Toc vcph total organic carbon analyzer

Inorganic carbon was removed by purging the acidified sample with a gas which is free from CO₂ and organic compounds. Samples were pre-filtered with 0.45 µm filter unit. Shimadzu TOC-V cph total organic carbon analyzer was used for the analysis of DOC. The UV absorbance (SUVA) at 254 nm (SUVA254) was measured using a Shimadzu UV-1800 spectrophotometer. The SUVA₂₅₄ parameter is defined as the UV absorbance at 254 nm measured in inverse meters (m⁻¹) divided by the DOC concentration (mg L⁻¹) and multiplied then with 100^{54 (link)}.

Bioreactor samples taken for the determination of optical density at 600 nm (OD₆₀₀), total biomass (g/L), and PHA (g/L) were prepared and analyzed as previously described (Ingram and Winterburn 2021 (link)). The cells were washed with n-hexane to remove any residual oils before analysis. Residual biomass was defined as the difference between the total biomass and PHA concentrations. For total nitrogen (TN), samples were initially prepared by taking 0.1 mL aliquots from the cell-free supernatants of the OD₆₀₀ samples, diluted to a final volume of 20 ml with HPLC grade-water, and filtered through 0.45-μm pore nylon syringe filters into sample vials (Fisher Scientific, UK). Analysis was performed using a TOC-VCPH Total Organic Carbon analyzer, coupled with a TNM-1 TN analyzer unit (Shimadzu, UK), as previously described (Urbina et al. 2018 (link); Wongsirichot 2020 ). Standards for the TN were made from NH₄Cl.

To prepare a graphite dissolved carbon standard, 0.0665 g of graphite (Ward’s Science) was suspended in 1 L of filtered seawater (FSW) collected offshore of Delaware. Homogeneity and particle disaggregation were ensured by sonication. In triplicate, 60 mL of the graphite/FSW mixture were vacuum filtered using pre-combusted GF/F filters (Whatman, 0.7 μm nominal pore size). Filtrate was immediately poured into 30 mL vials, acidified using 2 m HCl, and analyzed on a Shimadzu TOC-V CPH Total Organic Carbon Analyzer. The instrument was operated at 680 °C using UHP air as the carrier gas and calibrated using a five-point standard curve created with a potassium hydrogen phthalate (LabChem) standard. Concentrations were determined using the best three of five injections per sample. Acidified FSW samples (no graphite added) were analyzed in the same way and in the presence of pre-combusted glass beads, and graphite suspended samples were compared with these control samples to determine whether graphite was detected in the operationally-defined DOC. The difference in DOC concentrations between FSW and FSW with glass bead samples was not statistically different than 0 (0.6 ± 4 µm; n = 4). A Deep Sea Reference (Hansell Laboratory, DSR Lot # 07–07, 41–45 μm DOC) was also run as a quality control. A value of 47.8 ± 2.0 μm, n = 3 was measured for this reference standard.