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

palm) microalgal feedstocks have potential to achieve higher lipid yields per unit area than conventional crops without the need for arable land that could be used for food production [1–4]. Like terrestrial crop production, the predominant approach to large scale cultivation of algae has largely focused on genetic engineering and strain selection to identify single species that maximize lipid production when grown in monoculture under laboratory settings [1, 4, 5]. However, the high yields achieved by genetically or strain selected species in the lab rarely hold under field conditions, such as in large-scale open pond outdoor raceways that are the most economically viable method for generating large feedstock quantities [1, 6]. Indeed, algal feedstocks grown in outdoor raceway ponds have proven more difficult to maintain because of invasion by unwanted species of competing algae or cyanobacteria [7], invasion by herbivores that consume the crop [8], invasion by parasites and diseases that kill the focal algae [6] or environmental conditions that fluctuate beyond the tolerance limits of the focal species [9]. In response to the challenges of algal monoculture production, particularly in outdoor open raceway ponds, researchers have begun to consider how ecological engineering of diverse algal feedstocks can alleviate the challenges faced by monoculture feedstock production [10, 11]. Several studies have shown that when compared to the average monoculture, algal polycultures composed of multiple species can be designed to improve the total production of biomass, the

temporal stability of feedstock production in variable environments, and desirable biochemical properties of feedstocks that are upgraded to biocrude [12–16]. For example, Shurin et al. [14], found that particular polycultures were able to achieve high algal biomass yield than their most productive monocultures. Similarly, diverse algal feedstocks have shown to be on par or exceed nutrient use efficiencies of the average monocultures [13, 17, 18]. Additionally, Godwin et al. [17] demonstrated that biodiverse algal cultures were able to maintain higher levels of multi- functionality (simultaneously maintain high levels of yield, stability, invasion resistance, etc.) than any of the component species grown in monoculture. However, it is important to note that most of the aforementioned studies have been performed in laboratory settings. As such, it remains unclear whether the benefits that are sometimes conferred by biodiversity will hold under commercial scale outdoor cultivation where the risk of pests, parasites, and pathogens are more substantial than they are in the safety of the lab [6, 19–21]. One of the greatest risks to outdoor cultivation of algal feedstocks is their susceptibility to invasion by unwanted pathogens, particularly pathogens such as chytrid (phylum Cryptomy- cota) and aphelid (phylum Aphelida) fungi [6, 22]. These fungal pathogens penetrate host cells, consume intracellular contents so as to produce large quantities propagules that are released through the ruptured host cell wall, causing host cell mortality [23]. As they do so, the

fungi are able to quickly proliferate through algal cultures, often leading to culture crash and complete loss of feedstock productivity [22]. Currently, two main strategies are used to miti- gate the impact of infection. One involves a salvage harvest of the culture, followed by subse- quent disinfection of the culture tanks, prior to reestablishing the outdoor pond operation [6]. This approach leads to yield reductions in addition to increased operational costs, making the strategy economically infeasible. A second strategy of fungal control involves the application of chemical fungicides to reduce the risk of disease spread [6, 10, 24, 25]. While chemical fungi- cides have proven to be effective in the short term, they can be expensive, have the potential for target species to develop resistance, can pose certain human health risks, and can have unintended environmental impacts [26, 27]. Given these limitations, there has been recent interest in developing better methods of fungal control in algal feedstock systems [28, 29]. A recently proposed method for fungal control utilizes a concept from the field of Disease Ecology called the “dilution effect” [29]. The dilution effect occurs when an increase in host diversity leads to a reduction in the risk of disease within a community [30, 31]. One proposed mechanism for disease dilution occurs when less susceptible host species reduce the PLOS ONE Biodiversity, biofuel and disease: Ecological engineering in algal feedstocks PLOS ONE | https://doi.org/10.1371/journal.pone.0267674 April 28, 2022 2 / 15