A Novel Treatment Protects Chlorella at Commercial Scale from the Predatory Bacterium Vampirovibrio chlorellavorus

Abstract

The predatory bacterium, Vampirovibrio chlorellavorus, can destroy a Chlorella culture in just a few days, rendering an otherwise robust algal crop into a discolored suspension of empty cell walls. Chlorella is used as a benchmark for open pond cultivation due to its fast growth. In nature, V. chlorellavorus plays an ecological role by controlling this widespread terrestrial and freshwater microalga, but it can have a devastating effect when it attacks large commercial ponds. We discovered that V. chlorellavorus was associated with the collapse of four pilot commercial-scale (130,000 L volume) open-pond reactors. Routine microscopy revealed the distinctive pattern of V. chlorellavorus attachment to the algal cells, followed by algal cell clumping, culture discoloration and ultimately, growth decline. The “crash” of the algal culture coincided with increasing proportions of 16s rRNA sequencing reads assigned to V. chlorellavorus. We designed a qPCR assay to predict an impending culture crash and developed a novel treatment to control the bacterium. We found that (1) Chlorella growth was not affected by a 15 min exposure to pH 3.5 in the presence of 0.5 g/L acetate, when titrated with hydrochloric acid and (2) this treatment had a bactericidal effect on the culture (2-log decrease in aerobic counts). Therefore, when qPCR results indicated a rise in V. chlorellavorus amplicons, we found that the pH-shock treatment prevented the culture crash and doubled the productive longevity of the culture. Furthermore, the treatment could be repeatedly applied to the same culture, at the beginning of at least two sequential batch cycles. In this case, the treatment was applied preventively, further increasing the longevity of the open pond culture. In summary, the treatment reversed the infection of V. chlorellavorus as confirmed by observations of bacterial attachment to Chlorella cells and by detection of V. chlorellavorus by 16s rRNA sequencing and qPCR assay. The pH-shock treatment is highly selective against prokaryotes, and it is a cost-effective treatment that can be used throughout the scale up and production process. To our knowledge, the treatment described here is the first effective control of V. chlorellavorus and will be an important tool for the microalgal industry and biofuel research.

Real-time quantitative detection of Vampirovibrio chlorellavorus, an obligate bacterial pathogen of Chlorella sorokiniana

“…The reduction of disease severity in BAC-treated cultures contributed to the prolonged algal growth in the ARID reactors. This outcome is similar to that achieved by the low-pH treatment reported previously (Ganuza et al 2016); however, the use of BAC avoids exposure to strong acid, and for outdoor reactors is potentially less harmful to the environment. Because BAC is a general biocide, there is a tradeoff between its efficacy to abate pathogen attack by reducing the rate at which V. chlorellavorus accumulated in the reactors, and the negative effect of the biocide on algal growth.…”

“…within 24-48 h after pathogen presence if forewarned, indicated by a sharp decline in 750 nm optical density (O.D.). However, in a recent study, the duration of the cultivation run was increased substantially by the addition of acid to rapidly decrease the pH in the reactor from pH 7.5 to 3.5 for 15 min, with restoration to the normal pH of 7.5 (Ganuza et al 2016). Such a regime is not practical or environmentally sound for the treatment of large outdoor reactors.…”

Fatty Acids, Hydrocarbons and Terpenes of Nannochloropsis and Nannochloris Isolates with Potential for Biofuel Production

“…This pH-shock treatment has been applied before for effective control of V. chlorellavorus, a predatory bacterium that can destroy a Chlorella culture in just a few days [37]. This effective treatment with high selectivity and low cost can be considered throughout the scale-up and production process [37]. The pH-shock treatment applied in this study is simpler than the one executed by Ganuza et al [37] and patented by Ganuza and Tonkovich.…”

“…This effective treatment with high selectivity and low cost can be considered throughout the scale-up and production process [37]. The pH-shock treatment applied in this study is simpler than the one executed by Ganuza et al [37] and patented by Ganuza and Tonkovich. Application of antibiotics has been used by other authors to obtain axenic cultures from freshwater bodies [38].…”

“…Application of HCl proved to be an effective decontamination technique. This pH-shock treatment has been applied before for effective control of V. chlorellavorus, a predatory bacterium that can destroy a Chlorella culture in just a few days [37]. This effective treatment with high selectivity and low cost can be considered throughout the scale-up and production process [37].…”