Effects of phosphorus limitation on the epidemiology of a chytrid phytoplankton parasite

SUMMARY 1. The effect of phosphorus limitation of the diatom Asterionella formosa Hass. on growth, survival and epidemic development of its fungal parasite Rhizophydium planktonicum Canter emend. was estimated, using measurements of production and infectivity of the zoospores of the chytrid grown on host cultures with different phosphorus-limited growth rates.
2. Phosphorus-limited host cells were less susceptible to infection with zoospores of the parasite than non-limited host cells.
3. The sporangia on phosphorus-limited algae produced substantially less zoospores, but the development time of these sporangia was only slightly reduced.
4. As a result of these effects, Rhizophydium will reach lower growth rates at a given host density, and survival of the parasite will require higher host densities when Asterionella is phosphorus-limited.
5. The zoospore production remained high enough to enable the parasite to grow faster than the alga at sufficiently high host densities, both at limiting and non-limiting phosphorus levels.
6. In spite of the reduced growth rate of the parasite, phosphorus limitation of Asterionella was found to facilitate the development of a Rhizophydium epidemic. This was a consequence of the reduced algal growth rate at phosphorus limitation, which makes the host population more easily outgrown by the parasite.
7. Phosphorus limitation of the host could reduce the threshold host density required for the development of an epidemic by a factor of 2.5.     

Buoyancy regulation of Microcystis flos-aquae during phosphorus-limited and nitrogen-limited growth

The dominance of gas-vacuolate cyanobacteria is often attributedto their buoyancy and to their ability to regulate buoyancyin response to environmental conditions. Changes in absolutegas vesicles volume, carbohydrate content, protein content andcolony buoyancy of Microcystis flos-aquae were investigatedduring nitrogen-limited, phosphorus-limited and nutrient-repletegrowth. When nutrient-replete, M. flos-aquae cells consistentlyhad excess gas vesicles, which provided sufficient buoyancythat the influence of daily carbohydrate changes on cells uponfloatation was negligible. However, during nitrogen-limitedgrowth, gas vesicle volume per cell decreased significantlywith nitrogen exhaustion. The maximum decrease of gas vesiclevolume was up to 84–88%. At the same time, cellular carbohydratecontent had an accumulation trend. The decrease of gas vesiclebuoyancy together with the daily increase in carbohydrate aresuggested to explain the daily changes in the cell floatation.During phosphorus-limited growth, gas vesicle volume per celldecreased slightly (maximum to 22–32%), and they stillprovided sufficient buoyancy that most cells kept floating eventhough there were significant daily carbohydrate changes. Sincenitrogen limitation caused more significant buoyancy loss thanphosphorus limitation did, surface water blooms may disappearor appear frequently in nitrogen limited water bodies whilethey may persist a longer time in phosphorus limited water bodies.The quantitative analysis in buoyancy change by gas vesicles,carbohydrate and protein suggested that long-term buoyancy regulationwas mainly determined by changes of gas vesicle volume whereasshort-term buoyancy regulation was mainly determined by carbohydrateaccumulation and consumption. Both long-term and short-termbuoyancy regulation were influenced by cell nutrient status.Furthermore, gas vesicle volume per cell and protein contentchanged in the same way in both nitrogen-limited and phosphorus-limitedgrowth, which implied that the decrease of gas vesicles wereassociated with controls of total protein synthesis.     

The effect of degree and timing of nitrogen limitation on lipid productivity in Chlorella vulgaris

Improvements in lipid productivity would enhance the economic feasibility of microalgal biodiesel. In order to optimise lipid productivity, both the growth rate and lipid content of algal cells must be maximised. The lipid content of many microalgae can be enhanced through nitrogen limitation, but at the expense of biomass productivity. This suggests that a two-stage nitrogen supply strategy might improve lipid productivity. Two different nitrogen supply strategies were investigated for their effect on lipid productivity in Chlorella vulgaris. The first was an initial nitrogen-replete stage, designed to optimise biomass productivity, followed by nitrogen limitation to enhance lipid content (two-stage batch) and the second was an initial nitrogen-limited stage, designed to maximise lipid content, followed by addition of nitrogen to enhance biomass concentration (fed-batch). Volumetric lipid yield in nitrogen-limited two-stage batch and fed-batch was compared with that achieved in nitrogen-replete and nitrogen-limited batch culture. In a previous work, maximum lipid productivity in batch culture was found at an intermediate level of nitrogen limitation (starting nitrate concentration of 170 mg L^?1). Overall lipid productivity was not improved by using fed-batch or two-stage culture strategies, although these strategies showed higher volumetric lipid concentrations than nitrogen-replete batch culture. The dilution of cultures prior to nitrogen deprivation led to increased lipid accumulation, indicating that the availability of light influenced the rate of lipid accumulation. However, dilution did not lead to increased lipid productivity due to the resulting lower biomass concentration.     

Influence of nutrient limitation and growth rate on the outer membrane proteins of Klebsiella aerogenes NCTC 418

Four major proteins with molecular weights of 78 000, 37 000, 34 000 and 20 000 were present in the envelope of Klebsiella aerogenes when cultured at a high specific growth rate. However, at lower growth rates, the protein content and composition of the envelope depended on the imposed nutrient limitation. Under potassium-, carbon-, sulphur- and phosphorus-limited conditions, derepression of synthesis of limitation-specific proteins was observed, their apparent molecular weights being 90 000, 48 000, 41 000 and 36 000, respectively. Nitrogen-limited cells had no additional proteins. For a particular limiting nutrient, expression of the limitation-specific proteins was independent of the chemical or physical form in which the nutrient was supplied. Under potassium or sulphur limitation the specific proteins were present maximally at the lowest imposed growth rate, whereas under carbon limitation a maximum expression of these proteins was found at moderate growth rates. It is concluded that limitation-specific proteins which are associated with the outer membrane function in the uptake of limiting nutrients or, possibly, limitation-releasing compounds.     

Lipid accumulation in an oleaginous yeast (Candida 107) growing on glucose in single-stage continuous culture.

Lipid accumulation of Candida 107, grown at dilution rates from 0.03 to the maximum of 0.21/h, with carbon, nitrogen, phosphate, and magnesium limitations in a chemostat, was maximal at about 40% (wt/wt) with nitrogen-limited medium at a dilution rate of 0.06/h, giving an efficiency of substrate conversion of 22 g of lipid per g of glucose consumed. At higher dilution rates the lipid content decreased. With carbon-limited growth, the highest lipid content (14%, wt/wt) was at the maximum dilution rate. High lipid contents also occurred with phosphate + nitrogen as double limitations of growth, with the lipid content of the yeast (about 35%, wt/wt) continuing to be near maximum at dilution rates also near maximum (0.17/h), thus giving the highest specific rate of lipid formation of any growth conditions (0.59 g of lipid/g of yeast per h). However, the efficiency of substrate utilization was only 5.2 g of lipid formed per 100 g of glucose consumed. The composition of the fatty acyl residues within the lipid remained constant over many weeks if the steady-state conditions remained unchanged. With carbon-limited growth, the degree of unsaturation of the fatty acids markedly decreased as the dilution rate was increased, but with nitrogen limitation the reverse trend was seen. In all cases, linoleic and oleic acids were the principal fatty acyl residues affected, and their relative proportions always varied in opposite directions. When magnesium was a limiting nutrient, there was a considerable increase in the proportion of myristic acid produced within the lipid. Neutral lipids (predominantly triglycerides) varied from 66 to 92% of the total lipid from carbon- and nitrogen-limited growth; phospholipids (varying from 2 to 25%) were highest in nitrogen-limited growth. The fatty acyl residues within each lipid fraction showed the same variations with changing growth rates.     

PRODUCTION OF MUCILAGE BY THE ADRIATIC EPIPELIC DIATOM CYLINDROTHECA CLOSTERIUM (BACILLARIOPHYCEAE) UNDER NUTRIENT LIMITATION

The carbon partitioning of the epipelic diatom Cylindrotheca closterium (Ehrenberg) Reiman and Lewin isolated from the Adriatic Sea was studied in the laboratory under varying scenarios of nutrient limitation. Total number of cells, photosynthesis measured at 695 μmol photons·m ^{− 2}·s ^{− 1} irradiance (P_{695- μ mol}), chlorophyll ( a + c ) content, respiration, extracellular polymeric substances (EPS), total particulate carbohydrate (TPC), and dissolved carbohydrate were evaluated under nitrogen and phosphorus deficiencies in culture. The highest total number of cells was found in the control, whereas the nitrogen-limited treatment showed the lowest value. During the transition phase of growth, photosynthesis in the nitrogen-limited treatment was 3-fold lower than in the phosphorus-limited treatment and 4-fold lower than in the control. Differences in respiration rates and chlorophyll ( a + c ) content were even more marked. Dissolved carbohydrate remained the same in all the treatments, whereas during the transition and stationary phase, EPS presented the highest values under phosphorus limitation and the lowest in the control treatment. The production of EPS was closely linked to the periods of carbon assimilation (transition phase) in the nutrient depleted treatments, especially in the phosphorus-limited treatment. These results point out the relevance of the nutrient imbalance (nitrogen or phosphorus) in the production of EPS by the benthic or resuspended diatoms and suggest that these diatoms play an important role in nutrient-unbalanced systems like sediments or marine snow.     

Effects of phosphate limitation of growth on the cell-wall and lipid composition of Saccharomyces cerevisiae.

The phosphorus content of phosphate-limited Saccharomyces cerevisiae was only 71% of that of non-limited yeast. Walls prepared from phosphate-limited cells contained slightly less phosphorus than control walls. No evidence was obtained for the presence in these walls of uronic acid or succinyl residues. The carbohydrate content of walls of phosphate-limited yeast was less than that of non-limited walls, and this was reflected in a decreased glucan content. There was only a slight decrease in glucosamine content while the protein content increased. The major change in the lipid composition of phosphate-limited yeast was a decrease in both sterol esters and triacylglycerols. There was a decrease in total lipid content, but increased production of phosphatidylethanolamine and phosphatidylcholine. The phosphatidylserine content was decreased. These results suggest that there are fewer intracellular low-density vesicles in phosphate-limited yeast.     

Influence of cultivation conditions on lipid production by Cryptococcus albidus

Summary Cryptococcus albidus var. albidus CBS 4517 was able to accumulate lipid under nitrogen-limited as well as excess-nitrogen conditions. The highest lipid-producting capacity was, however, observed in nitrogen-limited cultivations. In nitrogen-limited batch cultures, a lipid content of 34% (w/w) in biomass and a maximum specific lipid productivity of 37 mg lipid/g lipid-free biomass·h, was determined. The yield of lipid from glucose was about 0.15 g/g in nitrogen-limited and 0.11 g/g in excess-nitrogen cultures.In a nitrogen-limited fed-batch culture, 12.4 g/l lipid was produced at 90 h of cultivation and the cells contained 46.3% (w/w) lipid.Higher lipid yield and cellular lipid content were observed when inorganic nitrogen sources were used compared with organic. The choice of carbon source was seen to influence growth as well as lipid production and the highest yields of lipid were obtained when glucose, maltose or mannitol was used.A cultivation temperature of 20°C provided the highest lipid productivity compared to 25°C and 30°C. Addition of citrate to the growth medium was seen to have a stimulating effect on the specific lipid productivity.     

Importance of the methyl-citrate cycle on glycerol metabolism in the yeast Yarrowia lipolytica

A novel approach to trigger lipid accumulation and/or citrate production in vivo through the inactivation of the 2-methyl-citrate dehydratase in Yarrowia lipolytica was developed. In nitrogen-limited cultures with biodiesel-derived glycerol utilized as substrate, the Δphd1 mutant (JMY1203) produced 57.7 g/L of total citrate, 1.6-fold more than the wild-type strain, with a concomitant glycerol to citrate yield of 0.91 g/g. Storage lipid in cells increased at the early growth stages, suggesting that inactivation of the 2-methyl-citrate dehydratase would mimic nitrogen limitation. Thus, a trial of JMY1203 strain was performed with glycerol under nitrogen-excess conditions. Compared with the equivalent nitrogen-limited culture, significant quantities of lipid (up to ∼31% w/w in dry weight, 1.6-fold higher than the nitrogen-limited experiment) were produced. Also, non-negligible quantities of citric acid (up to ∼26 g/L, though 0.57-fold lower than the nitrogen-limited experiment) were produced, despite remarkable nitrogen presence into the medium, indicating the construction of phenotype that constitutively accumulated lipid and secreted citrate in Y. lipolytica during growth on waste glycerol utilized as substrate.