Deoxygenation Treatment Strategy to Control Vampirovibrio chlorellavorus in Chlorella sorokiniana Cultures

Abstract

Researchers and algae growers have shown deep interest in the cultivation of Chlorella microalgae because of its significant growth potential, high biomass quality, and broad range of applications. Both small and large-scale cultivation systems (laboratory or outdoors) with Chlorella microalgae have been extensively studied, and promising results have been demonstrated in terms of cultivation procedures, growth media, growth parameters, harvesting, and processing; however, protection against biological contaminants such as the predatory Vampirovibrio chlorellavorus requires more investigation. Despite the high growth potential of Chlorella species, the impact of V. chlorellavorus infection on the culture productivity is significantly damaging and cannot be counterbalanced naturally. Thus, a pressing need to develop appropriate preventative and/or curative strategies for an optimal control of this type of predator has become one of the priorities in the cultivation process of Chlorella microalgae. The present research focusses on the management of dissolved oxygen (DO) during the nighttime (dark period) in co-cultures of Chlorella sorokiniana and V. chlorellavorus. We developed an unprecedented method to control the infection of C. sorokiniana by V. chlorellavorus. Because V. chlorellavorus is an obligate aerobe and C. sorokiniana is not, deoxygenating the culture assumably harms the predator and not the host. This research first examined the effect of deoxygenation on pathogen-free C. sorokiniana monoculture, and then on C. sorokiniana-V. chlorellavorus co-cultures. In the first section, initial experiments with pathogen-free C. sorokiniana cultures included different deoxygenation-aeration cycle times. Pure nitrogen gas was used to create anoxic conditions, and ambient air was used to reestablish aerobic conditions. In these initial experiments, C. sorokiniana tolerated anoxic conditions for extended time intervals as long as 8 hours. In the experiments with infection by V. chlorellavorus, aerated controls collapsed while the deoxygenated cultures sustained a normal growth cycle. Visual observation showed a healthy green C. sorokiniana culture in the treated cultures and a brown slime and collapsed culture in the aerated controls. In the second section, we evaluated the technical aspects of the deoxygenation practice and cost of nitrogen gas sparging as an appropriate method to create anoxic conditions in the cultivation system. In this experiment, DO concentrations were driven to low levels (0.2 ppm to 0.5 ppm) by sparging nitrogen gas for one hour at the beginning of the night (dark period), and then natural deoxygenation by dark respiration kept the oxygen concentration at a low level. The laboratory results showed that this method kept the DO levels low during the entire dark period and effectively controlled V. chlorellavorus infection in C. sorokiniana co-cultures. The cost of the deoxygenation method was estimated in outdoor experiments with pathogen-free C. sorokiniana cultures. Nitrogen sparging for one-hour at the beginning of the dark period maintained dissolved oxygen concentrations at low levels (<0.5 ppm) throughout the night. The total nitrogen injection per night per liter algae culture was then translated to annual commercial-scale raceway cost. Finally, the technical and economic feasibility of this process was evaluated for onsite nitrogen gas generators in commercial-scale reactors.