Results of HPLC derived photopigment concentrations for bioassays done in the North Inlet Estuary-Georgetown, South Carolina during 2014 (Photomixotrophy project)

Results of HPLC derived photopigment concentrations for bioassays done in the North Inlet Estuary Georgetown, South Carolina during 2014 (Photomixotrophy project)


INTRODUCTION
The HPLC method used at USC for photopigment separations is derived from the Van Heukelem et al. (1992Heukelem et al. ( , 1994 and  protocols. Two different reversephase C18 columns are connected in series. A single monomeric guard column is followed by a monomeric reverse-phase C18 column and a polymeric reverse-phase C18 column. This column configuration was originally devised to enhance photopigment separations from sediment samples containing numerous (>150) photopigment and pigment degradation products. Monomeric columns provide strong retention and high efficiency while polymeric columns select for similar compounds with minor differences in molecular structure (Van Heukelem et al. 1992. In addition to increasing the number of theoretical plates, the combination of both monomeric and polymeric columns optimizes photopigment separations based on two different molecular properties (coarse and fine structure). This method allows for the baseline separation of most major pigments including lutein/zeaxanthin and chlorophyll c3. However, chlorophylls c1 and c2 are not completely separated. Divinyl chlorophylls a an b are not completely resolved but occur as shoulders on the monovinyl chlorophylls a and b and can be visually identified in chromatograms.

EXTRACTION
SeaHARRE 4 samples were immediately frozen at -80 deg C upon receipt. For HPLC analysis, filters were placed in disposable polypropylene microfuge tubes (2 ml) and lyophilized (-50 deg C, 0.57 mbar, 12 h; Labconco FreeZone 2.5) to remove all water from the filters. After lyophilization, filters were cut into 6 equal sections and placed in microfuge tubes. Samples were extracted in 90% acetone (600 ul), and stored at -20 deg C for 18 -20 h. Each sample also received 50 ul of the synthetic carotenoid trans β-apo-8'-carotenal (Sigma, cat. no. 10810) in 90% acetone as an internal standard using a gas-tight syringe (Hamilton) and click dispenser (Hamilton PB600-1). After extraction, the extract was clarified using a 0.45 um PTFE filter (Gelman Acrodisc). A known volume of the extract (400 ul) was then dispensed into amber glass autosampler vials (2.0 ml) and sealed with PTFEsilicone caps.

HPLC ANALYSIS
The instrumentation consisted of a binary gradient pump (Shimadzu dual LC10-AT vp and Controller SCL-10A vp), temperature-controlled autosampler (Shimadzu SIL10-A vp) with a 500 ul injection loop, column oven (Shimadzu CTO-10AS vp), and photodiode array detector (PDA, Shimadzu SPD-M10A vp; 200 to 800 nm range). For the PDA, spectra (380 -700 nm) were obtained at 2.00 sec intervals for the duration of each run and photopigment peaks were quantified at 440 nm (± 4 nm). Two different reverse-phase C18 columns were connected in series. A single monomeric guard column (Rainin Microsorb, 0.46 x 1.5 cm, 3 um packing) was followed by a monomeric reverse-phase C18 column (Varian Microsorb-MV 100 -3, 0.46 x 10 cm, 3 um packing) and a polymeric reverse-phase C18 column (Vydac 201TP54, 0.46 x 25 cm, 5 um packing). The column oven maintained a constant 40 deg C for the duration of the gradient. A non-linear binary gradient, adapted from Van Heukelem et al. (1992), was used for pigment separations (Table 1). Solvent A consists of 80% methanol : 20% ammonium acetate (0.5 M adjusted to pH 7.2) and solvent B is composed of 80% methanol : 20% acetone (Table 1). Solvents were degassed with an in-line degasser (Shimadzu DGU 14A). All solvents were HPLC-grade and chemicals were analytical grade.
Just prior to the HPLC run, an ion-pairing (IP) solution (1.00 M ammonium acetate) was added to the vial in a ratio of 4 parts extract: 1 part ammonium acetate. Prior work has shown that there is negligible pigment degradation within 12 hours of adding the IP solution if the sample is placed in a refrigerated autosampler rack (4.0 deg C). However, the IP solution should not be added to the sample if the time until sample analysis is greater than 18 hours.

CALIBRATION
Peaks were identified based on retention time and spectral matches with pigment spectra obtained from liquid standards (DHI, Hørsholm, Denmark) ( Table 2). Peak areas for chromatograms are quantified using Shimadzu Client/Server 7.2.1 SP1 software. The PDA was calibrated using a multi-point calibration procedure for a range of injection volumes (25 -300 ul) of pigment standards (DHI, Denmark). Regressions were performed using known pigment concentration (Y) vs. the integrated peak area (X) and were of the form Y = mX + b; where m is the slope and b is the Y intercept. All regressions had an r2 > 0.98. The slope of the fitted line was used as the response factor for pigment concentration calculations. The concentrations of pigments for which standards were unavailable were estimated using the ratio method outlined in Jeffrey et al. (1997, p. 443-4)

VALIDATION
Carotenal blanks (trans β-apo-8'-carotenal in 90% acetone) were run after every 10 samples to verify peak time reproducibility, peak area precision, and instrument performance during the sequence run. Peaks were identified based on retention time and comparison of absorbance spectra with a spectral library derived from pure pigment standards (DHI, Denmark). Long-term quality control is achieved by analyzing pure standards for chlorophyll a and the Mixed Standard supplied by DHI, Denmark at monthly intervals. Instrument performance is measured and compared with previous measures to determine changes in performance metrics. Volumetric measuring devices are checked weekly. The performance metrics for this method are shown in Table 3. 6. DATA PRODUCTS Pigment concentrations were calculated for each identifiable peak using the following equation: where CPi is the pigment concentration in ug l-1, APi pigment peak area, FPi is the response factor, Vc is the injection volume (ul), Vm is the total extract volume (volume of added acetone + volume of internal standard in ml), R is the ratio of the volume of IP solution + Vm divided by Vm, Vf is the volume of seawater filtered (liters), Ac is the average peak area for carotenal standards, and As is the peak area of carotenal in the sample.

CONCLUSIONS
This method has been employed by USC for ca. 15 years to analyze a broad spectrum of sample types from marine and freshwater habitats. The execution of the method is straightforward and involves minimum manipulation of the samples and extracts, relatively inexpensive, and does not generate hazardous waste products. The primary weakness of the method is the inability to completely separate chlorophylls c1 and c2 and divinyl chlorophylls.

Description Units
Year   Phytoplankton, traditionally viewed as primary producers at the base of aquatic food webs, provide an energy source for higher trophic levels. However, some phytoplankton species function as both primary producers and heterotrophic secondary consumers. Phytoplankton that are photosynthetically competent but also take up and assimilate organic compounds are classified as facultative mixotrophs or, more simply, photomixotrophs. Unfortunately, we currently have few estimates of the proportion of the phytoplankton community that function as photomixotrophs, their rate of secondary production, or their temporal variation in abundance.
Current paradigms about trophodynamics in marine systems do not consider this potentially important alternative pathway for energy flow for phytoplankton. The implication is that we may be missing a significant, fundamental process that affects carbon cycling and trophodynamics in estuarine systems. Furthermore, changes in the DOC composition due to anthropogenic alterations may result in changes in phytoplankton community structure and possibly promote the proliferation of harmful algal bloom species. In terms of ecosystem function, even moderate rates of photomixotrophy could potentially alter our current understanding of phytoplankton productivity, overall C turnover, competitive interactions, and energy transfer in estuarine environments. This project will use a novel approach to provide quantitative measures of the in situ rates and magnitudes of facultative heterotrophy in natural, estuarine phytoplankton communities over seasonal time scales in a representative estuarine ecosystem. The project will utilize a unique 14C radiolabeling technique to quantify the in situ assimilation rates of DOC by estuarine photomixotrophs and estimate the amount of DOC converted to phytoplankton biomass by photomixotrophy over seasonal time scales. This information will provide new insights into carbon dynamics in estuaries, the contribution of DOC to estuarine food webs, and the importance of photomixotrophy in determining the structural and functional characteristics of estuarine phytoplankton communities. [