Biotechnology and Bioprocess Engineering 2021; 26(6): 898-909  
Single Cell Analysis of Microalgae and Associated Bacteria Flora by Using Flow Cytometry
Fabrizio Di Caprio, Simone Posani, Pietro Altimari, Alessandro Concas, and Francesca Pagnanelli
Fabrizio Di Caprio*, Simone Posani, Pietro Altimari, Francesca Pagnanelli
Department of Chemistry, University Sapienza of Rome, Piazzale Aldo Moro 5, Rome 00185, Italy
Tel: +39-06-4991-3333
E-mail: fabrizio.dicaprio@uniroma1.it
Alessandro Concas
Department of Mechanical, Chemical and Materials Engineering, University of Cagliari, Via Marengo 2, Cagliari 09123, Italy
Received: March 3, 2021; Revised: April 11, 2021; Accepted: April 22, 2021; Published online: December 31, 2021.
© The Korean Society for Biotechnology and Bioengineering. All rights reserved.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Microalgae are usually cultivated in mixed cultures with associated bacteria flora. Despite bacteria contaminants can strongly influence microalgal growth, they are rarely monitored, mainly because standardized and quick methods are missing. The aim of this study was to develop a quick flow cytometric method to quantify coenobia-forming microalgae (Tetradesmus obliquus) and associated bacteria. Staining conditions and sonication pretreatment were optimized to obtain the most accurate microalgal and bacterial cell counting. To pre-treat microalgae before counting, pulsed sonication (ton/toff = 30/60) was better than continuous sonication since it allows obtaining 100% single cells by minimizing cell lysis. On the contrary, sonication was not required for bacteria, because they were mainly found as single cells and only a relevant cell lysis was obtained when sonication was applied. To analyze samples as those tested in this study, bacteria should be analyzed directly after fixation and DNA staining, without sonication (duration of analysis: 90 min). Instead, sonication was mandatory for microalgae, while fixation and DNA staining could be avoided (duration of analysis: 30 min). Future studies should investigate the effect of the biological variability, that seems to be the most relevant factor affecting the accuracy and reproducibility of the flow cytometric analysis.
Keywords: microalgae, bacteria contamination, flow cytometry, single cell analysis, ultrasonication pre-treatment, process monitoring


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