Elsevier

Biotechnology Advances

Volume 36, Issue 2, March–April 2018, Pages 380-396
Biotechnology Advances

Research review paper
Axenic cultures for microalgal biotechnology: Establishment, assessment, maintenance, and applications

https://doi.org/10.1016/j.biotechadv.2017.12.018Get rights and content

Abstract

The impact of microalgae (including blue-green algae or cyanobacteria) on human life can be both beneficiary and deleterious. While microalgae can be cultivated and used as feedstocks for the production of bioenergy and high value-added products in nutraceuticals, pharmaceuticals, and aquaculture feeds, some microalgae cause harmful algal blooms (HABs) that cause large-scale mortality in aquatic environments around the world. Thus, with the development of microalgal biotechnology and increasing concern about HABs, research on microscopic algae has increased significantly. However, this growth of academic research and application fields has been hindered by difficulties in obtaining axenic cultures. Therefore, this review provides a brief explanation of diverse establishment techniques, along with their strengths and weaknesses, with the hope of facilitating successful axenic cultures. A compilation of research fields and relevant important findings is also presented to clarify the importance of pure algal cultures. Finally, several controversial and sometimes overlooked issues related to the establishment, maintenance, and utilization of axenic cultures are discussed.

Introduction

Microalgae (including blue-green algae or cyanobacteria for the scope of this review) are microscopic primary producers that have recently drawn huge research attention due to their compelling potential. They are an extremely heterogeneous group of organisms with minimum nutrient requirements, allowing them to thrive in almost every environment. While mostly photoautotrophic, some can also grow heterotrophically. The size of microalgae can range from less than a micrometer to a few hundred micrometers; they can have a prokaryotic or eukaryotic cell structure and occur in the form of a unicellular or colonial cell. Due to their simple nutrient requirements and ability to grow in wastewater, microalgae are also used in CO2 fixation and wastewater treatment to turn the remaining nutrients into a microalgal biomass and other bioactive compounds (Borowitzka, 2013, Huang et al., 2017, Markou and Nerantzis, 2013, Olguín, 2012, Prajapati et al., 2013). Overall, they are a promising resource for a wide range of product applications, such as nutraceuticals, pharmaceuticals, industrial chemicals, bioenergy, and aquaculture (Chisti, 2007, Mata et al., 2010, Stengel et al., 2011, U.S. Department of Energy, 2016, Wang et al., 2017, Xiao and Zheng, 2016).

An axenic culture means only a single species or strain is present and the culture is totally free of all other living organisms. Axenic microalgal cultures are required for applications where the coexistence of other organisms is undesirable, such as genome sequencing (Alvarenga et al., 2017, Metzker, 2010), identifying a biological producer of a bioactive compound prior to the large-scale production (Borowitzka, 2013), or constructing a bioremediation artificial consortia (Olguín, 2012, Subashchandrabose et al., 2011). Axenic cultures are also needed when elucidating the relationship between microalgae and other organisms using omics tools (Amin et al., 2015, Ramanan et al., 2016, Schönknecht et al., 2013). Greater insights into these relationships can then be useful for industrial, environmental, and other applications. The co-cultivation of microalgae with other organisms can enhance the microalgal biomass and productivity of associated valuable compounds or facilitate the harvesting process for mass production (Fuentes et al., 2016, Wang et al., 2016a).

Moreover, understanding the relationship between harmful algae and their associated bacteria can help expose the causes and identify a potential biological cure for alarming harmful algal blooms (HABs). Although the microalgae-bacteria relationship is extremely complicated and maintaining an axenic culture in an open system is nearly impossible, preliminary research on axenic conditions is the first step to reveal this relationship under simple, controlled conditions. Establishing an axenic culture is a process of eliminating unwanted organisms (contaminants) in order to obtain a viable culture of the desirable organism (quarry). However, the axenization strategy is usually ad hoc, and strongly dependent on the characteristics of both the quarry and the contaminants in each microbial community. Therefore, understanding and selecting compatible methods are critical for each situation.

Accordingly, this article systematically summarizes the main axenization methods, including their strengths and weaknesses. The importance and main findings of utilizing axenic cultures in certain research fields are also discussed, along with some new perspectives on axenic culture-related issues.

Section snippets

Axenic microalgal cultures: establishment, assessment, and maintenance

The isolation of unialgal cultures from environmental samples is a prerequisite for establishing an axenic culture. The first step for successful algal monoculture isolation is understanding and mimicking the natural conditions of the desirable species. In nature, microalgae form a heterogeneous complex of organisms that belong to many different phyla. Depending on their natural habitats, they exhibit distinct nutritional requirements and particular physiological conditions for optimum growth.

Axenic microalgal cultures: potentials and application fields

In their natural habitats, microalgae often live in harmony with a wide range of organisms; ranging from other microscopic organisms to plants and animals. Despite the intensive time and effort required to establish axenic cultures, the endeavor is worthwhile as they are essential in several research fields (Fig. 4).

Axenic microalgal cultures: are they genuine? Are their endophytes being ignored?

As there is no consistent definition of an “axenic culture”, this can sometimes cause confusion. A literature search produces a wide range of acceptable purity levels for axenic cultures, including “a culture containing only one species” (Lee, 2008), or “a laboratory-maintained single strain or algal species that is free of other algae, bacteria, or fungi” (Graham and Wilcox, 2000), or can be more strictly defined as “a culture of a single algal species, free of all contaminants, including

Concluding remarks

Although small, microalgae can offer a powerful solution for the current human energy crisis. However, microalgae can also have a widespread deleterious effect via uncontrollable HABs. Therefore, this review examines the necessity of axenic microalgal cultures for promoting the positive impact of microalgae on human life, while also mitigating their negative impact. Due to the vast variety of microalgal characteristics and their coexisting organisms, it is impossible to provide a universal

Acknowledgements

This work was supported by the Advanced Biomass R&D Center (ABC), a Global Frontier Program, funded by the Korean Ministry of Science and ICT (2010-0029723). We are also grateful to anonymous reviewers for their valuable comments.

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