Real-time quantitative detection of Vampirovibrio chlorellavorus, an obligate bacterial pathogen of Chlorella sorokiniana
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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.
KeywordsBiocide Melainabacteria Microalgal cultivation Polymerase chain reaction Vampirovibrionales
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.
Support for this research was provided from the Department of Energy Grant DE-EE0006269.
Compliance with ethical standards
Conflict of interest statement
The authors declare they have no conflicts of interest.
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