2.1.3. Staged Cultivation

Conditions for product accumulation are usually different from those for biomass accumulation [17], the strategy of two-stage process has been found to conquer this paradox. According to this method, there is a new ALE approach named as "chemical modulators based adaptive laboratory evolution" (CM-ALE). The first step used acetyl-CoA carboxylase (ACCase), as a pressure to increase the lipid and docosahexaenoic acid (DHA) productivity of strains by 50% and 90%, respectively. Then, the second step used a sesamol based on the stress of ACCase, to increase the cell growth rate and make the productivity of lipid and DHA up to 100% and 130%, respectively [18]. This demonstrated that the two-step CM-ALE can achieve mutual improvement between desired products and cell growth. The two-stage process was considered as the better approach for productivity improvement. However, the choice of pressure is indispensable with the strategy applied [19]. The understanding of the relationship between carbon metabolism and the ROS quenching mechanism can provide strategies for microalgal production [18].

In addition, heterotrophic cultivation has become a tempting option to increase cell density by overcoming microalgal dependency on light [20]. Therefore, ALE strategy domesticating autotrophic strains to heterotrophic types is a promising approach for high density cultivation of microalgae.

#### *2.2. Choice of Stress Conditions and Equipment*

During microbial ALE, a strain is cultivated under clearly defined conditions for prolonged periods of time. The selective stress serves as the foremost step for the success of ALE, which can be classified into environment stress and the nutrient stress [21]. Microbial characteristics should be considered for selecting pressure. The special properties of microalgal strains are anticipated to amplify to improve the productivity in industrial production. For example, *Chlorella vulgaris*, *Neochloris oleeoabundans* and *Scenedesmus obliquus* [22] are known as producing lipids, so pressure promoting lipid accumulation is preferentially selected, such as nitrogen starvation and high light.

The culture equipment is another important factor. The approaches for ALE usually include serial transfer, colony transfer or chemostat culture [17]. The chemostat is commonly used for the continuous addition of medium and simultaneous removal of culture broth [23]. In addition, ALE is suitable for automation in photobioreactor (PBR) to achieve continuous monitoring, improve experimental robustness, increase throughput and minimize manual labor [24,25].

#### **3. Increased Cell Growth Rate**

The microalgae are a group of prospective resources. Their cultivation is simple, and requires less fresh water and fertile land compared to the other terrestrial plants [26]. Microalgae can be used in the pharmaceuticals, nutraceuticals, biofertilizers, bioplastics, biofuels, cosmetics and feeds for aquaculture and poultry [27–29], consequently addressing environment pollution [30]. The productivity and yield are still the key indicators for the biotechnological and economic feasibility of microalgae [7]. Recently, genetic engineering is applied to improve microalgal growth for large-scale production, which can eventually realize the commercial utilization of microalgae [31,32]. However, the use of transgenic microalgae in outdoor aquaculture systems is still limited for security reasons. Therefore, increasing ideas focus on ALE training, which can avoid risks with high growth rate of microalgae. The existing stress methods for increasing cell growth rate are shown in Table 1.

**Table 1.** Summary of targeting increased growth rate.


Recently, the method to improve the growth rate is mainly performed through controlling light factors by using ALE. In the early process of exploring natural selection, to improve the performance of strains, *C. reinhardtii* was evolved for 1880 generations in liquid medium under continuous light. At the end of experiment, evolved cells had a growth rate that was 35% higher than the progenitor population [38]. This significant growth enhancement was largely due to the improvement of acetic acid metabolism, which showed that the utilization route of organic carbon in algae can provide direction for strain improvement. The process completely transformed the strain at the gene level. In other words,

continuous light can enhance the utilization of acetic acid for fundamental processes, such as DNA replication and protein translation. Although whether the genetic modification was stable or permanent at that time was a subject for debate, it has clearly demonstrated the ability of algae genomes to adapt to environmental changes, and the potential of this strategy for future microalgae engineering, which is later known as ALE. Since then, there have been many studies on regulating light to increase cell growth rates. *C. reinhardtii* was cultivated in TAP media with a light intensity of 50 μmol photons m<sup>−</sup><sup>2</sup> s<sup>−</sup>1, the final biomass concentration can reach 1.48 times of the starting strains [39].

Light-emitting diode (LED) is a novel light source, which has the advantages of high efficiency, reliability, long life and low power consumption [40]. For microalgal cultivation, LED allows for artificial control of the spectral output, light intensity and light frequency for light configuration [35]. Biological productivity and light capture efficiency are crucial indicators to evaluate the economic feasibility of production mode by LED technology. Although LED is a little expensive, LED-based PBRs will become practical for producing algal biomass. Using LED-based PBRs for *C. vulgaris* can provide a biomass productivity of up to 2.11 g L−<sup>1</sup> d−1, with a light yield of 0.81 g DCW/Einstein. This demonstrated that LED-based PBRs, combined with microalgae biotechnology, can efficiently convert carbon dioxide into biomass and valuable products.

#### **4. Improved Product Yield**

Microalgae provide an abundance of value-added products that can accumulate up to 10–70% of specific biochemical substances (such as lipids and carbohydrates). These components have different functions because of various features. Carotenoids from microalgae can be used in medicine, cosmetics and food; lipid from microalgae can be used as a raw material to produce biodiesel to replace fossil fuels; carbohydrates produced by microalgae can be processed into bioethanol; and astaxanthin from microalgae has strong antioxidant activity and is used in health products, food and feed industries. However, the yield of these active products is not large enough to meet the industrial demand without external interference, so ALE can be applied to obtain high-yield strains for commercial utilization. The existing stress methods for improving product yield are shown in Table 2.


**Table 2.** Summary of targeting increased product yield.

