Contents lists available at ScienceDirect Algal Research journal homepage: www.elsevier.com/locate/algal Iterative screening of an evolutionary engineered Desmodesmus generates robust field strains with pesticide tolerance Alina A. Corcoran a,b, ⁎ , Matthew A. Saunders a,c,1 , Adam P. Hanley a,c , Philip A. Lee a,d , Salvador Lopez a , Rebecca Ryan a,e , Christopher B. Yohn a,f a Sapphire Energy, Inc., 10996 Torreyana Rd, Suite 280, San Diego, CA 92121, USA b New Mexico Consortium, 100 Entrada Dr, Los Alamos, NM 87544, USA c Renew Biopharma, 11555 Sorrento Valley Road, San Diego, CA 92121, USA d Midland College, 3600 N Garfield, Midland, TX 79705, USA e Indigo Agriculture, 500 Rutherford Ave, Boston, MA 02129, USA f Unity Biotechnology, 3280 Bayshore Blvd, Brisbane, CA 94005, USA ARTICLE INFO Keywords: UV mutagenesis High throughput screening Crop protection Fluazinam Fungicide Raceway ABSTRACT Microalgae hold remarkable promise to produce food, fuel, and nutraceuticals. To satisfy industrial economics, microalgae are commonly cultivated outdoors in open ponds that are subject to invasions by a suite of pests including predators, competitors, and parasites. Although pests can be mitigated and/or controlled by biocides or pesticides, therapeutic doses of these chemicals often adversely affect the productivity and yield of the target crop. To combat this tradeoff imposed by pesticide treatment, if pesticides are to be used, robust strains with increased pesticide tolerance must be developed and validated for growth at commercial scales. In this study, we evolutionarily engineered strains of the green algae Desmodesmus armatus to tolerate treatment with Omega 500F®, a broad-spectrum agricultural fungicide that is used to treat pest invasions in open, outdoor microalgal ponds. An algal clone library generated through UV mutagenesis was screened for strains that exhibited the greatest growth rates under batch and semi-continuous culture in the lab with and without exposure to Omega. We then screened strains at increasing spatial (i.e., from 96-well plates to 33,000 L raceways) and temporal (i.e., from 5 days to months) scales to select for those strains that were most robust. Our process resulted in the cultivation of two robust field strains that demonstrated tolerance to repetitive dosing with Omega without apparent trade-offs in productivity. This work highlights the utility of non-GM methods, specifically UV muta- genesis, to improve cultivation strains for the production of biomass and bioproducts from microalgae. Moreover, it demonstrates the importance of iterative validation steps in facilitating a successful lab to field transition of engineered strains. 1. Introduction With largely untapped taxonomic and phenotypic diversity, micro- algae hold remarkable promise to produce food, fuel, and nutraceuticals at industrial scales, meeting growing societal demands for these com- modities. Microalgae exhibit high doubling rates and can be grown on non-arable land in natural or artificial ponds, generating high yields and reducing economic competition with traditional agricultural sys- tems [1]. Microalgae are also particularly amenable to laboratory evolution due to their rapid doubling rates and unicellular form [2,3]. To satisfy industrial economics, microalgae are commonly cultivated outdoors in open raceway ponds [4–6]. Unfortunately, with this approach, predators, competitors, and parasites can quickly invade ponds and decrease productivity and yield or decimate entire crops [7–9]. Pest pressure in ponds can be controlled biologically (e.g., by introducing herbivores; [10]), mechanically (e.g., filtering the pond culture; [11]), or chemically. In the latter case, chemicals may be in- troduced either to change environmental conditions such that pests are not favored [12,13] or to directly kill pests through the application of a biocide or pesticide [14–16]. Although pest pressure can be effectively mitigated by biocide and pesticide treatment, such treatment may come at a cost to algal fitness in the form of lower productivity/biomass and/ or altered environmental tolerances [17,18]. Development and valida- tion of robust algal field strains with increased tolerance or resistance to https://doi.org/10.1016/j.algal.2018.02.026 Received 4 December 2017; Received in revised form 6 February 2018; Accepted 22 February 2018 ⁎ Corresponding author at: Sapphire Energy, Inc., 10996 Torreyana Rd, Suite 280, San Diego, CA 92121, USA. 1 Co-first author. E-mail address: alina.corcoran@outlook.com (A.A. Corcoran). Algal Research 31 (2018) 443–453 2211-9264/ © 2018 Elsevier B.V. All rights reserved. T