# Network Component Analysis Can Identify Potential Axenisation Strategies Circumventing Antibiotic‐Use for Phototrophic Eukaryotic Microalgae

## TL;DR

This paper proposes antibiotic-free methods for creating pure microalgal cultures using network analysis of existing techniques.

## Contribution

It introduces tailored, antibiotic-free axenisation workflows for diatoms, dinoflagellates, and green algae using network component analysis.

## Key findings

- Filtration, washing, and micropicking are effective for diatoms without antibiotics.
- Micropicking, subculturing, and flow cytometry work well for dinoflagellates.
- Microscopy, cell counting, and sequencing are essential for verifying culture purity.

## Abstract

Axenisation of phototrophic eukaryotic microalgae has been studied for over a century, with antibiotics commonly employed to achieve axenic cultures. However, this approach often yields inconsistent outcomes and contributes to the emergence of antibiotic‐resistant microbes. A comprehensive analysis of previous reports on axenisation was necessary to identify alternate workflows tailored to each major microalgal group. Literature from scholarly databases was systematically recovered and network component analysis was performed to identify method‐clusters commonly reported for the axenisation of diatoms, dinoflagellates, and green algae. Promising workflows circumventing the use of antibiotics appeared to be filtration ↔ washing ↔ micropicking for diatoms, and micropicking ↔ subculturing ↔ flow cytometry for dinoflagellates. No clear workflow could emerge for green algae although Streak plating ↔ Flowcytometry → Ultrasonication was considered despite these methods appearing in different clusters. Furthermore, the literature suggests that a combination of microscopy (e.g., epifluorescence), cell counting (e.g., agar plating), and sequencing (16S and/or 18S) was essential to confirm the final purity of the mother culture. More systematic and high‐quality primary research is required to identify effective workflows for other microalgal divisions and fortify/contrast the ones proposed herein based on network component analysis.

Network component analysis of published axenisation methods reveals optimal, antibiotic‐free workflows for phototrophic eukaryotic microalgae. Distinct strategies are identified for diatoms, dinoflagellates, and green algae. Verification using microscopy, sequencing, and cell counting enhances reproducibility. This work promotes sustainable, targeted axenisation approaches across microalgal divisions.