Establishment of axenic cultures from cyanobacterium Aphanizomenon flos-aquae akinetes by micromanipulation and chemical treatment Sigitas Šulčius ⁎, Kristina Slavuckytė, Milda Januškaitė, Ričardas Paškauskas Laboratory of Algology and Microbial Ecology, Nature Research Centre, Akademijos str. 2, LT-08412 Vilnius, Lithuania abstract article info Article history: Received 7 September 2016 Received in revised form 16 January 2017 Accepted 18 January 2017 Available online xxxx Filamentous cyanobacteria are an environmentally and biotechnologically important species. However, the iso- lation and purification techniques for these organisms remain poorly developed and rarely used in studies of their biology, ecology and growth requirements, mainly due to the need of species- or strain-specific approaches and labour-intensive work. Here we propose a simple protocol for the establishment of an axenic (pure) culture of filamentous cyanobacterium Aphanizomenon flos-aquae from its akinetes. We tested the effect of different physical and chemical treatments on akinetes viability and germination time, removal of epiphytic and contam- inating bacteria and growth of the recovered cultures. The protocol consists of three steps: 1) capturing the A. flos-aquae akinetes using a micromanipulator, 2) akinete treatment with a TESC buffer, containing 1% of CTAB and 3) transferring the akinetes to the growth medium. We further demonstrate the increased growth of axenic A. flos-aquae compared to their bactericized counterparts, which provides insights into cyanobacteria-bacteria interactions. © 2017 Elsevier B.V. All rights reserved. Keywords: Nostocales Isolation Purification Pure cultures Bacterial contamination 1. Introduction The establishment and maintenance of axenic (pure) cultures of dif- ferent microorganisms, including cyanobacteria, is one of the greatest challenges in the field of fundamental and applied microbiology [1,2,3, 4]. The difficulties associated with the isolation and cultivation of axenic cyanobacterial strains limits their exploitation in biotechnological pro- cesses and uses for the production of various biologically active com- pounds, in particular, those of pharmaceutical and therapeutical value [5]. It also circumvents the definitive characterization of cultured strains for taxonomic classification and nomenclature [6,7]. Unlike in PCR- based studies, non-axenic strains may lead to a pronounced bias in the analysis of the genome sequencing data of cultured organisms. The iso- lation of pure cultures is also an obligate necessity to understand the biology and function of cultivated microorganisms as well as their eco- logical interactions with other species [8,9]. Hence, numerous studies over the last several decades attempted to develop more efficient methods for cell isolation, primarily aiming to increase precision, throughput and cell recovery rates [3,10,11,12,13]. Filamentous cyanobacteria are globally distributed photosynthetic microorganisms of great ecological and economic importance. In many natural environments they constitute a major component of the aquatic biomass playing a significant role in the food web dynamics and biogeo- chemical cycles [14]. Due to their capability to synthesize an array of natural bioactive compounds, cyanobacteria have great potential for multiple industrial applications. Commercially and biotechnologically, the most important filamentous species are Arthrospira platensis, Aphanizomenon flos-aquae and several members belonging to genera Nostoc and Anabaena [15,16,17]. However, the establishment of axenic cultures of these cyanobacteria remains rather difficult, and is considered to be time consuming and labour intensive work that requires strain-specific approaches [18]. Cyanobacteria are very morphologically diverse microorganisms ranging from simple unicellular to complex filamentous forms. The recent “polyphasic” approach subdivides all cyanobacteria into five subsections, where subsections IV and V include all cyanobacteria exhibiting cell differentiation. The prevalent types of differentiated cells are heterocytes, capable of atmospheric nitrogen fixation by the oxygen-sensitive nitrogenase enzyme complex, and akinetes. The later are the dormant stage cells produced exclusively by cyanobacteria belonging to the order Nostocales [19]. Akinetes develop from one or several neighbouring vegetative cells and possess a thick multi-layered cell wall, which makes akinetes more resistant to physical and chemical treatment, and enables the organism to survive in an unfavourable environment until the onset of suitable conditions for vegetative growth. The establishment of pure bacterial cultures relies on two funda- mental aspects. First, a pure culture has to be derived from the progeny of a single cell [10]. Second, it has to be free of all other free or cell surface attached contaminants, including eukaryotes, prokaryotes and viruses. Both conditions are hardly, if at all, achievable for many isolated Algal Research 23 (2017) 43–50 ⁎ Corresponding author. E-mail address: sigitas.sulcius@gmail.com (S. Šulčius). http://dx.doi.org/10.1016/j.algal.2017.01.006 2211-9264/© 2017 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Algal Research journal homepage: www.elsevier.com/locate/algal