Methanol Production by a Broad Phylogenetic Array of Marine Phytoplankton

Abstract

Methanol is a major volatile organic compound on Earth and serves as an important carbon and energy substrate for abundant methylotrophic microbes. Previous geochemical surveys coupled with predictive models suggest that the marine contributions are exceedingly large, rivaling terrestrial sources. Although well studied in terrestrial ecosystems, methanol sources are poorly understood in the marine environment and warrant further investigation. To this end, we adapted a Purge and Trap Gas Chromatography/Mass Spectrometry (P&T-GC/MS) method which allowed reliable measurements of methanol in seawater and marine phytoplankton cultures with a method detection limit of 120 nanomolar. All phytoplankton tested (cyanobacteria: Synechococcus spp. 8102 and 8103, Trichodesmium erythraeum, and Prochlorococcus marinus), and Eukarya (heterokont diatom: Phaeodactylum tricornutum, coccolithophore: Emiliania huxleyi, cryptophyte: Rhodomonas salina, and non-diatom heterokont: Nannochloropsis oculata) produced methanol, ranging from 0.8-13.7 micromolar in culture and methanol per total cellular carbon were measured in the ranges of 0.09-0.3%. Phytoplankton culture time-course measurements displayed a punctuated production pattern with maxima near early stationary phase. Stabile isotope labeled bicarbonate incorporation experiments confirmed that methanol was produced from phytoplankton biomass. Overall, our findings suggest that phytoplankton are a major source of methanol in the upper water column of the world's oceans.

Fig 1. Time-course plot of R. salina batch culture grown in biological triplicate showing methanol production (y-axis) over time (x-axis) in micromolar amounts in upper panel, and cell abundances and corresponding photosystem health (F_v/F_m) in lower panel.

Note, clear methanol production coinciding with the onset of stationary phase.

Fig 2. Time-course plot of N. oculata batch culture grown in biological triplicate showing methanol production (y-axis) over time (x-axis) in micromolar amounts in upper panel, and cell abundances and corresponding F_v/F_m in lower panel.

Note, highest methanol production where cell abundance levels off.

Fig 3. Time-course plot of P. tricornutum batch culture grown in biological triplicate showing methanol production (y-axis) over time (x-axis) in micromolar amounts in upper panel, and cell abundances and corresponding F_v/F_m in lower panel.

Note, spikes of methanol production and apparent loss coincidental with maximal cell abundance and drop in F_v/F_m.

Fig 4. Phaeodactylum tricornutum CCMP632 culture sample after 10 days growth (conditions detailed in Methods, with biological duplicates and technical triplicate measurements) were tested for background production of methanol after filtering through GF/F filters and freezing at -20°C for 24 hours.

Samples were processed with or without 4mM mercuric chloride (final concentration, as indicated), incubated for 10 minutes at room temperature then processed as detailed in Methods. A subset of samples was frozen at -20°C for 24 hours, another was measured immediately. Blanks consisting of negative media controls were processed in parallel. Asterisks indicate detection of background methanol at approximately 100 micromolar or less.