**Table 2.** *Cont*.


**Table 2.** *Cont*.

Because of the requirement to supply the global market, people are interested in extracting from algae and higher plants to gain β- carotenoids and lutein. Therefore, ALE has been applied to microalgae to produce a high content of carotenoids. It has been suggested that light plays a key role in the biosynthesis of carotenoids, through light signal sensing and downstream regulation [65]. It is well known that β-carotene can be overproduced in the marine microalga *Dunaliella salina*, in response to stressful light conditions [50]. Previous study explored the effect of red light-emitting diode (LED) lighting on growth rate and biomass yield, which identified the optimal photon flux for marine *D. salina* growth. The red-light photon stress alone at a high level was not capable of upregulating carotenoid accumulation. Therefore, combining red LED (75%) with blue LED (25%) allowed growth at a higher total photon flux, and increased β-carotene and lutein accumulation [50]. An efficient culture system with increased light energy efficiency and economy of operation can be developed in combination with genetically based methods, such as ALE for strain development.

*Haematococcus pluvialis* is considered as the best natural source to product astaxanthin. The antioxidant capacity of astaxanthin is the most outstanding than other carotenoids, which endows astaxanthin in suppressing tumor growth, improving body immunity and the scavenging of active oxygen and free radicals [66]. Many efforts are devoted to increase astaxanthin yield from *H. pluvialis*. More astaxanthin was obtained at the melatonin concentration of 5–15 mu mol/L, at 27–29 ◦C and light intensity of 198–216 μmol photons m<sup>−</sup><sup>2</sup> s<sup>−</sup><sup>1</sup> [67]. The application of nitrogen stress and excess light based on fed-batch culture can improve lipid and astaxanthin productivity to 457.1 and 2.0 mg L−<sup>1</sup> d−<sup>1</sup> [61]. The study revealed excess light can lead to more available carbon molecules to synthesize astaxanthin.

Microbial lipids often contain abundant polyunsaturated fatty acids, including eicosapenteanoic acid (EPA), DHA and arachidonic acid (AA), which can be used as the source of functional food and raw materials for green biofuels. ALE strategy has greatly improved the ability of microbial oil production, aiming to culture special microalgae strains with convenient, cost-saving and high-yield oil. The salt stress and nutrient osmotic pressure are widely used to increase lipid content in ALE [46].

These stressful conditions have largely changed the carbon flux to the expected products. 13C-MFA is used to be an accurate tool for describing the central carbon metabolism [68]. There is a conflict between lipid accumulation and cell growth; the massive accumulation of lipid is against for cell division. To resolve the conflict between cell growth and lipid accumulation, a general countermeasure is two-stage cultivation strategy. The first stage is for a rapid growth to gain the maximum biomass production, subsequently the second process for the lipid accumulation under various stress conditions at the expense of cell growth [17,69]. Studies have shown that *Parachlorella* sp. can be cultured in two stages, increasing the total FA productivity to 219.0 ± 10.7 mg L−<sup>1</sup> d−1, and the biomass reached by 80% [29]. Similarly, in the first stage, microalgae grew under red LED to obtain the maximum biomass; in the second phase, green LED was used for stress to produce a large amount of lipids [70].

However, two-stage culture strategy can also lead to large labor expenditure and economic consumption, so it is necessary to culture algae strains that can grow rapidly under stress for a long time. Therefore, transcriptome analysis of strains produced by ALE can be carried out to obtain two different metabolic responses to stress and reveal the different gene expression patterns among the strains. The understanding of microalgal evolution under stress is beneficial for the development of the strains with rapid growth under stress [71]. Based on gene expression patterns of different metabolic reactions under stress, the ALE can be applied to all lipid-rich microalgae.

#### **5. Enhanced Environmental Tolerance**

ALE, to enhance the environmental tolerance of strains (Table 3), is profitable for the industrial production of microalgae. For example, cyanobacteria, a group of Gramnegative prokaryotes capable of performing oxygenic photosynthesis [72], is a promising cell factory to convert carbon dioxide into useful chemicals by autotrophic mode. Since toxicity produced is a key challenge, when cyanobacteria is the host for photosynthetic production of chemicals, tolerance of cyanobacteria to solvents is required to improve by ALE. *Synechococcus elongatus* PCC 11801 was cultured in ALE stage, and a new strain resistant to n-butanol and 2, 3-butanediol was obtained, which also showed high tolerance to other alcohols. The evolved strain had high tolerance without obvious growth lag phenomenon compared with original strain. The mutation mechanism was revealed by whole genome sequencing [73].


**Table 3.** Summary of targeting increased stress tolerance.

Light factors of light source, light quality, light intensity and light cycle affect microalgal growth. Microalgae have different appropriate light intensities. The light compensation points of microalgae can only maintain cell basic metabolism without growth. The higher light intensity promotes microalgal growth. However, when the light intensity reaches the saturation point of photosynthesis, microalgal growth rate no longer increases. A further increase of light intensity will lead microalgae to suffer a photoinhibition. At present, studies indicated that enhanced light tolerance of the cyanobacterium *Synechocystis* by ALE could

increase the biomass. By combining repeated mutagenesis and exposure to increasing light intensities, the modified strains can grow rapidly under extremely high light intensities [75]. The ALE maximized photosynthesis and thus increased the accumulation of photosynthetic products, converting carbon dioxide into useful chemicals.

In addition, large-scale cultivation of microalgae is mostly used for environmental governance. The atmospheric CO2 levels reached the "dangerously high" levels of 400 ppm. One of the reasons for the large emission of greenhouse gases is the combustion of fuel in power plants. Efforts are underway to scrub CO2 from flue gas emitted from pulverized coal power plants using carbon sequestration and storage technology [83], which will make the production costly. Subsequently, microalgae are rapid converters of solar energy to biomass by assimilating atmospheric CO2 [84]. There has been demonstrated to use industrial flue for culturing microalgae [85], but flue gas supplementation at higher flow rates leads to the acidification of the growth medium, which typically cannot be tolerated by microalgae. Therefore, the microalgal tolerance is required to enhance to offset carbon emissions from fossil fuel combustion. A study showed that mixed biodiverse microalgal communities can be selected and adapted to tolerate growth in 100% flue gas from an unfiltered coal-fired power plant that contained 11% CO2 [80]. Cheng et al. have reported that the adaptive evolution against simulated flue gas containing 10% CO2, 200 ppm NOx and 100 ppm SOx can obtain a new strain, *Chlorella* sp. CV, which can tolerate simulated flue gas conditions and the maximum CO2 fixation rate was 1.2 g L−<sup>1</sup> d −1 [81]. It can be helpful to establish a new process for CO2 capture directly from industrial flue gas.

#### **6. Promoted Nitrogen and Phosphorus Removal in Wastewater**

Wastewater, resulting from various farming, domestic and industrial water operations, has been a key pollution of the environment for a long time in many countries or regions [86]. However, current methods for wastewater treatment have many problems, such as high energy consumption and cost, and heavy secondary pollution [87]. It is reported that some microalgae can utilize external organic carbon sources to remove nitrogen and phosphorus efficiently, and absorb heavy metals by functional groups on its cell surface [88–90]. Moreover, microalgae can utilize the nutrients from wastewater discharge to form beneficial products, including methane and hydrogen [22,91,92]. The application of removing nitrogen and phosphorus from wastewater by microalgae is developing rapidly [93]. The existing stress methods for increasing the ability of nitrogen and phosphorus removal in wastewater are shown in Table 4.



1 TN: total nitrogen; TP: total phosphorus; COD: chemical oxygen demand; DIN: dissolved inorganic nitrogen and DIP: dissolved inorganic phosphorus.

As mentioned above, light stress is used to improve the biomass and biodiesel production of microalgae [103]. Therefore, in wastewater treatment, light stress increases microalgal biomass to improve the removal rate of nitrogen and phosphate. Mahsa et al. have reported that the total biomass and protein concentrations of marine *Spirulina platensis* were observed under blue light, at around 100 μmol photons m<sup>−</sup><sup>2</sup> s<sup>−</sup><sup>1</sup> of 13.4 and 9.0 g <sup>L</sup>−1, respectively. However, the highest phosphate and ammonium removal were about 145 and 218 mg L−<sup>1</sup> under purple light, at around 100 μmol photons m<sup>−</sup><sup>2</sup> s<sup>−</sup>1, respectively [97]. Results showed that light intensity and wavelength, combined with semi-batch cultivation, can be designed to achieve the highest biomass and production, as well as to maximize the removal of phosphorous and ammonium.

Obviously, when microalgae absorb nitrogen, phosphorus and heavy metals in wastewater, their biomass and by-product yield are correspondingly increased. Previous research was to develop large-scale production to produce oil-rich algal biomass from wastewater. Almost all nutrients in sewage were consumed, and microalgal biomass and oil content could reach 2.0 g<sup>−</sup><sup>1</sup> L−<sup>1</sup> d−<sup>1</sup> and 25.25% (*w/w*) [97]; *Chlorella* cultivated in pig manure wastewater could also obtain 0.23 g m<sup>−</sup><sup>2</sup> d−<sup>1</sup> fatty acid production capacity [98]. In addition, bacteriostatic compounds against antibiotic-resistant bacteria have been discovered from microalgae during phytoremediation of swine wastewater [99]. Aigars et al. have reported phosphorus removal from municipal wastewater and the biomass were enhanced after four microalgal species were exposed to a phosphorus starvation medium, which indicated that the species, N/P molar ratio in the wastewater and P content of biomass could control the efficiency of phosphorus uptake [99]. *C. vulgaris* was continuously cultured for 15 days in municipal wastewater, in 3 temperature regimes. In this study, the analysis revealed that in alternating high–low temperature conditions, biomass production had the potential for biofuel production, with the highest lipid content (26.4% of total dry biomass) [95]. Therefore, some microalgae have the grea<sup>t</sup> potential to remove nutrient from wastewater and produce valuable compounds, simultaneously.

The technologies aim at promoting nitrogen and phosphorus metabolism in microalgae, by converting the nutrients into biomass [91]. There are two main pathways of nitrogen and phosphorus removal with microalgae for efficient wastewater treatment. One is performed through biochemical pathways for the uptake of nutrient components into the biomass for nucleic acids and proteins production. Additionally, the other can ingest phosphate for storing as an acid-insoluble polyphosphate granule [104].

Nowadays, microalgae technology is promising for wastewater treatment. However, adaptability metabolism of microalgae to extreme conditions in industrial wastewater is poorly understood. The microalgae harvesting in wastewater requires equipment investment and large amounts of energy, as the biomass concentration is usually below 0.6 g L−<sup>1</sup> after the treatment. Therefore, efficient microalgal systems to treat wastewater at a large-scale production are in urgen<sup>t</sup> needs.

#### **7. Industrial Application of ALE in Microalgae**

There are two mainly aspects about the industrial application of ALE in microalgae. Firstly, ALE is used as an optimal tool to improve the photosynthetic efficiency of microalgae in PBRs. The low and high light intensities limited the microalgal productivity at a large-scale production. ALE can develop an evolved microalgal strain with a new fixed trait, which can later be used as industrial strain for the enhanced production of the target metabolite. With the growing greenhouse effect, it is very exciting to produce biofuels, such as lipid, bioethanol, methane and hydrogen from microalgae via ALE.

Secondly, ALE has been applied to improve microalgal strains for industrial wastewater treatment. Since phosphorus and nitrogen in wastewater are the major nutrients causing eutrophication of aquatic ecosystems [99], it is necessary to search for a more effective means of nutrient removal. ALE can obtain microalgal strains for rapid nutrient loading, high tolerance to heavy metal ions and low nutrient effluent concentration [105]. At present, *Chlorella* sp. and *S. obliquus* are the most promising unicellular algae in wastewater treatment.

#### **8. Challenges and Prospects**

ALE is an emerging approach in artificial conditions, imitating the process of natural evolution to improve organisms, and being performed for various purposes at the laboratory level. The industrial microorganisms have become a key producer in various fields, including food, pharmaceuticals and other value-added chemical production [106]. ALE is one of the most effective approaches to eliminate obstacles and maximize productivity in bio-based processes, having advantages, such as the easy control of culture conditions, short generation time, easy manufacturing and storage of living fossil records for each period [107].

Meanwhile, based on these known influencing conditions, suitable combinations can make the effect more significant. Recent research showed that ALE can be combined with genetic engineering to improve the efficiency of gene transformation [108]. ALE was applied to streamline fitness recovery of genomically recoded *Escherichia coli*, for industrialscale protein production [108]. High salinity accelerated the synthesis of phospholipids to restore lipid, and ALE culture was therefore carried out to obtain an evolutionary strain of *Nitzschia* sp. to increase lipid content to 51.2% [109]. Therefore, future research can combine several factors to obtain higher returns.

However, ALE currently still faces many challenges. First is the formation of large mutant libraries and the need for large-scale screening of the required evolutionary bodies. Because the generation of genetic diversity has become a core technology for accelerating ALE, a high-quality mutant library is crucial to its success [6]. Second, with cell division, effective genes can disappear. The application of multi-omics analyses can promote the efficient data mining for the implementation of ALE experiments. The third is that the time span of ALE experiments is very long, and the end point is entirely up to the researcher's decision. The novel cultivation strategy, such as red LED light and phytohormone, can potentially accelerate ALE progress based on microalgal cell physiology. Last, the strains produced by ALE have multiple mutations, and the interpretation of genotypes requires tedious omics analysis. The upgrade and development of related software will provide more effective data for ALE performance.

Whatever the purpose, its essence is to continuously culture microalgae under certain pressure, screen out the most adaptable strains and constantly culture to meet our expectations. It is in response to "natural selection and survival of the fittest". With in-depth research to conquer those difficulties, ALE combined with systems biology and synthetic biology tools is a prominent strategy. It is acknowledged that ALE is bound to become a popular technology in microalgae for the future.

**Author Contributions:** Conceptualization, resources and supervision, H.S. and H.M.; writing— original draft preparation, J.W., Y.W. (Yuxin Wang), Y.W. (Yijian Wu), X.F. and Y.F.; writing—review and editing, J.W., C.Z., F.C., Y.C., H.S. and H.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** China Postdoctoral Science Foundation (No. 2021M703039) and the National Key Research and Development (R&D) Program of China (2019YFD0900201).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.
