Chlamydomonas Reinhardtii Strain Research Articles

Biosorption of cyanate by two strains of Chlamydomonas reinhardtii: evaluation of the removal efficiency and antioxidants activity

Two strains of the chlorophyte Chlamydomonas reinhardtii, a wild type (WT) and a transgenic strain (C.CYN) contained an exogenous cyanase gene (CYN), were used to investigate the growth and cyanate biosorption capability through the analysis of the adsorption equilibrium isotherm. The potential antioxidants activity of the algal strains was also investigated under cyanate concentration. The antioxidants activity of both C.CYN and WT were enhanced by the application of cyanate. Two adsorption isotherm models and the sorption kinetics were used to check the efficiency of the cyanate removal process. The results showed the biosorbent efficiency of Chlamydomonas in the removal of KCNO from aqueous solution. The C.CYN strain has great efficiency to remove cyanate as compared to the WT. The maximum percentage of cyanate removal was 83.75% for the C.CYN and 50% for the WT as treated with 0.8 mg.ml−1 KCNO. The data were adapted to the nonlinear Langmuir model on the basis of the coefficient of determination. The calculated qmax was 0.54 and 0.42 µg.mg−1 for C.CYN and WT which correlated to the experimental one (0.67 and 0.4 µg.mg−1, respectively). Our data highlight the application of the transgenic algal strain toward the removal of highly toxic materials as cyanate. Novelty statement The main objective of this work is to find out an efficient genetically-modified Chlamydomonas strain to remove the highly toxic cyanate compound from contaminated area. Moreover, to evaluate the biosorption ability of this transgenic strain with its wild one via two adsorption isotherm (the Langmuir and Freundlich) models. Also, to estimate the antioxidants activity of these strains under the cyanate toxicity through four different assays.

A Carbon Fixation Enhanced Chlamydomonas reinhardtii Strain for Achieving the Double-Win Between Growth and Biofuel Production Under Non-stressed Conditions.

The stressed cultivations are widely used in microalgae R&D for the biofuel production with the repress on growth to a certain degree, which limits the overall productivity. The balance between the growth and energy storage compounds accumulation is a target needing the combination of both strain selection or construction and culture optimization. Here, an engineered strain of Chlamydomonas reinhardtii, in which the chloroplast type glyceraldehyde-3-phosphate dehydrogenase (cGAPDH) was overexpressed and named as P3-GAPDH, was cultured on the Algal Station platform. Compared with wild type (WT), C. reinhardtii CC137c, in Tris-acetate-phosphate (TAP) medium, the highest density of WT and P3-GAPDH were 1.23 ± 0.13 and 1.74 ± 0.09 g L–1 within 96 h, and the maximum biomass productivity was 24.30 ± 1.65 and 28.54 ± 1.43 mg L–1 h–1, respectively. In terms of the energy storage compounds, both carbohydrate and fatty acids content doubled in P3-GAPDH, from 0.13 ± 0.02 to 0.26 ± 0.04 g L–1 for carbohydrate and from 0.08 ± 0.01 to 0.16 ± 0.01 g L–1 for fatty acids, among which poly unsaturated fatty acids increased by 65.8%. Together with the continuous monitor of the chlorophyll fluorescence dynamics parameters Fv/Fm and Fv’/Fm’ and pH of culture, enhanced Calvin cycle by overexpressed cGAPDH promoted the carbon conversion and subsequent energy storage compounds accumulation. C. reinhardtii P3-GAPDH strain showed the potential as a good chassis with high carbon conversion ability.

Enhancing resistance of Chlamydomonas reinhardtii to oxidative stress fusing constructs of heterologous antioxidant peptides into D1 protein

Algae emerge as leading actors for the development of new strategies to cope oxidative stress, thanks to their availability for genetic manipulation and simple cultivation. This study reports the design of novel mutant strains of the green photosynthetic alga Chlamydomonas reinhardtii, as a model system, able to produce bioactive peptides with high antioxidant capacity by means of a custom-made expression system. The D1 protein of Photosystem II is highly abundant in the chloroplast with a rapid steady-state turnover; the final step of D1 protein processing is thus exploited for the continuous release of antioxidant peptides into the organism. Three antioxidant peptides, isolated from different food sources, were successfully expressed in C. reinhardtii. We show that the in vitro antioxidant activity of algae extract is lower in transgenic lines, while the in vivo antioxidant activity is highly evident towards radical oxygen-generating substances than in the control strain. Moreover, the antioxidant mutant releasing the peptide designed from Hoki fish, showed a longer shelf-life and a better resistance to high light and heat stress than the control. These features can be exploited in the vision of the development of robust biocomponents for bioassay applications. In particular, we described the design of an optical bioassay for herbicides detection, showing the high potential of the proposed mutant strains as robust and sensitive biological recognition elements.

Effect of lhcsr gene dosage on oxidative stress and light use efficiency by Chlamydomonas reinhardtii cultures

Unicellular green algae, a promising source for renewable biofuels, produce lipid-rich biomass from light and CO2. Productivity in photo-bioreactors is affected by inhomogeneous light distribution from high cell pigment causing heat dissipation of light energy absorbed in excess and shading of the deep layers. Contrasting reports have been published on the relation between photoprotective energy dissipation and productivity. Here, we have re-investigated the relation between energy quenching (qE) activity, photodamage and light use efficiency by comparing WT and two Chlamydomonas reinhardtii strains differing for their complement in LHCSR proteins, which catalyse dissipation of excitation energy in excess (qE). Strains were analysed for ROS production, protein composition, rate of photodamage and productivity assessed under wide light and CO2 conditions.The strain lacking LHCSR1 and knocked down in LHCSR3, thus depleted in qE, produced O2 at significantly higher rate under high light, accompanied by enhanced singlet oxygen release and PSII photodamage. However, biomass productivity of WT was delayed in respect for mutant strains under intermittent light conditions only, implying that PSII activity was not the limiting factor under excess light. Contrary to previous proposals, domestication of Chlamydomonas for carbon assimilation rate in photo-bioreactors by down-regulation of photoprotective energy dissipation was ineffective in increasing algal biomass productivity.

Acclimation of Chlamydomonas reinhardtii to extremely strong light

Most photosynthetic organisms are sensitive to very high light, although acclimation mechanisms enable them to deal with exposure to strong light up to a point. Here we show that cultures of wild-type Chlamydomonas reinhardtii strain cc124, when exposed to photosynthetic photon flux density 3000 μmol m−2 s−1 for a couple of days, are able to suddenly attain the ability to grow and thrive. We compared the phenotypes of control cells and cells acclimated to this extreme light (EL). The results suggest that genetic or epigenetic variation, developing during maintenance of the population in moderate light, contributes to the acclimation capability. EL acclimation was associated with a high carotenoid-to-chlorophyll ratio and slowed down PSII charge recombination reactions, probably by affecting the pre-exponential Arrhenius factor of the rate constant. In agreement with these findings, EL acclimated cells showed only one tenth of the 1O2 level of control cells. In spite of low 1O2 levels, the rate of the damaging reaction of PSII photoinhibition was similar in EL acclimated and control cells. Furthermore, EL acclimation was associated with slow PSII electron transfer to artificial quinone acceptors. The data show that ability to grow and thrive in extremely strong light is not restricted to photoinhibition-resistant organisms such as Chlorella ohadii or to high-light tolerant mutants, but a wild-type strain of a common model microalga has this ability as well.

Exploring the potential of microalgae in the recycling of dairy wastes

Culturing microalgae using dairy-wastes offers the opportunity of producing valuable biomass for different industrial applications. The capability of four Chlorella species and a recombinant Chlamydomonas reinhardtii strain to mixotrophically grow in wastewaters from an Italian dairy factory was investigated. A robust algal growth could be efficiently sustained in these wastes, despite the abundance of d-Lactose (~4% w/v), that could not be metabolized by any microalgal species. Non axenic cocultivation of microalgae together with microbial communities from the dairy wastes resulted in a marked decrease of their pollution load, thus reducing the necessity of expensive treatments before their discharge in the municipal sewage system. Microalgae cultivated using dairy-wastes were characterized by a lipid content ranging from 12% to 21% (w/w), with Auxenochlorella protothecoides reaching the highest lipid productivity (~0.16 g/L/d) whereas the transplastomic C. reinhardtii strain expressing a thermostable β-glucosidase reached a recombinant enzyme productivity of 0.18 mg/L/d.

Experimental Calibration of a Biohydrogen Production Estimation Model

Abstract A dynamic physics-based model developed for the prediction of biohydrogen production in a compact tubular photobioreactor (PBR) was calibrated experimentally. The spatial domain in the model was discretized with lumped control volumes and the principles of classical thermodynamics, mass, species, and heat transfer were combined to derive a system of ordinary differential equations, whose solution was the temperature and mass fraction distributions across the entire system. Two microalgae species, namely, Acutodesmus obliquus and Chlamydomonas reinhardtii strain cc125, were cultured in triplicate with different culture media via indirect biophotolysis. Measured biomass and hydrogen concentrations were then used to adjust the specific microalgae growth and hydrogen production rates in the model based on residual sum of squares (RSS) and the direct search method. Despite its simplicity, the presented volume element model was verified to well predict both hydrogen and biomass concentration over time. The microalgae growth rate for each species was determined as 2.16 μalga,0 s−1 and 0.91 μalga,0 s−1 for A. obliquus and C. reinhardtii strain cc125, respectively, where μalga,0 is the specific growth rate of Scenedesmus almeriensis for given temperature and irradiance. The adjusted maximum hydrogen production rates for the local nonmutant A. obliquus and for C. reinhardtii strain cc125 were 1.3 × 10−7 s−1 and 4.1 × 10−7 s−1. Consequently, these hydrogen production rates were 59 times and 19 times smaller, respectively, than that for the mutant C. reinhardtii strain cc849.

Growth and hydrogen production by three Chlamydomonas strains cultivated in a commercial fertilizer

In the present investigation, we report the growth and hydrogen production of two wild type Chlamydomonas reinhardtii strains isolated from a tropical oxidation pond in Costa Rica. The performance of these two new isolates was compared to that of Chlamydomonas reinhardtii CC124. All the strains were grown both in conventional Tris-Acetate-Phosphate medium (TAP) and in a commercial fertilizer medium (NPK 20-20-20). The growth of the new two isolates in medium formulated with fertilizer was higher than that attained with the reference strain (CC124). However, the hydrogen production performance of the strain CC124 in TAP-S and fertilizer were comparable, while the two new strains performed better in fertilizer, although the total hydrogen production was lower than that achieved with CC124. By using fertilizer it is possible to reduce the cost of chemical reagents by about 63% compared to TAP. Another advantage of the fertilizer is that it does not contain sulfur, therefore it can be directly used for hydrogen production using the Melis protocol.

Survey of the anaerobic metabolism of various laboratory wild-type Chlamydomonas reinhardtii strains

Given the wide diversity of wild-type Chlamydomonas reinhardtii genotypes due to the presence of genes belonging to either of different gene haplotypes (Gallaher, S.D., Fitz-Gibon, S.R., Glaesener, A.G., Pellegrini, M. and Merchant, S.S. 2015, The Plant Cell, doi 10.1105/tpc.15.00508), we performed a phenotypic characterization of representative strains whose anaerobic metabolism has been studied either by our laboratory or by others. Our results reveal major differences in the levels of H2 production and in the pattern of fermentative metabolite generation, highlighting the activation of different fermentative pathways in each organism. Some of the differences could be attributed to differences in the haplotypes of the various genes involved in these pathways. Moreover, the nature of the metabolites produced under dark anaerobiosis versus sulfur-deprivation-induced anaerobiosis are different in the analyzed strains, emphasizing the differential activation of metabolic pathways promoted under the two conditions and highlighting the importance of utilizing appropriate C. reinhardtii strains for addressing specific research questions.

Expression of a Synthetic Gene for the Major Cytotoxin (Cyt1Aa) of Bacillus thuringiensis subsp. israelensis in the Chloroplast of Wild-Type Chlamydomonas.

Chlamydomonas reinhardtii (Chlamydomonas) strains that are toxic to mosquito larvae because they express chloroplast transgenes that are based on the mosquitocidal proteins of Bacillus thuringiensis subsp. israelensis (Bti) could be very useful in mosquito control. Chlamydomonas has several advantages for this approach, including genetic controls not generally available with industrial algae. The Bti toxin is produced by sporulating bacteria and has been used for mosquito control for >30 years without creating highly resistant mosquito populations. The suite of toxins is four main proteins: three Cry proteins and the cytotoxic Cyt1Aa (27 kDa). Cyt1Aa is not very toxic to mosquitoes by itself, but it prevents the development of resistance. The production of Cyt1Aa in other microbes, however, has been challenging due to its affinity for certain membrane phospholipids. Here we report on the production of recombinant Cyt1Aa (rCyt1A) in the chloroplast of photosynthetic Chlamydomonas at levels of at least 0.3% total protein. Live cell bioassays demonstrated toxicity of the rCyt1Aa Chlamydomonas to larvae of Aedes aegypti. We also expressed the chloroplast cyt1Aa gene in a wild-type Chlamydomonas strain (21 gr) that can grow on nitrate. These results have implications for developing a Chlamydomonas strain that will be toxic to mosquito larvae but will not induce strongly resistant populations.

Lipid accumulation during nitrogen and sulfur starvation in Chlamydomonas reinhardtii overexpressing a transcription factor

Algal triacylglycerol biosynthesis is of increasing interest for potential biodiesel production. In this study, we investigated the lipids content and gene expression changes in Chlamydomonas reinhardtii strain overexpressing a Dof-type transcription factor in response to nutrient deficiency. The untransformed strain grew in TAP complete medium and accumulated only 4.1% dry cell weight of fatty acids; while in the transgenic strain, the total fatty acids content increased to 15.58 and 17.02% in medium without sulfur or nitrogen, respectively. The major fatty acids found in the stressed microalgae were palmitic, oleic (all strains), and linoleic (transgenic strain). Additionally, we observed that the transgenic strain does not have a defect in carbon partitioning but redirect the carbon flux from starch to lipids. Among the most interesting results in the transcriptional profile, we found an overexpression of the enzymes lecithin cholesterol acyltransferase, glycerol-3-phosphatase acyltransferase, and phosphatidylcholine-sterol O-aciltransferase. As observed in this study, the combination of strategies (overexpression of a transcription factor and nutrient deficit) increases not only the content of total lipids in transgenic cells compared to an untransformed strain but, also the proportion of specific fatty acids that are desirable for biodiesel production.

Isolation, phenotypic characterization and genome wide analysis of a Chlamydomonas reinhardtii strain naturally modified under laboratory conditions: towards enhanced microalgal biomass and lipid production for biofuels

BackgroundMicroalgal strain development through genetic engineering has received much attention as a way to improve the traits of microalgae suitable for biofuel production. However, there are still some limitations in application of genetically modified organisms. In this regard, there has been recent interest in the isolation and characterization of superior strains naturally modified and/or adapted under a certain condition and on the interpretation of phenotypic changes through the whole genome sequencing.ResultsIn this study, we isolated and characterized a novel derivative of C. reinhardtii, whose phenotypic traits diverged significantly from its ancestral strain, C. reinhardtii CC-124. This strain, designated as CC-124H, displayed cell population containing increased numbers of larger cells, which resulted in an increased biomass productivity compared to its ancestor CC-124. CC-124H was further compared with the CC-124 wild-type strain which underwent long-term storage under low light condition, designated as CC-124L. In an effort to evaluate the potential of CC-124H for biofuel production, we also found that CC-124H accumulated 116 and 66% greater lipids than that of the CC-124L, after 4 days under nitrogen and sulfur depleted conditions, respectively. Taken together, our results revealed that CC-124H had significantly increased fatty acid methyl ester (FAME) yields that were 2.66 and 1.98 times higher than that of the CC-124L at 4 days after the onset of cultivation under N and S depleted conditions, respectively, and these higher FAME yields were still maintained by day 8. We next analyzed single nucleotide polymorphisms (SNPs) and insertion/deletions (indels) based on the whole genome sequencing. The result revealed that of the 44 CDS region alterations, 34 resulted in non-synonymous substitutions within 33 genes which may mostly be involved in cell cycle, division or proliferation.ConclusionOur phenotypic analysis, which emphasized lipid productivity, clearly revealed that CC-124H had a dramatically enhanced biomass and lipid content compared to the CC-124L. Moreover, SNPs and indels analysis enabled us to identify 34 of non-synonymous substitutions which may result in phenotypic changes of CC-124H. All of these results suggest that the concept of adaptive evolution combined with genome wide analysis can be applied to microalgal strain development for biofuel production.

Expression of multiple antihypertensive peptides as a fusion protein in the chloroplast of Chlamydomonas reinhardtii

Hypertension affects about a quarter of the global population and is a major risk factor in cardiovascular diseases. Current pharmacological treatments are considered useful but lead to secondary effects that end in low compliance. In this work, a transplastomic Chlamydomonas reinhardtii strain producing three antihypertensive peptides (AHP) as a fusion protein has been developed. The chimeric protein was coded by a synthetic gene and transferred to the plastome in a site-specific integration approach. Expression was mediated by the rbcL promoter. Molecular analysis confirmed the presence of the transgene inserted in the chloroplast genome. An ELISA quantification assay indicated that the amount of recombinant protein is 34.4 ng per mg of freeze-dried biomass. The RPLKPW and AINPSK peptides were identified by HPLC after in vitro digestion form biomass of the transplastomic C. reinhardtii strain. The antihypertensive effect of the recombinant protein was demonstrated after intragastric administration of the genetically modified strain to spontaneously hypertensive rats (SHR) at a dose of 10 mg of recombinantAHP3 protein per kg of body weight with the maximal decrease in blood pressure 6 h post-administration. These results suggested that this transplastomic strain can be used to obtain a large quantity of antihypertensive peptides which could be useful for the production of functional foods.

Flocculation of Chlamydomonas reinhardtii with Different Phenotypic Traits by Metal Cations and High pH.

Concentrating algal cells by flocculation as a prelude to centrifugation could significantly reduce the energy and cost of harvesting the algae. However, how variation in phenotypic traits such as cell surface features, cell size and motility alter the efficiency of metal cation and pH-induced flocculation is not well understood. Our results demonstrate that both wild-type and cell wall-deficient strains of the green unicellular alga Chlamydomonas reinhardtii efficiently flocculate (>90%) at an elevated pH of the medium (pH 11) upon the addition of divalent cations such as calcium and magnesium (>5 mM). The trivalent ferric cation (at 10 mM) proved to be essential for promoting flocculation under weak alkaline conditions (pH ∼8.5), with a maximum efficiency that exceeded 95 and 85% for wild-type CC1690 and the cell wall-deficient sta6 mutant, respectively. Near complete flocculation could be achieved using a combination of 5 mM calcium and a pH >11, while the medium recovered following cell removal could be re-cycled without affecting algal growth rates. Moreover, the absence of starch in the cell had little overall impact on flocculation efficiency. These findings contribute to our understanding of flocculation in different Chlamydomonas strains and have implications with respect to inexpensive methods for harvesting algae with different phenotypic traits. Additional research on the conditions (e.g., pH and metal ions) used for efficient flocculation of diverse algal groups with diverse characteristics, at both small and large scale, will help establish inexpensive procedures for harvesting cell biomass.

Evaluation of light energy to H2 energy conversion efficiency in thin films of cyanobacteria and green alga under photoautotrophic conditions

Cyanobacteria and green algae harness solar energy to split water and to fix CO2. Under specific conditions, they are capable of photoproduction of molecular hydrogen (H2). This study compares the light-energy-to-hydrogen-energy conversion efficiency (LHCE) in two heterocystous, N2-fixing cyanobacteria (wild-type Calothrix sp. strain 336/3 and the ΔhupL mutant of Anabaena sp. strain PCC 7120) and in the sulfur-deprived green alga, Chlamydomonas reinhardtii strain CC-124, after entrapment of the cells in thin Ca2+-alginate films. The experiments, performed under photoautotrophic conditions, showed higher LHCEs in the cyanobacteria as compared to the green alga. The highest efficiency of ca. 2.5% was obtained in films of the entrapped ΔhupL strain under low light condition (2.9Wm−2). Calothrix sp. 336/3 films produced H2 with a maximum efficiency of 0.6% under 2.9Wm−2, while C. reinhardtii films produced H2 most efficiently under moderate light (0.14% at 12.1Wm−2). Exposure of the films to light above 16Wm−2 led to noticeable oxidative stress in all three strains, which increased with light intensity. The presence of oxidative stress was confirmed by increased (i) degradation of chlorophylls and some structural carotenoids (such as β-carotene), (ii) production of hydroxylated carotenoids (such as zeaxanthin), and (iii) carbonylation of proteins. We conclude that the H2 photoproduction efficiency in immobilized algae and cyanobacteria can be further improved by entrapping cultures in immobilization matrices with increased permeability for gases, especially oxygen, while matrices with low porosity produced increased amounts of xanthophylls and other antioxidant compounds.

Effect of cadmium accumulation on green algae Chlamydomonas reinhardtii and acid-tolerant Chlamydomonas CPCC 121

Cadmium is one of the most dangerous metals found in wastewater since exposure to low concentrations are highly toxic for cellular functions. In this study, the effect of cadmium accumulation on Chlamydomonas reinhardtii and acid-tolerant strain CPCC 121 was investigated during 48 h under 100–600 μM of Cd and two pH conditions (4 and 7). The toxicity of accumulated Cd was determined by the change of cellular and photosynthetic parameters. Obtained results showed that the maximum capacity of Cd accumulation in algal biomass was reached for both strains at 24 h of exposure to 600 μM of Cd. Under this condition, C. reinhardtii showed a higher uptake of Cd compared to the strain CPCC 121, inducing a stronger cellular toxic impact. Chlamydomonas CPCC 121 showed a tolerance for Cd due to the exclusion of Cd at the cell wall surface, which was higher at pH 4 than pH 7. TEM images and EDX spectrum of Cd distribution within the cell confirmed the role of the cell wall as a barrier for Cd uptake. Although Cd2+ concentration was the highest in the medium, CPCC 121 was the most tolerant at pH 4, but was not enough efficient to be considered for the phycoremediation of Cd. At neutral pH, the efficiency of C. reinhardtii for the removal of Cd was limited by its toxicity, which was dependent to the concentration of Cd in the medium and the time of exposure.

Hydrogen Production by a Chlamydomonas reinhardtii Strain with Inducible Expression of Photosystem II.

Chlamydomonas reinhardtii cy6Nac2.49 is a genetically modified algal strain that activates photosynthesis in a cyclical manner, so that photosynthesis is not active constitutively in the presence of oxygen, but is turned on only in response to a metabolic trigger (anaerobiosis). Here, we further investigated hydrogen production by this strain comparing it with the parental wild-type strain under photoheterotrophic conditions in regular tris-acetate-phosphate (TAP) medium with a 10-h:14-h light/dark regime. Unlike the wild-type, whose level of H2 production remained low during illumination, H2 production in the mutant strain increased gradually with each subsequent light period, and by the final light period was significantly higher than the wild-type. The relatively low Photosystem II (PSII) activity of the mutant culture was shown by low fluorescence yield both in the dark (Fv/Fm) and in the light (δF/Fm’) periods. Measurement of oxygen evolution confirmed the low photosynthetic activity of the mutant cells, which gradually accumulated O2 to a lesser extent than the wild-type, thus allowing the mutant strain to maintain hydrogenase activity over a longer time period and to gradually accumulate H2 during periods of illumination. Therefore, controllable expression of PSII can be used to increase hydrogen production under nutrient replete conditions, thus avoiding many of the limitations associated with nutrient deprivation approaches sometimes used to promote hydrogen production.

Competitive growth experiments with a high-lipid Chlamydomonas reinhardtii mutant strain and its wild-type to predict industrial and ecological risks

Key microalgal species are currently being exploited as biomanufacturing platforms using mass cultivation systems. The opportunities to enhance productivity levels or produce non-native compounds are increasing as genetic manipulation and metabolic engineering tools are rapidly advancing. Regardless of the end product, there are both environmental and industrial risks associated to open pond cultivation of mutant microalgal strains. A mutant escape could be detrimental to local biodiversity and increase the risk of algal blooms. Similarly, if the cultivation pond is invaded by a wild-type (WT) microalgae or the mutant reverts to WT phenotypes, productivity could be impacted. To investigate these potential risks, a response surface methodology was applied to determine the competitive outcome of two Chlamydomonas reinhardtii strains, a WT (CC-124) and a high-lipid accumulating mutant (CC-4333), grown in mixotrophic conditions, with differing levels of nitrogen and initial WT to mutant ratios. Results of the growth experiments show that mutant cells have double the exponential growth rate of the WT in monoculture. However, due to a slower transition from lag phase to exponential phase, mutant cells are outcompeted by the WT in every co-culture treatment. This suggests that, under the conditions tested, outdoor cultivation of the C. reinhardtii cell wall-deficient mutant strains does not carry a significant environmental risk to its WT in an escape scenario. Furthermore, lipid results show the mutant strain accumulates over 200% more TAGs per cell, at 50 mg L−1 NH4Cl, compared to the WT, therefore, the fragility of the mutant strain could impact on overall industrial productivity.

Expression of a clostridial [FeFe]-hydrogenase in Chlamydomonas reinhardtii prolongs photo-production of hydrogen from water splitting

The high oxygen (O2) sensitivity of green algal [FeFe]-hydrogenases is a significant limitation for the sustained production of hydrogen gas (H2) from photosynthetic water splitting. To address this limitation we replaced the native [FeFe]-hydrogenases with a more O2-tolerant clostridial [FeFe]-hydrogenase CaI in Chlamydomonas reinhardtii strain D66ΔHYD (hydA1−hydA2−) that contains insertionally inactivated [FeFe]-hydrogenases genes. Expression and translocation of CaI in D66ΔHYD led to the recovery of H2 photoproduction at ~20% of the rates of the wild-type parent strain D66. We show for the first time that a bacterial [FeFe]-hydrogenase can be expressed, localized and matured to a catalytically active form that couples to photosynthetic electron transport in the green alga C. reinhardtii. The lower rates of O2 inactivation of CaI led to more sustained H2 photoproduction when cultures were challenged with O2 or kept under prolonged illumination at solar intensities. These results provide new insights into the requisites for attaining photobiological H2 production from water splitting using a more O2-tolerant hydrogenase.

A novel optical/electrochemical biosensor for real time measurement of physiological effect of astaxanthin on algal photoprotection

An optical/amperometric biosensor based on algal cells immobilized in calcium alginate gel was developed. Various Chlamydomonas reinhardtii strains mutated at the level of photosynthetic D1 protein were used as biomediators to quantify the capacity of the carotenoid xanthophylls to protect the photosynthetic apparatus from photoinhibition. The highly sensitive/selective biosensor was used for studies on cell physiology aimed to determine the antioxidant and light filtering effects of the xanthophyll astaxanthin. The biosensor was proved to be suitable for the determination of the exogenous supplied astaxanthin, showing in a short time a reliable response with a detection limit of 3μM. This technique revealed the photoprotective effect of astaxanthin-enriched extracts of Haematococcus pluvialis in algal cells. The results suggest that in algae astaxanthin exploits both filtering and antioxidant effects at different light intensities. This bioinspired approach can provide new insights into biological, biomedical, environmental and agricultural research applications and nutraceutical studies.