**1. Introduction**

Adaptive laboratory evolution (ALE) refers to obtaining the expected biological evolution under given laboratory conditions. As an innovative method, it is to make up for the neglect of molecular genetic mechanisms in Darwin's theory of evolution and its development. As an experimental method, it uses high-throughput sequencing of DNA as a tool to effectively simulate the evolutionary process of the selection (Figure 1).

Compared with natural selection, ALE is the process of implementing the "rules" of natural evolution for specific populations (mainly microorganisms) in the laboratory under controlled conditions, exerting pressure on them to obtain the required characteristics until the new strains with favorable mutations are developed [1]. Over the past few decades, ALEhasbeensuccessfullyusedtodevelopmicroorganismswiththerequiredphenotypes.

Microalgae have attracted extensive attention from all walks of life and penetrated various fields. For example, they are used for the production of bioactive compounds [2] and bioenergy [3,4], as well as in wastewater treatment facilities [5]. Therefore, microalgae are being intensively developed and utilized for various applications. To better utilize and control microalgal biomass and product yield, microalgae are suitable for ALE research with their advantages of fast growth rate, short generation time, easy to control in different cultivation systems and convenience preservation. Altering the environment for microalgal culture through ALE, we can use low-cost investment in exchange for the higher biomass concentration and product yield.

**Citation:** Wang, J.; Wang, Y.; Wu, Y.; Fan, Y.; Zhu, C.; Fu, X.; Chu, Y.; Chen,F.; Sun, H.; Mou, H. Application of Microalgal Stress Responses in Industrial Microalgal Production Systems. *Mar. Drugs* **2022**, *20*, 30. https://doi.org/10.3390/ md20010030

Academic Editors: Marco García-Vaquero and Brijesh K. Tiwari

Received: 29 November 2021 Accepted: 23 December 2021 Published: 26 December 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

**Figure 1.** Adaptive laboratory evolution for strains improvement. The serial dilution is applied for mutation selection during the propagation until the evolved strains obtained (**a**). The improved strains can be cultured for high-density cultivation and wastewater treatment (**b**). The strains undergo starting, midpoint and endpoint periods (**c**) with types of nucleotide deletion and disrupted DNA (**d**).

ALE is less common in microalgal experiments at the early stage. Recently, numerous studies explored the effects of environment conditions or chemical compounds on cell growth and product accumulation; the genetic materials are not changed in a short time [6]. The microalgal production still needs condition configurations for high productivity in each batch cultivation. However, ALE induces the accumulation of beneficial mutant genes, resulting in new genotypes more suitable for the stressful environment [7]. Later experiments revealed the adaptation mechanism of planktonic alga, *Skeletonema costatuni*, under strong light and a high temperature, which provided a foundation for ALE in microalgal engineering [8].

The improvement of microalgal strains is one of the major applications of ALE in microalgae (Figure 1). It can exert pressure on the process of microalgae growth and metabolism through batch or continuous culture, to make microalgae constantly adapt to the new environment, and evolve towards the beneficial mutation, including high biomass concentration and product yield. The serial dilution is applied to obtain the evolved strain, which usually continues for 3 months to 2 years. The fitness of strain undergoes starting, midpoint and endpoint periods. Then, the improved strains can be cultured in chemostat for high-cell density growth or wastewater treatment. Unlike genetic engineering, ALE does not need to know the genetic basis of the target phenotype in advance [9], the generated mutants are generally recognized as safe (GRAS) [10]. ALE with the above advantages, has been initially developed mainly for bacterial and fungal models, allowing cells containing beneficial random gene mutations to reproduce more rapidly under environmental stress. When this strategy is applied to microalgae, it is mainly used to improve the growth rate, product yield, stress tolerance and the ability of nitrogen and phosphorus removal in wastewater [7]. These can meet the industrial demand of microalgal production.

#### **2. Adaptive Laboratory Evolution Experimental Design**

In an ALE experiment, strains are cultured in a unique pressure under artificial environmental conditions for a long time. Therefore, the formation of evolved strains is promoted, and populations best adapted to the growth environment outperform the residual ones [11]. It is necessary for ALE to take various factors into consideration simultaneously in microalgae, such as the strain, stressful condition, cultivation condition and cultivation strategy.

## *2.1. Cultivation Modes*

A range of culture methods have been successfully used for ALE, including continuous culture, batch culture and staged culture.

## 2.1.1. Continuous Culture

The continuous culture can maintain process conditions with constant nutrient supply and cell densities [11]. Similar to "Bioreactor Batch Cultivations", the same bioreactor method can be used for continuous evolution experiments. The process of this training is that the addition of fresh nutrients into the medium during exponential growth at a suitable rate would allow the biomass to increase at a given rate indefinitely. A steady state can be achieved so that the microbial population grows at a constant rate in a constant environment [12]. However, there are also some disadvantages, such as the high expense and difficult control.

#### 2.1.2. Batch and Fed-Batch Culture

Batch culture refers to a method of culturing strains using a certain amount of medium in a closed reactor. The characteristic of this mode is to load culture medium and inoculate bacteria at the beginning of culture. The volume of culture medium and culture temperature during the process are maintained.

Fed-batch culture is a variation on batch culture, which is fed continuously or sequentially with substrate without removing any of the biomass. Compared with conventional batch culture, fed-batch culture has several advantages, including sufficient nutrients, decreased fermentation time and higher productivity [13]. However, the operation of the fed-batch culture is more complicated, which requires an appropriate feeding strategy in detail. Erythritol production by fed-batch culture of *Trichosporon* sp. resulted in a high constant productivity [14]. According to the general expression patterns, there were outgoing differences in gene expression profiles between the batch and fed-batch cultures that can be attributed to the fed-batch process [15]. Fed-batch culture has been applied widely to increase biomass and lipid productivity [16].
