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

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Abstract

Vampirovibrio chlorellavorus is an obligate, predatory bacterial pathogen of the genus Chlorella. It is recognized as an important pathogen of Chlorella sorokiniana, field isolate DOE 1412, a highly-favored microalga for cultivation in outdoor reactors in the arid USA Southwest for feedstocks used in biofuel production. To determine the V. chlorellavorus titer, based on gene copy number, required to cause infection and mortality of C. sorokiniana in an experimental outdoor reactor, a multiplexed quantitative polymerase chain reaction (qPCR) assay was developed for pathogen detection, based on the 16S and 18S ribosomal RNA gene of V. chlorellavorus and C. sorokiniana, respectively. The assay was further used to establish the optimal effective concentration of benzalkonium chloride required to achieve a below “disease-threshold”-bacterial titer, while minimizing biocidal effects on algal growth and enable economic biomass production. Reactors treated with 2.0 ppm benzalkonium chloride at four-day intervals throughout the cultivation cycle experienced runs of 22 days or longer, compared to 12 days for the untreated control. The qPCR assay was used to estimate disease severity over time using the Area Under the Disease Progress Stairs (AUDPS) metric, indicating a severity rating of 0.016 and 62.308 in biocide-treated and untreated cultures, respectively. The near-real time assay detected as few as 13 copies of V. chlorellavorus, allowing for the recognition of its presence in the reactor just before algal cell density decreased, an indication of pathogen attack, while also informing the timing of biocide applications to minimize DOE 1412 infection such that harvestable biomass could be produced.

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Acknowledgements

The authors would like to express appreciation to Caitlin C. Brown and Noel Kitchen for their support and invaluable contributions with culture maintenance and qPCR assay design and validation, to undergraduates Cassandra Galves and Stephen Lee for laboratory assistance, and to the ARID raceway team. The first author would like to thank Drs. J.K. Brown and K. Ogden for guidance and mentoring throughout this study, leading to completion of his Master’s Thesis.

Funding

Support for this research was provided from the Department of Energy Grant DE-EE0006269.

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  1. School of Plant Sciences, The University of Arizona, Tucson, AZ, 85721, USA

    Seth A. Steichen & Judith K. Brown

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  1. Seth A. Steichen
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Correspondence to Judith K. Brown.

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Electronic supplementary material

Supplemental Fig. 1

Benzalkonium chloride effects on DOE 1412 growth and Vampirovibrio chlorellavorus accumulation in field reactors. Growth data of experiments conducted from May 19 to Jun 9, 2016 reporting Top) culture temperature is displayed as recorded by continuous data logger from PW1 reactor unit and compared across a time course of 25 days of growth against Middle) the optical density (absorbance at 750 nm) of Chlorella sorokiniana culture in collocated PW reactor units either treated with BAC or a no treatment control (NT) as well as rain fill, quantified by the volume of water added to reactors. Bottom) V. chlorellavorus accumulation is displayed in both biomass and supernatant fractions by the ratio of 16S ribosomal RNA gene per algal cell 18S ribosomal RNA gene detected on a logarithmic scale which excludes initial time point samples of undetected 16S ribosomal RNA genes. (PDF 62 kb)

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Steichen, S.A., Brown, J.K. Real-time quantitative detection of Vampirovibrio chlorellavorus, an obligate bacterial pathogen of Chlorella sorokiniana. J Appl Phycol 31, 1117–1129 (2019). https://doi.org/10.1007/s10811-018-1659-z

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