Biodiversity and disease risk in an algal biofuel system: An experimental test in outdoor ponds using a before-after-control-impact (BACI) design

relative to algal monocultures (the ‘Control’ group) when they are exposed to an infectious fungal parasite (‘Before’ vs. ‘After’ infection). The Control treatment consisted of a monocul- ture of S. capricornutum, while the Impact treatment consisted of a polyculture of S. capricor- nutum, C. sorokiniana, and S. obliquus (Fig 1). The Control and Impact treatments were each replicated in 3 raceway ponds. Prior to their inoculation in the 400-L raceway ponds, each algal species was scaled up indi- vidually in laboratory cultures, first in 800-mL sterile glass columns containing BG-11 growth media, which were then transferred into 15-L plexiglass panels containing BG-11 growth media that were exposed to continuous light and a constant delivery of gaseous CO2 (S2 Fig in S1 Appendix). Once adequate cell densities were reached in laboratory cultures, each of the 400-L raceways was inoculated with the same starting concentration of dry algal biomass (0.084 g L-1). This resulted in 0.084 g L-1 of S. capricornutum being added to each of the three Control (monoculture) raceway ponds, and 0.028 g L-1 of each of the three species into the Treatment (polyculture) raceway ponds. The Before and After portion of the study represented sampling periods before and after a fungal parasite was intentionally introduced to the ponds. Raceway ponds were monitored for 23 days before a fungal parasite was introduced (sampling is described later in Section Experi- mental design). During this time period, through microscopic examination of algal samples

there was no significant sign of fungal infection detected in any of the raceway ponds. Then, on day 23 and again on day 25, each raceway pond was inoculated at less than 1% of total cell density with 75mL of Desmodesmus armatus from a nearby outdoor pond that was nearly 100% infected with a local fungal parasite that is a common pest at AzCATI (see Results for description of fungal parasite). The fungal parasite was intentionally introduced using a non- focal species of algae because (1) cultures of infected D. armatus are routinely maintained at AzCATI for study of the fungus, making it a readily available and controlled source of infec- tion, and (2) D. armatus is a morphologically distinct, competitively inferior species (based on prior field experience and observations) that can be used as a ‘tracer’. As a tracer species an equal amount of infection to all ponds was able to be introduced, after which, the ponds were monitored to show that the source of infection (D. armatus) was successful at initiating disease in other algal species; yet D. armatus did not establish itself in the ponds or alter the intended species composition of algae (as will be discussed later). On day 27, five days after the introduction of the fungus, each raceway pond was dosed with 1-ppm of Secure 1 with the active ingredient fluazinam, which is a pesticide that is com- monly used to control fungal pathogens during algal feedstock cultivation at AzCATI (U.S. Patent Application No. 14/351,540; Publication No. 20140378513; Published Dec. 25, 2014;

Sapphire Energy Inc., Applicant). By applying a pesticide that is normally used to control fun- gal proliferation during feedstock production, this study was intended to test whether algal polycultures offer any additional benefits for pest control above and beyond the traditional controls that are applied during feedstock growth. For the remaining 20 days after fungal infection (After period), the raceways were sampled and harvested utilizing the same methods as during the Before period (described next). Raceway sampling and processing Following the initial inoculation of algae in the raceway ponds, samples were collected from each pond every 2-days using a sterile 50-mL polypropylene centrifuge tubes by submerging the tubes in same well mixed portion of each raceway. From these samples, two sub-samples were taken. One 15-mL subsample was used to determine daily estimates of feedstock biomass by measuring ash free dry weight (AFDW). AFDW was measured utilizing the methodology PLOS ONE Biodiversity, biofuel and disease: Ecological engineering in algal feedstocks PLOS ONE | https://doi.org/10.1371/journal.pone.0267674 April 28, 2022 5 / 15