Application of deoxygenation-aeration cycling to control the predatory bacterium Vampirovibrio chlorellavorus in Chlorella sorokiniana cultures
Introduction
Outdoor raceways expose algae cultures to contamination, which can cause cultures to collapse, reduce biomass productivity, and increase maintenance and re-inoculation costs [1]. Several strategies have been suggested to reduce contamination of algae cultures: salvage harvest [2], pH shock treatment [3], use of chemical agents [[4], [5], [6], [7], [8], [9]], iron limitation [10], physical disruption and removal [11], and biological control [12,13], with cost, side effects, and efficacy having determined the feasibility of each strategy.
Species of the chlorophyte genus, Chlorella, belong to the Trebouxiophyceae (order, Chlorellales; family, Chlorellaceae), and are spherical or ellipsoidal unicellular species ranging from 2 to 15 μm in size [14]. A number of Chlorella species are cultivated for food, cosmetics, and vitamins, and some are of potential interest for producing biofuels because of their ability to produce 35 g/m2 d−1 or more of biomass [15], tolerate a wide range of growth conditions [16], can grow under both aerobic and anaerobic conditions [17], and have a specific growth rate as high as 5.9 d−1 [18]. However, several Chlorella species have been lucrative hosts of the predatory cyanobacterium, Vampirovibrio chlorellavorus, which parasitizes the algal host cells, leading to rapid death [19] and complete loss of harvestable biomass [20,21].
Vampirovibrio chlorellavorus [19,22,23] is a non-photoautotroph, obligate aerobic gram-negative cyanobacterium with a pleomorphic shape ranging from 0.3 μm (vibrios) to 0.6 μm (cocci) and lives attached to the surface of Chlorella spp. cells [19,[24], [25], [26]]. Under high temperatures, within several days after V. chlorellavorus presence has been detected in the Chlorella culture [20], cells have been observed to clump, collapse and lyse [24], resulting in substantially reduced biomass [21,27]. With the recent increased interest in Chlorella cultivation for biofuel production, this predatory bacterial pathogen has become of a great concern to the industry [3]. In 1966, Mamkaeva [28] first reported an unidentified bacterium that attacked Chlorella spp. In 1972, the bacterium was named Bdellovibrio chlorellavorus [22], and in 1980 the genus affiliation was amended to Vampirovibrio, resulting a name change to V. chlorellavorus [23]. A hallmark of species within the genus, Bdellovibrio, is that they enter the periplasm of their hosts. However, V. chlorellavorus attaches to Chlorella microalgae and ruptures the cell wall, a tissue tropism that might be associated with the particular cell wall properties of Chlorella host species [[24], [25], [26]]. Predatory bacteria like V. chlorellavorus have been found to attach to their algal host using a (proposed) electron-dense pad, and to inject effectors, enzymes, and plasmid DNA into the algal host cell, leading to cell lysis and loss of the cellular content [25,26].
Experimental and natural host range studies have shown that V. chlorellavorus infects a number of Chlorella species, including C. kessleri, C. sorokiniana, and C. vulgaris, whereas species of Ankistrodesmus, Chlorococcum, Scenedesmus, and Scotiella were found to be non-hosts of V. chlorellavorus [19,20,25,26]. These findings are consistent with the observation that in experimental outdoor raceways, designed to establish optimal conditions for commercial cultivation of C. sorokiniana, the bacterial pathogen V. chlorellavorus has been associated with lysis of cells and death of C. sorokiniana [14,29] when the optical density of C. sorokiniana was approximately ≥1.0 at 750 nm [21,27].
Strategies have been developed to reduce losses in Chlorella algal reactors by limiting infection by the bacterial pathogen. An acid-induced drop in pH in reactors containing susceptible Chlorella species co-cultured with V. chlorellavorus showed reduced rates of infection, extended duration of culture growth, and increased number of successive cultivation-runs [3]. Also, a treatment implemented to limit iron availability, resulted in decreased biomass ranging from zero to 9% in the iron-treated Chlorella cells infected with V. chlorellavorus, compared to cultures with no iron limitation, which showed a 72% decrease in biomass, indicating that a shortage of iron could limit pathogen attack [10].
The V. chlorellavorus is an obligate aerobe [26]; however, Chlorella species can grow under aerobic or anaerobic conditions [17]. Thus, deoxygenation was considered as an option for limiting the rates of V. chlorellavorus infection that would not significantly reduce the growth of C. sorokiniana. However, although anoxic-oxic cycles might be expected to negatively affect V. chlorellavorus growth, such conditions could possibly alter the composition of beneficial microorganisms associated with the algal culture. Because C. sorokiniana is known to be capable of growing axenically [3] and can tolerate anaerobic conditions [17], the proposed hypothesis was that C. sorokiniana growth might not be significantly reduced when cultured in a partially-deoxygenated environment, and at the same time, such conditions might be detrimental to the mutualistic, non-pathogenic phycosphere bacterial community. To guard against possible negative effects of deoxygenation on the non-pathogenic bacteria, algal cultures were aerated with ambient air during the dark period after deoxygenation cycles.
Nitrogen gas sparging is cost-effective and environmentally-compatible [[30], [31], [32], [33]] for deoxygenating water. Deoxygenation with the timed injection of nitrogen gas can be accomplished in closed bioreactors, and also in the deep canal of the testbed utilized here, referred to as the Algae Raceway Integrated Design (ARID) [34,35], by covering the canal to enhance the effects of deoxygenation and/or aeration cycling.
The goal of this study was to evaluate cyclical deoxygenation-oxygenation as a method to limit the growth of V. chlorellavorus to sub-pathogenic levels in suspension cultures of the algal host, C. sorokiniana, based on the inability of V. chlorellavorus to grow anaerobically, and ability of C. sorokiniana to thrive in aerobic or anaerobic environments. The first objective was to determine the tolerance of C. sorokiniana, DOE1412 [36], to various deoxygenation cycle times. The second objective was to define a deoxygenation cycling regime under which the effects of deoxygenation could be tested to evaluate its potential to abate V. chlorellavorus infection of C. sorokiniana, measured as algal culture phenotype, biomass production, and V. chlorellavorus titer determined by quantitative polymerase chain reaction (PCR) amplification. The rationale for the study was based on the hypothesis that if the effective V. chlorellavorus pathogen population size, below a to-be-determined threshold, at which infection occurred, could be manipulated by periodically deoxygenating the algal culture. Finally, in this study, a quantitative PCR assay developed previously [27] was used to monitor V. chlorellavorus accumulation under aerobic and deoxygenated conditions, to determine the relative threshold conducive to pathogenicity.
Section snippets
Pathogen-free cultures and co-cultures
The C. sorokiniana monoculture was cultivated in modified BG-11 solid growth medium containing 0.1 g/L urea, 0.012 g/L MgSO4, 0.035 g/L NH4H2PO4, 0.175 g/L KCl, 0.005 g/L FeCl3 [37], with trace minerals [38]. Individual algal cells were selected and transferred from the solid media and cultivated in 50 mL, 250 mL, then in 20 L carboy reactors with continuous agitation by injection of ambient air. All cultures were grown under 200 μmole/m2 s−1 light intensity with 16-h and 8-h light and dark
Effect of deoxygenation cycling on V. chlorellavorus-free C. sorokiniana
The effect of deoxygenation-aeration cycles on the growth of V. chlorellavorus-free C. sorokiniana was monitored in the treated cultures (TR1, TR2, and TR3) and compared to the nonaerated positive control C1, and the aerated negative control C2 (Exp. 1–3).
In Exp. 1, with 2-h/2-h and 4-h/4-h deoxygenation/aeration cycles, the algal growth rate in the TR3 replicate was much higher than the other two treatment replicates (Fig. 2-A). This artifact occurred because all sides of the middle flask
Conclusion
The deoxygenation-aeration cycles did not significantly inhibit C. sorokiniana growth in the experiments with V. chlorellavorus-free C. sorokiniana cultures (Exp.1–3). In the C. sorokiniana-V. chlorellavorus co-cultures (Exp.4), the aerated controls collapsed; however, the deoxygenation treatment provided protection of C. sorokiniana cells against attack and predation by V. chlorellavorus. These results provide a robust demonstration that deoxygenation-aeration cycling can dramatically limit
Acknowledgements
The authors are grateful for the U.S. Department of Energy and Regional Algal Feedstock Testbed (RAFT) project, University of Arizona, for supporting this study.
Conflict of interest
This work was supported by the U.S. Department of Energy Bioenergy Technologies Office Regional Algal Feedstock Testbed (RAFT) Project [grant number DE-EE0006269]. All the authors declare that there are no conflicts of interest. No conflicts, informed consent, human or animal rights applicable. All the authors agree to authorship and submission of the manuscript for peer review.
We did file a patent on this process. I am not sure if I need to state this in the paper.
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Cost minimization of deoxygenation for control of Vampirovibrio chlorellavorus in Chlorella sorokiniana cultures
2019, Algal ResearchCitation Excerpt :The volume in the reactors was adjusted to the initial fill level by the addition of water, after each sampling time. Algae growth was evaluated by measuring the DO concentrations and the culture optical density at 750 nm wavelength (OD750) which was then converted to AFDW biomass (g/L) as described previously [25]. The cultures and water samples were regularly inspected using a light microscope to monitor the progression of infection, which was confirmed based on presence of lysed C. sorokiniana cells, algal cell discoloration from grass-green to brownish, change in odor, and the accumulation of froth on the culture surface and reactor walls.
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