Nitrogen-depleted Cultures Research Articles

Nanoscale elemental and morphological imaging of nitrogen-fixing cyanobacteria.

Nitrogen-fixing cyanobacteria bind atmospheric nitrogen and carbon dioxide using sunlight. This experimental study focused on a laboratory-based model system, Anabaena sp., in nitrogen-depleted culture. When combined nitrogen is scarce, the filamentous prokaryotes reconcile photosynthesis and nitrogen fixation by cellular differentiation into heterocysts. To better understand the influence of micronutrients on cellular function, 2D and 3D synchrotron X-ray fluorescence mappings were acquired from whole biological cells in their frozen-hydrated state at the Bionanoprobe, Advanced Photon Source. To study elemental homeostasis within these chain-like organisms, biologically relevant elements were mapped using X-ray fluorescence spectroscopy and energy-dispersive X-ray microanalysis. Higher levels of cytosolic K+, Ca2+, and Fe2+ were measured in the heterocyst than in adjacent vegetative cells, supporting the notion of elevated micronutrient demand. P-rich clusters, identified as polyphosphate bodies involved in nutrient storage, metal detoxification, and osmotic regulation, were consistently co-localized with K+ and occasionally sequestered Mg2+, Ca2+, Fe2+, and Mn2+ ions. Machine-learning-based k-mean clustering revealed that P/K clusters were associated with either Fe or Ca, with Fe and Ca clusters also occurring individually. In accordance with XRF nanotomography, distinct P/K-containing clusters close to the cellular envelope were surrounded by larger Ca-rich clusters. The transition metal Fe, which is a part of nitrogenase enzyme, was detected as irregularly shaped clusters. The elemental composition and cellular morphology of diazotrophic Anabaena sp. was visualized by multimodal imaging using atomic force microscopy, scanning electron microscopy, and fluorescence microscopy. This paper discusses the first experimental results obtained with a combined in-line optical and X-ray fluorescence microscope at the Bionanoprobe.

Testing for terrestrial and freshwater microalgae productivity under elevated CO2 conditions and nutrient limitation

BackgroundMicroalgae CO2 fixation results in the production of biomass rich in high-valuable products, such as fatty acids and carotenoids. Enhanced productivity of valuable compounds can be achieved through the microalgae’s ability to capture CO2 efficiently from sources of high CO2 contents, but it depends on the species. Culture collections of microalgae offer a wide variety of defined strains. However, an inadequate understanding of which groups of microalgae and from which habitats they originate offer high productivity under increased CO2 concentrations hampers exploiting microalgae as a sustainable source in the bioeconomy.ResultsA large variety of 81 defined algal strains, including new green algal isolates from various terrestrial environments, were studied for their growth under atmospheres with CO2 levels of 5–25% in air. They were from a pool of 200 strains that had been pre-selected for phylogenetic diversity and high productivity under ambient CO2. Green algae from terrestrial environments exhibited enhanced growth up to 25% CO2. In contrast, in unicellular red algae and stramenopile algae, which originated through the endosymbiotic uptake of a red algal cell, growth at CO2 concentrations above 5% was suppressed. While terrestrial stramenopile algae generally tolerated such CO2 concentrations, their counterparts from marine phytoplankton did not. The tests of four new strains in liquid culture revealed enhanced biomass and chlorophyll production under elevated CO2 levels. The 15% CO2 aeration increased their total carotenoid and fatty acid contents, which were further stimulated when combined with the starvation of macronutrients, i.e., less with phosphate and more with nitrogen-depleted culture media.ConclusionGreen algae originating from terrestrial environments, Chlorophyceae and Trebouxiophyceae, exhibit enhanced productivity of carotenoids and fatty acids under elevated CO2 concentrations. This ability supports the economic and sustainable production of valuable compounds from these microalgae using inexpensive sources of high CO2 concentrations, such as industrial exhaust fumes.

Lipid recovery from Nannochloropsis gaditana using the wet pathway: Investigation of the operating parameters of bead milling and centrifugal extraction

The aim of this work is to track and optimize lipid recovery from Nannochloropsis gaditana in wet extraction operations. No significant differences in biomass concentration were found when disrupting microalgal suspensions of up to 30 g/L dry weight, but disruption efficiency differed depending on their physiological states. It took 5.8 min in a bed milling device to disrupt 80% of the cells in a nitrogen-depleted culture (10–30 g/L), compared to 4.8 min for a nitrogen-replete culture (10–30 g/L). The fatty acids released were then recovered by two different methods: one using a centrifugal partition extractor device and the other using a continuous centrifugal extractor device. For the latter, Box-Behnken RSM analysis showed that the interaction between biomass concentration and solvent inlet rate had the greatest influence on lipid recovery. Up to 84% of the triacylglycerol was recovered using 7.9 g/L of algal suspension at 5.4 mL/min, and treated with 8.9 mL/min of 2-methyl-tetra-hydrofuran.

Electrical characterization of phytoplankton suspensions using impedance spectroscopy

This study used impedance spectroscopy measurements to extract the electrical properties of phytoplankton cells in suspension. Experimental measurements were acquired, and the single-shell model was applied to extract the specific membrane capacitance, cytoplasm permittivity, and conductivity of assumingly spherical cells in suspension utilizing Maxwell’s mixture theory of a controlled volume fraction of cells. The impedance of suspensions of algae was measured at different frequencies ranging from 3 kHz to 10 MHz and impedance values were compared to investigate differences between two types of cells by characterizing their change in cytoplasm permittivity and specific membrane capacitance. Differentiation between healthy control and nitrogen-depleted cultured algae was attempted. The extracted specific membrane capacitances of Chlamydomonas and Selenastrum were 15.5 ± 3.6 and 40.6 ± 12.6 mF m− 2 respectively. Successful differentiation based on the specific membrane capacitance of different algae species was achieved. However, no significant difference was noticed between nitrogen-abundant and nitrogen-depleted cultures.

Heterocephalacria mucosa sp. nov., a new basidiomycetous yeast species isolated from a mangrove forest in Thailand.

Strains of yeast were isolated under a nitrogen-depleted culture condition from decaying tree bark (strain N-12.1) and from mangrove forest water (strain 1-7W.1) sampled at different locations within a mangrove forest site in Ranong province, Thailand. They were found to be genetically and phenotypically different from any currently recognised yeast species. Phylogenetic analysis of nucleotide sequence of three genes, the internal transcribed spacer region 1 and 2 plus 5.8S ribosomal RNA gene (ITS), D1/D2 domain of the large subunit ribosomal RNA gene (LSU D1/D2) and the small subunit of the ribosomal RNA gene (SSU), revealed that these two strains were related to but distinguished from Heterocephalacriaarrabidensis. Several distinct physiological characteristics of these two strains were detected, namely inability to assimilate glycerol, dl-lactic acid, succinic acid, citric acid, d-gluconic acid, and ability to grow well at 25 °C, which were different from those of H. arrabidensis. Accordingly, the name Heterocephalacria mucosa sp. nov., is proposed to accommodate this novel species. The MycoBank number is MB 828624.

Modulation of microcystin congener abundance following nitrogen depletion of a Microcystis batch culture

Microcystins are a group of cyclic peptides which are commonly produced by freshwater cyanobacteria and are harmful to humans and other animals when ingested. As the toxicity of microcystins varies according to their structure, it is important to understand the environmental variables which influence microcystin congener abundance. Because of the differential levels of nitrogen in amino acids incorporated into microcystins, nitrogen concentration might play an important role. During this study, Microcystis CAWBG11 was grown in batch culture until nitrogen-deplete. Over the duration of the study, the relative abundance of the microcystin congeners present shifted. The relative abundance of the arginine-containing microcystins decreased, whilst the congeners which did not contain arginine increased. These changes coincided with a large decrease in nitrate concentration to 0.35 % of initial levels. Linear regression of log10 microcystin content and nitrate concentration revealed slopes which were dependent upon the number of arginine residues present in the toxin. This modulation in microcystin congener relative abundance was likely to be due to scarcity of the nitrogen-rich arginine during periods of nitrogen limitation. During the experimental period the relative abundance of congeners with a single arginine residue at position-2 did not change (p > 0.05), whilst the relative abundance of the congeners with a position-4 arginine decreased significantly (p ≤ 0.001). Our results suggested that there arginine in the microcystin structure may be selectively modulated. Furthermore, the relative toxicity of the nitrogen-depleted culture was found to be reduced (15 % lower) compared with the starting culture.

The effect of light, salinity, and nitrogen availability on lipid production by Nannochloropsis sp.

We examined responses of batch cultures of the marine microalga Nannochloropsis sp. to combined alterations in salinity (13, 27, and 40 g/l NaCl) and light intensity (170 and 700 μmol photons/m(2)·s). Major growth parameters and lipid productivity (based on total fatty acid determination) were determined in nitrogen-replete and nitrogen-depleted cultures of an initial biomass of 0.8 and 1.4 g/l, respectively. On the nitrogen-replete medium, increases in light intensity and salinity increased the cellular content of dry weight and lipids due to enhanced formation of triacylglycerols (TAG). Maximum average productivity of ca. 410 mg TFA/l/d were obtained at 700 μmol photons/m(2)·s and 40 g/l NaCl within 7 days. Under stressful conditions, content of the major LC-PUFA, eicosapentaenoic acid (EPA), was significantly reduced while TAG reached 25% of biomass. In contrast, lower salinity tended to improve major growth parameters, consistent with less variation in EPA contents. Combined higher salinity and light intensity was detrimental to lipid productivity under nitrogen starvation; biomass TFA content, and lipid productivity amounted for only 33% of DW and ca. 200 mg TFA/l/day, respectively. The highest biomass TFA content (ca. 47% DW) and average lipid productivity of ca. 360 mg TFA/l/day were achieved at 13 g/l NaCl and 700 μmol photons/m(2)·s. Our data further support selecting Nannochloropsis as promising microalgae for biodiesel production. Moreover, appropriate cultivation regimes may render Nannochloropsis microalgae to produce simultaneously major valuable components, EPA, and TAG, while sustaining relatively high biomass growth rates.

Influences of nitrogen deficiency on the transcript levels of ammonium transporter, nitrate transporter and glutamine synthetase genes in Isochrysis galbana (Isochrysidales, Haptophyta)

L.-K. Kang, S.-P.L. Hwang, G.-C. Gong, H.-J. Lin, P.-C. Chen and J. Chang. 2007. Influences of nitrogen deficiency on the transcript levels of ammonium transporter, nitrate transporter and glutamine synthetase genes in Isochrysis galbana (Isochrysidales, Haptophyta). Phycologia 46: 521–533. DOI: 10.2216/06-44.1Three DNA fragments belonging to genes involved in nitrogen uptake and assimilation, including a nitrate transporter gene (IgNrt2), an ammonium transporter gene (IgAmt), and a glutamine synthetase gene (IgglnII), were obtained from the marine haptophyte, Isochrysis galbana. Based on alignments of the deduced amino acid sequences, the IgNRT2 fragment shared 47% identity with the Cylindrotheca fusiformis (Bacillariophyceae) nitrate transporter, the IgAMT fragment shared 48% identity with the C. fusiformis ammonium transporter, and the IgGSII fragment shared 61% identity with Skeletonema costatum (Bacillariophyceae) glutamine synthetase. Southern hybridization indicated that both IgNrt2 and IgAmt may have more than one copy in the genome of I. galbana, but only a single copy was detected for IgglnII. The transcript abundances of these three genes under various nitrogen-supply conditions were monitored simultaneously by quantitative real-time polymerase chain reaction (PCR). In the presence of ammonium, both IgNrt2 and IgAmt were severely repressed with mean mRNA concentrations of 0.67 and 3.45 µmol (mol 18S rRNA)−1. IgNrt2 and IgAmt mRNA transcripts increased 41-fold and 8.3-fold, respectively, in the presence of nitrate, and increased 160-fold and 15-fold, respectively, in the nitrogen-depleted condition. The mRNA level of IgglnII was low in cells grown in the presence of ammonium [3.98 µmol (mol 18S rRNA)−1], but mild increases in the range of 2- to 4-fold were observed in cells grown in both nitrate-containing and nitrogen-depleted cultures. Our results demonstrated that the expression patterns of IgNrt2, IgAmt, and IgglnII shared a common trend with their homologous genes in diatoms and green algae under various nitrogen-supply conditions. This group of genes could be used as indicators in natural environments to estimate the degree of nitrogen deficiency and chemical forms of nitrogenous nutrients in use on a species-specific basis.

High hopanoid/total lipids ratio in Frankia mycelia is not related to the nitrogen status.

Vesicles are specific Frankia structures which are produced under nitrogen-limiting culture conditions. Hopanoids are the most abundant lipids in these vesicles and are believed to protect the nitrogenase against oxygen. The amounts and quality of each hopanoid were estimated in different Frankia strains cultivated under nitrogen-depleted and nitrogen-replete conditions in order to detect a possible variation. Studied Frankia strains nodulating Eleagnus were phylogenetically characterized by analysis of the nifD-K intergenic region as closely related to genomic species 4 and 5. Phylogenetically different strains belonging to three infectivity groups were cultivated in the same medium with and without nitrogen source for 10 d before hopanoid content analysis by HPLC. Four hopanoids together accounted for 23-87% and 15-87% of the total lipids under nitrogen-replete and nitrogen-depleted culture conditions, respectively. Two of the hopanoids found, bacteriohopanetetrols and their phenylacetic acid esters, have previously been described in Frankia Two new hopanoids, moretan-29-ol and a bacteriohopanetetrol propionate, have also been identified. The moretan-29-ol and bacteriohopanetetrols were found to be the most abundant hopanoids whereas the bacteriohopanetetrol propionate and phenylacetates were present at a concentration close to the limit of detection. The ratio of (bacteriohopanetetrols + moretan-29-ol)/(total lipids) varied in most of the strains between nitrogen-depleted and nitrogen-replete culture conditions. In most of the strains, the hopanoid content was found to be slightly higher under nitrogen-replete conditions than under nitrogen-depleted conditions. These results suggest that remobilization, rather than neosynthesis of hopanoids, is implicated in vesicle formation in Frankia under nitrogen-depleted conditions.

Characterization of HetR protein turnover in Anabaena sp. PCC 7120.

The hetR gene plays an important role in heterocyst development and pattern formation in heterocystous cyanobacteria. The hetR gene from Anabaena sp. PCC 7120 was overexpressed in Escherichia coli. Antibodies raised against the recombinant HetR protein (rHetR) were used to characterize metabolism of the HetR of Anabaena sp. PCC 7120 in vivo. HetR was present at a low level when Anabaena sp. PCC 7120 was grown in the presence of combined nitrogen. Shifting from nitrogen repletion conditions to nitrogen depletion conditions led to a two fold increase of HetR in total cell extracts, and most of HetR was located in heterocysts. The amount of HetR in total cellular extracts increased rapidly after shifting to nitrogen depletion conditions and reached a maximum level 3 h after the shift. Isoelectrofocusing electrophoresis revealed that the native HetR had a more acidic isoelectric point than did rHetR. After combined nitrogen was added to the nitrogen-depleted cultures, the degradation of HetR depended on culture conditions: before heterocysts were fully developed, HetR was rapidly degraded; after heterocysts were fully developed, HetR was degraded much more slowly. The distribution of HetR in other species of cyanobacteria was also studied.

Characterization of the growth and lipid content of the diatom Chaetoceros muelleri

Chaetoceros muelleri (Schutt) was cultured on a thermal gradient plate, subjected to two media types with a range of specific conductances, and evaluated for growth and neutral lipid accumulation. Growth was measured directly by daily changes in cell numbers and indirectly by changes in optical density at 750 nm. C. muelleri exhibited a growth rate of at least two doublings day-1 over broad temperature (20 to 35 °C) and conductance ranges (10 to over 60 mS cm-1) and the optimum growth rate approached 4.0 doublings day-1 at 30 °C and a conductance of 25 mS cm-1. Intracellular neutral lipid storage was evaluated with fluorometry and epifluorescent microscopy using the fluorochrome Nile Red. Gravimetric analysis revealed a total lipid content in nitrogen-depleted cultures of C. muelleri of over 400 mg L-1, five to seven times that observed in nitrogen-replete cultures. Based on its high growth rate, tolerance to a broad range of temperatures and specific conductances, and large quantity of intracellular lipid, C. muelleri may have potential for exploitation as a renewable precursor to liquid fuels or as a lipid source.

Changes in carotenoids but not in D1 protein in response to nitrogen depletion and recovery in a cyanobacterium

In Synechococcus PCC 6301 (Anacystis nidulans) the imposition of nitrogen stress resulted in substantial losses of phycobiliproteins, lesser changes in chlorophyll-proteins and a dramatic change in carotenoid composition. In nitrogen-depleted cultures carotenoids continued to be synthesised, with the increase being accounted for by zeaxanthin with β-carotene content declining slightly. In these cultures zeaxanthin accounted for 75% of the carotenoid present compared to 43% in nitrogen-replete cells. Amounts of D1, a protein associated with the Photosystem II reaction centre, were similar in nitrogen-replete and nitrogen-starved cells; this retention was in accord with those of β-carotene and chlorophyll. On nitrate replenishment, zeaxanthin was not produced for 36 h, by which time β-carotene level had increased to restore the carotenoid composition characteristic of an exponential culture, and normal phycocyanin and chlorophyll levels had also been recovered. Throughout, the ratio of β-carotene to chlorophyll remained more-or-less constant.

Changes in carotenoids but not in D1 protein in response to nitrogen depletion and recovery in a cyanobacterium

In Synechococcus PCC 6301 (Anacystis nidulans) the imposition of nitrogen stress resulted in substantial losses of phycobiliproteins, lesser changes in chlorophyll-proteins and a dramatic change in carotenoid composition. In nitrogen-depleted cultures carotenoids continued to be synthesised, with the increase being accounted for by zeaxanthin with β-carotene content declining slightly. In these cultures zeaxanthin accounted for 75% of the carotenoid present compared to 43% in nitrogen-replete cells. Amounts of D1, a protein associated with the Photosystem II reaction centre, were similar in nitrogen-replete and nitrogen-starved cells; this retention was in accord with those of β-carotene and chlorophyll. On nitrate replenishment, zeaxanthin was not produced for 36 h, by which time β-carotene level had increased to restore the carotenoid composition characteristic of an exponential culture, and normal phycocyanin and chlorophyll levels had also been recovered. Throughout, the ratio of β-carotene to chlorophyll remained more-or-less constant.

Glucose transport throughout fermentation by Saccharomyces cerevisiae NCYC 1108

The rate of fermentation of glucose by a polyploid strain of Saccharomyces cerevisiae growing in a defined salts medium depends on the availability of NH4++. Its decline after exhaustion of the nitrogen source corresponded with the ability of the cells to accumulate the glucose analogue 2-deoxyglucose. Addition of NH4++to a nitrogen-depleted culture stimulated both glucose utilization and 2-deoxyglucose uptake. Since stimulation was inhibited by cycloheximide, maintenance of glucose transport during fermentation is dependent on protein synthesis.

Relative effects of nitrogen or phosphorus depletion and light intensity on the pigmentation, chemical composition, and volume of Pyrenomonas salina (Cryptophyceae)

The nitrogen-rich phycobiliproteins are efficient light-harvesting pigments of cryptophytes, cyanobacteria, and rhodophytes. Whereas it is well established that certain cyanobacteria mobilize their phycobiliproteins in response to nitrogen deprivation, the effect of nitrogen stress on cryptophyte physiology has been studied only rarely. We compared the effects of nitrogen and phosphorus depletion on the biovolume, chemical composition, and pigment composition of the marine cryptophyte Pyrenomonas salina, grown in batch cultures under light-limiting and light-saturating conditions. Cellular phycoerythrin content declined during the late exponential growth phase in nitrogen-depleted cultures, and the cells were nearly devoid of phycoerythrin (< 3 % of the phycoerythrin content of nitrogen-replete cells) at the beginning of stationary growth. Phycoerythrin content also decreased during late exponential phase in phosphorus-depleted cultures, but these latter cells contained more than 10 times the phycoerythrin concentration of nitrogen-depleted cells at the same growth stage. Chlorophyll a content declined in nitrogen-depleted cultures, but at a slower rate than the decrease in phycoerythrin, such that the phycoerythrin to chlorophyll a ratio decreased by more than 95 O/O from the early exponential to early stationary growth phase. In nitrogen-depleted cultures grown at a low light intensity, the decline in phycoerythrin content preceded the decline in chlorophyll a content, and resulted in only slight changes in cellular nitrogen content and biovolume. These data indicate that phycoerythrin is preferentially lost from P. salina during nitrogen deficiency, and support the hypothesis that, similar to certain cyanobacteria, P. salina responds to nitrogen deprivation by mobilizing phycoerythrin in order to help sustain cellular nitrogen requirements.

Regulation of Trichophyton rubrum proteolytic activity

Trichophyton rubrum is the most common dermatophyte of humans and normally colonizes the superficial layers of the epidermis (stratum corneum). Several proteinases with a possible role in the metabolism of host proteins have been purified from this fungus. The regulation of these enzymes and their role in fungal metabolism were studied at the biochemical level. General proteolytic (azocollytic) activity was repressed when log-phase cultures of T. rubrum were grown in a minimal medium that contained readily metabolized sources of carbon, nitrogen, sulfur, and phosphorus. When either carbon, nitrogen, or sulfur was deleted from this minimal medium, azocollytic activity was derepressed. In all cases a high-molecular-weight activity (Mr, greater than 200,000) was expressed. A 71,000-Mr proteinase was observed in nitrogen-depleted cultures, and proteolytic species of Mr 124,000 and 27,000 were secreted in sulfur-depleted cultures. The addition of either inorganic (MgSO4, Na2SO3, NaS2O3) or organic (methionine, cysteine) sulfur to the sulfur-depleted medium repressed the expression of azocollytic activity. In contrast, keratinolytic activity was not repressed by carbon, nitrogen, or sulfur but instead was induced when a protein source was included in the minimal medium. Stationary-phase cultures of T. rubrum secreted all proteolytic activities constitutively. Unlike log-phase cultures, the stationary-phase cultures secreted azocollytic, elastinolytic, and keratinolytic activity in minimal medium. These activities fell in the carbon-, nitrogen-, and phosphorous-depleted media but remained high in sulfur-depleted medium. The following model is proposed for the regulation of T. rubrum proteolytic activity. In the initial stages of infection, T. rubrum grows logarithmically. In this state, proteolytic activity is derepressed whenever carbon, nitrogen, or sulfur is lacking in the fungal milieu. The general proteinases produced would act on the nonkeratinous proteins in the stratum corneum. There are probably peptidases, as yet unidentified, that would cleave the peptides generated by the initial proteolysis into amino acids. These amino acids would provide the cell with a source of carbon, nitrogen, and sulfur. Under these conditions, the expression of general proteinases would be repressed, whereas specific keratinases would be induced in this nutrient-rich environment. Disease may occur when the fungus reaches stationary phase, when proteinases are secreted constitutively. These enzymes may directly or indirectly incite a host response, resulting in the inflammatory manifestations of dermatophytosis.

Nitrate Reduction in the Marine Brown Alga Giffordia mitchellae (Harv.) Ham.

Summary Nitrate reductase (NADH-nitrate oxidoreductase, E.C. 1.6.6.2) and glutamine synthetase (L-glutamate: ammonia ligase (ADP), EC 6.3.1.2) have been preliminarily characterized for the first time in a Brown Alga, Giffordia mitchellae (Harv.) Ham. Nitrate reductase activity could be estimated in an «in vivo» assay system only. For determination of glutamine synthetase activity solely the transferase reaction (L-γ-glutamylhydroxamate formation) gave reliable results. In nitrogen-starved algal cultures nitrate reductase is inducible by nitrate, a lag phase of approx. 5 hours preceding induction. Ammonium ions rapidly inactivate nitrate reductase, inactivation is overcome by addition of excess nitrate to ammonium-containing cultures. Nitrate reductase activity in Giffordia is markedly reduced under the influence of glutamine administered to the cultures. Glutamine synthetase activity is not affected by glutamine in the medium. However NO3--and NH4+-ions exert a significant influence on the activity level of this enzyme in nitrogen-depleted cultures. Nitrate leads to an temporary enhancement of the activity level, whereas ammonium reduces glutamine synthetase activity. Nitrate reductase activity exhibits pronounced diurnal fluctuations. This rhythmicity, occurring under continuous light and constant temperature conditions, is initiated by addition of nitrate to nitrogen-depleted algae.