Fast repetition rate fluorometer (FRRf) is a beneficial method for measuring photosystem II photophysiology and primary productivity. Here we describe a protocol to measure PSII photophysiology of epizoic alga, Colacium sp. on substrate zooplankton using cuvette-type FRRf.
Fast repetition rate fluorometer (FRRf) is a beneficial method for measuring photosystem II (PSII) photophysiology and primary productivity. Although FRRf can measure PSII absorption cross-section (σPSII), maximum photochemical efficiency (Fv/Fm), effective photochemical efficiency (Fq′/Fm′), and non-photochemical quenching (NPQNSV) for various eukaryotic algae and cyanobacteria, almost all FRRf studies to date have focused on phytoplankton. Here, the protocol describes how to measure PSII photophysiology of an epizoic alga Colacium sp. Ehrenberg 1834 (Euglenophyta), in its attached stage (attached to zooplankton), using cuvette-type FRRf. First, we estimated the effects of substrate zooplankton (Scapholeberis mucronata O.F. Müller 1776, Cladocera, Daphniidae) on baseline fluorescence and σPSII, Fv/Fm, Fq′/Fm′, and NPQNSV of planktonic Colacium sp. To validate this methodology, we recorded photophysiology measurements of attached Colacium sp. on S. mucronata and compared these results with its planktonic stage. Representative results showed how the protocol could determine the effects of calcium (Ca) and manganese (Mn) on Colacium sp. photophysiology and identify the various effects of Mn enrichment between attached and planktonic stages. Finally, we discuss the adaptability of this protocol to other periphytic algae.
Chlorophyll variable fluorescence is a useful tool for measuring algal photosystem II (PSII) photophysiology. Algae respond to various environmental stresses, such as excess light and nutrient deficiency, by altering their PSII photophysiology. Fast repetition rate fluorometer (FRRf) is a common method for measuring PSII photophysiology1,2 and estimating primary productivity1,3,4, which enables monitoring phytoplankton PSII photophysiology, as well as primary productivity across wide spatial and temporal scales5,6,7. FRRf can simultaneously measure PSII (σPSII) absorption cross section, reaction center ([RCII]) concentration, maximum photochemical efficiency (Fv/Fm), effective photochemical efficiency (Fq′/Fm′), and non-photochemical quenching (NPQNSV) (Table 1). In general, Fv/Fm and Fq′/Fm′ are defined as PSII activity8, while NPQNSV is defined as relative heat-dissipated energy9.
Importantly, single turnover (ST) flashes of FRRf fully reduce the primary quinone electron acceptor, QA, but not the plastoquinone pool. Conversely, multiple turnover (MT) flashes from a pulse amplitude modulation (PAM) fluorometer can reduce both. The ST method has a clear advantage over the MT method when identifying the possible origins of NPQNSV by simultaneously measuring recovery kinetics of Fv/Fm, Fq′/Fm′, NPQNSV, and σPSII10. To date, several types of FRRf instruments, such as submersible-type, cuvette-type, and flow-through-type, are commercially available. The submersible-type FRRf enables in situ measurements in oceans and lakes, while the cuvette-type FRRf is suitable for measuring small sample volumes. The flow-through type is commonly used to continuously measure the photophysiology of phytoplankton in surface waters.
Given the development of PAM fluorometers, including the cuvette-type, for a broad range of subjects11, PAM fluorometers are still more common than FRRfs in algal photophysiology research12. For example, although the sample chamber structure and cuvette capacity between these tools only differs slightly, the cuvette-type PAM has been applied to phytoplanktons13,14,15, benthic microalgae16,17,18, ice algae19, and epizoic algae20, while the cuvette-type FRRf has been applied primarily to phytoplanktons21,22,23 and a limited number of ice algal communities24,25. Given its effectiveness, cuvette-type FRRf is equally applicable to benthic and epizoic algae. Therefore, expanding its application will provide considerable insight into PSII photophysiology, particularly for lesser-known epizoic algal photophysiology.
Epizoic algae have received little attention, with few studies examining their PSII photophysiology20,26, most likely due to their minor roles in aquatic food webs27,28. However, epibionts, including epizoic algae, can positively influence zooplankton community dynamics, such as increasing reproduction and survival rates29,30, as well as negatively impact processes, such as increasing sinking rate29,31 and vulnerability to visual predators32,33,34,35,36. Therefore, exploring the environmental and biological factors controlling epibiont dynamics in zooplankton communities is crucial.
Among epizoic algae, Colacium Ehrenberg 1834 (Euglenophyta) is a common, freshwater, algal group32,37,38,39 with various life stages, including attached (Figure 1A–D), non-motile planktonic (Figure 1E,F), and motile planktonic stages40,41. During the non-motile planktonic stage, cells live as single-cell planktons, aggregated colonies, or one-layer sheet colonies, covered by mucilage42. In the attached stage, Colacium sp. uses mucilage excreted from the anterior end of the cell37,39,41 to attach to substrate organisms (basibionts), particularly microcrustaceans41,43. Their life cycle also involves detaching from the molted exoskeleton or dead basibiont and swimming with their flagella to find another substrate organism39. Both planktonic and attached stages can increase their population size by mitosis40. Although their attached stage is hypothesized to be an evolutionary trait for gathering resources, such as light44 and trace elements41,45,46, or as a dispersion strategy27, little experimental evidence is available about these aspects37,41,44 and the key attachment mechanisms are largely unknown. For example, Rosowski and Kugrens expected that Colacium obtains manganese (Mn) from substrate copepods41, concentrated in the exoskeleton47.
Here, we describe how to measure PSII photophysiology of planktonic algae and the related application method for targeting attached algae (attaching to zooplankton) with Colacium sp. cells using the cuvette-type FRRf. We use the Act2 system equipped with three light-emitting diodes (LEDs) that provide flash excitation energy centered at 444 nm, 512 nm, and 633 nm48. Here, 444 nm (blue) corresponds to the absorption peak of chrophyll a (Chl-a), while 512 nm (green) and 633 nm (orange) correspond to the absorption peaks of phycoerythrin and phycocyanin, respectively. The fluorescent signal detection peak is 682 nm with 30 nm half bandwidth. Since it is difficult to find the planktonic stage of Colacium sp. in natural environments, their attached stage was collected for the experiments. Among the numerous substrate organisms,Scapholeberis mucronata O.F. Müller 1776 (Branchiopoda, Daphniidae; Figure 1A,B,G) is one of the simplest to handle due to their slow swimming speed, large body size (400-650 µm), and unique behavior (hanging upside down on the water surface). Therefore, this protocol uses Colacium sp. attached on S. mucronata as a case study of the Colacium-basibiont system. To avoid fluorescence derived from the gut contents, S. mucronata was starved. As a previous study reported that the fluorescence signal from gut contents (ingested algae) displays a five-fold decrease after 40 min49, we expected that 90 min starvation would be enough to minimize the possibility of gut content fluorescence affecting the FRRf measurement with minimum effects of experimental stress to Colacium sp., such as nutrient deficiency. Furthermore, this protocol was applied to clarify the attaching mechanism of Colacium sp. and determine how two metals, calcium (Ca) and manganese (Mn) affect the photophysiology of both planktonic and attached stages. Calcium plays key roles in the photosynthetic pathways50 in multiple ways, and both metals are required to construct the oxygen-evolving complexes of the PSII51. As calcium and manganese are highly concentrated in the carapace of crustacean zooplankton47, we hypothesize that Colacium sp. photophysiology might respond more prominently to Ca and Mn enrichment during the planktonic stage if this life stage obtains these elements from S. mucronata during the attached stage.
1. Sampling
2. Effects of S . mucronata on baseline fluorescence
3. Effects of substrate organism on Chl- a fluorescence
4. Photophysiology of Colacium sp. (attached stage)
5. Photophysiology of Colacium sp. (planktonic stage)
6. Effects of Ca and Mn addition on photophysiology of Colacium sp.
There was no significant effect of baseline fluorescence (Figure 5) or Chl-a fluorescence (Figure 6) by S. mucronata up to 5 individuals (inds.) mL−1. However, Fv/Fm and NPQNSV were significantly affected when S. mucronata was 7.5 inds·mL−1. Therefore, for measuring the photophysiology of Colacium sp. during the attached stage, we chose S. mucronata with the higher burden of Colacium sp. in order to reach sufficient Colacium sp. abundance (>50 cells·mL−1) and a low number of S. mucronata (≤5 inds·mL−1) in the cuvette.
Table 3 shows seasonal variation in photophysiology of Colacium sp. during the attached stage. Although sampling temperature varied, their photophysiology remained relatively constant. σPSII varied from 3.42 nm2 to 3.76 nm2 (mean 3.60 nm2), Fv/Fm varied from 0.52 to 0.60 (mean 0.55), and NPQNSV varied from 0.66 to 0.85 (mean 0.82). To validate these results, we further investigated variations in Colacium sp. photophysiology during the planktonic stage for the stationary phase in the AF-6 medium (Table 4). Mean Fv/Fm and NPQNSV for the attached stage were similar to those of the planktonic stage when incubated in AF-6 medium.
To determine the effect of Ca and Mn on Colacium sp. photophysiology in both the attached and planktonic stages, we performed Ca and Mn enrichment experiments. Samples were taken from the reed area of Lake Biwa on May 7, 2021. For the attached stage of Colacium sp. under dark conditions, there was no significant difference in photophysiological parameters among treatments, except for NPQNSV between Mn and Ca treatments at 3 h, where Ca < Mn (Figure 7A,C,E). Further, σPSII′, Fq′/Fm′, and NPQNSV responses to increasing light during the attached stage showed no clear differences among treatments (Figure 8A,C,E and Figure 9A,C,E). However, NPQNSV tended to be lower in the Ca treatment than the control at a low light intensity at 21 h (11 and 25 µmol photon·m−2·s−1, Figure 9E). For the planktonic stage, σPSII was significantly lower in the Mn than Ca treatment at 3 h (Figure 7B). Fq′/Fm′ was significantly higher, but NPQNSV was lower in the Mn treatment than control at 21 h (Figure 7D,F). Under increasing light, Mn tended to decrease σPSII and increase Fq′/Fm′ during the planktonic stage, compared to the control at 3 h (Figure 8D). Similarly, Mn significantly reduced NPQNSV during the planktonic stage compared to the control at 21 h (Figure 9F). Similar to the attached stage, calcium slightly improved NPQNSV for the planktonic stage under increasing light (Figure 9F). However, Ca decreased Fq′/Fm′ and increased NPQNSV for the planktonic stage compared to Mn treatment under 44-200 µmol photon·m−2·s−1 at 3 h (Figure 8D,F).
Figure 1: Colacium sp. and substance organism Scapholeberis mucronata. (A) Infected S. mucronata. (B) Infected S. mucronata fixed with glutaraldehyde. (C) Attached Colacium cells on living S. mucronata. (D) Attached Colacium cells on the molted carapace. (E, F) Colacium sp. of planktonic (palmella) stage. (G) Non-infected S. mucronata. Arrows indicate Colacium cells. Scale bars: 200 µm (A, B, and G), 10 µm (C, E, and F), and 100 µm (D). Please click here to view a larger version of this figure.
Figure 2: Washing zooplankton by pipetting under filtered lake water (FLW). Please click here to view a larger version of this figure.
Figure 3: Act2Run software user interface. (A) Exposure time to each actinic light step; (B) Number of steps and photon flux of actinic light; (C) Combination of excitation wavelength; (D) Saturation and relaxation flashlet sequence; (E) Frequencies and intensities of the water jacket and sample mixing pumps; (F) Photon flux of excitation flash at 444 (denoted as 450 here), 512 (530), and 633 nm (624), photomultiplier tube (PMT) voltage, and replicates and interval of sequence. Please click here to view a larger version of this figure.
Figure 4: Fluorescence reading of the algal sample on Act2Run software. The red and blue lines indicate raw fluorescence signal by a sequence of flashlet and curve fitting in both saturation and relaxation phases, respectively. See Kolber et al.2 for more details. Please click here to view a larger version of this figure.
Figure 5: The effect of S. mucronata density on baseline fluorescence. The small dots represent replicates (n = 3). The results of the ANOVA test are also shown. Please click here to view a larger version of this figure.
Figure 6: The effects of S. mucronata densities on (A) FO (B) σPSII, (C) Fv/Fm, and (D) NPQNSV for Colacium sp. during the planktonic stage. The small dots represent replicates (n = 3). Colacium sp. was cultured in AF-6 medium. The results of ANOVA and Tukey post-hoc test are also presented. Please click here to view a larger version of this figure.
Figure 7: Responses of (A,B) absorption cross-section, (C,D) PSII photochemistry, and (E,F) non-photochemical quenching of (A,C,E) attached stage and (B,D,F) planktonic stage of Colacium sp. at 3 h and 21 h after Ca and Mn addition. The small dots represent replicates (n = 4). The results of ANOVA and Tukey post-hoc test are also presented. * p < 0.05. Please click here to view a larger version of this figure.
Figure 8: Rapid-light responses of (A, B) absorption cross-section, (C,D) PSII photochemistry, and (E,F) non-photochemical quenching of Colacium sp. in attached and planktonic stages to stepwise light protocol at 3 h after Ca and Mn addition. C, control; Ca, 200 µM Ca; Mn, 40 µM Mn. Significant differences between (a) C and Ca, (b) C and Mn, and (c) Ca and Mn at each PAR flux, with a significance level of p < 0.05 shown in each panel. Error bar, Mean SD (n = 4). Please click here to view a larger version of this figure.
Figure 9: Rapid-light responses of (A,B) absorption cross-section, (C,D) PSII photochemistry, and (E,F) non-photochemical quenching of Colacium sp. in attached and planktonic stages to stepwise light protocol at 21 h after Ca and Mn addition. C, control; Ca, 200 µM Ca; Mn, 40 µM Mn. Significant differences between (a) C and Ca, (b) C and Mn, and (c) Ca and Mn at each PAR flux, with a significance level of p < 0.05 shown in each panel. Error bar, Mean SD (n = 4). Please click here to view a larger version of this figure.
Term | Definition | Units | |
Baseline fluorescence | Fo value without Chl-a fluorescence | ||
F' | Fluorescence at zeroth flashlet of a single turnover measurement when C>0 | ||
Fo (ʹ) | Minimum PSII Fluorescence yield (under background light) at zeroth flashlet | ||
Fv (ʹ) | Fm(ʹ) − Fo(ʹ) | ||
Fm (ʹ) | Maximum PSII Fluorescence yield (under background light) | ||
Fv/Fm | Maximum PSII photochemical efficiency under dark | ||
Fqʹ/Fmʹ | Maximum PSII photochemical efficiency under background light, (Fmʹ− F)/(Fmʹ) | ||
NPQNSV | Normalized Stern-Volmer quenching, FOʹ/(Fmʹ − FOʹ) | ||
[RCII] | Concentration of reaction center | ||
RσPSII (ʹ) | Probability of an RCII being closed during the first flashlet of a single turnover saturation phase (under background light) | ||
σPSII (ʹ) | Functional absorption cross section of PSII for excitation flashlets (under background light) | nm2 |
Table 1: Terms used in this protocol.
Component | Quantity |
NaNO3 | 140 mg·L−1 |
NH4NO3 | 22 mg·L−1 |
MgSO4·7H2O | 30 mg·L−1 |
KH2PO4 | 10 mg·L−1 |
K2HPO4 | 5 mg·L−1 |
CaCl2·2H2O | 10 mg·L−1 |
CaCO3 | 10 mg·L−1 |
Fe-citrate* | 2 mg·L−1 |
Citric acid* | 2 mg·L−1 |
Biotin | 0.002 mg·L−1 |
Vit. B1 | 0.01 mg·L−1 |
Vit. B6 | 0.001 mg·L−1 |
Vit. B12 | 0.001 mg·L−1 |
Trace metals | 1 mL·L−1 |
(FeCl3·6H2O) | (1.0 mg·mL−1) |
(MnCl3·4H2O) | (0.4 mg·mL−1) |
(ZnSO4·7H2O) | (0.005 mg·mL−1) |
(CoCl2·6H2O) | (0.002 mg·mL−1) |
(Na2MoO4) | (0.004 mg·mL−1) |
(Na2-EDTA) | (7.5 mg·mL−1) |
Table 2: Recipe for AF-6 medium. Adjust pH to 6.6. Dissolve Fe-citrate and citric acid in warm H2O separately and add HCl (1 mL·L−1) after mixing both reagents. Contents of trace metals are shown in parenthesis.
Sampling date | Sample No. | Water temp. (°C) |
S. mucronata density (inds.·mL−1) |
Colacium sp. cell density (inds.·mL−1) |
σPSII (nm2) | Fv/Fm | NPQNSV |
April 27/2020 | No. 1 | 14.2 | 4.5 | 77 | 3.42 | 0.60 | 0.66 |
SE | 0.22 | 0.01 | 0.04 | ||||
May 21/2020 | No. 2 | 19.4 | 2 | 282.5 | 3.62 | 0.54 | 0.85 |
SE | 0.16 | 0.02 | 0.06 | ||||
No.3 | 19.4 | 2 | 250.5 | 3.55 | 0.56 | 0.77 | |
SE | 0.09 | 0.01 | 0.02 | ||||
No.4 | 19.4 | 5 | 204.5 | 3.76 | 0.52 | 0.94 | |
SE | 0.12 | 0.00 | 0.02 | ||||
June 18/2020 | No.5 | 22.4 | 2.5 | 474 | 3.62 | 0.54 | 0.85 |
SE | 0.16 | 0.02 | 0.06 | ||||
No.6 | 22.4 | 2 | 410 | 3.55 | 0.56 | 0.77 | |
SE | 0.09 | 0.01 | 0.02 | ||||
No.7 | 22.4 | 2.5 | 441 | 3.76 | 0.52 | 0.94 | |
SE | 0.12 | 0.00 | 0.02 | ||||
July 20/2020 | No. 8 | 27.5 | 5 | 109 | 3.49 | 0.58 | 0.74 |
SE | 0.10 | 0.00 | 0.00 | ||||
Mean | 3.60 | 0.55 | 0.82 | ||||
S.D. | 0.120349 | 0.03 | 0.10 |
Table 3: Photophysiology of Colacium sp. attached on S. mucronata.
Sampling date | Sample No. | Medium | Growth temperature (°C) | σPSII (nm2) | Fv/Fm | NPQNSV |
May 21/2020 | No. 1 | AF-6 | 19.4 | 2.72 | 0.65 | 0.53 |
SE | 0.03 | 0.00 | 0.01 | |||
June 18/2020 | No. 2 | AF-6 | 22.4 | 3.07 | 0.55 | 0.84 |
SE | 0.08 | 0.02 | 0.07 | |||
July 20/2020 | No.3 | AF-6 | 27.5 | 2.90 | 0.58 | 0.73 |
SE | 0.06 | 0.01 | 0.02 | |||
Mean | 2.90 | 0.59 | 0.70 | |||
S.D. | 0.18 | 0.05 | 0.16 |
Table 4: Photophysiology of Colacium sp. planktonic stage. Each sample was measured during the stationary phase.
This protocol demonstrated for the first time that photophysiology of Colacium sp. during the attached stage in a natural environment is comparable to its planktonic stage in AF-6 medium. Additionally, gut contents of starved S. mucronata did not affect baseline and Chl-a fluorescence when density was ≤5 inds·mL−1 (Figure 5 and Figure 6). These results suggest this protocol can measure photophysiology of Colacium sp. during the attached stage without correction under low substrate organism abundance. However, results from steps 3.2.1-3.2.8 showed that the highest S. mucronata abundance affected Fv/Fm and NPQNSV significantly, but not FO and σPSII (Figure 4). Here, it's possible higher organism density exacerbated physical stress on Colacium sp. individuals and subsequently decreased photosynthetic activity. For measurements under a high abundance of substrate organisms or other species, the effects of substrate organism density on the baseline and Chl-a fluorescence requires further attention.
FRRfs have been used to examine the impact of nutrient manipulation on the linear electron flow and non-photochemical quenching of phytoplankton22,56,57. The primary results show that Ca and Mn enrichment differed significantly between Colacium sp. life stages (Figure 7, Figure 8, and Figure 9). Specifically, manganese clearly improved the (maximum) photochemical yield of PSII (Fv/Fm and Fq′/Fm′) and decreased the heat dissipation (NPQNSV)50 of planktonic stages under dark (Figure 7D,F) and light conditions (Figure 8D,F and Figure 9D,F). These outcomes can stem from reduced antenna size on PSII, σPSII, and σPSII′ (Figure 7B and Figure 8B), which reduces excess light absorption58,59. Measuring antenna size in addition to energy flow between PSII complexes would allow more precise measurements of the algal response10. This protocol also allows the examination of photosynthesis limitations by other resources. For example, nitrogen and phosphorus limitations have been examined in various phytoplankton communities, but not in epizoic algae, despite predicted effects on Colacium41 and marine epizoic diatoms60,61. In addition to nutrients, the light environment can further influence epizoic algae distribution44.
As shown in Figure 7, Figure 8, and Figure 9, cuvette-type FRRf enables us to simultaneously examine nutrient and light effects without long incubation times and measurement effort. This stepwise light protocol (step 6.1.5) can also draw rapid-light curves of relative electron transport rates (rETR = Fq′/Fm′× light) vs. light as an analog for production vs. light curves62. However, although linear electron flow in PSII can be estimated from photophysiological parameters by the FRRf, it is not necessarily analogous to the carbon fixation rate63,64. For estimating carbon-based primary production, electron requirement per CO2 fixation (Фe, C), which can vary temporally and spatially5,48, should be examined when assessing subject communities.
If the plankton net is clogged by debris, prescreen by a larger mesh, such as a 5-mm mesh net, or pick zooplanktons directly from the lake water using a pipette without filtration. It should be noted that some damage might occur to the attached algae even when the filtration is conducted gently using a relatively large (200 µm) mesh size. Although the results show that the standard deviation of the PSII parameters was small (Table 3), and the mean values of the parameters were very similar to those of the cultured planktonic stage (Table 4), sampling without filtration might be ideal.
Another limitation of this study was deriving σPSII. Actinic light and Chl-a fluorescence attenuation can exert a major influence/distortion on the FRRfs, which relies upon optically thin samples for the accurate σPSII determination65. Although we showed that S. mucronata did not affect the σPSII of planktonic Colacium cells, that should be examined related to the σPSII of the attached Colacium cells. Furthermore, a spectral correction factor (SCF) would be needed for the σPSII in situ66 estimation as the excitation wavelength of the ACT2 system (444 nm) differs from the spectral distribution of the in situ light environment. In general, the filter pad technique is used to measure the Chl-a specific absorption spectrum to calculate the SCF. This procedure is necessary to estimate algal primary productivity by the FRRfs. As we could not harvest enough attached Colacium cells through the study period, the Chl-a specific absorption spectrum should be examined in future studies.
Implementing cuvette-type FRRf should depend on substrate size as periphytic algae require a substrate attachment. For example, studies of algae on indestructible substances, such as rocks67, larger organisms26,68, or symbiotic algae, including Symbiodinium associated with hard corals10,69,70, might require the submersible-type FRRf69. Conversely, if the basibiont is small enough to suspend itself in a cuvette, a cuvette-type FRRf might be sufficient in addition to a cuvette-type PAM, such as benthic algae16,17,18. Indeed, recent studies have explored a cuvette-type FRRf for measuring the photophysiology of ice algae24,25. Furthermore, turning on the excitation flash at 512 and 633 nm enables the application to cyanobacteria with different PSII antenna pigments, phycoerythrins and phycocyanins, and thus different absorption peaks with Chl-a71. As current FRRf models incorporating multi-excitation wavelengths are useful tools for examining cyanobacteria photophysiology and productivity7,66,72, these ought to be useful methods for assessing benthic cyanobacteria, if the effects of sample thickness on photophysiological parameters would be improved65. In future studies, FRRfs should be aimed at a wider range of subject organisms to shed further insight on the complex mechanisms of algal photophysiology across various habitats.
The authors have nothing to disclose.
The work was supported by the Collaborative Research Fund from Shiga Prefecture entitled "Study on water quality and lake-bottom environment for the protection of the soundness of water environment" under the Japanese Grant for Regional Revitalization and the Environment Research and Technology Development Fund (No. 5-1607) of the Ministry of the Environment, Japan. https://www.kantei.go.jp/jp/singi/tiiki/tiikisaisei/souseikoufukin.html. The authors would like to thank Enago (www.enago.jp) for the English language review.
Acrodisc syringe filter | Pall Corporation, Ann Arbor, MI, USA | 0.2 μm pore size | |
Act2Run | CTG Ltd., West Molesey, UK | ||
Biotin | Wako | 023-08711 | AF-6 medium |
CaCl2·2H2O | Wako | 031-25031 | AF-6 medium |
CaCO3 | Wako | 036-00382 | AF-6 medium |
Citric acid | Wako | 036-05522 | AF-6 medium |
CoCl2·6H2O | Wako | 036-03682 | AF-6 medium |
Concentrated Chlorella | Recenttec, Tokyo, Japan | 20 mg C·mL−1 ; store at 4 °C | |
FastOcean Act2 | CTG Ltd., West Molesey, UK | ||
Fe-citrate | Wako | 093-00952 | AF-6 medium |
FeCl3·6H2O | Wako | 091-00872 | AF-6 medium |
HCLP-880PF | Nippon Medical and Chemical Instruments Co., Ltd., Osaka, Japan |
With LED light bulbs | |
K2HPO4 | Wako | 160-04292 | AF-6 medium |
KH2PO4 | Wako | 167-04241 | AF-6 medium |
MgSO4·7H2O | Wako | 137-00402 | AF-6 medium |
MnCl3·4H2O | Wako | 139-00722 | AF-6 medium |
Na2EDTA | Wako | 343-01861 | AF-6 medium |
Na2MoO4 | Wako | 196-02472 | AF-6 medium |
NaNO3 | Wako | 191-02542 | AF-6 medium |
NH4NO3 | Wako | 015-03231 | AF-6 medium |
Plankton Counter | Matsunami Glass, Osaka, Japan | S6300 | |
Pylex test tube | CTG Ltd., West Molesey, UK | With rim, 16 x 100 mm | |
Vit. B1 | Wako | 203-00851 | AF-6 medium |
Vit. B12 | Wako | 226-00343 | AF-6 medium |
Vit. B6 | Wako | 165-05401 | AF-6 medium |
ZnSO4·7H2O | Wako | 264-00402 | AF-6 medium |