency in E. coli ^[54][91]. Similar effects can be observed for the combination of BAs with other physical or chemical mutagens, such as UV radiation and AAs. The repair mechanisms activated by the mutagens increase the probability of the BAs being introduced into the DNA ^[48][26]. Similar mechanisms can be assumed using BAs to induce random mutations to microalgae ^[53][90]; however, further research is necessary in this field.

The structure of AMs is very similar to metabolites that appear naturally in the cell, but they cannot fulfill their function. AMs, such as 5′fluoro-deoxyuridine (5′FDU) or 2-Desoxy-D-glucose, are inhibiting essential enzymes or mechanisms necessary for DNA replication ^[55][56][27,92]. AMs tend to have multiple mutating and cytotoxic effects, e.g., the pyrimidine analog 5′FDU. After biotransformation, 5′FDU inhibits the enzymatic transformation of cytosine nucleosides into their deoxy derivative and the incorporation of thymidine nucleotides into the DNA strand ^[56][92].

AMs have been successfully used as chemical mutagens for many bacteria and fungi species ^[55][56][57][27,92,93]. In combination with a physical mutagen, such as UV light, good mutagenesis results have been reported in recent studies ^[55][27]. However, applying AMs to microalgal cells is a future field of research.

IAs wedge between the DNA base pairs due to their particular shape. Streisinger et al. ^[58][94] recognized that this interaction often occurs in regions with repeated base pairs (e.g., CCCCC) during DNA replication. The bonds are reversible and non-covalent.

This intercalating leads to the deformation of base pairs, resulting in the untwisting and lengthening of the DNA strands. These structural modifications to the DNA affect many functions, such as transcription, replication and repair mechanisms, and may inhibit them or be mutagenic ^[59][95].

Acridine and its derivatives are the most widely used and studied DNA IAs. IAs can be mono-intercalators, bis-intercalators or both (such as echinomycin), often depending upon the length of the alkyl chain separating the chromophores ^[60][61][96,97].

Mono-intercalators appear either as frameshift mutations in bacteria or as non-mutagens. Bis-intercalators act as “petite” mutagens, e.g., in Saccharomyces cerevisiae, suggesting that they may be more likely to target mitochondrial than nuclear DNA. IA often introduces frameshifting mutations, which they are commonly used for ^[59][95]. Petite mutants are described by Ephrussi ^[62][98], as cells having defective or altered mitochondrial DNA, resulting in very small (“petite”) colonies ^[63][99]. In microalgae and other eukaryotes, IAs seem to introduce mutations, especially in the mitochondrial genome ^[61][64][97,100].

Most IAs, such as echinomycin and acridine and its derivatives, have so far mainly been studied for bacteria, bacteriophage and yeast. A wider use for the random mutagenesis of microalgae is still pending.

A vast number of other chemicals are described in fundamental biology literature ^[7][12][51,56], for example, deaminating agents (e.g., nitrite) or hydroxylating agents (e.g., hydroxylamine), which replace the amino group of bases with a hydroxyl group and cause alterations in base pairing. Cross-linking agents (e.g., psoralen) or adduct-forming agents (e.g., acetaldehyde) bind covalently to DNA bases and, thus, complicate DNA replication. Other chemical mutagens include mycotoxins (e.g., aflatoxin B1), which can cause indirect damage to metabolites ^[7][12][51,56].

Chemical mutagens applied on microalgae.

Mutagen	Mutagen Concentration, Time of Exposure	Method, Exposure Time, Source, Distance, Recovery Time	Reference Microalgae	Mutation Results			References
			Mutated trait	WT *	M **
	EMS		EMS 0.1–1.2 Mfor 60 min		Nannochloropsis sp.	Mutated trait	fatty acid methyl esters g/g of dry wt]	WT *	0.123	M **	0.238		[101]
EMS		EMS 0.4–1 g/Lfor 60–120 min	EMS 0.1–1.2 M for 60 min	Haematococcus pluvialis	Nannochloropsis sp.	total carotenoid; Astaxanthin[g/g of dry wt]	fatty acid methyl esters [g/g of dry wt]	0.02; 0.005	0.123	0.02;0.019	0.238	[102]	[65]
		EMS 0.4–1 g/L for 60–120 min	EMS 300 mM for 60 min	Haematococcus pluvialis	Chlorella vulgaris	total carotenoid; Astaxanthin [g/g of dry wt]	protein content [g/g of dry wt]	0.02; 0.005	0.353	0.02; 0.019	0.455	[66]	[34]
		EMS 300 mM for 60 min	EMS 0.2–0.4 M for 2 h in darkness	Chlorella vulgaris	protein content [g/g of dry wt]	violaxanthin [mg/L culture]	0.353	1.64	0.455	5.23	[67]	[103]
		EMS 0.2–0.4 M for 2 h in darkness	EMS 0.1–0.2 M	Chlorella vulgaris	Phaeodactylum tricornutum	violaxanthin [mg/L culture]	total carotenoids [g/g dry wt]	1.64	0.009	5.23	0.011	[68]	[104]
		EMS 0.1–0.2 M	EMS 0.2 M for2 h in the dark	Phaeodactylum tricornutum	Dunaliella tertiolecta	total carotenoids [g/g dry wt]	Zeaxanthin [μg/106·cells]	0.009	0.131	0.011	0.359	[69]	[105]
		EMS 0.2 M for 2 h in the dark	EMS 20–40 µL/mL for 2 h	Dunaliella tertiolecta	Chlamydomonasreinhardtii	Zeaxanthin [μg/106·cells]	fatty acid methyl esters yield [%]	0.131	6.53	0.359	7.56	[70]	[106]
		EMS 20–40 µL/mL for 2 h	EMS 0.2 M for2 h in the dark	Chlamydomonas	Dunaliellasalina	reinhardtii	fatty acid methyl esters yield [%]	carotenoid synthesis [Mol Car/Mol Chl]	6.53	0.99	7.56	1.24	[71]	[107]
		EMS 0.2 M for 2 h in the dark	EMS 100 μ mol mL−1, for 30 min	Dunaliella	Chlorella	salina	sp.	carotenoid synthesis [Mol Car/Mol Chl]	lipid content (g/g of dry wt]; productivity [g/(L·d)]	0.99	0.247; 0.1536	1.24	0.356; 0.2487	[72]	[108]
	EMS 100 μ mol mL−1, for 30 min		Chlorella sp.	EMS 0.4M, for 60 min	lipid content [g/g of dry wt]; productivity [g/(L·d)]	Coelastrum sp.	0.247; 0.1536	Astaxanthin content [g/L]	0.356; 0.2487	0.0145	[73]	0.0283	[109]
	EMS 0.4 M, for 60 min	EMS + UV	Coelastrum sp.	UV + EMS 25 mM for 60 min	Astaxanthin content [g/L]	Chlorella vulgaris	0.0145	lipid content [%]	0.0283	100	[74]	167	[85]
EMS + UV	UV 5–240 s, 245 nm + EMS 0.24 mol/L for 30 min		UV + EMS 25 mM for 60 min	Nannochloropsis salina	Chlorella vulgaris	fatty acid methyl ester [g/g of dry wt]	lipid content [%]	0.175	100	0.787	167	[110]	[46]
	UV 5–240 s, 245 nm + EMS 0.24 mol/L for 30 min	MNNG	Nannochloropsis salina	MNNG 0.1 mM for 60 min	fatty acid methyl ester [g/g of dry wt]	Haematococcus pluvialis	0.175	Total carotenoid content [g/L]	0.787	~0.067	[75]	0.089	[80]
MNNG	MNNG 5 µg/mL for 60 min		MNNG 0.1 mM for 60 min	Chlorella sp.	Haematococcus pluvialis	max. growth rate under alkaline conditions [ d−1]	Total carotenoid content [g/L]	0.064	~0.067	0.554	0.089	[111]	[41]
	MNNG 5 µg/mL for 60 min		MNNG 0.02 mol/Lfor 60 min	Chlorella sp.	Nannochloropsisoceanica	max. growth rate under alkaline conditions [ d−1]	Total lipidcontent [g/g]Lipid productivity [g/(L·d)]	0.064	0.241; 0.0065	0.554	0.299; 0.0086	[76]	[33]
	MNNG 0.02 mol/L for 60 min		MNNG 0.1–0.2 M	Nannochloropsis	Phaeodactylum tricornutum	oceanica	Total lipid content [g/g] Lipid productivity [g/(L·d)]	total carotenoids [g/g dry wt]	0.241; 0.0065	0.009	0.299; 0.0086	0.011	[50]	[104]
	MNNG 0.1–0.2 M		MNNG 0.2 mg/mL	Phaeodactylum tricornutum	Chlorella sorokiniana	total carotenoids [g/g dry wt]	Lutein content [g/L]	0.009	0.025	0.011	0.042	[69]	[83]
	MNNG 0.2 mg/mL		Chlorella sorokiniana	MNNG 0.25–0.5 mM	Lutein content [g/L]	Botryosphaerella sp.	0.025	lipid[g dry wt/(m2 day)]; biomass productivity [g dry wt/(m2·day)]	0.042	1.0; 3.2	[44]	1.9; 5.4	[84]
	MNNG 0.25–0.5 mM	NMU	Botryosphaerella sp.	NMU 5 mM for60–90 min	lipid [g dry wt/(m2 day)]; biomass productivity [g dry wt/(m2·day)]	Nannochloropsis oculata	1.0; 3.2	Total fatty acid [g/g dry wt]	1.9; 5.4	0.0634	[45]	0.0762	[82]
NMU	DES + UV	NMU 5 mM for 60–90 min	UV 7–11 min 254 nm +DES 0.1–1.5% (V/V) 40 min	Nannochloropsis oculata	Haematococcus pluvialis	Total fatty acid [g/g dry wt]	astaxanthin content [mg/L]	0.0634	~0.031	0.0762	~0.089	[43]	[81]
DES + UV	5BU	UV 7–11 min 254 nm + DES 0.1–1.5% (V/V) 40 min	5BU 1 mM for 48 h	Haematococcus pluvialis	Chlamydomonas reinhardtii	astaxanthin content [mg/L]	O2 tolerance [%]	~0.031	100	~0.089	1400	[42]	[112]
5BU	5′FDU	5BU 1 mM for 48 h	5′FDU 0.25 and 0.50 mM for 1 week	Chlamydomonas reinhardtii	Chlorella vulgaris	O2 tolerance [%]	fatty acids 16:0; 18:0; 20:0 [% of total fatty acids]	100	27.9; 3.9; 11.9	1400	46.9; 5.5; 18.5	[77]	[68]
5′FDU	Acriflavin	5′FDU 0.25 and 0.50 mM for 1 week	Acriflavin 2–8 μg/mL for 1–3 d in darkness	Chlorella vulgaris	Chlamydomonas reinhardtii zyklo	fatty acids 16:0; 18:0; 20:0 [% of total fatty acids]	Loss of respiratory rate [nmol O2/(min·107 cells)] through loss of mitochondrial DNA	27.9; 3.9; 11.9	23.2	46.9; 5.5; 18.5	3.7	[26]	[100]
Acriflavin	Acriflavin 2–8 μg/mL for 1–3 d in darkness	Chlamydomonas reinhardtii zyklo	Loss of respiratory rate [nmol O2/(min·107 cells)] through loss of mitochondrial DNA	23.2	3.7	[64]

* Wildtype, ** Mutant.

3. Further Approaches in Random Mutagenesis

Recently, combined mutagenesis approaches have generated high interest as results indicated that they have a higher success rate than individual approaches. For instance, Wang et al. ^[42][81] applied a two-step random mutagenesis protocol to Haematococcus pluvialis cells using first UV irradiation, then EMS and DES mutagenesis, causing astaxanthin production to increase by a factor of 1.7 compared to the wild strain. Beacham et al. ^[75][110] used a reverse protocol for Nannochloropsis salina, starting with exposure to EMS, followed by UV irradiation, yielding a three-fold increase in cellular lipid accumulation. Comparable results were achieved by Sivaramakrishnan and Incharoensakdi ^[78][113], who exposed Scenedesmus sp. to UV irradiation in combination with oxidative stress by H₂O₂.

Other approaches can be used to select desired microalgal cells if the results obtained by random mutagenesis are insufficient. Among them, Adaptive Laboratory Evolution (ALE) is commonly used to adapt the physiology of cells to specific process conditions, such as high temperatures ^[79][114]. Its principle is based on natural selection, as presented in the Darwinian Theory, on the laboratory bench ^[80][115], and includes extensive cultivation in a specifically designed lab environment so that enhanced phenotypes can be selected after a long period of time ^[81][116]. The environmental conditions that can be altered include light irradiation, lack of nutrients, such as nitrogen, osmotic, temperature and oxidative stress ^[80][82][83][115,117,118]. Connecting the results of ALE with whole genome sequencing and “omics” methods enables gene functions to be discovered easily ^[81][116]. However, ALE does not prevent gene instability that might occur more often than in randomly mutated cells ^[79][82][114,117].

Additional environmental factors can be applied on microalgae; for example, Miazek et al. ^[84][119] reviewed the use of metals, metalloids and metallic nanoparticles to enhance cell characteristics. Moreover, phytohormones or chemicals acting as metabolic precursors have already been applied to microalgae ^[85][120]. A discussion of the methods used in the latter case exceeds the scope of this resviearchw.

More recently, a new technique was developed, known as Space Mutation Breeding (SMB). This technique may have direct or indirect effects on the growth and metabolic activities of microalgae, due to the unusual environment of space, characterized by high-energy ionic radiation, space’s magnetic field, ultra-high vacuum and microgravity ^[86][121]. The SMB technique provides some advantages, such as the great improvement in species’' qualities in a short time ^[87][122]. This was achieved by Chen Zishuo et al. ^[86][121], with a seawater Arthrospira platensis mutant, yielding a sugar content 62.26% higher than the wild type.

Reference