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 Bdisease-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.

Microalgae as a biofuel source are of great interest. Bacterial phycosphere inhabitants of algal cultures are hypothesized to contribute to productivity. In this study, the bacterial composition of the Chlorella sorokiniana phycosphere was determined over several production cycles in different growing seasons by 16S rRNA gene sequencing and identification. The diversity of the phycosphere increased with time during each individual reactor run, based on Faith's phylogenetic diversity metric versus days post-inoculation (R = 0.66, P < 0.001). During summer months, Vampirovibrio chlorellavorus, an obligate predatory bacterium, was prevalent. Bacterial sequences assigned to the Rhizobiales, Betaproteobacteriales and Chitinophagales were positively associated with algal biomass productivity. Applications of the general biocide, benzalkonium chloride, to a subset of experiments intended to abate V. chlorellavorus appeared to temporarily suppress phycosphere bacterial growth, however, there was no relationship between those bacterial taxa suppressed by benzalkonium chloride and their association with algal productivity, based on multinomial model correlations. Algal health was approximated using a model-based metric, or the 'Health Index' that indicated a robust, positive relationship between C. sorokiniana fitness and presence of members belonging to the Burholderiaceae and Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium clade. Bacterial community composition was linked to the efficiency of microalgal biomass production and algal health.

Algal growth demands a continuous source of water of appropriate salinity and nutritional content. Fresh water sources are scarce in the deserts of the Southwestern United States, hence, salt water algae species are being investigated as a renewable biofuel source. The use of produced water from oil wells (PW) could offset the demand for fresh water in cultivation. Produced water can contain various concentrations of dissolved solids, metals and organic contaminants and often requires treatment beyond oil/water separation to make it suitable for algae cultivation. The produced water used in this study was taken from an oil well in Jal, New Mexico. An F/2-Si (minus silica) growth media commonly used to cultivate Nannochloropsis salina 1776 (NS 1776) was prepared using the produced water (F/2-Si PW) taking into account the metals and salts already present in the water. NS 1776 was seeded into a bioreactor containing 5L of the (F/2-Si PW media. After eleven days the optical density at 750 nm (an indicator of algal growth) increased from 0 to 2.52. These results indicate algae are able to grow, though inhibited when compared with non-PW media, in the complex chemical Neal 2 conditions found in produced water. Savings from using nutrients present in the PW, such as P, K, and HCO 3-, results in a 44.38% cost savings over fresh water to mix the F/2-Si media.

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Vampirovibrio chlorellavorus is recognized as a pathogen of commercially-relevant Chlorella species. Algal infection and total loss of productivity (biomass) often occurs when susceptible algal hosts are cultivated in outdoor open pond systems. The pathogenic life cycle of this bacterium has been inferred from laboratory and field observations, and corroborated in part by the genomic analyses for two Arizona isolates recovered from an open algal reactor. V. chlorellavorus predation has been reported to occur in geographically-and environmentally-diverse conditions. Genomic analyses of these and additional field isolates is expected to reveal new information about the extent of ecological diversity and genes involved in host-pathogen interactions. The draft genome sequences for two isolates of the predatory V. chlorellavorus (Cyanobacteria; Ca. Melainabacteria) from an outdoor cultivation system located in the Arizona Sonoran Desert were assembled and annotated. The genomes were sequenced and analyzed to identify genes (proteins) with predicted involvement in predation, infection, and cell death of Chlorella host species prioritized for biofuel production at sites identified as highly suitable for algal production in the southwestern USA. Genomic analyses identified several predicted genes encoding secreted proteins that are potentially involved in pathogenicity, and at least three apparently complete sets of virulence (Vir) genes, characteristic of the VirB-VirD type system encoding the canonical VirB1-11 and VirD4 proteins, respectively. Additional protein functions were predicted suggesting their involvement in quorum sensing and motility. The genomes of two previously uncharacterized V. chlorellavorus isolates reveal nucleotide and protein level divergence between each other, and a previously sequenced V. chlorellavorus genome. This new knowledge will enhance the fundamental understanding of trans-kingdom interactions between a unique cosmopolitan cyanobacterial pathogen and its green microalgal host, of broad interest as a source of harvestable biomass for biofuels or bioproducts.