# Elusive Recurrent Bacterial Contamination in a Diatom Culture: A Case Study
## 1 Introduction  
Diatom algae are a major primary producer in the biosphere, fixating approximately 20% of all organic carbon on the planet. They have also been used in a number of biotechnological applications, including biofuel production [1], biosynthesis of various pharmaceuticals and food supplements [2], drug delivery systems [3, 4], biodegradation of toxic compounds [5], fish feed [6], and other applications recently reviewed in, e.g., [7]. There is also a tantalizing promise made by diatom biosilification capability: these algae feature siliceous cell walls with finely patterned nanostructures. Should the mechanism behind the production of these cell walls be understood and reproduced, it would have a large impact on nano-scale engineering. Finally, genetic engineering has been successfully used on diatoms (see e.g., [8] for a review of methods and applications), further expanding the range of their possible roles in biotechnology. All these applications require an understanding of diatom metabolism and genetics that can be provided by genome sequencing studies. A number of such studies have been performed, some using model species [9, 10] and others focusing on promising candidates for biofuel production [1, 11]. One of these studies, co-authored by authors of this work, was done on _Fragilaria radians_ ([PERSON]) [PERSON]. [PERSON] (_=Synteda acus_ subsp. _radians_ ([PERSON]) [PERSON]) isolated from Lake Baikal, a major player in the lake ecosystem, and potentially a model freshwater diatom [12]. Diatom algae in natural habitats are colonized by numerous bacteria that interact with their host in complex ways, ranging from simple consumption of diatom-produced polysaccharides to algicidal activity [13, 14, 15]. This close relationship with bacteria has always been a feature of diatoms, as evidenced by a high amount of bacterium-derived genesin their genomes, most likely acquired via horizontal genetic transfer (HGT) throughout evolutionary history [16]. On the other hand, genomic sequencing and other application may require the production of axenic biomass, which is possible in culture. _F. radians_ strains used in the genomic project were axenized using a previously developed protocol [17]. Axenization strategy involved filtration through a polycarbonate membrane with a pore size of 5 \(\upmu\)m (to remove free bacteria), detergent treatment (20 \(\upmu\)g/mL Triton X-100), cell treatment with the selected antibiotic (5 \(\upmu\)g/mL ciprofloxacin, 18 h incubation), repeated filtration, and monoclonal culturing of diatom cells. Axenic diatom culture was grown in DM medium [18] in 100 mL Erlenmeyer flasks up to a density of 10\({}^{4}\) cells/mL and then moved to 20 L bottles for further biomass growth. Since the cultures could have been recolonized by bacteria at some point between axenization and sequencing, their purity was further controlled by light microscopy (using DAPI staining) right before DNA isolation, and by measuring the abundance of non-organellar 165 rRNA sequences in read libraries. Both of these methods have detected no contamination, and a number of libraries was produced using 454, Illumina HiSeq and PacBio sequencers. Later, these libraries were used to assemble, annotate, and eventually publish _F. radians_ genome [12] and transcriptome [19]. Two strains were used for whole-genome and transcriptome sequencing. Initially, strain G9 isolated from South Baikal was sequenced from 2010 to 2012 to produce a series of shotgun libraries on Roche 454 sequencer, and also used in 2013 to produce a single Illumina MiSeq 2 \(\times\) 250 paired-end library. These data were used in the assembly that was published in [12]. By that time, strain G9 was lab-cultured for close to 10 years, leading to decrease in cell size and viability. It was replaced with a new strain, dubbed Ax BK280, which was also isolated from South Baikal and axenized according to the same protocol.