Microalgae are the support base of the marine food chain and are the natural food of filter feeders. Algae are essential for the production of live food for larval culture. Non-axenic microalgal cultures are commonly used, however, axenic may be required under different biotechnological uses. Chaetoceros is one of the most widely used diatom genera in aquaculture, and in this research axenic and non-axenic cultures of three species of microalgae Chaetoceros calcitrans, Chaetoceros muelleri and Chaetoceros sp. We tried to culture in axenic conditions Chaetoceros by using a cocktail of antibiotics (ampicillin, neomycin, kanamycin, and streptomycin). In order to determine the axenicity level produced by the antibiotic cocktail since no growth was observed, a metagenomic analysis using next generation sequencing was used with total DNA of the three Chaetoceros species.
The cultures were harvested in the stationary phase and total the DNA was extracted by means of the commercial protocol DNeasy® Blood and Tissue (QIAGEN®, Valencia, CA). The quantification and evaluation of the quality and integrity of the DNA were carried out, then samples were sent to be sequenced with the MiSeq Benchtop Sequencer platform (Illumina) to the University of Georgia, Georgia, USA. The sequenced reads were assembled using SOAPdenovo software (Li et al., 2009).
Upon assembly completion, a fasta file was generated for further annotation analysis. With the resulting contigs, the NCBI nucleotide (nt) database (https://www.ncbi.nlm.nih.gov/) was blasted to separate bacterial from eukaryote sequences. With the contigs related to bacteria, phyla, genera and species identification was carried out.
The number of blast hits to the nt database corresponding to the Bacteria, Archaea, Eukaryote and Virus Kingdoms of three Chaetoceros species for axenic cultures: 47,486, 14, 38,745 and 27 respectively for C. calcitrans, 14,105, 13, 23,805 and 35 for C. muerelli, and 70,721, 24, 68,816 and 54 for Chaetoceros sp. And for non-axenic cultures: 82,849, 9, 13,692 and 52 for C. calcitrans, 17,631, 11, 32,962 and 31 for C. muerelli, and 48,037, 22, 41,934 and 37 for Chaetoceros sp.
The group of cytophaga-favobacter-bacteroides (CFB) filum bacteria, alpha-Proteobacteria and gamma-Proteobacteria were the most abundant in the three species of microalgae. However, regarding axenic cultures in the species C. calcitrans and Chaetoceros sp., the group of CFB bacteria was more abundant compared to the non-axenic cultures, with the exception of the microalgae species C. muerelli in non-axenic cultures it presented more abundance. The response of cultures to antibiotics was species-specific and other specific protocols and antibiotics should be tested for biotechnological uses with axenic cultures.