Application of deoxygenation-aeration cycling to control the predatory bacterium Vampirovibrio chlorellavorus in Chlorella sorokiniana cultures

Abstract A previously untested approach was evaluated to enable management of the predatory bacterium, Vampirovibrio chlorellavorus , a pathogen of Chlorella sorokiniana , in suspension cultures grown in a laboratory test reactor. Because V. chlorellavorus is an obligate aerobic bacterium, whereas C. sorokiniana grows under aerobic and anaerobic conditions, deoxygenation of the culture was expected to be detrimental to the pathogen, but not to the algal host. The effect of deoxygenation on the uninfected (healthy) C. sorokiniana suspension cells, compared to the C. sorokiniana - V. chlorellavorus co-culture, was studied in relation to biomass, dissolved oxygen, ratio of C. sorokiniana to V. chlorellavorus DNA, and visual and light microscopic observations. Preliminary experiments were conducted to test the effects of different deoxygenation-aeration cycling regimes on performance of V. chlorellavorus -free C. sorokiniana cultures. To an aerobic culture, pure nitrogen gas was introduced to create anoxic conditions, followed by the injection of ambient air to re-establish an aerobic environment. Under this repeated cycling regime, C. sorokiniana was shown to tolerate the anoxic conditions for extended timespans that ranged from 2 to 8 h over a 5-day test period. The analogous aerobic-anoxic cycling with the C. sorokiniana - V. chlorellavorus co-cultures resulted in ‘near-normal’ growth cycle and harvestable biomass, whereas the continuously-aerated (aerobic) co-cultures that were grown without the deoxygenation step in the cycle collapsed in 3 days. Visual and light microscopic observations revealed intact C. sorokiniana cells were present in the deoxygenated cultures, compared to the aerobically-grown, brown-colored algal cultures consisting of collapsed cells. Quantitative polymerase chain reaction analysis showed continuous increases in the ratio of V. chlorellavorus (16S rDNA) to C. sorokiniana (18S rDNA) DNA in the aerated co-cultures, with greater increases during dark periods, while the pathogen-to-host DNA ratio in the deoxygenated co-cultures was relatively low and algal cells did not collapse, as would be expected following pathogen attack.