Bacterial response to spatial gradients of algal-derived nutrients in a porous microplate
Acrylic and polydimethylsiloxane (PDMS) molds preparationThe incubating device for the porous microplate was designed using a CAD software (Solidworks, Dassault Systèmes) and the exported drawing files were used to laser cut 1/4” and 1/8” acrylic sheet (Universal Laser Systems; Supplementary Fig. S2). After washing the cut acrylic parts with deionized water, they were attached by acrylic (Weld-On) and epoxy (3 M) adhesives that were followed by a curing process for ~18 h. Polydimethylsiloxane (PDMS) (Sylgard 184, Dow Corning) was cast onto the acrylic mold and cured at 80 °C for at least 3 h. The PDMS mold was carefully detached from the acrylic surface by dispensing isopropyl alcohol (VWR) into the area between the PDMS and the acrylic molds (Fig. 2a).Fig. 2: Synthesis and characterization of porous microplate.a Procedure to build a porous microplate using polydimethylsiloxane (PDMS) and acrylic molds. b Image of the microplate with an array of culture wells (wall thickness: 0.9 mm). c Scanning electron microscopy image of nanoporous copolymer HEMA–EDMA.Full size imagePorous microplate preparationSynthesis of copolymer HEMA–EDMA was based on previously described protocols [30, 31] and details are given as follows. Prepolymer solution HEMA − EDMA was prepared by mixing 2-hydroxyethyl methacrylate (HEMA; monomer, 24 wt.%, Sigma-Aldrich), ethylene glycol dimethacrylate (EDMA; crosslinker, 16 wt.%, Sigma-Aldrich), 1-decanol (porogen, 12 wt.%, Sigma-Aldrich), cyclohexanol (porogen, 48 wt.%, Sigma-Aldrich) and 2,2-dimethoxy-2-phenylacetophenone (DMPAP; photoinitiator, 1 wt.%). The solution was stored at room temperature without light exposure until further use. Glass slides (75 × 50 mm2, VWR) were chemically cleaned by sequentially soaking in 1 M hydrochloric acid and 1 M sodium hydroxide for one hour, followed by rinsing with deionized water and air drying. The prepolymer solution was cast onto the PDMS mold and a glass slide was placed on the mold. The solution was then polymerized under ultraviolet light with a wavelength 365 nm for 15 min by using a commercial UV lamp (VWR). The photopolymerized device was detached from the PDMS mold and stored in a jar containing methanol (VWR) until further use (Fig. 2a). The jar was refilled with new methanol twice in order to remove the remaining porogen and uncrosslinked monomers from the hydrogel.Upon each incubation experiment with the porous microplate, each device was decontaminated by replacing the solvent with 70% alcohol (VWR) and storing it for 24 h. They were immersed in a pre-autoclaved jar for two weeks with f/2 medium with omitted silicate, where the jar was refilled once with a new sterile medium to adjust its pH for the algal culture and remove any solvent remaining in the hydrogel. Before inoculating microbial cells, each microplate was taken out from the jar and the media remaining on the top surface was removed by absorbing it with a pre-sterilized wipe to minimize the chance for cross-contamination between wells (Fig. 2b).Scanning electron microscopyPhotopolymerized HEMA − EDMA was removed from methanol and dried in air for at least one week to evaporate the excess solvent. A ~5 × 5 mm2 specimen was collected from the dried copolymer and attached to a pin stub. The stub was loaded on a scanning electron microscope (SEM; MERLIN, Carl Zeiss), and the specimen was characterized with imaging software (SmartSEM, Carl Zeiss) with 16,270X magnification and an operating voltage of 1 kV. The SEM imaging was performed at the Electron Microscopy Facility in the MIT Materials Research Science and Engineering Centers (MRSEC; Fig. 2c).Strains and culturing conditionsAxenic P. tricornutum CCMP 2561 was acquired from the National Center for Marine Algae and Microbiota (NCMA) and shown to be axenic via epifluorescence microscopy and sequencing of the 16 S rRNA gene [11]. P. tricornutum was maintained in f/2 medium with 20 g L−1 commercially available sea salts (Instant Ocean, Blacksburg) and with omitted silicate, which we will refer to as f/2-Si [11, 16]. Batch cultures were grown at 20 °C with a 12 h light/12 h dark diurnal cycle and a light intensity of 200 μmol photons m−2 s−1 (Exlenvce). Every 2–3 weeks, axenic cultures were monitored for bacterial contamination by streaking culture samples on marine broth agar [33], that tests for contamination by bacteria that can grow on agar media and is not definitive. Every 6–12 months, every axenic and bacterial co-culture of P. tricornutum was inspected for the absence/presence of bacteria by staining the cellular DNA with 0.1% v/v SYTO BC Green Fluorescent Acid Stain (Thermofisher, Supplementary Fig. S1).Bacterial community samples (referred to as “phycosphere enrichments”) were obtained from mesocosms of P. tricornutum and maintained as previously described [11, 16]. Briefly, an outdoor P. tricornutum mesocosm sample in natural seawater was collected in Corpus Christi, TX and filtered with 0.6–1 µm pores to remove larger algal cells. The bacterial filtrates were inoculated to an axenic algal culture, maintained in f/2-Si media for ~3 months, and washed with a sterile medium to enrich for phycosphere-associated bacteria. These enriched communities were subsequently co-cultured with P. tricornutum in f/2-Si media for ~4 years prior to the start of the experiments.Two bacterial strains, Marinobacter sp. 3-2 and Algoriphagus sp. ARW1R1, were isolated from the phycosphere enrichment samples (Supplementary Table S1). The isolates were either maintained by growing on marine broth agar plates at 30 °C or by co-culturing with P. tricornutum through inoculation of a single colony into the axenic culture.
P. tricornutum culture in porous microplateThree baseline experiments were designed to study how the alga P. tricornutum interacts with its associated bacteria in the porous microplate (Fig. 1). For experiments assessing the algal growth in the microplate, axenic P. tricornutum was acclimated to a copolymer environment in advance by inoculating a stationary phase-culture to a separate microplate. After acclimation for 4 days, the culture was diluted to ~1 × 106 cells ml−1 and transferred to the experimental microplate. Three replicated microplates were placed in a single transparent covered container (128 × 85 × 10 mm3, VWR) which was filled with ~25 ml f/2-Si medium to keep the microplate hydrated throughout the incubation period of 20 days with an initial culture volume of 75 µl (Fig. 1a). The procedures were conducted under a biosafety cabinet to prevent any biological contamination. The cells were incubated under the same conditions as described above for the batch cultures (temperature, light intensity, diurnal cycle).Growth of P. tricornutum was measured by counting cells using a hemocytometer (Electron Microscopy Sciences) or flow cytometry (described later). Specific growth rates were calculated from the natural log of the cell densities in triplicate sampled during an exponential growth phase (day 3 for the batch culture, day 5 for the porous microplate system; Fig. 3a).Fig. 3: Cultivation of P. tricornutum in the porous microplate.a Schematic of a microplate for algal cultivation. b Growth curve and maximal growth rate (inset) comparing the porous microplate with flask culture. Error bars, standard deviation of triplicates. c Cell abundance at center (n = 3) and surrounding (n = 18) wells after incubation. Asterisks denote statistical differences with following levels (two-tailed t-test): ***P More
