This study introduces an evaluation methodology tailored for bioreactors, with the aim of assessing the stress experienced by algae due to harmful contaminants released from antifouling (AF) paints. We present an online monitoring system equipped with an ultra-sensitive sensor that conducts non-invasive measurements of algal culture's optical density and physiological stage through chlorophyll fluorescence signals. By coupling the ultra-sensitive sensor with flash-induced chlorophyll fluorescence, we examined the dynamic fluorescence changes in the green microalga Chlamydomonas reinhardtii when exposed to biocides. Over a 24-h observation period, increasing concentrations of biocides led to a decrease in photosynthetic activity. Notably, a substantial reduction in the maximum quantum yield of primary photochemistry (FV/FM) was observed within the first hour of exposure. Subsequently, we detected a partial recovery in FV/FM; however, this recovery remained 50% lower than that of the controls. Integrating the advanced submersible sensor with fluorescence decay kinetics offered a comprehensive perspective on the dynamic alterations in algal cells under the exposure to biocides released from antifouling coatings. The analysis of fluorescence relaxation kinetics revealed a significant shortening of the fast and middle phases, along with an increase in the duration of the slow phase, for the coating with the highest levels of biocides. Combining automated culturing and measuring methods, this approach has demonstrated its effectiveness as an ultrasensitive and non-invasive tool for monitoring the physiology of photosynthetic cultures. This is particularly valuable in the context of studying microalgae and their early responses to various environmental conditions, as well as the potential to develop an AF system with minimal harm to the environment.

• Klíčová slova
• Antifouling coatings, Bioreactor, Chlorophyll fluorescence spectroscopy, Microalgae, Toxicity, Ultrasensitive sensor,
• MeSH
• bioreaktory * MeSH
• chemické látky znečišťující vodu analýza   MeSH
• Chlamydomonas reinhardtii * účinky léků   metabolismus   MeSH
• chlorofyl metabolismus   MeSH
• dezinficiencia farmakologie   MeSH
• fluorescence MeSH
• fotosyntéza účinky léků   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• chemické látky znečišťující vodu MeSH
• chlorofyl MeSH
• dezinficiencia MeSH

INTRODUCTION: Polyunsaturated fatty acids (PUFAs) are essential nutrients that humans obtain from their diet, primarily through fish oil consumption. However, fish oil production is no longer sustainable. An alternative approach is to produce PUFAs through marine microalgae. Despite the potential of algae strains to accumulate high concentrations of PUFAs, including essential fatty acids (EFAs), many aspects of PUFA production by microalgae remain unexplored and their current production outputs are frequently suboptimal. METHODS: In this study, we optimized biomass and selected ω-3 PUFAs production in two strains of algae, Schizochytrium marinum AN-4 and Schizochytrium limacinum CO3H. We examined a broad range of cultivation conditions, including pH, temperature, stirring intensity, nutrient concentrations, and their combinations. RESULTS: We found that both strains grew well at low pH levels (4.5), which could reduce bacterial contamination and facilitate the use of industrial waste products as substrate supplements. Intensive stirring was necessary for rapid biomass accumulation but caused cell disruption during lipid accumulation. Docosahexaenoic acid (DHA) yield was independent of cultivation temperature within a range of 28-34°C. We also achieved high cell densities (up to 9 g/L) and stable DHA production (average around 0.1 g/L/d) under diverse conditions and nutrient concentrations, with minimal nutrients required for stable production including standard sea salt, glucose or glycerol, and yeast extract. DISCUSSION: Our findings demonstrate the potential of Schizochytrium strains to boost industrial-scale PUFA production and make it more economically viable. Additionally, these results may pave the way for smaller-scale production of essential fatty acids in a domestic setting. The development of a new minimal culturing medium with reduced ionic strength and antibacterial pH could further enhance the feasibility of this approach.

• Klíčová slova
• bioreactors, docosahexaenoic acid, growth, health supplements, optimization, process automation, unicellular eukaryote,
• Publikační typ
• časopisecké články MeSH

Crocosphaera and Cyanothece are both unicellular, nitrogen-fixing cyanobacteria that prefer different environments. Whereas Crocosphaera mainly lives in nutrient-deplete, open oceans, Cyanothece is more common in coastal, nutrient-rich regions. Despite their physiological similarities, the factors separating their niches remain elusive. Here we performed physiological experiments on clone cultures and expand upon a simple ecological model to show that their different niches can be sufficiently explained by the observed differences in their photosynthetic capacities and rates of carbon (C) consumption. Our experiments revealed that Cyanothece has overall higher photosynthesis and respiration rates than Crocosphaera. A simple growth model of these microorganisms suggests that C storage and consumption are previously under-appreciated factors when evaluating the occupation of niches by different marine nitrogen fixers.

• Klíčová slova
• Carbon consumption, Niche separation, UCYN-B, UCYN-C,
• Publikační typ
• časopisecké články MeSH

Photosynthetic light reactions proceed in thylakoid membranes (TMs) due to the activity of pigment-protein complexes. These complexes are heterogeneously organized into granal/stromal thylakoids (in plants) or into recently identified cyanobacterial microdomains (MDs). MDs are characterized by specific ratios of photosystem I (PSI), photosystem II (PSII), and phycobilisomes (PBS) and they are visible as sub-micrometer sized areas with different fluorescence ratios. In this report, the process of long-term plasticity in cyanobacterial thylakoid MDs has been explored under variable growth light conditions using Synechocystis sp. PCC6803 expressing YFP tagged PSI. TM organization into MDs has been observed for all categorized shapes of cells independently of their stage in cell cycle. The heterogeneous PSI, PSII, and PBS thylakoid areas were also identified under two types of growth conditions: at continuous light (CL) and at light-dark (L-D) cycle. The acclimation from CL to L-D cycle changed spatial distribution of photosystems, in particular PSI became more evenly distributed in thylakoids under L-D cycle. The process of the spatial PSI (and partially also PSII) redistribution required 1 week and was accompanied by temporal appearance of PBS decoupling probably caused by the re-organization of photosystems. The overall acclimation we observed was defined as TM plasticity as it resembles higher plants grana/stroma reorganization at variable growth light conditions. In addition, we observed large cell to cell variability in the actual MDs organization. It leads us to suggest that the plasticity, and cell to cell variability in MDs could be a manifestation of phenotypic heterogeneity, a recently broadly discussed phenomenon for prokaryotes.

• Klíčová slova
• cyanobacteria, membrane organization, microdomains and rafts, phenotypic heterogeneity, photosynthesis, photosystems, phycobilisomes decoupling, thylakoid membrane,
• Publikační typ
• časopisecké články MeSH

Crocosphaera is a major dinitrogen (N2)-fixing microorganism, providing bioavailable nitrogen (N) to marine ecosystems. The N2-fixing enzyme nitrogenase is deactivated by oxygen (O2), which is abundant in marine environments. Using a cellular scale model of Crocosphaera sp. and laboratory data, we quantify the role of three O2 management strategies by Crocosphaera sp.: size adjustment, reduced O2 diffusivity, and respiratory protection. Our model predicts that Crocosphaera cells increase their size under high O2 Using transmission electron microscopy, we show that starch granules and thylakoid membranes are located near the cytoplasmic membranes, forming a barrier for O2 The model indicates a critical role for respiration in protecting the rate of N2 fixation. Moreover, the rise in respiration rates and the decline in ambient O2 with temperature strengthen this mechanism in warmer water, providing a physiological rationale for the observed niche of Crocosphaera at temperatures exceeding 20°C. Our new measurements of the sensitivity to light intensity show that the rate of N2 fixation reaches saturation at a lower light intensity (∼100 μmol m-2 s-1) than photosynthesis and that both are similarly inhibited by light intensities of >500 μmol m-2 s-1 This suggests an explanation for the maximum population of Crocosphaera occurring slightly below the ocean surface.IMPORTANCECrocosphaera is one of the major N2-fixing microorganisms in the open ocean. On a global scale, the process of N2 fixation is important in balancing the N budget, but the factors governing the rate of N2 fixation remain poorly resolved. Here, we combine a mechanistic model and both previous and present laboratory studies of Crocosphaera to quantify how chemical factors such as C, N, Fe, and O2 and physical factors such as temperature and light affect N2 fixation. Our study shows that Crocosphaera combines multiple mechanisms to reduce intracellular O2 to protect the O2-sensitive N2-fixing enzyme. Our model, however, indicates that these protections are insufficient at low temperature due to reduced respiration and the rate of N2 fixation becomes severely limited. This provides a physiological explanation for why the geographic distribution of Crocosphaera is confined to the warm low-latitude ocean.

• Klíčová slova
• Crocosphaera, carbon, cell flux model, daily cycle, iron, light, nitrogen, nitrogen fixation, oxygen, photosynthesis, temperature,
• MeSH
• fixace dusíku * MeSH
• kyslík metabolismus   MeSH
• sinice cytologie   metabolismus   účinky záření   MeSH
• škrob metabolismus   MeSH
• světlo * MeSH
• teplota * MeSH
• transmisní elektronová mikroskopie MeSH
• tylakoidy metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• kyslík MeSH
• škrob MeSH

Photosynthetic organisms developed various strategies to mitigate high light stress. For instance, aquatic organisms are able to spend excessive energy by exchanging dissolved CO2 (dCO2) and bicarbonate ( HCO 3 - ) with the environment. Simultaneous uptake and excretion of the two carbon species is referred to as inorganic carbon cycling. Often, inorganic carbon cycling is indicated by displacements of the extracellular dCO2 signal from the equilibrium value after changing the light conditions. In this work, we additionally use (i) the extracellular pH signal, which requires non- or weakly-buffered medium, and (ii) a dynamic model of carbonate chemistry in the aquatic environment to detect and quantitatively describe inorganic carbon cycling. Based on simulations and experiments in precisely controlled photobioreactors, we show that the magnitude of the observed dCO2 displacement crucially depends on extracellular pH level and buffer concentration. Moreover, we find that the dCO2 displacement can also be caused by simultaneous uptake of both dCO2 and HCO 3 - (no inorganic carbon cycling). In a next step, the dynamic model of carbonate chemistry allows for a quantitative assessment of cellular dCO2, HCO 3 - , and H+ exchange rates from the measured dCO2 and pH signals. Limitations of the method are discussed.

• Klíčová slova
• carbonate chemistry, computational modeling, cyanobacteria, futile cycles, photosynthesis,
• Publikační typ
• časopisecké články MeSH

Phototrophic microorganisms are promising resources for green biotechnology. Compared to heterotrophic microorganisms, however, the cellular economy of phototrophic growth is still insufficiently understood. We provide a quantitative analysis of light-limited, light-saturated, and light-inhibited growth of the cyanobacterium Synechocystis sp. PCC 6803 using a reproducible cultivation setup. We report key physiological parameters, including growth rate, cell size, and photosynthetic activity over a wide range of light intensities. Intracellular proteins were quantified to monitor proteome allocation as a function of growth rate. Among other physiological acclimations, we identify an upregulation of the translational machinery and downregulation of light harvesting components with increasing light intensity and growth rate. The resulting growth laws are discussed in the context of a coarse-grained model of phototrophic growth and available data obtained by a comprehensive literature search. Our insights into quantitative aspects of cyanobacterial acclimations to different growth rates have implications to understand and optimize photosynthetic productivity.

• Klíčová slova
• computational biology, growth model, infectious disease, light limitation, microbiology, photoinhibition, phototrophic growth laws, proteome allocation, resource allocation, systems biology,
• MeSH
• biotechnologie MeSH
• fotosyntéza genetika   MeSH
• fototrofní procesy genetika   MeSH
• proteom genetika   MeSH
• sinice genetika   růst a vývoj   metabolismus   MeSH
• světlo MeSH
• Synechocystis genetika   růst a vývoj   MeSH
• velikost buňky MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteom MeSH

This is a simple protocol for the quantitative determination of phycobiliprotein content in the model cyanobacterium Synechocystis. Phycobiliproteins are the most important components of phycobilisomes, the major light-harvesting antennae in cyanobacteria and several algae taxa. The phycobilisomes of Synechocystis contain two phycobiliproteins: phycocyanin and allophycocyanin. This protocol describes a simple, efficient, and reliable method for the quantitative determination of both phycocyanin and allophycocyanin in this model cyanobacterium. We compared several methods of phycobiliprotein extraction and spectrophotometric quantification. The extraction procedure as described in this protocol was also successfully applied to other cyanobacteria strains such as Cyanothece sp., Synechococcuselongatus, Spirulina sp., Arthrospira sp., and Nostoc sp., as well as to red algae Porphyridium cruentum. However, the extinction coefficients of specific phycobiliproteins from various taxa can differ and it is, therefore, recommended to validate the spectrophotometric quantification method for every single strain individually. The protocol requires little time and can be performed in any standard life science laboratory since it requires only standard equipment.

• MeSH
• fykobiliproteiny metabolismus   MeSH
• rostlinné proteiny metabolismus   MeSH
• sinice patogenita   MeSH
• spektrofotometrie metody   MeSH
• Synechocystis patogenita   MeSH
• Publikační typ
• audiovizuální média MeSH
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fykobiliproteiny MeSH
• rostlinné proteiny MeSH

This is a protocol for quantitative determination of storage and total carbohydrates in algae and cyanobacteria. The protocol is simple, fast and sensitive and it requires only few standard chemicals. Great advantage of this protocol is that both storage and total saccharides can be determined in the cellular pellets that were already used for chlorophyll and carotenoids quantification. Since it is recommended to perform the pigments measurement in triplicates, each pigment analysis can generate samples for both total saccharide and glycogen/starch content quantification. The protocol was applied for quantification of both storage and total carbohydrates in cyanobacteria Synechocystis sp. PCC 6803, Cyanothece sp. ATCC 51142 and Cyanobacterium sp. IPPAS B-1200. It was also applied for estimation of storage polysaccharides in Galdieria (IPPAS P-500, IPPAS P-507, IPPAS P-508, IPPAS P-513), Cyanidium caldarium IPPAS P-510, in green algae Chlorella sp. IPPAS C-1 and C-1210, Parachlorella kessleri IPPAS C-9, Nannochloris sp. C-1509, Coelastrella sp. IPPAS H-626, Haematococcus sp. IPPAS H-629 and H-239, and in Eustigmatos sp. IPPAS H-242 and IPPAS C-70.

• Klíčová slova
• Carbohydrates, Chlorella, Colorimetry, Haematococcus, Polysaccharides, Spectrophotometry, Sugars, Synechocystis,
• Publikační typ
• časopisecké články MeSH

Synechocystis sp. PCC 6803 is a widely used model cyanobacterium, whose substrains can vary on both genotype and phenotype levels. Previously described phenotypic variations include ability of mixotrophic growth, ability of movement on agar plates and variations in pigments composition or cell size. In this study, we report for the first time significant variation among Synechocystis substrains in complex cellular traits such as growth rate, photosynthesis efficiency, cellular dry weight and cellular composition (including protein or carbohydrates content). We also confirmed previously reported differences in cell size. Synechocystis cultures were cultivated in controlled environment of flat panel photobioreactors under red, blue and white light of intensities up to 790 μmol(photons) m-2 s-1, temperatures 23°C-60°C, input CO2 concentrations ranging from 400 to 15 000 ppm and in BG11 cultivation medium with and without addition of NaCl. Three Synechocystis substrains were used for the comparative experiments: GT-L, GT-B (Brno, CZ) and PCC-B (Brno, CZ). Growth rates of Synechocystis GT-B were inhibited under high intensities of red light (585-670 nm), and growth rates of both substrains GT-B and PCC-B were inhibited under photons of wavelengths 485-585 nm and 670-700 nm. Synechocystis GT-B was more sensitive to low temperatures than the other two tested substrains, and Synechocystis GT-L was sensitive to the presence of NaCl in the cultivation media. The results suggest that stress sensitivity of commonly used Synechocystis substrains can strongly vary, similarly as glucose tolerance or motility as reported previously. Our study further supports the previous statement that emphasizes importance of proper Synechocystis substrains selection and awareness of phenotypical differences among Synechocystis substrains which is crucial for comparative and reproducible research. This is highly relevant for studies related to stress physiology and development of sustainable biotechnological applications.

• MeSH
• fenotyp MeSH
• fyziologický stres * MeSH
• Synechocystis fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH