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A Novel Treatment Protects Chlorella at Commercial Scale From the Predatory Bacterium Vampirovibrio Chlorellavorus

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A Novel Treatment Protects Chlorella at Commercial Scale From the Predatory Bacterium Vampirovibrio Chlorellavorus

Eneko Ganuza et al. Front Microbiol.

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.

Keywords: Chlorella; Micractinium inermum; Vampirovibrio chlorellavorus; algae industry; crop protection; pH-shock; predatory bacterium.

Figures

FIGURE 1
FIGURE 1
(A) Growth of Chlorella culture during three different scale up cycles in outdoor mixotrophic commercial reactors. Each cycle (divided by the dash line) represents the seed production (60,000 L) and transfer (denoted by arrows) to a commercial-scale reactor (130,000 L volume). Bars represent Vampirovibrio chlorellavorus infection as detected by qPCR assay in each cycle. The Ct value, or cycle threshold, decreases as the target abundance increases. (B) Bacterial community structure from whole culture samples of same three runs based on 16s rRNA gene sequencing. The detection of V. chlorellavorus sequences coincide with a decline or arrest in the growth of Chlorella shown in (A).
FIGURE 2
FIGURE 2
The comparison between the whole culture bacterial community structure (16s rRNA gene sequencing assay) of a batch culture of Chlorella and the corresponding phycosphere portion. Sequencing data was not available for days 2–4.
FIGURE 3
FIGURE 3
Phase contrast micrographs of Chlorella commercial cultures heavily infested with Vampirovibrio-like cells at two different stages (A) Appearance of the first empty or ghost cells. (B) Culture clumping. Micrographs produced by R. A. Andersen.
FIGURE 4
FIGURE 4
The pH-shock treatment (A) duration, (B) pH level, (C) residual acetate concentration, and (D) type of titrant did not affect Chlorella growth in flasks (250 mL, n = 2). Unless stated otherwise the pH treatment was conducted using hydrochloric acid (HCL) to reduce pH to 3.5 for 15 min in the presence of 0.5 g/L acetate and then inoculated aseptically into the Chlorella culture. Error bars represent 1 standard deviation.
FIGURE 5
FIGURE 5
The symptoms associated with Vampirovibrio chlorellavorus infection such as (A) bacterial attachment to the algae (B), clumping of algae cells and (C) change in the coloration of the culture (C) were reverted by the pH-shock treatment (D–F, respectively). Pictures and micrographs correspond to a simultaneous side by side comparison of raceways (1000 L, C,F) containing untreated (left) or pH-treated (right) culture derived from an industrial scale reactor (130,000 L). Micrograph 5A was produced by J. Wilkenfeld.
FIGURE 6
FIGURE 6
The impact of pH-shock treatment (close circles) on (A) Chlorella growth (B) bacterial attachment to the algae phycosphere (C) total aerobic bacterial count and (D) 16s rRNA Vampirovibrio chlorellavorus sequencing. Data correspond to a simultaneous side by side comparison in raceways (1000 L, n = 1) of pH-treated and untreated culture originated from in an industrial scale reactor (130,000 L).
FIGURE 7
FIGURE 7
Impact of the pH-shock treatment on (A) Chlorella cell dry weight, (B) bacterial attachment, and (C) qPCR assay Ct values for Vampirovibrio chlorellavorous from outdoor pilot scale reactors (1000 L, n = 1) inoculated with a contaminated Chlorella culture. The Ct value, or cycle threshold, in (C) decreases as the target abundance increases. Lysates prepared for qPCR on day 1 from the pH treated reactor failed to amplify.
FIGURE 8
FIGURE 8
Longevity of a Chlorella culture was prolonged by applying the pH treatment repeatedly to the same culture upon transfer, at the beginning of at least two consecutive batch cycles.
FIGURE 9
FIGURE 9
Summary of cytoplasmic pH regulation in response to the pH in the surrounding media for members of phylum Chlorophyta and Cyanobacteria reported in the literature (seven and four members, respectively). Chlorophyta includes Chlorella kessleri (El-Ansari and Colman, 2015), Chlorella pyrenoidosa and Scenedesmus quadricauda (Lane and Burris, 1981), Chlorella saccharophila (Gehl and Colman, 1985), Chlorella vulgaris and Chlorella fusca (Küsel et al., 1990) and Dunaliella parva (Gimmler et al., 1988). Cyanobacteria include Agmenellum quadruplicatum and Gloeobacter violaceus (Belkin et al., 1987), Anacystis nidulans, (Falkner and Horner, 1976) and Synechococcus sp. (Kallas and Castenholz, 1982). Note that only one Cyanobacterium (A. quadruplicatrum) did grow at a pH below 6.0. All other data points are expressed as mean values with error bars representing 1 standard deviation.

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References

    1. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. (1990). Basic local alignment search tool. J. Mol. Biol. 215 403–410. 10.1016/S0022-2836(05)80360-2 - DOI - PubMed
    1. Basso L., Basso T., Rocha S. (2011). “Ethanol production in Brazil: The industrial process and its impact on yeast fermentation,” in Biofuel Production-Recent Developments and Pospects, ed. Bernardes M. A. S., editor. (Rijeka: InTech Open Access Publisher; ), 85–100.
    1. Bates L., Maechler M., Bolker B., Walker S. (2015). Fitting linear mixed-effects models using lme4. J. Stat. Softw. 67 1–48. 10.18637/jss.v067.i01 - DOI
    1. Beijerinck M. (1890). Kulturversuche mit Zoochlorellen, Lichenengonidien und anderen niederen Algen. Bot. Ztg. 48 729.
    1. Belkin S., Mehlhorn R. J., Packer L. (1987). Proton gradients in intact cyanobacteria. Plant Physiol. 84 25–30. 10.1104/pp.84.1.25 - DOI - PMC - PubMed

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