Culturing the Cells:
The cyanobacterium Synechococcus PCC7942 was originally obtained from the Pasteur Culture Collection (PCC). Acaryochloris marina (MBIC11017) (Miyashita & Chihara) was a gift from A.W.D. Larkum (UTS-Sydney, NSW, Australia). The green alga Chlorella vulgaris (Beyerinck [Beijerinck]), the diatom Chaetoceros sp. and Isochrysis sp. (Prymnesiales, Haptophyta) were from the Phuket Marine Biological Centre, Laem Panwa, Phuket 83000. Synechococcus and Chlorella grew well in BG-11 medium (Allen 1973) and Chlorella also grew well in seawater supplemented with nitrate, phosphate and trace elements as for BG-11. No added vitamins were needed. Chaetoceros, Isochrysis and Acaryochloris were grown in seawater supplemented with BG-11 trace elements, 100 mmol m-3 sodium silicate, 200 mmol m-3 KH2PO4 and 1 mol m-3 sodium nitrate. f/2 vitamins were added as described by McLachlan (1973).
Synechococcus, Acaryochloris, Chlorella, Chaetoceros and Isochrysis were grown in 250 and 500 mL conical flasks, shaken and stirred daily. Cultures of all four phototrophic microbes were kept on shelves fitted with overhead fluorescent lights (Panasonic 36 W daylight, colour temperature 6500 K: TIS 956-2533) in continuous light at about 27 oC. The light intensity in the culture room was approximately 100-150 µmol photon m-2 s-1 (PPFD 400 – 700 nm), measured using a Li-Cor photon flux meter Model LI-189 (Li-Cor Corp, Lincoln, Nebraska, USA).
Chemicals
DMSO (Dimethylsulphoxide, dimethyl sulfoxide, (CH3)2SO) was from WINNEX (Thailand) Co. Ltd, Bangkok, Thailand. Acetone (CH3)2CO 99.5 AR/ACS was from LOBA Chemie PVT. LTD, Mumbai, India. 90% acetone and 100% DMSO were neutralised with magnesium carbonate.
Preparation of Cells for Experiments:
Synechococcus, Acaryochloris, Chlorella, Chaetoceros and Isochrysis cultures were filtered onto Whatman GF/C glass fibre disks (Whatman International, Maidstone, England, U.K.) using a Millipore apparatus designed for 25 mm filters as described by Ritchie and Runcie (2013). The inside diameter of the Millipore filtration apparatus was 16.2 mm and so the disks of microbial cells adhering to the glass-fibre filter had a surface area of 206.12 × 10-6 m2. The Synechococcus, Acaryochloris, Chlorella, Chaetoceros and Isochrysis-impregnated disks provided highly reproducible material for experiments. The disks were not allowed to dry out and were placed in a darkened Petri dish with a layer of filter paper moistened with seawater or BG-11 medium as appropriate before making absorptance measurements. Excessive delays in measurements were avoided. Glass fibre filters eventually block if overloaded with cells. Full loading where no more cells could be loaded onto the glass fibre filters varied from one species to another: ≈ 200 mg Chl a m-2 for Synechococcus, Acaryochloris and Chlorella but < 100 mg Chl a m-2 for Chaetoceros and Isochrysis because of the mucilaginous nature of the cells.
Scanning Dual Beam Spectrophotometry:
A standard dual beam scanning spectrophotometer was used for routine chlorophyll determinations (Shimadzu UV-1601, UV-Visible Spectrophotometer, Shimadzu Corporation, Kyoto, Japan, Software: UV-Probe 2.21, Shimadzu Corporation, Kyoto, Japan). Routine chlorophyll determinations were made in MgCO3-neutralised 90% acetone or DMSO (Jeffrey and Humphrey 1975; Ritchie 2006; Ritchie et al. 2021; Ritchie et al. 2022) using quartz cuvettes. Chlorella is a recalcitrant alga and heating at 55 oC was usually required to effectively extract chlorophylls. Fewer problems were encountered with the other algae. DMSO is a far better quantitative extractant than acetone. Following our recent practice 850 nm was used as the zero wavelength rather than 750 nm. Routine scans from 850 to 600 nm were used for data acquisition and exported as EXCEL.csv files.
Integrating Sphere Spectrometer:
A UV Vis Spectrophotometer Model: AE-s90-2D, Serial Number: AT161006, A & E Lab (UK) Co. Ltd fitted with a 60 mm (2 1/2-inch) integrating sphere was used as described in detail in Ritchie and Sma-Air (2020a, b) to measure the optical properties of cell suspensions. The spectrophotometer was run using UV-VIS Analyst version 5.43, prom Version 0.000, Copyright 2013 Macroeasy Technologies Ltd, License number UV 20081012-001-8828-FFF.
Absorptance Measurements using RAT:
A RGB-diode based leaf absorptance RAT meter was designed by Aquation Pty Ltd, Umina Beach, Australia for the measurement of absorptance of leaves at the same light wavelengths as used by PAM fluorometers (Ritchie and Runcie 2014). The RAT was fitted with a three-colour “RGB” LED as the light source [SML-LX1610RGBW/A diode light source (Lumex Inc., 290 E. Helen Rd, Palatine, IL 60067-6976, USA)] (Carreres-Prieto et al. 2020). The bandwidths of the RGB diode used in the RAT meter were blue, 445±15 nm bandwidth; green, 525±15 nm bandwidth and red, 625±15 nm bandwidth. Intensities of each colour can be individually adjusted by changing the current to each diode. We have found that the RAT was not only useful for measuring the absorptance properties of leaves and thalli of lichens and macroalgae algae for which it was designed (Ritchie and Runcie 2014) but was also particularly well suited for absorptance readings on algal films mounted upon glass fibre disks (Cebrian et al. 1999; Ritchie 2013; Ritchie and Runcie 2013). The RAT measures transmittance of this light through a specimen to obtain T% and also measures reflectance (R %) using a diode set at 45o to the light beam in an arrangement based upon Schultz (1996) (see Figure 1). Absorptance is calculated as Abt%λ = 100-T%λ-R% λ (Runcie and Durako 2004). The meter is calibrated using a black and a white (0 and 100% reflectance respectively) standard card for the particular light source being used (red, green, blue or a RGB combination of sources giving “white” light) following the factory calibration instructions. Some difficulties were encountered in the case of Acaryochloris because it absorbs 625 nm light so poorly (Figure 2) for low loadings of the alga on the glass fibre disks, giving zero apparent absorptance for loadings up to about 30 mg Chl d m-2 if the standard white polyester card was used. Satisfactory results on Acaryochloris were obtained if a blank glass fibre filter disk was used as a zero rather than the factory supplied white plastic card (see Supplementary Figure). A blank glass fibre disk was also found to be much more satisfactory as a zero for Isochrysis than the factory-supplied white card.
Commercial “White Light” diodes are typically configured to have a very high level of blue light and very little green and red light compared to sunlight and so can be misleading for photosynthetic work: in the present study an RGB adjustable for Blue, Green and Red light was used set at blue 50%/green 100%/red 100% to better represent sunlight (Larkum et al. 2018; Carreres-Prieto et al. 2020). Calibration steps involved firstly measuring 100% transmittance with no sample, 0% reflectance with the black card and 100% reflectance with the white standard. The RAT needs to be re-calibrated for each coloured light source.
Saturating absorptance vs. chlorophyll curves were fitted using a simple exponential saturating model. Curves were determined for blue, green and red light and for “white” light. Curves could be determined easily for the algae in the present study with Chl a as their primary photosynthetic pigment as previously described for a selection of vascular plants and lichen (Ritchie and Runcie 2014). More caution was needed to make satisfactory measurements of the chlorobacterium, Acaryochloris marina, because of the Chl d + a pigmentation of the organism (Supplementary Figure).
Statistics:
Unless otherwise stated all values quoted are means ± 95% confidence limits with the number of data points quoted in brackets. Using least squares methods and error bars of the fitted parameters Abt%λ, ∞ and exponential constant k were determined and the asymptotic errors determined by matrix inversion using EXCEL®. Zar (2014) was used as the standard statistical reference text.