The container cleaning technique was adopted from Bruland and Franks (1979). All plastic and glass containers were washed with pure water and soaked in 3 N hydrochloric acid for at least 2 weeks at ambient temperature. The acid-washed containers were rinsed with Milli-Q water and dried in a laminar-flow air bench. For those experiments requiring sterile conditions, acid-washed containers were placed in pouches and autoclaved at 121°C for 30 min followed by a 45 min dry cycle. Alternatively, purchased sterile containers Nunc™ Cell Culture Treated Flasks with Filter Caps were used.

Of eight sources of natural waters screened, Pacific Ocean oligotrophic water was initially selected for our use due to its extremely low dissolved iron concentration (pM). This was replaced later by Scripps Pier seawater because of its easier accessibility and comparable L. polyedrum growth patterns observed in the two sources of seawater. Seawater from Scripps Pier (32.153°N, 117.115°W, San Diego, CA) was collected (pH 8.2–8.4, 980 mOsm/Kg H₂O, total dissolved iron 3–4 nM) and filtered immediately through 0.22 mm pore size membrane. The filtered seawater was mixed with approximately 0.005% metal free hydrochloric acid, autoclaved for 30 min, cooled at ambient temperature for a day, and stored in 5°C until use. The pH of the autoclaved seawater was ensured to be between 8.0 and 8.2 at ambient temperature. L1 nutrient was added to sterile seawater per manufacturer's instruction (L1 medium). For those cultures requiring controlled iron concentrations in media, L1 trace element solution without FeCl₃ and Na₂EDTA was prepared in house (Guillard and Hargraves, 1993) and added along with other L1 nutrients (nitrate, phosphate, silicate, vitamins) to sterile seawater (L1-Fe medium). Serial dilutions were made on L1 medium with L1-Fe medium to prepare growth medium with defined total iron concentrations, [Fe]_T.

The component solutions were prepared in three separate containers, one with 15 g NaCl, 0.75 g KCl, 1 g NH₄Cl in approximately 500 mL water, one with 12.4 g MgSO_4^.7H₂O in small volume of water, and one with 3.0 g of CaCl_2^.2H₂O in a small volume of water. The three solutions were mixed and 0.1 g disodium ß-glycerol phosphate was added. The pH was adjusted to 8.0–8.2 and volume was adjusted to 1 L. The solution was autoclaved at 121°C for 30 min.

The L. polyedrum strain used in this study was isolated from Venice Beach, California, and kindly provided to us by Avery Tatters (University of Southern California). L. polyedrum cells were maintained in sterile 1L Erlenmeyer flasks containing L1 medium and capped with a ventilation sponge. Cultures were exposed to 100 μmol photons m⁻² s⁻¹ on a 12-h alternating light and dark cycle at a temperature of 20 ± 2°C (standard growth condition). The upper portion of a culture containing healthy cells was diluted 1/5 with L1 medium every 3 weeks. Algal growth was monitored by direct cell count using an inverted microscope (Nikon Eclipse TE2000-U, 4× objective). To prepare cells in controlled iron medium, cultures containing 10⁴-10⁵ cells per mL was placed in a 15-mL Falcon tubes and centrifuged for a few seconds to pellet cells which were gently washed three times and re-suspended to an appropriate volume with controlled iron medium.

Non-axenic L. polyedrum cells were grown to 10⁵ cells/mL in L1 medium. Antibiotics 0.1% (v/v) (Penicillin 5 units/mL, Streptomycin 5 μg/mL, Neomycin 10 μg/mL) were added to a non-axenic culture and incubated for 24 h under the standard growth condition. Antibiotic-treated cultures were placed in 15 mL Falcon tubes and gently centrifuged for a few seconds. The pelleted cells were washed three times and re-suspended with L1 medium containing 0.05% (v/v) antibiotics in a sterile culture flask to appropriate volume. Antibiotics 0.05% (v/v) was added every 3 days to maintain the culture “axenic.” The absence of cultureable bacteria in the antibiotic treated L. polyedrum was tested by spreading a 10 μL sample of the culture on a marine broth plate (5 g/L peptone, 1 g/L yeast extract, 15 g/L agar in 75% seawater) followed by incubation at 25°C for 2–3 days. The absence of any visible bacterial colonies was considered indicative of an “axenic” culture of L. polyedrum.

The model bacterium used was Marinobacter algicola DG893 (GenBank code NZ_ABCP00000000.1) since it has been isolated by Green et al. (2004) and well characterized as a producer of the photoactive siderophore vibrioferrin (Amin et al., 2007). The mutant ΔpvsAB-DG893 was made as described by Amin et al. (2012a) by 872 base pair deletion from the wild type to knock out two siderophore biosynthesis genes, pvsA and pvsB. To monitor bacterial growth, two methods were used: Optical density at 600 nm was applied to apparently turbid samples which generally contained ≥10⁷/mL of bacterial cells. Enumeration of bacteria by serial dilution was applied for samples containing lower numbers of bacteria cells (<10⁷/mL). For enumeration techniques, serial dilution was made on those samples with sterile seawater, each diluted sample was spread on marine broth plates, and the number of colony forming units (CFU) recorded from the lowest diluted sample plate. CFU in original sample per mL was calculated by (CFU in diluted sample)/(volume plated in mL) × dilution factor.

One liter of marine broth was prepared (5 g/L peptone, 1 g/L yeast extract, 15 g/L agar in 75% seawater, pH 8.2, sterile) and dispensed into sterile tubes with a 5 mL volume. A few colonies of bacteria picked from stock plates were transferred into the marine broth tubes and shaken at 25°C for 1–2 days until they reached log phase. The cells were collected by centrifugation, washed three times and re-suspended in sterile medium to an optical density (OD600) of 0.3 (stock bacteria solution).

The method was adopted from Alexander and Zuberer (1991) to detect siderophore production by DG893. Using acid washed glassware, the following stock solutions were prepared: 10 mM HDTMA in water, 2 mM CAS in water, 1 mM FeCl₃.6H₂O in water, 50 mM piperazine anhydrous buffer (pH 5.6), and 0.2 M 5-sulfosalicylic acid in water. CAS solution was prepared by pouring a mixture of 15 mL of CAS and 3 mL of FeCl_3.6H₂O slowly into 12 mL of HDTMA followed by addition of 50 mL of piperazine. The final volume was adjusted to 200 mL with water. CAS shuttle solution was prepared by adding 20 μL of 5-sulfosalicylic acid to every mL of CAS solution (used within 1 day of preparation). Samples to be tested were centrifuged to remove particles and 0.5 mL supernatant was mixed with 0.5 mL of CAS shuttle solution followed by incubation in the dark at ambient temperature for 10 min. A color change from dark purple to clear red indicated siderophore production.

The following materials were purchased from Sigma-Aldrich: Chrome azurol S (CAS, C-1018, MW605.28), antibiotics (Penicillin/Streptomycin/Neomycin, P4083-100 mL), peptone (P-1265), iron(III) chloride hexahydrate (FeCl_3^.6H₂O, 10025-77-1, MW270.30), manganese(II) chloride tetrahydrate (MnCl_2^.4H₂O, 13446-34-9, MW197.91), hexadecyltrimethylammonium bromide (HDTMA, H6269-100G, MW364.45), 1,4-diazacyclohexane, diethylenediamine (Piperazine, Sigma-Aldrich, P45907-100G, MW 86.14), 5-sulfosalicylic acid (390275, MW218. 18), zinc sulfate heptahydrate (ZnSO_4^.7H₂O, 1088830500, MW287.54), cobalt(II) chloride hexahydrate (CoCl_2^.6H₂O, 8025400010, MW129.83), copper(II) sulfate pentahydrate (CuSO_4^.5H₂O, 209198-5G, MW249.69), sodium molybdate dehydrate (Na₂MoO_4^.2H₂O, 331058-5G, MW241.95), selenous acid (H₂SeO₃, 211176-10G, MW128.97), nickel(II) sulfate hexahydrate (NiSO_4^.6H₂O, 227676-100G, MW262.85), sodium orthovanadate (Na₃VO₄, 450243-10G, MW183.91), potassium chromate (K₂CrO₄, 216615-100G, MW194.19), salicylaldoxime (SA, 84172-100G). The following materials were purchased from Fisher Scientific: trace metal-free hydrochloric acid (Optima, A466-250), yeast extract (BP9727-500), agar (BP1423-500), Casamino Acids (BP1424-500), Nunc^TM Cell Culture Treated Flasks with Filter Caps (12-565-57), ethylenediaminetetraaceticacid, tetrasodium salt dihydrate (Na₄EDTA^.2H₂O, BP121-500, MW416.2), boric acid (AAJ67202A1). Other materials were purchased as follows: 0.22 μm filter membrane (Millipore, MillexGV, SLGV033RS), L1 medium kit (Bigelow, MKL150L), pure water (Barnstead water system, 18.2 mΩ). Vibrioferrin (VF, MW434.35) was isolated and purified in house according to Amin et al. (2007).

The growth of DG893 and mutant ΔpvsAB-DG893 in L1 medium containing different total iron concentrations [Fe]_T (1,000, 100, 10, and 0 nM) was monitored. All media were divided in two parts, one maintaining [EDTA] at 10,000 nM (L1 nutrient level) with variable [Fe]_T, and the other reducing [EDTA] along with [Fe]_T to maintain molar ratio of Fe: EDTA of 1:1. All media were prepared with and without sterile Casamino Acids. In each medium, ca. 10⁶ cells/mL of DG893 or mutant ΔpvsAB-DG893 were added and shaken in the dark at 30°C. The bacterial growth in each medium was monitored for 5 days by optical density at 600 nm. Neither WT DG893 nor the mutant grew in simple seawater or L1-Fe medium without a carbon source (Figure 1A). Addition of iron to seawater or L1-Fe medium also did not maintain growth (Figure 1B). However upon addition of Casamino Acids, both DG893 and the mutant grew and their growth increased with increasing [Fe]_T in media (Figure 1C). There was no remarkable difference in growth pattern between DG893 and the mutant, consistent with the report by Amin et al. (2012a). In summary, both DG893 and the mutant require a carbon source to grow to a detectable level in seawater medium.

The ability to produce the siderophore vibrioferrin (VF) by DG893 but not by the mutant was confirmed by the CAS-dye assay. Since siderophore biosynthesis is generally turned on under low iron concentrations and turned off under high concentrations (Braun, 1995; Braun et al., 1998; Miethke and Marahiel, 2007), WT was grown in L1 medium containing various [Fe]_T with 0.3% Casamino Acids at 30°C in dark for 24 h to find the approximate [Fe]_T under which DG893 produces VF. The WT did not produce VF in the media with [Fe]_T ≥10 μM but did so with a [Fe]_T of 1 μM or less. The mutant ΔpvsAB-DG893 did not show signs of siderophore production under any of the conditions tested. The results suggest that under normal seawater conditions, where [Fe]_T is estimated to be nM to pM, that siderophore biosynthesis in DG893 is likely turned on.

The difficulty in preparing and maintaining axenic cultures of many marine algae has been reported in the past (Green et al., 2004; Jauzein et al., 2015; Liu et al., 2017). In fact, Lupette et al. (2016) noted that completely axenic cultures of a model green algal species could not be maintained despite the use of antibiotic treatment protocols. We were partially successful in our efforts to prepare axenic L. polyedrum. Initially, non-axenic L. polyedrum cultures containing 10⁵ cells/mL were treated with antibiotics (1% v/v Penicillin 50 units/mL, Streptomycin 50 μg/mL, Neomycin 100 μg/mL) for 24 h under the normal growth conditions. The treated cells were washed and re-suspended with sterile L1 media and while the “axenicity” of the culture at this point was confirmed, bacterial colonies reappeared after only a few days. An attempt was made to keep 1% antibiotics in the culture longer than 24 h, however, dinoflagellate cells began to disintegrate after 2 days suggesting that 1% antibiotics must be removed from the culture within 24 h. The fact that the negative control and sterile L1 medium without phytoplankton cells remained bacteria-free for weeks ruled out a possibility of culture contamination. We believe that the antibiotics are most likely eliminating only free-living bacteria leaving attached bacteria unaffected. The next effort of preparing axenic cultures was made by immediate dilution of antibiotic-treated cultures. The rationale of this attempt was to minimize attached bacteria on L. polyedrum cells by dilution. The antibiotic treated L. polyedrum culture was diluted 200-fold with sterile L1 medium and incubated under standard growth conditions. The diluted culture remained bacteria-free for the first 10 days, however microbial growth was observed on the 12th day as the number of L. polyedrum cells also increased. Although this dilution method suggested that the culture stayed bacteria free for up to 10 days, the L. polyedrum cell count was too dilute to be useful for further study. The next attempt at axenic culture preparation was made with three sequential antibiotic treatments. The non-axenic culture was treated with 1% antibiotics for 24 h, washed three times, and re-suspended with sterile L1 medium. The cells were allowed to recover under normal growth conditions for a day and then again treated with 1% antibiotics. This procedure was repeated three times. This procedure also failed as the dinoflagellate cells began to disintegrate. Finally, using a similar procedure but reducing the concentration of antibiotics to 0.1% for the first treatment and adding 0.05% subsequently every 2–3 days resulted in L. polyedrum cultures without visible cell damage that appeared to be free of free-living bacteria for more than a month.

A matrix containing thirty cultures of L. polyedrum in L1 medium containing different numbers of DG893 cells in the initial inoculum (10⁷, 10⁶, 10⁵, 10⁴, 10³, 0 CFU/mL) and different [Fe]_T (10⁴, 10³, 10², 10¹, and 0 nM) was set up. The same matrix was set up with the mutant ΔpvsAB-DG893. Bacterial growth was monitored by enumeration of bacteria by serial dilution and phytoplankton growth was monitored by direct cell counts under the microscope. Regardless of the starting bacterial cell number, both DG893 wild type and mutant grew in all the binary culture matrices over 28 days to eventually reach a bacterial population of ca. 10⁵ CFU/mL. The group of media containing 1,000 nM [Fe]_T showed the greatest growth of L. polyedrum regardless of the starting bacterial inoculum (Figure 2A). The group of media containing ≤10 nM [Fe]_T showed very slow L. polyedrum growth regardless of starting bacterial inoculum. No significant differences in L. polyedrum growth co-cultured with DG893 or the mutant were observed, which left a question as to whether VF secreted by DG893 would have a significant effect on L. polyedrum growth. It was speculated that under these growth conditions, the amount of VF being produced by DG893 was insufficient to see detectable changes in L. polyedrum growth. This was later confirmed (vide infra) when excess VF artificially added to the culture produced pronounced L. polyedrum growth. Finally, the growth pattern of axenic L. polyedrum was similar to that of a non-axenic culture. This observation left open another question as to whether DG893 and L. polyedrum were commensally associated. However this also was confirmed by experiments described below where after subsequent culturing the “axenic” L. polyedrum ceased to grow while binary L. polyedrum DG893 kept growing exponentially (Figure 3).

L. polyedrum grown in L1 media containing various [Fe]_T and DG893 (Figure 2A) for 28 days were diluted with appropriate media to adjust L. polyedrum to 10² cells/mL in the 2nd subsequent batch culture. The L. polyedrum growth in this 2nd subsequent culture was monitored for another 28 days under each growth condition. The result showed that the 2nd subsequent batch culture L. polyedrum in the higher two [Fe]_T (10,000 and 1,000 nM) maintained their exponential growth while those in the lower three [Fe]_T did not grow (Figure 2B). Although the cells under the latter conditions did not completely die out, the few cells that survived had their swimming activity under the microscope clearly reduced.

The growth of “axenic” and binary cultures of L. polyedrum with DG893 in L1 was compared over several subsequent batch cultures. No significant difference in growth was observed in the 1st culture, however, a clear difference was observed in the 2nd subsequent batch culture of L. polyedrum with and without DG893. While L. polyedrum with DG893 grew fully in both the 1st and 2nd subsequent batch cultures, “axenic” L. polyedrum ceased to grow (Figure 3). When DG893 was added to the 2nd subsequent “axenic” batch culture, L. polyedrum cells began to regrow exponentially demonstrating that DG893 could rescue “axenic” L. polyedrum.

The non-axenic L. polyedrum was maintained in L1 medium and assumed to contain not only siderophore-producing bacteria but also other unknown bacteria. The non-axenic culture was divided in two parts, one diluted to 1/5 with L1 medium and other diluted to 1/5 with L1-Fe medium (batch subculture 1). When the cell count reached approximately 10⁴ cells/mL, both cultures were further diluted with appropriate medium. Therefore, one culture maintained [Fe]_T at L1 level throughout the subsequent batch cultures while the other contained successively reduced [Fe]_T. The L. polyedrum growth was monitored for 28 days for each subculture. After the 6th subculture where [Fe]_T in the medium was estimated to be 2 nM, L. polyedrum stopped growing completely (Figure 4). On 29th day of the 6th subculture, an iron supplement to give a [Fe]_T of 5,850 nM was added to the culture and growth was continued to be monitored for additional 28 days. The results indicate that the L. polyedrum culture which stopped growing under 2 nM [Fe]_T, could be rescued by addition of iron after 28 days (Figure 5).

Earlier it was stated that when L. polyedrum was co-cultured with either DG893 or the mutant ΔpvsAB-DG893, little difference was observed in its growth pattern. We speculated that this may have been due to the small amount of VF produced by the equilibrium population of DG893 in the binary culture. To test this idea, samples of the supernatant of the media from the WT DG893 grown under ideal conditions and containing detectable amounts of VF by CAS-dye assay, as well as the mutant ΔpvsAB-DG893 which did not, respectively, were added to L. polyedrum cultures and growth under these different conditions was compared. It should be noted that while we traditionally grow DG893 and the mutant in marine broth containing Casamino Acids, these were found to be toxic to L. polyedrum cells. In this experiment, we instead used ASW with succinic acid (at from 0.001 to 0.1%) as a carbon source (pH 8.2) to grow DG893 and the mutants prior to their supernatant being added to L. polyedrum cultures. L. polyedrum growth was enhanced the most by the addition of the DG893 supernatant. The mutant supernatant also showed a slightly positive effect on L. polyedrum growth compared to media without bacterial supernatant, however the degree of growth was not as large as that induced by the DG893 supernatant (Figure 6). These results suggest that VF has potential influence on L. polyedrum growth, but that other extracts from bacteria (vitamin B₁₂?) may also have a potential effect on their growth. Further experiments were performed using purified VF itself. When purified VF at 57 μM was directly added to a L. polyedrum culture in L1 media containing 100 nM [Fe]_T, L. polyedrum reached cell counts approximately 1.5 times as large as those in media without VF.

“Axenic” L. polyedrum cells were resuspended in L1 media containing 100, 1,000, 10,000 nM [Fe]_T and incubated under the standard growth conditions for 2 days to collect their organic extracts. The L. polyedrum cultures were divided in two parts, one of which was filtered through a 0.22 μm membrane to collect only organics and the other was kept without filtration to keep phytoplankton cell debris. The L. polyedrum supernatant with and without cells was added to the stock DG893 containing 0.3% Casamino Acids, and the mixture shaken at 30°C for several days. The control was prepared in the same manner without L. polyedrum supernatant and cells. In the media containing 1,000 and 10,000 nM [Fe]_T, DG893 growth was enhanced by the presence of L. polyedrum supernatant and was even more pronounced with the supernatant containing cell debris (Figure 7). In media with 100 nM [Fe]_T, only a subtle difference was observed in DG893 growth with and without L. polyedrum supernatant.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.