Costanza Baldisserotto

Morphophysiological analyses of Neochloris oleoabundans (Chlorophyta) grown mixotrophically in a carbon-rich waste product

Neochloris oleoabundans is considered one of the most promising oil-rich microalgae because of its ability to store lipids under nitrogen starvation. However, high biomass densities, required for applications on medium to large scale, are not reached in this condition of growth. As previous studies on other microalgae have shown that mixotrophy allows to obtain higher biomass in comparison to autotrophic cultures, we performed morphophysiological analyses in order to test the mixotrophic growth capability of N. oleoabundans. A carbon-rich manure derived from the apple vinegar production (AWP) was added to the medium. Cells were also cultivated under nutrient starvation (tap water), to observe the expected lipids accumulation, and combining AWP to water, to test the potential of this waste in a low-cost culture system. The results highlighted that AWP in the medium allowed to obtain the highest final cell density. Moreover, starch granules were stored inside chloroplast at the beginning of the experiment. The presence of AWP did not induce variations on light harvesting complex II (LHCII)–photosystem II (PSII) assembly, even if an interesting promotion of pigment synthesis in cells was observed. On the other hand, in starved cells, chloroplast degeneration, pigment content decrease, altered LHCII–PSII assembly and accumulation of high amount of lipid globules were observed, irrespective of the presence of AWP. The results suggest that mixotrophy promotes growth in N. oleoabundans and open up the possibility of using waste products from agri-food industries for this purpose. After growth, cells could be transferred under nutrient starvation to induce lipid accumulation.

Responses of Trapa natans L. floating laminae to high concentrations of manganese

Summary.The present study focuses on the responses of floating laminae of the Mn-tolerant hydrophyte Trapa natans L. to 1 mM Mn and their ability to accumulate the metal. Studies were carried out first on young floating laminae belonging to the second verticil of 30-day-old plants which originated from fruits that had been maintained in a 1 mM Mn-treated environment and again on the young floating laminae after 10 days of further treatment with 1 mM Mn. Mn storing was observed from the first days after germination, but only 10-day-treated laminae showed the capability to hyperaccumulate the element inside specialised cells (>20000 μg/g [dry weight]). Electron microscopy and the Folin–Ciocalteu reaction for phenolics revealed deposits of chelated material inside vacuoles of the first palisade layer and of idioblasts in the spongy tissue. X-ray microanalysis indicated that the deposits were Mn chelated with phenolic compounds. Numerous trichomes were observed at the lower epidermis of 10-day-treated laminae. They were rich in phenolics and characterised by Mn concretions at their base. As they are associated with a high concentration of the metal in culture water and sediments, trichomes may constitute a morphological differentiation for the secretion of Mn-chelating molecules into the culture water, as a probable “avoidance” mechanism. Finally, monitoring of the photosynthetic apparatus showed that photosynthetic function was not impaired, though differences in development occurred.

High salinity alters chloroplast morpho-physiology in a freshwater Kirchneriella species (Selenastraceae) from Ethiopian Lake Awasa

Plants differ in their ability to tolerate salt stress. In aquatic ecosystems, it is important to know the responses of microalgae to increased salinity levels, especially considering that global warming will increase salinity levels in some regions of the Earth, e.g., Ethiopia. A green microalga, Kirchneriella sp. (Selenastraceae, Chlorophyta), isolated from freshwater Lake Awasa in the Rift Valley, Ethiopia, was cultured in media amended with 0, 0.4, 1.9, 5.9, and 19.4 g NaCl·L(-1) adjusted with NaCl to five salinity levels adjusted with NaCl. Growth was monitored for 3 mo, then samples were collected for photosynthetic pigment determinations, microspectrofluorimetric analyses, and micro- and submicroscopic examinations. The best growth was found at 1.9 g NaCl·L(-1). In the chloroplast, excess NaCl affected the coupling of light harvesting complex II and photosystem II (LHCII-PSII), but changes in thylakoid architecture and in the PSII assembly state allowed sufficient integrity of the photosynthetic membrane. The mucilaginous capsule around the cell probably provided partial protection against NaCl excess. On the whole, the microalga is able to acclimate to a range of NaCl concentrations, and this plasticity indicates that Kirchneriella sp. may survive future changes in water quality.

Adaptive modifications of the photosynthetic apparatus in Euglena gracilis Klebs exposed to manganese excess

Summary.Asynchronous cultures of wild-type Euglena gracilis were tested for their morphophysiological response to 10 mM MnSO4. Growth was only moderately slowed (15%), while oxygen evolution was never compromised. Inductively coupled plasma analyses indicated that the Mn cell content doubled with respect to controls, but no signs of localised accumulation were detected with X-ray microanalysis. Evident morphological alterations were found at the plastid level with transmission electron microscopy and confocal laser scanning microscopy. An increase in the plastid mass, accompanied by frequent aberrations of chloroplast shape and of the organisation of the thylakoid system, was observed. These aspects paralleled a decrease in the molar ratio of chlorophyll a to b and an increase in the fluorescence emission ratio of light-harvesting complex II to photosystem II, the latter evaluated by in vivo single-cell microspectrofluorimetry. These changes were observed between 24 and 72 h of treatment. However, the alterations in the pigment pattern and photosystem II fluorescence were no longer observed after 96 h of Mn exposure, notwithstanding the maintenance of the large plastid mass. The response of the photosynthetic apparatus probably allows the alga to limit the photooxidative damage linked to the inappropriately large peripheral antennae of photosystem II. On the whole, the resistance of Euglena gracilis to Mn may be due to an exclusion–tolerance mechanism since most Mn is excluded from the cell, and the small amount entering the organism is tolerated by means of morphophysiological adaptation strategies, mainly acting at the plastid level.

Revised assignment of room-temperature chlorophyll fluorescence emission bands in single living cells of Chlamydomonas reinhardtii

Room temperature (RT) microspectrofluorimetry in vivo of single cells has a great potential in photosynthesis studies. In order to get new information on RT chlorophyll fluorescence bands, we analyzed the spectra of Chlamydomonas reinhardtii mutants lacking fundamental proteins of the thylakoid membrane and spectra of photoinhibited WT cells. RT spectra of single living cells were characterized thorough derivative analyses and Gaussian deconvolution. The results obtained suggest that the dynamism in LHCII assembly could be sufficient to explain the variations in amplitudes of F680 (free LHCII), F694 (LHCII-PSII) and F702 (LHCII aggregates); F686 was assigned to the PSII core. Based on the revised assignments and on the variations observed, we discuss the meaning of the two fluorescence emission ratios F680/(F686 + F694) and F702/(F686 + F694), showing that these are sensitive parameters under moderate photoinhibition. In the most photoinhibited samples, the RT spectra tended to degenerate, showing characteristics of mutants that are partly depleted in PSII.

Acclimation to darkness in the marine chlorophyte Koliella antarctica cultured under low salinity: hypotheses on its origin in the polar environment

In order to obtain new insights on the origin and physiology of the marine chlorophyte Koliella antarctica, the response of the microalga was studied at a salinity of 0.2 in the light and during a 60-day dark period. In light conditions, the alga grows and maintains a functional cell organization. In darkness, the chloroplast-to-chromoplast transition, previously described during dark-acclimation in K. antarctica under a salinity of 34 was only partially triggered; thylakoid lamellae became re-organized into short bundles, but storage substructures were almost completely missing. Microspectrofluorometry, pigment analyses, and morphological observations revealed dark-induced degradation of photosystem II (PSII) with relative stability in the light-harvesting complex (LHCII). Chromatin condensation, mitochondrion fragmentation and material digestion in vacuoles were similar to morphological hallmarks of programmed cell death (PCD), but only 30% of cells underwent cell death and, at the end of the experiment, only 1–2% of cells were TUNEL-positive. Rapid recovery in culture growth after exposure to light showed that the changes apparent in the rest of cells were reversible. Taking into account the response of the plastid and assuming an adaptive benefit of PCD, our results are consistent with the hypothesis that K. antarctica evolved from an Antarctic freshwater ancestor.

Degreening of the unicellular alga Euglena gracilis: thylakoid composition, room temperature fluorescence spectra and chloroplast morphology

Thylakoid dismantling is one of the most relevant processes occurring when chloroplasts are converted to non-photosynthetically active plastids. The process is well characterised in senescing leaves, but other systems could present different features. In this study, thylakoid dismantling has been analysed in dividing cells of the unicellular alga, Euglena gracilis, cultured in darkness. Changes in photosynthetic pigments and in the abundance of LHC and PSII core proteins (D2 and CP43) showed that: (i) during the 0-24 h interval, the decline in LHCII was faster than that in the PSII core; (ii) during the 24-48 h interval, PSII and LHCII were strongly degraded to nearly the same extent; (iii) in the 48-72 h interval, the PSII core proteins declined markedly, while LHCII was maintained. These changes were accompanied by variations in room temperature fluorescence emission spectra recorded from single living cells with a microspectrofluorimeter (excitation, 436 nm; range 620-780 nm). Emission in the 700-715 nm range was proposed to derive from LHCI-II assemblages; changes in emission at 678 nm relative to PSII matched PSII core degradation phases. Overall, the results suggest that, in degreening E. gracilis, thylakoid dismantling is somewhat different from that associated with senescence, because of the early loss of LHCII. Moreover, it is proposed that, in this alga, disruption of the correct LHCI-II stoichiometry alters the energy transfer to photosystems and destabilises membrane appression leading to the thylakoid destacking observed using transmission electron microscopy.

Growth, morphology and photosynthetic responses of Neochloris oleoabundans during cultivation in a mixotrophic brackish medium and subsequent starvation

The green microalga Neochloris oleoabundans is able to grow in both low and high salinity media and is largely studied for its capability to accumulate lipids under starvation. Moreover, N. oleoabundans is a mixotrophic alga, and then organic carbon addition can promote its growth. This research aims to study the morpho-physiological aspects, with a particular attention on the photosynthetic response, both during mixotrophic growth and starvation in brackish media, more sustainable than freshwater cultivation. In the first step, the alga was cultivated mixotrophically in a brackish medium added with an apple waste product; in the second one, cells were starved also to verify lipid induction. Results indicate that growth is highly promoted during the first week of mixotrophic cultivation, while photosynthetic pigments and lipids are over-produced during the following three weeks of cultivation. In parallel, in mixotrophic cultures the maximum PSII quantum yield was enhanced during the exponential phase of growth. Interesting changes affected the mixotrophic cultures with respect to the partitioning of absorbed light energy. Starvation of both 7-day-grown mixotrophic and autotrophic cultures caused growth inhibition, pigments and photosynthesis downshifting, and concomitantly promoted evident lipid synthesis.

Salinity promotes growth of freshwater Neochloris oleoabundans UTEX 1185 (Sphaeropleales, Chlorophyta): morphophysiological aspects

Baldisserotto C., Ferroni L., Giovanardi M., Boccaletti L., Pantaleoni L. and Pancaldi S. 2012. Salinity promotes growth of freshwater Neochloris oleoabundans UTEX 1185 (Sphaeropleales, Chlorophyta): morphophysiological aspects. Phycologia 51: 700–710. DOI: 10.2216/11-099.1 Neochloris oleoabundans was described as a freshwater unicellular green microalga; however, some literature suggested that it was an edaphic and halotolerant alga. Neochloris oleoabundans was studied so far for its high lipid content, especially under nitrogen starvation, for possible industrial applications. Information on the morphophysiological characteristics of the alga and its photosynthetic apparatus in different culture conditions still remained incomplete. In the present work, its growth was compared using low-salinity and brackish media with increasing nitrogen supply. The morphophysiological aspects, with a special attention on its photosynthetic apparatus, were analysed through light and transmission electron microscopy, photosynthetic pigment quantification, PSII maximum quantum yield measurements and evaluations of the chlorophyll-protein assembly state. In contrast to what has been reported in previous work on the positive effect of nitrate on N. oleobundans growth, we found negative or negligible effects in our samples cultivated in low-salinity or brackish media, respectively. Brackish conditions induced a better growth of the alga, which showed some morphological variations (cell volume enlargement, cell wall thickening, increased stromatic starch and polyphosphate grains). Furthermore, brackish cultured algae were characterized by a strong increase in cellular chlorophylls and carotenoids. Fluorimetric analyses pointed to the absence of disturbance to the photosynthetic apparatus and to a higher photosynthetic efficiency in brackish cultured samples with respect to controls in the low-salinity medium, indicating a somehow better photosynthetic performance. Interestingly, the behaviour of the F680/F685+694 ratio pointed to a possible positive correlation between nitrogen supply and PSII core stability. On the whole, morphological, biochemical and biophysical results confirmed the higher acclimatized growth of N. oleoabundans in brackish media, which seem more suitable for algal growth than low-salinity ones.

Photosystem II organisation in chloroplasts of Arum italicum leaf depends on tissue location

The growth of plants under stable light quality induces long-term acclimation responses of the photosynthetic apparatus. Light can even cause variations depending on the tissue location, as in Arum italicum leaf, where chloroplasts are developed in the lamina and in the entire thickness of the petiole. We addressed the question whether differences in plastids can be characterised in terms of protein–protein interactions in the thylakoid membranes. Thylakoid assembly was studied in the palisade and spongy tissue of the lamina and in the outer parenchyma and inner aerenchyma of the petiole of the mature winter leaf of Arum italicum. The chlorophyll–protein complexes were analysed by means of blue-native-PAGE and fluorescence emission spectra. The petiole chloroplasts differ from those in the lamina in thylakoid composition: (1) reaction centres are scarce, especially photosystem (PS) I in the inner aerenchyma; (2) light-harvesting complex (LHC) II is abundant, (3) the relative amount of LHCII trimers increases, but this is not accompanied by increased levels of PSII–LHCII supercomplexes. Nevertheless, the intrinsic PSII functionality is comparable in all tissues. In Arum italicum leaf, the gradient in thylakoid organisation, which occurs from the palisade tissue to the inner aerenchyma of the petiole, is typical for photosynthetic acclimation to low-light intensity with a high enrichment of far-red light. The results obtained demonstrate a high plasticity of chloroplasts even in an individual plant. The mutual interaction of thylakoid protein complexes is discussed in relation to the photosynthetic efficiency of the leaf parts and to the ecodevelopmental role of light.