Chlorophyll Autofluorescence Research Articles

Mechanism of up-regulated H2O2 BPA-derived production and production of (poly)phenols by two seaweeds of the genus Ulva.

The present study provides information on the effects of BPA on ROS production-related phenomena in the chlorophytes Ulva rigida and U. intestinalis, and on the mechanism they establish against BPA toxicity, at environmentally relevant concentrations (0.1-3 μg L-1). Up-regulated H2O2 generation seems to be a key factor causing oxidative damage. Interspecific differences, in terms of the mechanism and the temporal response to BPA toxicity were observed. BPA effects on U. rigida were more intense and appeared earlier (on 1D at 0.1 μg L-1) compared to U. intestinalis and mostly after 7D (LOEC: 0.3 μg L-1, Terminal time, Tt: 7D). In U. rigida, on 1-5D, the 'mosaic' type effect patterns ('models' 3A/3B) with 'unaffected' and 'affected' areas (dark content, positive H2DCF-DA staining signal/H2O2 production and chlorophyll autofluorescence signal loss) indicated a time-dependent manner. After 7D, only U. rigida cells with dark content formed aggregates, showing positive H2O2 production ('model' 4) or in some cells oxidative damages triggering retrograde signaling in the neighboring 'unaffected' areas ('model' 5). H2O2 overproduction (CTCF ratio) in U. rigida, on 1D at the lowest concentration and after 7D at 0.3-1/3 μg L-1, respectively, seems to stimulate (poly)phenolic production, in a dose- and time-dependent manner. U. intestinalis did not display severe BPA impact (i.e., 'models' 4, 5) at any exposures, although at a later time indicated a lower LOEC (0.1 μg L-1, Tt: 9D) than that in U. rigida. In U. intestinalis, H2O2 production does not appear to stimulate high (poly)phenolic amounts.

Fluorescence Microscopy with Deep UV, Near UV, and Visible Excitation for In Situ Detection of Microorganisms.

We report a simple, inexpensive design of a fluorescence microscope with light-emitting diode (LED) excitation for detection of labeled and unlabeled microorganisms in mineral substrates. The use of deep UV (DUV) excitation with visible emission requires no specialized optics or slides and can be implemented easily and inexpensively using an oblique illumination geometry. DUV excitation (<280 nm) is preferable to near UV (365 nm) for avoidance of mineral autofluorescence. When excited with DUV, unpigmented bacteria show two emission peaks: one in the near UV ∼320 nm, corresponding to proteins, and another peak in the blue to green range, corresponding to flavins and/or reduced nicotinamide adenine dinucleotide (NADH). Many commonly used dyes also show secondary excitation peaks in the DUV, with identical emission spectra and quantum yields as their primary peak. However, DUV fails to excite key biosignature molecules, especially chlorophyll in cyanobacteria. Visible excitation (violet to blue) also results in less mineral autofluorescence than near UV, and most autofluorescence in the minerals seen here is green, so that red dyes and red autofluorescence of chlorophyll and porphyrins are readily distinguished. The pairing of DUV and near UV or visible excitation, with emission across the visible, represents the most thorough approach to detection of labeled and unlabeled bacteria in soil and rock.

Application of confocal microscopy and flow cytometry to identify physiological responses of Prorocentrum micans to the herbicide glyphosate

The physiological response of the dinoflagellate P. micans to the effect of the herbicide glyphosate at a concentration of 25–200 μg L−1 was evaluated. It has been shown that P. micans is able to grow due to the consumption of dissolved organic phosphorus formed as a result of the mineralization of glyphosate by bacteria. The addition of glyphosate to the medium inhibits the photosynthetic activity of cells; there is a pronounced inhibition of the relative electron transfer rate along the electron transport chain and the maximum quantum efficiency of the use of light energy. Morphological and ultrastructural changes in P. micans cells were evaluated at sublethal (150 μg L−1) and lethal (200 μg L−1) glyphosate concentrations. It has been shown that at a herbicide concentration of 150 μg L−1, the first signs of apoptosis appear in most P. micans cells: a decrease in lateral light scattering, cytoplasmic retraction, partial destruction of cytoplasmic organelles, a change in the morphology of nuclei, mitochondria, a change in the potential of mitochondrial membranes, and a decrease in the autofluorescence of chlorophyll in cells. At a glyphosate concentration of 200 μg L−1, P. micans showed signs of a late stage of apoptosis: violation of the integrity of intracellular organelles and chromatin organization, fragmentation of nuclei, condensation of cytoplasm, disorganization of chloroplasts in the cells, and the release of cell contents beyond the cell membrane. The effectiveness of using flow cytometry and laser scanning confocal microscopy methods for identifying signs and stages of cell apoptosis when exposed to glyphosate is discussed.

Rhizospheric nano-remediation salvages arsenic genotoxicity: Zinc-oxide nanoparticles articulate better oxidative stress management, reduce arsenic uptake, and increase yield in Pisum sativum (L.)

Pea (Pisum sativum L.), a legume, has a high nutritional content, but arsenic (As) in the agro-ecosystem poses a significant bottleneck to its yield, especially in South East Asia, by severely hampering ontogeny. The present study proposes a rhizospheric nano-remediation strategy to evade As-genotoxicity and improve crop yield using biogenic zinc-oxide nanoparticles (ZnONPs). Similar to any other source of environmental stress, As-toxicity caused rapid oxidative bursts with deterioration in morpho-physiological attributes (germination rate, shoot length, and root length decreased by 62 %, 16 %, and 14.9 % respectively in the negative control, over normal control). Reactive oxygen species (ROS) accumulation (12.8 and 9-fold increase in leaves and roots) overburdened antioxidative defense, and loss of cellular homeostasis resulted in membrane damage (82.75 % increase) and electrolyte-leakage (2.6-fold increase) in negative control. The study also reveals a significant increase in nuclear area, nuclear fragmentation, and micronuclei formation in root tip cells under As-stress, indicating severe genomic instability and increased programmed cell death (3.3-fold increase in early apoptotic cells) due to leaky plasma membrane and unrepaired DNA damage. Application of ZnONPs significantly reduced As-toxicity in peas due to its adsorption in the rhizosphere, causing diminished As-uptake and better antioxidant response. Improved phytochelatin synthesis enhanced vacuolar sequestration of arsenic, which reduced As-interference. Comparatively better flowering time (7.74–19.36 % reduction in flowering delay) with greater transcript abundance of GIGANTIA (GI), CONSTANS (CO), and FLOWERING LOCUS T (FT) genes; better photosynthetic activity (1.3–1.9-fold increased chlorophyll autofluorescence); increased pollen viability; lesser genotoxicity (decreased tail DNA in comet assay) was noticed. A maximum increase of 37.5 % in pod number and seed zinc content (1.67-fold) was observed while seed arsenic content decreased under ZnONPs treatment. However, the highest dose of ZnONPs (400 mg L−1) induced NP-toxicity in pea plants under our experimental conditions, while optimum stress-alleviation was observed up to 300 mg L−1.

Quantitative Viability Assessment of Microalgae for Advanced Wastewater Treatment by Flow Cytometry

The nutrient uptake capacity of microalgae can be applied in advanced wastewater treatment processes. This study was set to develop a rapid and effective method for assessing the viability (i.e., live or dead) of microalgae applied for advanced wastewater treatment using a flow cytometry. Especially, a methodology to selectively evaluate the quantitative viability of only microalgae present in wastewaters having the co-culture of microalgae and bacteria was proposed. The live and dead cells of microalgae could be discriminated using chlorophyll autofluorescence and the SYTOX Green method developed in this study. Cell viability in live and dead conditions of microalgae showed about 90% accuracy, and about 40% viability ratio was derived in half-live and half-dead conditions. This result shows the potential to quantitatively evaluate the activity of microalgae in the wastewater environment.

Single and multispecies microalgae toxicological tests assessing the impact of several BPA analogues used by industry

BPA is a hazard for human and environmental health and recently BPA was added to the Candidate List of substances of very high concern by European Chemical Agency (ECHA). In accordance with this proposal, the authorities have encouraged the replacement of BPA by BPA analogues; however, little is known about the impact of these compounds on the environment. Due to this situation five BPA analogues (BPS, BPAP, BPAF, BPFL and BPC) were chosen in order to study their effects on marine primary producers. Three marine microalgae species (Phaeodactylum tricornutum, Tetraselmis suecica and Nannochloropsis gaditana) were selected for single and multispecies tests concerning the ecotoxicological effects of these BPA analogues. Microalgae were exposed to BPs over 72 h at different dosages (5, 20, 40, 80, 150 and 300 μM). Responses such as: growth, ROS production, cell complexity, cell size, autofluorescence of chlorophyll a, effective quantum yield of PSII and pigment concentrations were assessed at 24, 48 and 72 h. The results revealed that BPS and BPA showed lower toxicity to microalgae in comparison with BPFL > BPAF > BPAP and >BPC for the endpoints studied. N. gaditana was the least sensitive microalgae in comparison to P. tricornutum and T. suecica. However, a different trend was found in the multispecies tests where T. suecica dominated the microalgae community in relation to N. gaditana and P. tricornutum. The results of this work revealed for first time that present day BPA analogues are a threat and not a safe substitute for BPA in terms of the marine phytoplanktonic community. Therefore, the results of their impact on aquatic organisms should be shared.

Physiological responses of plants to in vivo X-ray damage from X-ray fluorescence measurements: insights from anatomical, elemental, histochemical, and ultrastructural analyses.

X-ray fluorescence spectroscopy (XRF) is a powerful technique for the in vivo assessment of plant tissues. However, the potential X-ray exposure damages might affect the structure and elemental composition of living plant tissues, leading to artefacts in the recorded data. Herein, we exposed in vivo soybean (Glycine max (L.) Merrill) leaves to several X-ray doses through a polychromatic benchtop microprobe X-ray fluorescence spectrometer, modulating the photon flux density by adjusting either the beam size, current, or exposure time. Changes in the irradiated plant tissues' structure, ultrastructure, and physiology were investigated through light and transmission electron microscopy (TEM). Depending on X-ray exposure dose, decreased K and X-ray scattering intensities and increased Ca, P, and Mn signals on soybean leaves were recorded. Anatomical analysis indicated the necrosis of epidermal and mesophyll cells on the irradiated spots, where TEM images revealed the collapse of cytoplasm and cell wall breaking. Furthermore, the histochemical analysis detected the production of reactive oxygen species and the inhibition of chlorophyll autofluorescence in these areas. Under certain X-ray exposure conditions, e.g. high photon flux density and long exposure time, XRF measurements may affect the soybean leaves structures, elemental composition, and cellular ultrastructure, inducing programmed cell death. Our characterization shed light on the plant's responses to the X-ray-induced radiation damage and might help to establish proper X-ray radiation limits and novel strategies for in vivo benchtop-XRF analysis of vegetal materials.

Different responses of Chlorella vulgaris to silver nanoparticles and silver ions under modulation of nitric oxide.

Silver nanoparticles (Ag-NPs) are widely used in daily life because of their antibacterial properties. A fraction of Ag-NPs are released into the ecosystem during their production and utilization. The toxicity of Ag-NPs has been reported. However, it is still disputed whether the toxicity is mainly due to the released silver ions (Ag+). In addition, few studies have reported the response of algae to metal nanoparticles under modulation of nitric oxide (NO). In this study, Chlorella vulgaris (C. vulgaris) was used as a model organism to study the toxic effects of Ag-NPs and Ag+ released from Ag-NPs on algae under the modulation of NO. The results showed that the biomass inhibition rate of Ag-NPs (44.84%) to C. vulgaris was higher than that of Ag+ (7.84%). Compared with Ag+, Ag-NPs induced more severe damage to photosynthetic pigments, photosynthetic system II (PSII) performance, and lipid peroxidation. More serious damage to cell permeability led to higher internalization of Ag under Ag-NPs stress. Application of exogenous NO reduced the inhibition ratio of photosynthetic pigments and chlorophyll autofluorescence. Further, NO reduced the MDA levels by scavenging reactive oxygen species induced by Ag-NPs. NO modulated the secretion of extracellular polymers and hampered the internalization of Ag. All these results showed that NO alleviates the toxicity of Ag-NPs to C. vulgaris. However, NO did not improve the toxic effects of Ag+. Our results provide new insights into the toxicity mechanism of Ag-NPs to algae modulated by the signal molecule NO.

Improvements for Tissue-Chopping-Based Immunofluorescence Staining Method of Chloroplast Proteins

Immunofluorescence staining is a very common method for the subcellular localization study of proteins. A tissue-chopping-based immunofluorescence staining method for chloroplast proteins overcomes the restriction of plant cell wall, makes the operation simpler, and uses less experimental materials. Here we provide some improvements for this method. We found that the stained tissues can be directly observed with a confocal microscope without tissue lysis. Samples maintained at a low temperature (0-4 °C) throughout the process can reduce the intensity of chlorophyll autofluorescence and the background signal. A low temperature is also good for the storage of the sample. Fluorescence signal of the stained samples can be kept for several weeks if they are stored at -20 °C. FtsZ is an essential component of the chloroplast division apparatus. We demonstrated this method with the immunofluorescence staining of FtsZ1 in wildtype Arabidopsis and some chloroplast division mutants. We also successfully tested this method by the immunofluorescence staining of FtsZ1 in many other plants, including woody plants. With these procedures, the performance of tissue-chopping-based immunofluorescence staining method are further improved.

Mercury Content and Amelioration of Its Toxicity by Nitric Oxide in Lichens.

Mercury (Hg) content measured in five epiphytic lichen species collected in Slovakia mountain forests ranged from 30 to 100 ng/g DW and was species-specific, decreasing in the order Hypogymnia > Pseudevernia > Usnea > Xanthoria > Evernia prunastri (but polluted sites had no impact on Hg amount in Xanthoria). Evernia was therefore used to study the impact of short-term exogenous Hg (100 µM, 24 h) and possible amelioration of Hg toxicity by nitric oxide (NO) donor sodium nitroprusside (SNP). NO was efficiently released from SNP as detected by two staining reagents and fluorescence microscopy and reduced Hg-induced ROS signal and absorption of Hg by thalli of Evernia prunastri. At the same time, NO ameliorated Hg-induced depletion of metabolites such as ascorbic acid and non-protein thiols, but not of free amino acids. The amount of metabolites, including soluble phenols, was reduced by excess Hg per se. On the contrary, NO was unable to restore Hg-stimulated depletion of chlorophyll autofluorescence but mitigated the decline of some macronutrients (K and Ca). Data confirm that accumulation of Hg in the epiphytic lichens is species-specific and that NO is a vital molecule in Evernia prunastri that provides protection against Hg-induced toxicity with considerable positive impact on metabolic changes.

The Effect of LED and HPS Assimilation Lighting on Leaf Anatomy, Chlorophyll and Carotenoid Autofluorescence Signals, and Some Physiological and Chemical Leaf Traits Related to the Productivity of Cucumber (Cucumis sativus L.) in High-Wire Cultivation

Supplemental lighting with light-emitting diode (LED) lamps and/or high-pressure sodium (HPS) lamps was applied to increase the activity of the photosynthetic apparatus and thus productivity of greenhouse cucumber (Cucumis sativus L.) in a high-wire growing system. The colocalisation of the chlorophyll of PSII (located mainly in grana) and carotenoid fluorescence signals in chloroplasts of cucumber leaves was studied under confocal microscopy. Leaf anatomy and some chemical quality traits (dry matter, chlorophyll, carotenoids, total soluble solids, total sugars and nitrate reductase activity) as well as selected chlorophyll fluorescence parameters were also investigated and subjected to the multidimensional principal component analysis together with the data on fruit yield. Under LED lighting, a lower correlation between the occurrence of chlorophyll and carotenoid fluorescence signals was observed, especially in older (lower-located) leaves, which may have resulted from changes in the distribution of carotenoids within chloroplasts and/or relative concentrations of chlorophyll and carotenoids. Compared to toplighting with HPS lamps, most commonly used in commercial greenhouse cucumber production, the application of LED interlighting, especially in combination with LED toplighting, led to the increase in chlorophyll and carotenoid content and photosynthetic performance index in older leaves, which was related to the increased cucumber productivity.

Estimating Biomass and Vitality of Microalgae for Monitoring Cultures: A Roadmap for Reliable Measurements.

Estimating algal biomass is a prerequisite for monitoring growth of microalgae. Especially for large-scale production sites, the measurements must be robust, reliable, fast and easy to obtain. We compare the relevant parameters, discuss potential hurdles and provide recommendations to tackle these issues. The focus is on optical density and in vivo autofluorescence of chlorophyll, which have proven to be ideal candidates for monitoring purposes. Beyond biomass, cell vitality is also crucial for maintaining cultures. While maximizing biomass yield is often the primary consideration, some applications require adverse growth conditions for the synthesis of high-quality compounds. The non-invasive technique of pulse-amplified modulated (PAM) fluorescence measurements provides an ideal tool and is increasingly being employed due to ever more affordable devices. We compared three devices and studied the robustness of the dark fluorescence yield of photosystem II (Fv/Fm) at various cell densities. Although the so-called inner filter effects influence the fluorescence signal, the resulting Fv/Fm remain stable and robust over a wide range of cell densities due to mutual effects.

Single Cell Plant Model of Equisetum arvense for the Study Antihistamine Effects of Azulene and Sesquiterpene Lactones

Single cell of vegetative microspore from spore-bearing plant field Equisetum arvense L. has been presented as a single-cell experimental model for the screening of native compounds acting as antihistamine agents. The effects of azulene, sesquiterpene lactones austricine, gaillardine, grosshemine, inulicine, and desacetylinulicine as well as sesquiterpene alcohol ledol, on the content of histamine in germinating horsetail microspores has been investigated by the fluorescent method. It has been shown using microspectrofluorimetry that these compounds are able to regulate the germination of microspores to varying degrees, as assessed by the autofluorescence of chlorophyll, in a medium without and in the presence of 0.5–1% sodium sulfate as a salt stress factor. A fluorescent histochemical reaction to histamine with ortho-phthalic aldehyde in cells and secretory mucilage revealed the ability of the compounds studied to reduce the level of this biogenic amine depending on their structure in the following order: grosshemine &gt; azulene &gt; austricine &gt; ledol. Gaillardine, inulicine, and desacetylinulicine showed weak antihistamine activity

Cover Picture: ChemBioEng Reviews 2/2022

ChemBioEng ReviewsVolume 9, Issue 2 p. 125-125 Cover PictureFree Access Cover Picture: ChemBioEng Reviews 2/2022 First published: 11 April 2022 https://doi.org/10.1002/cben.202270201AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Graphical Abstract Methods such as confocal laser scanning microscopy (CLSM) and micro computed tomography (µCT) allow us to get deeper insights into growth forms of various organisms and interactions with different materials – or, in summary, structures of life itself. The cover picture of this volume presents a multi-faceted combination of structures and organisms to highlight the possibilities of CLSM and µCT. Upper left: growth of the cyanobacterium Desmonostoc muscorum on loofah (CLSM recording of autofluorescence of chlorophyll a (red) and lignin (green)); upper right: brewers' spent grain (µCT recording); lower left: callus cells of Ocimum sanctum Rama (CLSM recording of cells stained with calcofluor white, fluorescein diacetate and propidium iodide); lower right: growth of the cyanobacterium Nostoc sp. on a carrier (Type HXF14KLL, Hel-X; Stöhr GmbH & Co.KG, Markrodach, Germany; µCT recording). These images not only show the beauty of life on a microscopic scale, they also allow the analysis of interactions between agglomerating cells and cells adhering to surfaces. Information about the structure of materials such as loofah, brewers' spent grain or the carrier provide indication of their suitability for specific applications. In summary, a small insight into the possibilities of the presented methods is given here, which outlines a large field of applications. Copyright: Judith Stiefelmaier and Susanne Schaefer, Dorina Strieth, Jens Weiermüller, Jakob Walther, Jonas Chodorski, Ulrich Bröckel, Roland Ulber Volume9, Issue2April 2022Pages 125-125 RelatedInformation

Ecotoxicological responses and removal of submerged macrophyte Hydrilla verticillate to multiple perfluoroalkyl acid (PFAA) pollutants in aquatic environments

The ubiquitous existence of perfluoroalkyl acids (PFAAs) in aquatic environments might pose toxic potential to ecosystems. To assess the ecotoxicological responses and removal of submerged macrophyte to multiple PFAA pollutants in aquatic environments, a typical submerged macrophyte, Hydrilla verticillate, was exposed to solutions with 12 typical PFAAs in the present study. The results showed that PFAAs at concentrations higher than 10 μg/L had significantly passive effects on biomass, relative growth rates, chlorophyll contents, and chlorophyll autofluorescence. PFAAs could induce the accumulation of hydrogen peroxide and lipid peroxidation in H. verticillate. Significant upregulation of CAT was observed in treatments with more than 10 μg/L PFAAs (p < 0.05). The results also showed that 13.53–20.01% and 19.73–37.72% of PFAAs could be removed in treatments without plants and with H. verticillate, respectively. The removal rates of PFAAs were significantly correlated with perfluoroalkyl chain length in treatments with H. verticillate. The removal of PFAAs was suggested to be related to the uptake of plant tissues and biosorption of microbiota. Furthermore, the dominant microbiota and biomarkers were identified in water and biofilm. Betaproteobacteriales was the most dominant microbiota at the order level. The presence of PFAAs could significantly increase the relative abundance of Micrococcales, Verrucomicrobiales, Rhizobiales, Sphingomonadales, Roseomonas, Cyanobium_PCC_6307, and Synechococcales. Our results provide scientific basis for evaluating the ecotoxicological responses and removal of submerged macrophytes in response to multiple PFAA pollutants at environmentally relevant levels, thereby providing insights into PFAA management and removal.

Live Cell Imaging of ATP Dynamics in Plant Cells.

Adenosine triphosphate (ATP) is a central metabolite that functions as the energy currency in a living cell. Therefore, visualizing cellular ATP dynamics provides the fundamental information necessary to understand the molecular events involving life phenomena. Live cell imaging technologies using fluorescence (FL)-based indicators have been developed to analyze the dynamics of various biological processes, such as intracellular ATP synthesis and consumption. However, the application of FL-based indicators to plant cells is limited due to the presence of strong chlorophyll autofluorescence, which drastically worsen the signal-to-noise ratio. The bioluminescent (BL) indicators that do not require excitation light could overcome this problem. In this chapter, we introduce a methodology to analyze ATP dynamics in plant cells using BL ATP indicators.

A microfluidic system for viability determination of microalgae upon disinfectant treatment under continuous flow

The extensive use of quaternary ammonium compounds (QACs) has raised concerns regarding their environmental fate and potential risks to the ecosystem. As sensitive pollution indicators, green microalgae could readily monitor the aquatic toxicity of QACs as reflective of the changes in cell viability. Recent microfluidic-based systems have been designed for environmental biomonitoring and ecotoxicity studies while overall information of cell viability cannot be directly visualized under flowing conditions. In the present study, we developed a multifunctional microfluidic platform with the integration of analytical techniques including laser speckle contrast imaging and fluorescence spectroscopy for monitoring algal activity in response to QAC treatment. The biocidal efficiency of a representative QAC benzalkonium bromide (BAB) on a typical aquatic algae Chlorella vulgaris was determined by collecting the bio-speckles and chlorophyll autofluorescence in real-time, where dose-dependent and time-dependent decrease of algal growth was found with the increase of BAB concentration and interaction time. The integrated system was capable of rapid detection of the aquatic toxicity of QACs along with macroscopical visualization of algal activities under flowing conditions in time-course, which could be extended to future implementation for broad ecotoxicity analysis of versatile environmental samples.

Phenotypic changes in microalgae at acidic pH mediate their tolerance to higher concentrations of transition metals.

Acclimatory phenotypic response is a common phenomenon in microalgae, particularly during heavy metal stress. It is not clear so far whether acclimating to one abiotic stressor can alleviate the stress imposed by another abiotic factor. The intent of the present study was to demonstrate the implication of acidic pH in effecting phenotypic changes that facilitate microalgal tolerance to biologically excess concentrations of heavy metals. Two microalgal strains, Desmodesmus sp. MAS1 and Heterochlorella sp. MAS3, were exposed to biologically excess concentrations of Cu (0.50 and 1.0mg L‒1), Fe (5 and 10mg L‒1), Mn (5 and 10mg L‒1) and Zn (2, 5 and 10mg L‒1) supplemented to the culture medium at pH 3.5 and 6.7. Chlorophyll autofluorescence and biochemical fingerprinting using FTIR-spectroscopy were used to assess the microalgal strains for phenotypic changes that mediate tolerance to metals. Both the strains responded to acidic pH by effecting differential changes in biochemicals such as carbohydrates, proteins, and lipids. Both the microalgal strains, when acclimated to low pH of 3.5, exhibited an increase in protein (<2-fold) and lipid (>1.5-fold). Strain MAS1 grown at pH 3.5 showed a reduction (1.5-fold) in carbohydrates while strain MAS3 exhibited a 17-fold increase in carbohydrates as compared to their growth at pH 6.7. However, lower levels of biologically excess concentrations of the selected transition metals at pH 6.7 unveiled positive or no effect on physiology and biochemistry in microalgal strains, whereas growth with higher metal concentrations at this pH resulted in decreased chlorophyll content. Although the bioavailability of free-metal ions is higher at pH 3.5, as revealed by Visual MINTEQ model, no adverse effect was observed on chlorophyll content in cells grown at pH 3.5 than at pH 6.7. Furthermore, increasing concentrations of Fe, Mn and Zn significantly upregulated the carbohydrate metabolism, but not protein and lipid synthesis, in both strains at pH 3.5 as compared to their growth at pH 6.7. Overall, the impact of pH 3.5 on growth response suggested that acclimation of microalgal strains to acidic pH alleviates metal toxicity by triggering physiological and biochemical changes in microalgae for their survival.

Dynamic macromolecular composition and high exudation rates in Prochlorococcus

AbstractEvery living cell is composed of macromolecules such as proteins, DNA, RNA, and pigments. The ratio between these macromolecular pools depends on the allocation of resources within the organism to different physiological requirements, and in turn affects biogeochemical cycles of elements such as carbon, nitrogen, and phosphorus. Here, we present detailed measurements of the macromolecular composition of Prochlorococcus MIT9312, a representative strain of a globally abundant marine primary producer, as it grows and declines due to nitrogen starvation in laboratory batch cultures. As cells reached stationary stage and declined, protein per cell decreased by ~ 30% whereas RNA per cell and pigments per cell decreased by ~ 75%. The decline stage was associated with the appearance of chlorotic cells, which had higher forward scatter (a proxy for cell size) but lower chlorophyll autofluorescence, as well as with changes in photosynthetic pigment composition. Specifically, during culture decline divinyl‐chlorophyll‐like pigments emerged, which were not observed during exponential growth. These divinyl‐chlorophyll‐like pigments were also observed in natural samples from the Eastern Mediterranean. Over 90% of the carbon fixed by Prochlorococcus MIT9312 (but not of a different strain, NATL2A) was released into the growth media under these laboratory conditions. Variations in RNA/protein indicate that, broadly defined, the macromolecular composition of Prochlorococcus MIT9312 is more similar to eukaryotic phytoplankton than to marine heterotrophic bacteria, possibly due to the significant investment in photosynthetic machinery of phototrophs.

Automated Online Flow Cytometry Advances Microalgal Ecosystem Management as in situ, High-Temporal Resolution Monitoring Tool.

Microalgae are emerging as a next-generation biotechnological production system in the pharmaceutical, biofuel, and food domain. The economization of microalgal biorefineries remains a main target, where culture contamination and prokaryotic upsurge are main bottlenecks to impair culture stability, reproducibility, and consequently productivity. Automated online flow cytometry (FCM) is gaining momentum as bioprocess optimization tool, as it allows for spatial and temporal landscaping, real-time investigations of rapid microbial processes, and the assessment of intrinsic cell features. So far, automated online FCM has not been applied to microalgal ecosystems but poses a powerful technology for improving the feasibility of microalgal feedstock production through in situ, real-time, high-temporal resolution monitoring. The study lays the foundations for an application of automated online FCM implying far-reaching applications to impel and facilitate the implementation of innovations targeting at microalgal bioprocesses optimization. It shows that emissions collected on the FL1/FL3 fluorescent channels, harnessing nucleic acid staining and chlorophyll autofluorescence, enable a simultaneous assessment (quantitative and diversity-related) of prokaryotes and industrially relevant phototrophic Chlorella vulgaris in mixed ecosystems of different complexity over a broad concentration range (2.2–1,002.4 cells ⋅μL–1). Automated online FCM combined with data analysis relying on phenotypic fingerprinting poses a powerful tool for quantitative and diversity-related population dynamics monitoring. Quantitative data assessment showed that prokaryotic growth phases in engineered and natural ecosystems were characterized by different growth speeds and distinct peaks. Diversity-related population monitoring based on phenotypic fingerprinting indicated that prokaryotic upsurge in mixed cultures was governed by the dominance of single prokaryotic species. Automated online FCM is a powerful tool for microalgal bioprocess optimization owing to its adaptability to myriad phenotypic assays and its compatibility with various cultivation systems. This allows advancing bioprocesses associated with both microalgal biomass and compound production. Hence, automated online FCM poses a viable tool with applications across multiple domains within the biobased sector relying on single cell–based value chains.