Association of Vampirovibrio chlorellavorus with decline and death of Chlorella sorokiniana in outdoor reactors

The outdoor ARID raceway was established for optimizing the cultivation of microalgae for biofuel production. During the summers of 2014 and 2015, discoloration was observed in cultures of Chlorella sorokiniana (DOE1412), which shifted from a vibrant green color to yellow, followed by cell clumping, decline in density, and rapid death, resulting in 40-60% reduced biomass production. Total DNA was purified from the raceway samples and subjected to polymerase chain reaction (PCR) amplification using degenerate primers that amplify the 16S rRNA gene of eubacteria. BLASTn analysis of the cloned amplicon sequences revealed the presence of the Gram-negative, predatory bacterium, Vampirovibrio chlorellavorus. Scanning electron microscopic examination showed an abundance of coccoid cells, 0.3-0.6m in diameter, some of which were attached to C. sorokiniana cells. PCR amplification indicated the presence of V. chlorellavorus in raceway vessels, water lines, connective tubing, and in early, scaled-up DOE1412 cultures used to inoculate the raceway. Based on PCR detection, the decontamination of the equipment and water line with Wal-Clean more effectively eliminated V. chlorellavorus and delayed the onset of attack, compared to the chlorine disinfectant, trichloromelamine (TCM). Total DNA was isolated from soil samples collected monthly from the nearby Rillito River during 2014-2015 and subjected to PCR amplification using primers designed to amplify the 16S rRNA and 18S rRNA gene of V. chlorellavorus and C. sorokiniana, respectively. Results indicated that V. chlorellavorus and Chlorella spp. were present in most of the riverbed samples nearly year round, suggesting a possible naturally occurring reservoir of the predatory bacterium.