Light-induced changes in the morphology and fluorescence of Arthrospira platensis

1 Introduction

The advantages of Arthrospira platensis (AP) - like high protein content, vitamins, and other biologically active ingredients [1, 2], a cell wall, which is easy to digest, together with the possibility that the biomass is easy to harvest [3], and used without any risk [4] - led to intensive large-scale cultivation all around the world [5–7].

Like green plants, the cyanobacteria carry out oxygenic photosynthesis, a unique process of sunlight energy conversion. The nature of the light-harvesting complex in cyanobacteria is well investigated. Their photosynthetic apparatus is organized in layers of lipoprotein membranes (thylakoids) and aqueous phases, the cytosol [8]. Oxygenic photosynthesis drives biosynthesis at the expense of inorganic nutrients. In a controlled environment, the growth of AP depends mainly on the quality and quantity of light [9–11]. In principle, the density of the culture increases with increasing intensity of the light source –the photon flux density (PFD). The highest concentrations and biomass productivity can be obtained when there are no limitations by nutrients (C, N, P, S) or abiotic factors such as temperature or pH [12–15]. The maximum PFD that can be applied without cell damage depends further on how much light-harvesting complexes are present in AP. For AP from the stationary phase with low average light intensity, the maximum possible PFD is much lower than for AP that are in the growth phase with very high average light intensity. It is also relevant how much light illuminates the respective AP spiral (here, the reactor geometry plays a major role). In general, too strong PFD can induce two harmful effects: (i.) photoinhibition is seen in a cyanobacterial culture as a reduction in photosynthetic rate [16], and (ii.) photooxidation, which has lethal effects on the cells, and which can lead to total loss of the culture [16]. The effect of photoinhibition is thought to be responsible for a part of growth reduction [17]. It is generally accepted that photooxidation occurs when the level of light absorbed by the photosynthetic apparatus exceeds the rate by which it is consumed in photosynthetic reactions [18, 19]. Under such conditions, excess electrons that accumulate in the photosynthetic electron transport chain may induce overproduction of reactive oxygen species, which in turn might cause inhibition of photosynthesis and lastly damages membrane lipids, proteins, and other macromolecules [20]. This phase of the growth curve was described by Huang et al. as “phase 5”, the death phase [21].

The photosynthetic efficiency (in Roux bottles) starts to decrease as soon as the PFD surpasses 20 kLux or 380μmol/(m² . sec) [21], the PFD on an open pond outdoors at midday in summer is around 1400μmol/(m² . sec) [17]. However, if HCO 3 - in the cytosol or CO₂ in the carboxysome is available in sufficient quantities and very high cell densities are maintained, AP can withstand PFD up to 8000μmol/(m² . sec) [22]. When HCO 3 - - depleted AP is illuminated with PFD above the light compensation point, a different picture emerges. Here, low HCO 3 - concentrations in combination with a high PFD will lead to the formation of reactive oxygen species [23], which can damage the membrane or septa, so that their integrity and morphology changes [24]. Singlet oxygen was the most predominant oxygen species generated during high light stress, while superoxide and hydroxyl radicals played a minor role in the photodynamic damage of AP [23].

2 Material and methods

AP cells (strain: SAG 21.99) used for the experiments were obtained from the, The Culture Collection of Algae at Goettingen University“, Goettingen, Germany. The AP suspension was cultured in freshwater supplemented with Zarrouk medium (16.8 g NaHCO₃, 0.5 g K₂HPO₄, 2.5 g NaNO₃, 1 g K₂SO₄, 1 g NaCl, 0.2 g MgSO₄.7H₂O, 0.04 g CaCl₂, 0.01 g FeSO₄.7H₂O, 0.08 g Na₂EDTA and 1 mL of trace metal solution. The trace metal solution consisted of (per liter): 2.86 g H₃BO₃, 1.81 g MnCl₄.4H₂O, 0.22 g ZnSO₄.4H₂O, 0.0177 g Na₂MoO₄, 0.079 g CuSO₄.5H₂O) in the back-up bioreactor at a PFD of 100μmol/(m²  _* sec). This growth medium was initially sterilized for 15 minutes at 121 °C in an HV-50 autoclave (SYSMEX VX-95, Sysmex, Norderstedt, Germany). For the experiments, AP cells were used as inoculum from the backup bioreactor, in which the AP had already reached the stationary growth phase. The bioreactor was inoculated with 0.25 g/L AP cells and was aerated with ambient air (1,000 L/h per 2 L reactor volume), which was pumped through a membrane filter (Millipore; 0.45μm pore size, 10 cm diameter) and with an air bubble diameter of 1.5 cm. The bioreactor was illuminated with a LED lamp (AP673L, Valoya, Helsinki, Finland) set to 50μmol/(² _. sec) at the bioreactor surface. 24 h later, the first sample was taken (Figs. 1 and 2 A and C each). Thereafter, the AP culture was exposed to a high PFD of 12,000μmol/(m² _. sec) for 1 h (Figs 1 and 2 B and D each) and 3 hs (Fig. 3).

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Fig. 1

Live cell microscopy (Axio Scope, Zeiss, Jena, Germany) of Arthrospira platensis (primary magnification 1 : 100). (A) A spiral of normal AP (phase contrast mode), (B) a spiral of photo-oxidated AP (phase contrast mode), (C) A spiral of normal AP (light transmission mode), and (D) a spiral of photo-oxidated AP (light transmission mode).

Samples were examined with bright field and phase contrast microscopy (Axio Scope, Zeiss Microimaging GmbH, Germany; BZ-X810, Keyence, Japan) as well as with laser scanning microscopy (Axio Observer.Z1/7, Zeiss Microimaging GmbH, Germany). Geometry measurements of the trichomes were carried out with the ImageJ software (National Institute of Health, USA) 25 –27].

In addition, samples of the same AP cultures were subjected to chlorophyll-a fluorescence measurements by pulse-amplitude modulation (PAM) fluorometer using the cuvette configuration (WATER-PAM fluorometer; Walz, Germany), to assess the photosynthetic capacity of AP in vivo [28].

Figure 1 shows microscopic images of AP - aligned as spiral, the typical morphology of aggregated AP - from a well-growing AP culture under HCO 3 - restriction but with aeration of ambient air at a high flow rate (1,000 L/h per 2 L reactor volume) at a low PFD of 50μmol/(² .sec) at the bioreactor surface (Fig. 1A, 1C). Figures 1B and 1D show AP from the same culture after exposure to a high PFD of 12,000μmol/(m² _. sec) for 1 h which led to an almost complete HCO 3 - - depletion, in the following called photo-oxidated AP (Fig. 1B, 1D).

The photo-oxidated AP showed a completely different appearance (Fig. 1B, D), the granular structure in the AP was nearly lost. Akao et al. [24] described such AP as “transparent”. The phenomenon was discussed to be due to the perforation of the cell wall with a gradual release of intracellular organic matter (IOM, like proteins and polysaccharides) in contrast to spontaneous disruption of the cell wall, complete release, and loss of structural integrity.

Confocal laser scanning microscopy (CLSM) images show - beyond the transparency effect (Fig. 2A and 2B) - that the intensity of fluorescent structures (e.g. chlorophyl-a and phycobiliproteins) dramatically decreased indicating a change in the structural integrity of the pigments (Fig. 2C - 2F). This can lead to a decrease or finally to a loss of function of the antenna for the photons and so to the loss of the photosynthetic activity. Finally, the long-term exposure (3 hours) to a high photon flux density of 12,000μmol/(m² _. sec) results in the formation of debris as shown in Figs. 3A and 3B.

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Fig. 2

Live cell confocal laser scanning microscopy images of normal Arthrospira platensis (2A, 2C, 2E) appear with high intensity of fluorescent structures while in photo-oxidated Arthrospira platensis significantly lower intensities were detected (B, D, F). Image stacks were taken at 100-fold primary magnification with a ZEISS LSM800 in the AIRYSCAN- (B and C, excitation: maxim projection, emission: 650–700 nm) and transmission mode (2A and 2B). The detail image in 2C and 2D was contrast-enhanced to visualize the intracellular structures. Figures 2E and 2F show fluorescence intensity profiles along the line selections shown in 2C and 2D.

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Fig. 3

Representative phase contrast images of debris formation of Arthrospira platensis after exposure to a high photon flux density of 12,000μmol/(m² _. sec) for 3 hours (Phase contrast microscopy, primary magnification 1 : 40).

The photosynthetic activity can be measured using the Pulse Amplitude Modulation (PAM) fluorometer technique [26]. Over a wide range of physiological conditions, the quantum yield of non-cyclic electron transport was found to be directly proportional to the product of the photochemical fluorescence quenching (qQ) and the efficiency of excitation capture by open Photosystem II (PS II) reaction centers (F_v/F_m). The effective photochemical yield of the photosystem II (YII) with 0.360±0.008 (maximum yield F_v/F_m: 0.43±0.01) for the normal AP was significantly higher (p < 0.01) than for the photo-oxidated AP with 0.002±0.002 (maximum yield F_v/F_m: 0.002±0.002). This shows that the quantum yield of photo-oxidated AP was about 180-fold lower than that of the normal AP, which is well in line with the assumption that most of the antennas of these AP are destructed.

These observations demonstrate that under the procedures described, and very high PFD, not only a decrease in the photosynthetic efficiency, but beyond, also the destruction of AP can occur. In a bioreactor, this would lead to a clear decrease of the growth yield and over longer illumination to the death of the culture. Since AP is not only a rich source of dietary proteins but also of several pharmacologically relevant compounds, potential light-induced AP damage should be considered by manufacturers.