DATASET: Low-Cost Screening of Algae for Extreme Tolerance to pH, Temperature, Salinity, and Light

Low-Cost Screening of Algae for Extreme Tolerance to pH, Temperature, Salinity, and Light Authors: Barbara Saucedo, Kalisa Kang, Lauren May, Abhishek Gupta, Évellin do Espirito Santo, Stephen Mayfield, João Vitor Dutra Molino* Affiliation: 1 - Division of Biological Sciences, University of California San Diego, La Jolla, California, United States of America. 2 - Algenesis Inc., 1238 Sea Village Dr., Cardiff, CA, United States of America *Correspondence: Joao Vitor Dutra Molino ([e-mail]) ORCIDs Barbara Saucedo: https://orcid.org/0009-0000-0886-0839, [e-mail] Kalisa Kang: https://orcid.org/0009-0006-1939-8129, [e-mail] Lauren May: https://orcid.org/0009-0006-5023-5969, [e-mail] Abhishek Gupta: https://orcid.org/0000-0003-0136-3340, [e-mail] Évellin do Espirito Santo: https://orcid.org/0000-0002-9597-7118, [e-mail] Stephen Mayfield: https://orcid.org/0000-0001-7642-9047, [e-mail] João Vitor Dutra Molino: https://orcid.org/0000-0003-2475-9807, [e-mail] Figure 1: Demonstration of pH gradient formation on HSM nitrate media using KOH (basic) and phosphoric acid (acidic) solutions. Includes schematic diagrams and time-lapse images showing gradient development and stabilization over 9 days, visualized with a universal pH indicator. Figure 2: Growth and competitive assays of recombinant Chlamydomonas strains (Cin, Cre, Cpa) on pH gradient plates. Strains harboring antibiotic resistance vectors (bleomycin/hygromycin) exhibit distinct pH tolerance profiles. Includes individual and co-culture growth patterns. Figure 3: NaCl gradient plate preparation and growth of Chlamydomonas strains. Describes agar replacement with NaCl-rich media to create a salinity gradient and subsequent strain inoculation. Strains show differential salt tolerance based on vector-mediated resistance. Figure 4: Normalized chlorophyll fluorescence measurements of Chlamydomonas strains under increasing NaCl concentrations (0–400 mM). Fluorescence data, as a proxy for growth, reveal strain-specific salt tolerance profiles. Figure 5: Competitive growth assays of Chlamydomonas strain mixtures (Cin/Cre and Cpa/Cre) on NaCl gradient plates. Highlights tolerance zones and selective survival under salinity stress. Figure 6: Temperature-tolerance assay using a thermocycler-applied gradient (low to high). Growth of Chlamydomonas strains (Cin, Cre, Cpa) is assessed via post-incubation spotting on agar plates. Figure 7: Competitive growth of strain mixtures post-temperature screening on selective media (zeocin/hygromycin). Demonstrates colony recovery under antibiotic selection, validating strain-specific thermal tolerance. Figure 8: Light tolerance assay under shaded and direct high-intensity gradients. Strains exhibit differential growth inhibition at ~3000 µE/m²/s, with tape-protected regions as viability controls. Figure 9: Selection of Chlamydomonas colonies from high-light regions. Colonies are plated on selective media (zeocin/hygromycin), confirming strain identity via antibiotic resistance. Supplementary Figure 1.Universal pH indicator color scale (pH 4.0–10.0) used to visualize pH gradients on agar plates. Supplementary Figure 2.Position of the acidic (red) front over time in pH gradient plates, showing rapid expansion followed by stabilization between days 3 and 17. Supplementary Figure 3.Selective colony recovery from pH gradient transition zones in competitive co-cultures of Cin/Cre and Cpa/Cre, demonstrating strain-specific fitness under pH stress. Supplementary Figure 4.Colony recovery from NaCl gradient transition zones showing Cre dominance over Cin and Cpa dominance over Cre under salinity stress. Supplementary Figure 5.Competitive growth of Chlamydomonas strain mixtures under a light gradient, revealing differential tolerance to high-light conditions. Supplementary Figure 6.Schematic workflow of competitive gradient assays, including strain mixing, gradient exposure, recovery, and selective plating. Supplementary Figure 7.Plasmid maps of recombinant vectors used in this study; full sequences are available on Zenodo. Supplementary Videos Supplementary Video 1.Time-lapse of stable pH gradient formation over two weeks in phosphate, citrate, and combined buffer systems. Supplementary Video 2.Time-lapse showing unstable pH gradient dynamics in phosphate medium following localized acid addition. Videos: Supplementary Video 1: Time-lapse of pH gradient formation in phosphate and citrate buffer systems over two weeks, showing stable color transitions (red to blue). Supplementary Video 2: Dynamic pH gradient variation in phosphate media after acid addition, highlighting instability over two weeks.