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

abundance of more susceptible host through interspecific competition [32]. That is, the pres- ence of less susceptible hosts ‘dilute’ the risk of disease establishment and spread throughout the community. If the dilution effect were to operate in algal feedstocks, then it might be possi- ble to ecologically engineer the composition of feedstocks to have species that have differential fungal resistance, niche complementarity, and high measures of productivity (e.g. temporal stability, yield, etc.). In turn, one might be able to simultaneously safeguard against disease out- breaks while enhancing feedstock yields through time [29]. But while many studies have shown the operation of dilution effects in natural systems, the idea has not been tested using algal feedstock cultivation [33, 34]. In this study, the question of whether ecologically engineered multi-species consortia of algae would be more resistant to crop failure caused by fungal pathogens than single species monocultures is addressed. It is hypothesized that due to a dilution effect, ecologically engi- neered algal polycultures would maintain higher measures of feedstock production compared to monocultures of the most productive species when both were simultaneously exposed to a fungal pathogen. This hypothesis was tested by conducting a statistically rigorous, well repli- cated BACI experiment (before vs. after fungal infection, monocultures as controls vs. polycul- tures as the impact treatment) in outdoor raceway ponds. Algal monocultures and

polycultures were grown in replicate raceways and were subjected to routine harvests, inten- tionally challenged with a fungal pathogen, and sampled regularly to estimate feedstock pro- duction metrics and fungal infection. Materials and methods Microalgal species selection As the goal of this experiment was to not to simply test whether any multi-species consortia could buffer against disease risk, but rather whether the best algal feedstock monoculture and multi-species consortia, would differ in terms of productivity metrics in outdoor ponds in response to disease, results from prior experiments were used to determine the model algal species. From a three-phase series of prior experiments the best monoculture and multi-spe- cies consortia were identified. Below details of the following three phases are outlined: I) experimental biculture comparisons of yield from 55 species pool, II) laboratory comparison of yield, temporal stability, and nutrient use efficiency monocultures and polycultures best six species from Phase I in laboratory, and III) field comparison of yield, stability, and pest invasa- bility between single best monoculture and polyculture from Phase II in field experiment. Phase I involved 55 species of freshwater green algae that are included in the U.S. Depart- ment of Energy’s Aquatic Species Program and were identified in U.S. EPA’s 2007 National Lakes Assessment as being the most widespread and abundant species across North America (so they would not pose a risk to natural habitats in the event of a release). Using these species,

an initial set of laboratory experiments compared the yield of species bicultures to that of monocultures to screen for increased biomass production in cultures with co-occurring algal species [35]. Results from this work lead to the selection of six species that were found to rou- tinely be involved in increased biomass production of algal polycultures. During Phase II, labo- ratory experiments were used to evaluate whether polycultures of these six species would increase biomass production and temporal stability compared to each species grown alone in monoculture [16, 17, 36]. Phase III transitioned to field-based experiments in which the single best monoculture and polyculture consortia from Phase II were compared for production of algal biomass and biocrude yield, temporal stability, risk of invasive algae, potential for culture crash, and ability to maintain more of these functions at higher levels [12]. Based on the results of Phases I-III the single best monoculture (Selenastrum capricornutum) identified for algal PLOS ONE Biodiversity, biofuel and disease: Ecological engineering in algal feedstocks PLOS ONE | https://doi.org/10.1371/journal.pone.0267674 April 28, 2022 3 / 15