Fe nutrition demand and utilization by the green alga Dunaliella bardawil

The Fe nutritional demands, requirements and mechanisms of uptake by Dunaliella bardawil as well as potential Fe sources were studied. A comparison between Fe uptake from bacterial siderophores and from synthetic ferric chelates revealed algal growth response and chlorophyll synthesis to increasing concentrations and availability at a range of 0.01 μM–5 μM, as well as differences in efficiency. Furthermore, chloroplast ultrastructure, as observed by TEM, was affected by Fe deficiency, as was chlorophyll content. Ferric reduction is involved in the Fe uptake mechanism of Fe-stressed D. bardawil. Nutrient solution with controlled levels of free Fe²⁺ as well as spectrophotometric assays were used to measure Fe³⁺ reduction. This study shows that D. bardawil utilizes Fe³⁺ via a reduction mechanism, similar to that of strategy-I higher plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

MASSIVE ACCUMULATION OF PHYTOENE INDUCED BY NORFLURAZON IN DUNALIELLA BARDAWIL (CHLOROPHYCEAE) PREVENTS RECOVERY FROM PHOTOINHIBITION1

The effects of nanomolar to micromolar concentrations of the herbicide norflurazon were studied in Dunaliella bardawil Ben-Amotz et Avron, a β-carotene-accumulating halotolerant alga. The large amount of β-carotene which Dunaliella bardawil can contain, around 8% of the algal dry weight, is reduced to 0.2% by treatment with 100 nm norflurazon. Simultaneously, phytoene is accumulated to a similar level of about 8%. The gradual increase in phqtoene content, in response to increasing norflurazon concentrations, corresponds to the decrease in β-carotene, with no evident change in other isoprenoid intermediates. Carotene-rich Dunaliella bardawil is substantially resistant to high-intensity photoinhibition. This resistance is lost in cells grown to contain low & carotene and in the nor-urazon-treated phytoene-rich cells. These obseruations are in agreement with the hypothesis that the accumulated β-carotene in Dunaliella bardawil portects the cells against injury by excessive irradiation.     

Effect of iron on growth and lipid accumulation in Chlorella vulgaris

The economic feasibility of algal mass culture for biodiesel production is enhanced by the increase in biomass productivity and storage lipids. Effect of iron on growth and lipid accumulation in marine microalgae Chlorella vulgaris were investigated. In experiment I, supplementing the growth media with chelated FeCl3 in the late growth phase increased the final cell density but did not induce lipid accumulation in cells. In experiment II, cells in the late-exponential growth phase were collected by centrifugation and re-inoculated into new media supplemented with five levels of Fe3+ concentration. Total lipid content in cultures supplemented with 1.2 x 10(-5) mol L(-1) FeCl3 was up to 56.6% biomass by dry weight and was 3-7-fold that in other media supplemented with lower iron concentration. Moreover, a simple and rapid method determining the lipid accumulation in C. vulgaris with spectrofluorimetry was developed.     

STIMULATION OF GROWTH IN SCENEDESMUS OBLIQUUS (CHLOROPHYCEAE) BY HUMIC ACIDS UNDER IRON LIMITED CONDITIONS1,2

Stimulatory affects of humic acids of molecular weight 30,000 or greater on iron-starved Scenedesmus obliquus (Turp.) Kütz. in association with bacteria were studied by growth and Fe uptake experiments. Humic acids stimulated growth of Fe-starved cells by decreasing the lag phase and extending the growth phase. Humic acids stimulated increased algal growth in medium containing EDTA as well as in medium containing no EDTA, indicating humic acids are not stimulating algal growth under Fe limiting conditions by creating a soluble Fe pool. Humic acids decreased Fe availability to Fe-starved S. obliquus. Iron bound to humic acids is unavailable for uptake by Fe-starved cells indicating growth stimulation is not due to chelation effects alone. Stimulation of growth is not a membrane phenomenon as humic acids show the same stimulatory effect when in contact with cells or separated by dialysis membrane. Humic acids also stimulate growth in the dark, with and without aeration, indicating use as a heterotrophic substrate. A photoheterotrophic mechanism is indicated by increased algal growth caused by illuminating cultures, containing humic acids but excluding CO₂.     

Establishment of Agrobacterium tumefaciens-mediated genetic transformation in Dunaliella bardawil

Dunaliella bardawil, a unicellular microalga, grows in relatively high concentrations of salt and has so far been refractory to Agrobacterium-mediated transformation. An inverse relationship between salt concentration and hygromycin resistance was observed. Co-cultivation at 0.2?M NaCl allowed growth of both D. bardawil and A. tumefaciens. Lowering salt concentrations also enabled the use of lower concentrations of hygromycin, the selection agent. Cells resistant to 100?mg?l^?1 hygromycin were selected and growth of Agrobacterium was completely eliminated in these cells using cefotaxime/potassium clavulanate. The concentration of sodium chloride was gradually increased to 1.0?M with simultaneous reduction of hygromycin concentration for better growth of D. bardawil. Agrobacterium was unable to survive in the growth medium used for Dunaliella. Expression of β-glucuronidase (uidA), green fluorescent protein (GFP) and hygromycin phosphotransferase (hpt) in the hygromycin-resistant culture was detected using X-gluc as substrate and Western blotting using GFP antibodies and RT-PCR respectively. Cells growing in 1.0?M NaCl (in the absence of hygromycin) retained their ability to grow in hygromycin even after 18 months of cultivation. These cells expressed GFP and PCR for hpt gene was positive. The stability of the integrated transgene and resistance to hygromycin in three different transformation events were ascertained periodically. Southern blotting of DNA extracted from hygromycin resistant cells (HRC) that were 15–18 months old established the presence of the integrated transgene in the DNA of D. bardawil. Results of the present study substantiate A. tumefaciens-mediated transformation of the unicellular marine alga D. bardawil. Agrobacterium tumefaciens-mediated transgene integration along with the massive outdoor cultivation methods used for D. bardawil may allow the commercial synthesis of secondary metabolites and heterologous proteins.     

Influence of outbreak of macroalgal blooms on phosphorus release from the sediments in Swan Lake Wetland,China

Macroalgal blooms have occurred worldwide frequently in coastal areas in recent decades, which dramatically modify phosphorus (P) cycle in water column and the sediments. Rongcheng Swan Lake Wetland, a coastal wetland in China, is suffering from extensive macroalgal blooms. In order to verify the influence of macroalgal growth on sediment P release, the sediments and filamentous Chaetomorpha spp. were incubated in the laboratory to investigate the changes of water quality parameters, P levels in overlying water, and sediments during the growth period. In addition, algal biomass and tissue P concentration were determined. In general, Chaetomorpha biomasses were much higher in high P treatments than in low P treatments. Compared with algae+low P water treatment, the addition of sediments increased the algal growth rate and P accumulation amount. During the algal growth, water pH increased greatly, which showed significant correlation with algal biomass in treatments with high P (P < 0.05). P fractions in the sediments showed that Fe/Al–P and organic P concentrations declined during the algal growth, and great changes were observed in algae+low P water+sediment treatment for both. As a whole, the sediments can supply P for Chaetomorpha growth when water P level was low, and the probable mechanism was the release of Fe/Al–P at high pH condition induced by intensive Chaetomorpha blooms.     

Modification of Protein Synthesis by Vitamin B12 in the Marine Algal Flagellate Neochloris pseudoalveolaris*

SYNOPSIS. A strain of the marine algal flagellate Neochloris pseudoalgeolaris required only an inorganic medium for growth; however, in media containing vitamin B₁₂ it became greener, protein synthesis was modified, and the levels of DNA and RNA increased. By isotope tracer technics, transmethylation of the methyl group of methionine was found to play a role in modulation of protein synthesis governed by the vitamin. These results indicate that vitamin B₁₂ stimulates synthesis of chloroplasts in this algal strain.     

THE EFFECTS OF IRON AND COPPER AVAILABILITY ON THE COPPER STOICHIOMETRY OF MARINE PHYTOPLANKTON1

We studied the interactive effects of iron (Fe) and copper (Cu) availability on the growth rates, Cu quotas, and steady‐state Cu‐uptake rates (ρ_ssCu) of 12 phytoplankton (from four classes and two marine environments). A mixed‐effect statistical model indicated that low Fe significantly decreased phytoplankton growth rates. In contrast, lowering Cu levels only decreased the growth rates of the oceanic phytoplankton. Under Fe/Cu sufficiency, the Cu quotas ranged from 0.36 to 3.8 μmol Cu · mol^?1 C. Copper levels in the growth medium had a significant positive effect on the Cu quotas, and this effect was dependent on the algal class. Under Fe/Cu sufficiency, the highest average Cu quotas were observed for the Bacillariophyceae, followed by the Cyanophyceae, Prymnesiophyceae, and lastly the Dinophyceae. Similar taxonomic trends were observed for the ρ_ssCu. Although the Cu:C ratios were not significantly higher in oceanic strains, there are five independent lines of evidence supporting a more important role of Cu in the physiology of the oceanic phytoplankton. The mixed‐effect model indicated a significant Cu effect on the growth rates and ρ_ssCu of the oceanic strains, but not the coastal strains. In addition, lowering the Cu concentration in the media decreased the Cu quotas and ρ_ssCu of the oceanic strains to a greater extent (5.5‐ and 5.4‐fold, respectively) than those of the coastals (3.8‐ and 4.7‐fold, respectively). Iron limitation only had a significant effect on the Cu quotas of the oceanic strains, and this effect was dependent on Cu level and taxonomic class. Our results highlight a complex physiological interaction between Fe and Cu in marine phytoplankton.     

Responses of Two Coastal Algae (Skeletonema costatum and Chlorella vulgaris) to Changes in Light and Iron Levels1

Iron (Fe) is essential for phytoplankton growth and photosynthesis, and is proposed to be an important factor regulating algal blooms under replete major nutrients in coastal environments. Here, Skeletonema costatum, a typical red-tide diatom species, and Chlorella vulgaris, a widely distributed Chlorella, were chosen to examine carbon fixation and Fe uptake by coastal algae under dark and light conditions with different Fe levels. The cellular carbon fixation and intracellular Fe uptake were measured via ¹⁴C and ⁵⁵Fe tracer assay, respectively. Cell growth, cell size, and chlorophyll-α concentration were measured to investigate the algal physiological variation in different treatments. Our results showed that cellular Fe uptake proceeds under dark and the uptake rates were comparable to or even higher than those in the light for both algal species. Fe requirements per unit carbon fixation were also higher in the dark resulting in higher Fe: C ratios. During the experimental period, high Fe addition significantly enhanced cellular carbon fixation and Fe uptake. Compared to C. vulgaris, S. costatum was the common dominant bloom species because of its lower Fe demand but higher Fe uptake rate. This study provides some of the first measurements of Fe quotas in coastal phytoplankton cells, and implies that light and Fe concentrations may influence the phytoplankton community succession when blooms occur in coastal ecosystems.     

BORON AS A GROWTH REQUIREMENT FOR DIATOMS1

A boron requirement has been shown for 12 species of marine pennate diatoms, 4 species of marine centric diatoms, and S freshwater diatom species. It can be concluded that boron is essential for the growth of most, probably all, diatoms. It is much easier to demonstrate a requirement for the marine species than for the freshwater species. Some species of marine algal flagellates also require boron for growth; others apparently do not.     

Anaerobic digestates are useful nutrient sources for microalgae cultivation: functional coupling of energy and biomass production

We investigated the extent to which nitrogenous and phosphorus nutrients from liquid anaerobic digestates could be recycled for photosynthetic growth of a microalga, Scenedesmus sp. AMDD. Digestates recovered from the anaerobic digestion of cow manure and swine manure and a co-digestion of swine manure and algal biomass were diluted in distilled water and used for algal growth with and without supplemental CO₂ addition. Nutrient assimilation and final biomass yield were retarded in all but the swine manure/algae co-digestate cultures supplemented with high CO₂. Swine manure digestate cultures supplemented with the typical complement of micronutrients normally added with a commonly used growth medium or with Fe/EDTA failed to grow any better than unamended controls. When the culture medium was prepared by blending swine manure digestate with 25 or 50 % algal biomass digestate, diluting it with lake water or by supplementing with magnesium, nutrient assimilation and final algal biomass yields were maximized, indicating that magnesium was critically limiting for algal growth in swine manure digestates. Magnesium amendment thus appears to be essential if nutrients from swine manure digestates are recycled for algal growth. No such requirement is necessary for recycling nutrients from digestates generated wholly or in part from algal biomass.     

Biodiversity effects on productivity and stability of marine macroalgal communities: the role of environmental context

The influence of biodiversity on ecosystem functioning has been the focus of much recent research, but the role of environmental context and the mechanisms by which it may influence diversity effects on production and stability remain poorly understood. We assembled marine macroalgal communities in two mesocosm experiments that varied nutrient supply, and at four field sites that differed naturally in environmental conditions. Concordant with theory, nutrient addition promoted positive species richness effects on algal growth in the first mesocosm experiment; however, it tended to weaken the positive diversity relationship found under ambient conditions in a second experiment the next year. In the field experiments, species richness increased algal biomass production at two of four sites. Together, these experiments indicate that diversity effects on algal biomass production are strongly influenced by environmental conditions that vary over space and time. In decomposing the net biodiversity effect into its component mechanisms, seven of the eight experimental settings showed positive complementarity effects (suggesting facilitation or complementary resource use) countered by negative selection effects (i.e. enhanced growth in mixture of otherwise slow growing species) to varying degrees. Under no conditions, including nutrient enrichment, did we find evidence of positive selection effects commonly thought to drive positive diversity effects. Species richness enhanced stability of algal community biomass across a range of environmental settings in our field experiments. Hence, while species richness can increase production, enhanced stability is also an important functional outcome of maintaining diverse marine macroalgal communities.     

Lipid content in 19 brackish and marine microalgae: influence of growth phase, salinity and temperature

Algal lipids provide essential fatty acids for higher trophic levels in the marine food web, and understanding the fatty acid composition in phytoplankton is critical for evaluating its value as a diet. Nineteen microalgal species, mainly originating from the Baltic Sea, covering major algal classes were grown in different growth conditions. Samples were taken during both the exponential and stationary growth phases and analysed regarding their fatty acid methyl esters and free fatty acids. Our results show that across all screened species, total fatty acids increased significantly from exponential to stationary growth phase. Furthermore, it was observed that warm-water species contained more lipids and differed in their lipid profile as compared with the cold-water species. Brackish water species also showed a slightly higher lipid content than the marine species, but their lipid profile was not significantly different. Plotting changes in lipids against changes in cell nitrogen revealed a significant dependency between decrease in cell nitrogen and increase in lipids across all tested species.     

Production of phytoene by herbicide-treated microalgae Dunaliella bardawil in two-phase systems

In the present study we have optimized the concentration of the bleaching herbicide norflurazon to obtain Dunaliella bardawil cells able to accumulate phytoene without losing viability. The highest concentration of phytoene 10.4 g/gChl was obtained for a concentration of norflurazon of 10 microg/mL. Norflurazon-treated Dunaliella bardawil cells are able to accumulate high concentrations of phytoene if the carotenogenic pathway is stimulated, but the lack of colored carotenoids make these cells particularly sensitive to high light intensities and to UVB radiation, so other stimuli, such as nitrogen starvation, have to be used to force the accumulation of phytoene. Detailed time-course evolution of the carotenoids lutein, violaxanthin, zeaxanthin, phytene and beta-carotene and the photosynthetic pigment chlorophyll was followed upon transfer of Dunaliella bardawil cells to nitrogen starvation in presence and absence of norflurazon. The combined use of the carotenogenic pathway inhibitor norflurazon and biphasic aqueous/organic systems to force the excretion of phytoene into the culture medium has been investigated. Cells cultured in the biphasic system were viable and able to produce phytoene during 3 days. Futhermore the productivity increased from 0.14 g/gChl . h in the aqueous culture to 0.18 g/gChl . h in the biphasic system. About 15% of the total phytoene produced by Dunaliella bardawil was excreted and immediately partionated into the organic phase. The concentration of phytoene in the decane phase was 2.05 g/gChl after 72 h, this means that about 47 g of phytoene per litre of culture were in the organic phase.     

The effect of temperature and algal biomass on bacterial production and specific growth rate in freshwater and marine habitats

We analyzed heterotrophic, pelagic bacterial production and specific growth rate data from 57 studies conducted in fresh, marine and estuarine/coastal waters. Strong positive relationships were identified between 1) bacterial production and bacterial abundance and 2) bacterial production and algal biomass. The relationship between bacterial production and bacterial abundance was improved by also considering water temperature. The analysis of covariance model revealed consistent differences between fresh, marine and estuarine/coastal waters, with production consistently high in estuarine/coastal environments. The log-linear regression coefficient of abundance was not significantly different from 1.00, and this linear relationship permitted the use of specific growth rate (SGR in day⁻¹) as a dependent variable. A strong relationship was identified between specific growth rate and temperature. This relationship differed slightly across the three habitats. A substantial portion of the residual variation from this relationship was accounted for by algal biomass, including the difference between marine and estuarine/coastal habitats. A small but significant difference between the fresh- and saltwater habitats remained. No significant difference between the chlorophyll effect in different habitats was identified. The model of SGR against temperature and chlorophyll was much weaker for freshwater than for marine environments. For a small subset of the data set, mean cell volume accounted for some of the residual variation in SGR. Pronounced seasonality, fluctuations in nutrient quality, and variation of the grazing environment may contribute to the unexplained variation in specific growth.     

Etude de la fertilité des eaux marines au moyen de tests biologiques effectués avec des cultures d'algues I. Comparaison des méthodes d'estimation

Several methods of growth potential estimation of marine productivity are discussed: (1) simultaneous measurements of nutrient content and organic production, (2) chemical composition of algal cells, (3) bioassay with algal cultures. This last method appears to be the most convenient; it allows to study the role of a nutrient component both when utilized alone and when combined in a pool of nutrients. Several aspects of bioassay technology are discussed: test species, chemical enrichments and growth estimation of test cultures.     

Siderophore-mediated iron uptake in two clades of Marinobacter spp. associated with phytoplankton: the role of light

Iron is an essential element for oceanic microbial life but its low bioavailability limits microorganisms in large areas of the oceans. To acquire this metal many marine bacteria produce organic chelates that bind and transport iron (siderophores). We have previously shown that algal-associated heterotrophic bacteria belonging to the γ-proteobacterial Marinobacter genus release the siderophore vibrioferrin (VF). The iron-VF complex was shown to be both far more photolabile than all previously examined photolabile siderophores and to generate a photoproduct incapable of re-chelating the released iron. Thus, the photo-generated iron was shown to be highly bioavailable both to the producing bacterium and its algal partner. In exchange, we proposed that algal cells produced dissolved organic matter that helped support bacterial growth and ultimately fueled the biosynthesis of VF through a light-dependent “carbon for iron mutualism”. While our knowledge of the importance of light to phototrophs is vast, there are almost no studies that examine the effects of light on microbial heterotrophs. Here, we characterize iron uptake mechanisms in “algal-associated” VF-producers. Fe uptake by a VF knock-out mutant mimics the wild-type strain and demonstrates the versatility of iron uptake mechanisms in Marinobacter VF-producers. We also show that VF-producers selectively regulate a subset of their siderophore-dependent iron uptake genes in response to light exposure. The regulation of iron uptake and transport genes by light is consistent with the light driven algal–bacterial “carbon for iron mutualism” hypothesis in the marine environment.     

Paleoecology of Pennsylvanian phylloid algal buildups in south Guizhou,China

Pennsylvanian phylloid algal reefs are widespread and well exposed in south Guizhou, China. Here we report on reefs ranging from 2 to 8 m thickness and 30–50 m lateral extension. Algae, the main components, display a wide spectrum of growth forms, but are commonly cyathiform (cup-shaped) and leaf-like (undulate plates). The algal reef facies is dominated by boundstone. Algal thalli form a dense carpet whose framework pores are filled with marine cement and peloidal micrite. The peloidal matrix is dense, partly laminated or clotted with irregular surfaces and often gravity defying. Algal reefs in Guizhou differ from examples reported to date by the high biodiversity of organisms other than phylloids: e.g., the intergrowth of algae with corals (some of which are twice the size of algal thalli) and numerous large brachiopods. This contrasts to previous views that phylloid algal “meadows” dominated the actual seafloor, excluding other biota. Also, the pervasive marine cements (up to 50%) including botryoidal cement are noteworthy. Algal reefs developed at platform margins, a depositional environment similar to that of modern Halimeda mounds in Java, Australia and off Bahamas, and to that of time-equivalent examples reported from the Canadian Arctic Archipelago. Whereas nutrients appear decisive in the growth of Halimeda reefs, algal reefs reported herein seemingly grew under conditions of low nutrient levels. Overall, algal reefs in Guizhou challenge previous views on growth forms, diversity patterns, and depositional environments and add to the spectrum of these partly puzzling biogenic structures.     

Multiple addition bioassay of Tjeukemeer water

Multiple addition bioassay of water from the eutrophic lake Tjeukemeer revealed that in 1979 from the end of May until the end of November, when the lake was dominated by Oscillatoria agardhii, N and not P was the principal factor limiting algal growth. During periods with very low concentrations of Tot-Fe_diss in the lake, chelated Fe also limited algal growth in the bioassays. The results of this study further suggested that the concentrations of trace metals have reached levels toxic to phytoplankton. Trace metals inhibited growth particularly when the lake received relatively humus-poor IJsselmeer water with a lower metal binding capacity.     

Effects of snow removal and algal photoacclimation on growth and export of ice algae

Net growth of ice algae in response to changes in overlying snow cover was studied after manipulating snow thickness on land-fast, Arctic sea ice. Parallel laboratory experiments measured the effect of changing irradiance on growth rate of the ice diatom, Nitzschia frigida. After complete removal of thick snow (≥9 cm), in situ ice algae biomass declined (over 7–12 days), while removal of thin snow layers (4–5 cm), or partial snow removal, increased net algal growth. Ice bottom ablation sometimes followed snow removal, but did not always result in net loss of algae. Similarly, in laboratory experiments, small increases in irradiance increased algal growth rate, while greater light shifts suppressed growth for 3–6 days. However, N. frigida could acclimate to relatively high irradiance (110 μmol photons m² s⁻¹). The results suggest that algal loss following removal of a thick snow layer was due to the combination of photoinhibition and bottom ablation. The smaller relative increase in irradiance after removal of thin or partial snow layers allowed algae to maintain high specific-growth rates that compensated for loss from physical mechanisms. Thus, the response of ice algae to snow loss depends both on the amount of change in snow depth and algal photophysiology. The complex response of ice algae growth and export loss to frequently changing snow fields may contribute to horizontal and temporal patchiness of ecologically and biogeochemically important variables in sea ice and should be considered in predictions of how climate change will affect Arctic marine ecosystems.