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. Plasmids: Details on recombinant vectors (pAH04mCherry, pJPCHx1_mVenus, pJP32PHL7) used in Chlamydomonas strains, including antibiotic resistance markers. Vectors are available at the Chlamydomonas collection. Supplementary Figure 2: Graph showing temporal stability of the acidic (red) front position on pH gradient plates over 17 days, highlighting initial expansion and stabilization. Supplementary Figure 3: Colony recovery assays from pH gradient transition zones. Compares survival of Cre (hygromycin-resistant) vs. Cin/Cpa (bleomycin-resistant) in competitive co-cultures. Supplementary Figure 4: Colony recovery assays from NaCl gradient transition zones. Demonstrates Cre dominance over Cin but Cpa dominance over Cre under salinity stress. Supplementary Figure 5: Competitive growth of Chlamydomonas mixtures under light gradients. Cre outperforms Cin in high-light regions, while Cpa competes with Cre under similar conditions. 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.