Modulation of photosynthetic activity and photoprotection in Haematococcus pluvialis cells during their conversion into haematocysts and back.

The engagement of different photoprotective mechanisms in the cells of the carotenogenic astaxanthin-accumulating chlorophyte Haematococcus pluvialis (i) under favorable conditions, (ii) in the course of stress-induced haematocyst formation and (iii) during recovery from the stress was studied. To this end, we followed the changes in primary photochemistry, electron flow at the acceptor side of photosystem II, and non-photochemical quenching (NPQ) using PAM chlorophyll fluorimetry. A general trend recorded in the stressed cells undergoing transition to haematocysts (and reversed during recovery from the stress) was a gradual reduction of the photosynthetic apparatus accompanied by down-regulation of energy-dependent photoprotective mechanisms such as NPQ, along with the accumulation of astaxanthin. On this background, a transient up-regulation of the photosynthetic activity was detected at the intermediated stages (20-50 h of the stress exposure) of haematocyst formation. This phenomenon was tentatively related with the peak of metabolic activity found earlier in the forming haematocysts. The role of secondary carotenogenesis coupled with a reversible transition from 'active' (energy-dependent) to 'passive' photoprotective mechanisms in the extremely high stress tolerance of carotenogenic phototrophs is discussed.

Nondestructive monitoring of carotenogenesis in Haematococcus pluvialis via whole-cell optical density spectra.

We investigated the feasibility of rapid, nondestructive assay of carotenoid-to-chlorophyll (Car/Chl) ratio and total carotenoids (Car) in cell suspensions of the carotenogenic chlorophyte Haematococcus pluvialis Flotow under stressful conditions. Whole-cell spectra are characterized by variable nonlinear contributions of Car and chlorophylls (Chl), with a strong influence of Car packaging and sieve effect inherent to stressed H. pluvialis cells. Nevertheless, nondestructive assay of Car/Chl in the range of 0.55-31.2 (Car content up to 188 mg L(-1); 5.4 % of the cell dry weight) turned to be achievable with a simple spectrophotometer lacking an integrating sphere upon deposition of the cells on glass fiber filters. The scattering-corrected optical density (OD) in the blue-green region of the whole-cell spectrum, normalized to that in the red maximum of Chl absorption (OD500/OD678), was tightly related (r (2) = 0.96) with the Car/Chl ratio found in extracts. Some features such as the amplitude and position of the minimum of the normalized first-derivative OD whole-cell spectra also exhibited a strong (r (2) > 0.90) nonlinear correlation with Car/Chl. These spectral indices were also tightly related with Car, but the slope of the relationship varied with the stressor intensity. The importance of calibration over the widest possible range of pigment contents and a correct choice of biomass load per filter are emphasized. The advantages and limitations of nondestructive monitoring of carotenogenesis in H. pluvialis are discussed in view of its possible application in optical sensors for laboratory cultivation and mass production systems of the algae.

Evaluation of impaired beta-cell function in nonobese-diabetic (NOD) mouse model using bioluminescence imaging.

Insulin-producing pancreatic ß cells are functionally impaired or destroyed in diabetes mellitus. The onset of type 1 diabetes (T1D) represents the culmination of a prolonged prediabetic phase of immune-mediated ß-cell destruction. To assess the in vivo metabolic status of these cells, we used the ATP-sensitive firefly luciferase bioluminescence imaging approach, as a noninvasive probe to monitor pathological alterations in ß-cell function in the nonobese-diabetic (NOD) mouse model of T1D. Hence, we generated the ToIß-NOD transgenic mice in which doxycycline-inducible luciferase gene is selectively expressed in ß cells. A sharp reduction in bioluminescence emitted in vivo from ß cells at the early stages, preceded by several weeks of a limited reduction in ß-cell mass. Since this decline could be due to the ongoing inflammatory process occurring in vivo, we exposed control islets to inflammatory cytokines and observed a dramatic decrease in luciferase luminescence, which appears to be due in part to a decrease in protein levels and a drop in intracellular ATP levels. This is the first evidence that selective expression of the luciferase gene represents a sensitive method for noninvasive in vivo monitoring of early ß-cell dysfunction, subtle metabolic changes, such as endogenous ATP levels, indicative of a pathological condition in a tissue at the cellular level.

Isolation and characterization of a novel chytrid species (phylum Blastocladiomycota), parasitic on the green alga Haematococcus.

A parasite was found in cultures of the green microalga Haematococcus pluvialis that grew epibiotically on algal cells and caused epidemics resulting in damage to the host cultures. The parasite was isolated into axenic culture on solid and liquid media. It was demonstrated to be the sole causative agent of the epidemics. According to its life cycle and phylogenetic analysis based on 18S ribosomal DNA sequences, the pathogen appears to represent a novel chytrid fungus closely related to the vascular plant pathogen Physoderma (Blastocladiomycota), although it differs from all other known chytrids by its infective stage, a wall-less propagule endowed with amoeboid motion and lacking the group's typical flagellum.

On the relative efficiency of two- vs. one-stage production of astaxanthin by the green alga Haematococcus pluvialis.

Haematococcus pluvialis under stress conditions overproduces the valuable red ketocarotenoid astaxanthin. Two proposed strategies for commercial production are under current analysis. One separates in time the production of biomass (optimal growth, green stage) and pigment (permanent stress, red stage), while the other uses an approach based on continuous culture under limiting stress at steady state. The productivities, efficiencies and yields for the pigment accumulation in each case have been compared and analyzed in terms of the algal basic physiology. The two-stage system indoors yields a richer astaxanthin product (4% of dry biomass) with a final astaxanthin productivity of 11.5 mg L(-1) day(-1), is more readily upscalable and amenable to outdoors production. Furthermore, each stage can be optimized for green biomass growth and red pigment accumulation by adjusting independently the respective ratio of effective irradiance to cell density. We conclude that the two-stage system performs better (by a factor of 2.5-5) than the one-stage system, and the former is best fit in an efficient mass production setup.