Advances in the biotechnology of hydrogen production with the microalga Chlamydomonas reinhardtii.

Biological hydrogen production is being evaluated for use as a fuel, since it is a promising substitute for carbonaceous fuels owing to its high conversion efficiency and high specific energy content. The basic advantages of biological hydrogen production over other "green" energy sources are that it does not compete for agricultural land use, and it does not pollute, as water is the only by-product of the combustion. These characteristics make hydrogen a suitable fuel for the future. Among several biotechnological approaches, photobiological hydrogen production carried out by green microalgae has been intensively investigated in recent years. A select group of photosynthetic organisms has evolved the ability to harness light energy to drive hydrogen gas production from water. Of these, the microalga Chlamydomonas reinhardtii is considered one of the most promising eukaryotic H2 producers. In this model microorganism, light energy, H2O and H2 are linked by two excellent catalysts, the photosystem 2 (PSII) and the [FeFe]-hydrogenase, in a pathway usually referred to as direct biophotolysis. This review summarizes the main advances made over the past decade as an outcome of the discovery of the sulfur-deprivation process. Both the scientific and technical barriers that need to be overcome before H2 photoproduction can be scaled up to an industrial level are examined. Actual and theoretical limits of the efficiency of the process are also discussed. Particular emphasis is placed on algal biohydrogen production outdoors, and guidelines for an optimal photobioreactor design are suggested.

Effects of fluid-flow regimes on Chlorella sorokiniana cultivation in cascade photobioreactors with either flat or wavy bottoms.

Computational fluid dynamics (CFD) was used to investigate cascade photobioreactors (cascade PBRs) with two different bottom configurations-flat and wavy-to establish the effect that fluid-flow regimes exert on the photosynthetic productivity of Chlorella sorokiniana. In the flat-bottom PBR, areal biomass productivities decreased from 6.8 to 4.2 g·m-2·d-1 when the flow rate of a culture per unit of lane width was increased from 33 to 132 L·m-1·min-1. We found that this decrease in the areal productivity was the result of a decrease in the volumetric photon flux densities (volumetric PFDs), which was caused by an increase in the depth of the culture in the lane. Through CFD calculation and long-exposure photography, the flow of the culture in the wavy-bottom PBR was characterized in an upper straightforward section and underneath the swirling section. Under identical conditions of flow rate and volumetric PFD (66 L·m-1·min-1 and 50 µmol·m-3·s-1, respectively), the cell growth accelerated in the wavy-bottom PBR with areal productivity that reached 6.5 g·m-2·d-1-productivity was 5.1 g·m-2·d-1 in the flat-bottom PBR. The swirling flow in the wave troughs held the culture for longer periods in the illuminated lane, and the resultant extended period of mixing improved the photosynthetic productivity.

Productivity correlated to photobiochemical performance of Chlorella mass cultures grown outdoors in thin-layer cascades.

This work aims to: (1) correlate photochemical activity and productivity, (2) characterize the flow pattern of culture layers and (3) determine a range of biomass densities for high productivity of the freshwater microalga Chlorella spp., grown outdoors in thin-layer cascade units. Biomass density, irradiance inside culture, pigment content and productivity were measured in the microalgae cultures. Chlorophyll-fluorescence quenching was monitored in situ (using saturation-pulse method) to estimate photochemical activities. Photobiochemical activities and growth parameters were studied in cultures of biomass density between 1 and 47 g L(-1). Fluorescence measurements showed that diluted cultures (1-2 g DW L(-1)) experienced significant photostress due to inhibition of electron transport in the PSII complex. The highest photochemical activities were achieved in cultures of 6.5-12.5 g DW L(-1), which gave a maximum daylight productivity of up to 55 g dry biomass m(-2) day(-1). A midday depression of maximum PSII photochemical yield (F (v)/F (m)) of 20-30% compared with morning values in these cultures proved to be compatible with well-performing cultures. Lower or higher depression of F (v)/F (m) indicated low-light acclimated or photo-inhibited cultures, respectively. A hydrodynamic model of the culture demonstrated highly turbulent flow allowing rapid light/dark cycles (with frequency of 0.5 s(-1)) which possibly match the turnover of the photosynthetic apparatus. These results are important from a biotechnological point of view for optimisation of growth of outdoor microalgae mass cultures under various climatic conditions.

Evaluation of Vision in Gnathological and Orthodontic Patients with Temporomandibular Disorders: A Prospective Experimental Observational Cohort Study.

OBJECTIVES: Temporomandibular disorders (TMDs), orthodontic diseases, and vision dysfunctions seem to be strictly related. The purpose of this study was to prove the relationship, to evaluate the prevalence and the distribution of vision defects in dysfunctional and orthodontic patients, and to establish the type of the relationship. MATERIALS AND METHODS: A total of 100 patients with TMDs were selected and studied through epidemiological analyses of the following factors: gnathological parameters (temporomandibular joint pathologies according to Diagnostic Criteria for Temporomandibular Disorders); occlusal and skeletal parameters (overjet, overbite, dental class, transversal discrepancies, and mandibular asymmetry); and orthoptic parameters (refractive defects and oculomotor diseases). A prospective experimental observational cohort study was conducted. A comparison with the average frequency of vision defects of the Italian population was performed. The prevalence of vision defects was evaluated. All gnathological and orthodontic parameters were associated with the orthoptic ones. A descriptive and statistical analysis of the data was carried out with the Statistical Package for the Social Sciences software; z test (P < 0.05), frequency analysis (frequency >50%), chi-square test, and Student's t test (P < 0.05) were performed. The scientific consistency was evaluated by using the scientific criteria of Bradford Hill. RESULTS: The comparison with the Italian population showed a higher frequency of refractive defects in the study sample (P < 0.001). The most frequent vision defects were phorias (92%) and tropia (3%). The increased frequency of ocular convergence reduction in the presence of disc displacement with reduction was significant (n = 28; 60%; P < 0.05). In the presence of asymmetry, low frequencies of astigmatism (n = 18; 30%) were observed compared to its absence (n = 22; 54%) (P < 0.05) and high frequencies of motor ocular deviations (n = 59; 100%) were observed compared to its absence (n = 36; 88%) (P < 0.05). In the presence of headache, low frequencies of emmetropia (n = 13; 22%) and higher frequencies of hyperopia (n = 18; 30%) were observed (P < 0.05). Two of five scientific criteria of Bradford Hill were met. CONCLUSION: It seems to emerge a possible positive relationship between TMD and vision defects. In particular, the most interesting associations were found between functional or skeletal orthognathic alterations and oculomotor dysfunctions. However, it was not possible to establish the type of relationship.

Interplay between light intensity, chlorophyll concentration and culture mixing on the hydrogen production in sulfur-deprived Chlamydomonas reinhardtii cultures grown in laboratory photobioreactors.

Relationships between light intensity and chlorophyll concentration on hydrogen production were investigated in a sulfur-deprived Chlamydomonas reinhardtii culture in a laboratory scale photobioreactor (PBR) equipped with two different stirring devices. In the first case, the culture was mixed using a conventional magnetic stir bar, while in the second it was mixed using an impeller equipped with five turbines. Experiments were carried out at 70 and 140 micromol photons m(-2) s(-1) in combination with chlorophyll concentrations of 12 and 24 mg L(-1). A high light intensity (140 micromol photons m(-2) s(-1), supplied on both sides of the PBR) in combination with a low chlorophyll concentration (12 mg L(-1)) inhibited the production of hydrogen, in particular in the culture mixed with the stir bar. An optimal combination for hydrogen production was found when the cultures were exposed to 140 micromol photons m(-2) s(-1) (on both sides) and 24 mg L(-1) of chlorophyll. Under these conditions, the hydrogen production output rate reached about 120 mL L(-1) in the culture mixed with the stir bar, and rose to about 170 mL L(-1) in the one mixed with the impeller. These outputs corresponded to a mean light conversion efficiency of 0.56% and 0.81%, respectively. However, the efficiency increased to 1.08% and 1.64%, respectively, when maximum hydrogen rates were considered. The better performance of the dense cultures mixed with an impeller was mainly attributed to an intermittent illumination pattern to which the cells were subjected (time cycles within 50-100 ms) which influenced the hydrogen production (1) directly, by providing the PSII with a higher production of electrons for the hydrogenase and (2) indirectly, through a higher synthesis of carbohydrates. The fluid dynamics in the PBR equipped with the impeller was characterized. The better mixing state achieved in the PBR of the new configuration makes it a useful tool for studying the hydrogen production process involving photosynthetic microorganisms, and provides a better insight into the physiology of the process.

Effects of temperature on the astaxanthin productivity and light harvesting characteristics of the green alga Haematococcus pluvialis.

The green alga Haematococcus pluvialis, which accumulates astaxanthin at an optimal temperature of 20°C, was cultivated under temperatures of 20°C, 23.5°C, 27°C, and 30.5°C, in order to assess the effects on algal metabolism during the growth phase. The culture growth rate declined with above-optimal increases in temperature, and the final maximum cell concentration at 30.5°C reached only 35% of that attained at 20°C. On the contrary, the biomass productivity was increased under all the high-temperature conditions, probably reflecting the metabolism switch from cell duplication to energy accumulation that is typically observed in algal cultures subjected to environmental stress. Moreover, an increase in the light-harvesting capability of the alga was observed by means of the total pigment balance and the photosynthesis-intensity (PI) curve measured under the different cultivation conditions. Cultures kept at higher temperatures were able to better harvest and utilize the impinging light due to photo-acclimation. Finally, the differences in the astaxanthin metabolism were elucidated by subjecting the cultures to nitrogen starvation at 20°C and 27°C. In the culture at 27°C, a 1.4-fold increase in the astaxanthin productivity was observed when compared to that at 20°C, and the latter required almost two-fold more energy for the astaxanthin production compared with the 27°C culture.

Outdoor H2 production in a 50-L tubular photobioreactor by means of a sulfur-deprived culture of the microalga Chlamydomonas reinhardtii.

In the past decade, H2 production using the green microalga Chlamydomonas reinhardtii has been extensively studied under laboratory-scale photobioreactors, while information on outdoor cultures is still lacking. In this paper, the results of experiments conducted with sulfur-deprived cultures of C. reinhardtii carried out in a 50-L horizontal tubular photobioreactor are presented. Hydrogen production experiments were carried out under both artificial and direct solar light. In both cases, the H2 output attained was 18-20% of what obtained in the laboratory. However, no significant changes in the H2 production were observed when cells grown outdoors were tested under laboratory conditions. Chlorophyll fluorescence measurements showed that outdoor cultures were subjected to strong photo-inhibition, due to the combination of high solar light intensity and sulfur-deprivation. Indeed, H2 production was only achieved outdoors when cultures were previously acclimated to sunlight, a condition that caused a number of physiological changes, namely: (i) a decrease in the chlorophyll content per unit of dry weight; (ii) an increase in the photosynthesis and respiration rates, and (iii) a higher induction of the xanthophyll cycle pigments as compared to non-acclimated cultures. It was concluded that the reduced H2 output achieved in the 50-L photobioreactor was due to the different illumination pattern to which the cultures were exposed (one-sided vs. two-sided illumination provided in the laboratory), as well as to the great difference in the mixing times (60 min vs. 15.5s achieved in the lab-scale photobioreactor). To the very best of our knowledge this is the first time that H2 production with green algae has been achieved by means of solar light.

Sustained H2 production in a Chlamydomonas reinhardtii D1 protein mutant.

In the present investigation, a detailed biochemical analysis of the high H2 producer D1 protein mutant strain L159I-N230Y of Chlamydomonas reinhardtii, carrying a double amino acid substitution, was made. The leucine residue L159 was replaced by isoleucine, and the N230 asparagine was replaced by tyrosine. The performance of this strain was compared to that of the cc124 strain. The mutant showed a sustained capacity to donate electrons by means of direct biophotolysis for H2 production, as demonstrated by the higher efficiency of utilization of the hydrogenase enzyme when carried out under anaerobic conditions. The latter property was maintained also under sulfur deprivation. Furthermore, when compared to the cc124, the mutant showed a higher amount of D1 protein content, a higher carbohydrate storage capacity and a sustained PSII direct contribution to the H2 production during sulfur deprivation. The addition of DCMU to the cells showed that as much as 7.0 mL H2 liter of culture h⁻¹ were produced by means of direct biophotolysis. The maximum apparent light-to-hydrogen conversion efficiency expressed on PAR (photosynthetically active radiation) reached 3.22%, while PSII efficiency to perform direct biophotolysis was calculated to be 2.03%. These values are significantly higher than what has been reported in the literature.