Scenedesmus

We used 14 isofemale lines (hereafter referred as clones) of D. magna and one clone each of six other Daphnia species (Tables 1 and 3). Unless otherwise stated, Daphnia clones were kept in standard medium (ADaM, [54 ] modified by using only 5% of the recommended Selenium dioxide concentration) at 20°C and fed with the chemostat cultured unicellular algae, Scenedesmus obliquus.
The parasites used were single genotypes of P. ramosa, C1, C14 and C19, characterized as clones in Luijckx et al. [20 (link)] and originated from D. magna populations in Moscow (Russia), Tvärminne (Finland) and Gaarzerfeld (Germany), respectively. Spore suspensions of Pasteuria were obtained by homogenizing infected D. magna in ADaM and quantifying spore density. The status of resistant or susceptible D. magna were defined previously [20 (link)]. The infection status of two further Finnish D. magna clones ('Kela-39-09' and 'Kela-18-10') exposed to Pasteuria clones were tested with the same protocol. All infections in these experiments were done with naïve individuals born to naïve mothers, kept under high food conditions. These conditions were applied because they are known to minimize the triggering of immune effect [34 (link),35 (link),55 (link)].

The green microalgal strains Scenedesmus dimorphus (417), Selenastrum minutum (326) and Chlorella protothecoides (25) were purchased from UTEX The Culture Collection of Algae at the University of Texas at Austin (in parenthesis is the UTEX id); while an uncharacterized polyculture of algae cultured in municipal wastewater, was retrieved from a bioreactor installed at a combined heat and power plant (Umeå Energi, Umeå).
For the lipid extraction method comparison, two independent experiments were carried out. In experiment 1, S. dimorphus (417), S. minutum (326) and C. protothecoides (25) were grown in Proteose medium [21] (link) and the polyculture of endogenous algae was cultured in untreated final municipal wastewater treatment plant effluent. All cultures were grown in bottles with 1 liter of volume sparged with sterile air at a flow of 170 ml/min and subjected to approximately 12 hours of natural light from a window in Umeå, Sweden (March to April, 2012, 63°49′30′′N). After four weeks of growth, biomass was harvested by centrifugation at 3584 g for 10 min and pellets were stored at −20°C overnight. Experiment 1 had two replicates.
In experiment 2, the same algae were grown in untreated municipal wastewater treatment plant influent. All cultures were grown in bottles of 1 liter of volume sparged with flue gases at a flow of 170 ml/min containing approximately 10% CO₂, from a combined heat and power plant (Umeå Energi, Umeå). S. dimorphus (417), S. minutum (326) and the algal polyculture were grown for four days while C. protothecoides was grown for 11 days in a greenhouse in Umeå in April 2013 at an average temperature of 19°C, receiving approximately 16 hours of natural light a day at an average PAR (photosynthetic active radiation) intensity of 715 µEm⁻²s⁻¹. The PAR was measured and recorded every 5 minutes using a LiCor 1400 datalogger connected to a spherical light sensor LI 193 (LiCor Lincoln, Nebraska USA). The algae were harvested as mentioned in experiment 1 and immediately subjected to lipid extraction. Experiment 2 had four replicates.
For the cell disruption method comparison, the same algae were cultured in autoclaved (121°C, 20 min.) municipal wastewater treatment plant effluent. The growth conditions were the same as described for experiment 1.
For the oven dried experiment, biomass was harvested from an uncharacterized polyculture of microalgae grown in a 650 l bioreactor with municipal wastewater and treated flue gas, 3 l/min containing approximately 10% CO₂, from a combined heat and power plant (Umeå Energi, Umeå). The bioreactor was placed in a greenhouse on the roof of the combined heat and power plant. The algal polyculture was grown under a batch regime in February 2012 at an average temperature of 19°C receiving approximately 10 hours of natural light a day at an average PAR (photosynthetic active radiation) intensity of 361 µEm⁻²s⁻¹. Samples of algae, 12.73±0.31 mg (mean ± SE) dry weight harvested in the morning, were pelletized by centrifugation at 3584 g and stored overnight at −20°C.

The microalgal species were acquired as freeze-dried powder. Chlorella vulgaris, Artrhospira platensis, and Haematococcus pluvialis in red and green phase, and Nannochloropsis sp. were supplied by the company MICOPERI BLUE GROWTH^® (Ravenna, Italy). Dunaliella salina was provided by the company Algalimento (Santa Lucía de Tirajana, Gran Canaria) and Scenedesmus almeriensis was provided by the Company AlgaRes Srl (Rome, Italy). Each biomass was stored at −20 °C before characterization and analysis. Solvents such as ethanol, acetone, methanol, acetonitrile, isooctane, and chloroform were purchased by Sigma-Aldrich (Saint Louis, MO, USA) with a chromatographic grade; sulfuric and boric acids were purchased at ACS grade, and water was purchased at u-HPLC grade. For each investigation, three sample of microalgae biomass were treated (n = 3). Results are reported as average value ± standard deviation. Statistical analysis (One-Way ANOVA was performed using the statistical software SigmaStat 4.0 (Systat Software Inc., San Jose, CA, USA).

A 16 L photobioreactor (PBR) (Wheaton, UK) was filled with untreated municipal wastewater (MWW) collected at a local municipal wastewater treatment plant (Vakin, Umeå). The wastewater was filtered through paper tissue to remove large particles^{26 (link)}.
Two microalgal strains isolated in northern Sweden, Chlorella vulgaris (13–1) and Scenedesmus obliquus (B2-2)^{27 (link)}, were used to grow microalgal biomass, and one batch was cultivated per algal strain. The PBR with MWW was inoculated with approximately 125 mL of 14-day-old culture of either C. vulgaris (13–1) or S. obliquus (B2-2) grown in BBM medium²⁸ . The PBR was then kept in a 16:8 light:dark regime receiving 130 µmol m⁻² s⁻¹ photosynthetically active radiation on its surface using fluorescent lamps. The PBR was stirred at approximately 75 rpm and kept at room temperature for 8 days, at which point the culture was in the late exponential-early stationary growth phase.

Cobble substrate covered with periphytic algae was collected from a reach of Clear Creek (Jefferson County, Colorado, USA) which has high levels of Zn contamination from historical mining activities. In order to produce non-contaminated cultures used as control and acute exposure groups, substrate was placed in 1 cm of water containing Guillard's growth medium (Guillard 1975 ) fortified with 1.36 mM silicon using dissolved sodium meta-silicate 9-hydrate in 11.2 cm × 6 cm polyvinyl chloride (PVC) troughs. Solutions were renewed daily. Pumps (Rio 600, Taam Inc., Camarillo, California, USA) provided a recirculating flow of 757 L/hr. Periphytic algae was cultured on 6.25 cm², unglazed porcelain tiles (Cinca Tile Co., Fiães, Portugal). Tiles were arranged close together in culture trays to limit algal growth to the top surface, thereby producing uniform mats. Cultures received 12 h cycles of wide spectrum (Ecolux Plant & Aquarium Wide Spectrum, F40PL/AQ-ECO, General Electric Inc., Boston MA, USA) light. Culture tanks were positioned under two rows of the paired T12 florescent bulbs such that each tile was 120 cm and 150 cm (± 15 cm) from each pair of lights. Zn-cultured periphyton tiles (i.e., chronic exposure group) were prepared as above but with zinc sulfate (ZnSO₄) added to growth media of Zn-contaminated cultures to a concentration of 1600 µg/L in order to produce concentrations similar to those found in a survey of Colorado mountain streams (Schmidt et al. 2011 ). Surface area of culture tanks was scrubbed weekly, and a subset (20%) of tiles were replaced with new acid washed tiles to ensure periphytic algae had ample surface area to colonize. All cultures were maintained for three weeks prior to use in any exposure testing or analysis. Throughout the growth period, one water sample was taken from control and Zn treated troughs once every four days to measure aqueous Zn. Periphyton communities were dominated by Scenedesmus spp. (personal communication, Sarah Spaulding, United States Geological Survey, Boulder, Colorado, USA).
Immediately following the three-week growth period, a subset of tiles cultured in Zn (n = 4) and non-contaminated cultures (n = 4) were processed for subcellular fractionation to assess chronic exposure to Zn as well as growth in control conditions. Using a flow-through system, a subset of non-contaminated cultures was then bathed in 1600 µg/L Zn for either 15 min, 24 h, or 48 h to reflect episodic exposure likely to occur downstream of metal-contaminated landscapes as well as approximate common techniques used when preparing contaminated algae used in dietary toxicity trials (Irving et al. 2003 (link); Conley et al. 2009 (link); Xie et al. 2010 (link)). The flow-through system consisted of an 850 mL, circular artificial stream (Brinkman and Johnston 2008 (link)). Aqueous Zn samples were collected from the artificial stream at 0 min, 15 min, 24 h, and 48 h to ensure stable Zn exposure. Following acute exposure, four tiles from each group were processed for subcellular fractionation. All preparation methods/exposure regimes are listed in Table 1.Table 1

Exposure regimes/preparation methods used

Method	Description
Control	Cultured in non-contaminated Guillard’s growth media
Zn-cultured	Cultured in Guillard’s growth media contaminated with 1600 µg/L
15 min bathed	Cultured in non-contaminated Guillard’s growth media then bathed in 1600 µg/L for 15 min
24 h bathed	Cultured in non-contaminated Guillard’s growth media then bathed in 1600 µg/L for 24 h
48 h bathed	Cultured in non-contaminated Guillard’s growth media then bathed in 1600 µg/L for 48 h

The Multi-Color-PAM fluorometer used in the present study, like all PAM devices applies pulse-modulated ML and a special window-amplifier that is selective for the fluorescence excited by individual µs pulses of ML, so that the measurement of the ML-excited fluorescence is not disturbed by the fluorescence excited by much stronger AL or MT (Schreiber 1986 (link)). Hence, as ML intensity is constant during measurements, the ML-excited fluorescence may be considered a measure of relative fluorescence yield that varies between a minimal value of Fo (dark-adapted sample, primary acceptor Q_A fully oxidized) and Fm (Q_A fully reduced in the absence of non-photochemical quenching). In contrast, fluorescence intensity may vary indefinitely, depending on the intensity of the applied non-modulated actinic illumination. The output of the Multi-Color-PAM is a voltage signal that can vary between 0 and 6 Volt. The amplitude of this signal not only depends on the relative fluorescence yield of the sample, but also on chlorophyll content, the ML color, the chosen settings of ML intensity, and amplifier gain as well as on the choice of optical detector filters. Hence, while the signals are always proportional to fluorescence yield, the units with which the data are presented are arbitrary. In the present study, the instrument settings were generally optimized for maximal output signals of the various samples amounting to 3–5 Volt. The time-dependent fluorescence changes are plotted as relative fluorescence yield in arbitrary units, using the original voltage values for 540ex signals and appropriately rescaled values for 720ex signals (see section below on “Rescaling for comparison of 720ex and 540ex data”).
In the present study, the fluorescence responses induced by strong 540 nm multiple turnover pulses (MT), as measured with variously colored pulse-modulated ML, play a central role. The MT-induced rise of fluorescence yield consists of an initial “photochemical” phase from Fo to a first intermediate level I₁ (O-I₁ rise with rate proportional to quanta absorption by PSII) and two consecutive “thermal” phases, to a second intermediate level I₂ and to a peak P (Delosme 1967 (link), Schreiber 1986 (link), Neubauer and Schreiber 1987 (link), Schreiber and Neubauer 1987 (link); for reviews, see Schreiber 2004 , Lazar 2006 (link), Stirbet and Govindjee 2012 (link)). As the actinic effect of the pulse-modulated ML is negligibly small, the physiological reactions induced in measurements with variously colored ML are equal and solely caused by the 540 nm MT. This is particularly true in view of the applied optical geometry (see Fig. 1), with which light gradients and their effects are minimized. Hence, any differences that are observed in the MT-induced responses using different colors of ML must be due to heterogeneous origins of the excited pulse-modulated fluorescence. In particular, such differences may be expected between the responses assessed with FR and visible ML, as the PSI/PSII excitation and resulting F(I)/F(II) ratios are substantially higher with FR than with visible light. In supplementary figure S1, PSI and PSII action spectra of the unicellular green alga Scenedesmus obliquus are shown (Schreiber and Vidaver 1974 ), from which a PSI/PSII excitation ratio spectrum was derived (figure S2), which is close to unity between 660 and 680 nm and thereafter increases by about a factor of ten. It may be assumed that the Chlorella used in the present study displays similar PSI/PSII excitation properties. The obtained results show, however, that the ratio of F(I)/F(II) derived from F > 765 measurements with 720ex and 540ex does not follow the expected PSI/PSII excitation ratios, which will be dealt with in the Discussion (see section on “Apparently “too small” F(I)/F(II) excitation ratio with 720ex”).