High carotenoid mutants of Chlorella vulgaris show enhanced biomass yield under high irradiance

Microalgae represent a carbon-neutral source of bulk biomass, for extraction of high-value compounds and production of renewable fuels. Due to their high metabolic activity and reproduction rates, species of the genus Chlorella are highly productive when cultivated in photo-bioreactors. However, wild-type strains show biological limitations making algal bioproducts ex-pensive compared to those extracted from other feedstocks. Such constraints include inhomoge-neous light distribution due to high optical density of the culture, and photoinhibition of the sur-face-exposed cells. Thus, the domestication of algal strains for industry makes it increasingly important to select traits aimed at enhancing light-use efficiency while withstanding excess light stress. Carotenoids have a crucial role in protecting against photooxidative damage and, thus, represent a promising target for algal domestication. We applied chemical mutagenesis to Chlorella vulgaris and selected for enhanced tolerance to the carotenoid biosynthesis inhibitor norflurazon. The NFR (norflurazon-resistant) strains showed an increased carotenoid pool size and enhanced tolerance towards photooxidative stress. Growth under excess light revealed an improved carbon assimilation rate of NFR strains with respect to WT. We conclude that domestication of Chlorella vulgaris, by optimizing both carotenoid/chlorophyll ratio and resistance to photooxidative stress, boosted light-to-biomass conversion efficiency under high light conditions typical of photobiore-actors. Comparison with strains previously reported for enhanced tolerance to singlet oxygen, reveals that ROS resistance in Chlorella is promoted by at least two independent mechanisms, only one of which is carotenoid-dependent

Combined resistance to oxidative stress and reduced antenna size enhance light-to-biomass conversion efficiency in Chlorella vulgaris cultures

Background: Microalgae are efficient producers of lipid-rich biomass, making them a key component in developing a sustainable energy source, and an alternative to fossil fuels. Chlorella species are of special interest because of their fast growth rate in photobioreactors. However, biological constraints still cast a significant gap between the high cost of biofuel and cheap oil, thus hampering perspective of producing CO2-neutral biofuels. A key issue is the inefficient use of light caused by its uneven distribution in the culture that generates photoinhibition of the surface-exposed cells and darkening of the inner layers. Efficient biofuel production, thus, requires domestication, including traits which reduce optical density of cultures and enhance photoprotection. Results: We applied two steps of mutagenesis and phenotypic selection to the microalga Chlorella vulgaris. First, a pale-green mutant (PG-14) was selected, with a 50% reduction of both chlorophyll content per cell and LHCII complement per PSII, with respect to WT. PG-14 showed a 30% increased photon conversion into biomass efficiency vs. WT. A second step of mutagenesis of PG-14, followed by selection for higher tolerance to Rose Bengal, led to the isolation of pale-green genotypes, exhibiting higher resistance to singlet oxygen (strains SOR). Growth in photobioreactors under high light conditions showed an enhanced biomass production of SOR strains with respect to PG-14. When compared to WT strain, biomass yield of the pale green+ sor genotype was enhanced by 68%. Conclusions: Domestication of microalgae like Chlorella vulgaris, by optimizing both light distribution and ROS resistance, yielded an enhanced carbon assimilation rate in photobioreactor

A microalgal-based preparation with synergistic cellulolytic and detoxifying action towards chemical-treated lignocellulose

High-temperature bioconversion of lignocellulose into fermentable sugars has drawn attention for efficient production of renewable chemicals and biofuels, because competing microbial activities are inhibited at elevated temperatures and thermostable cell wall degrading enzymes are superior to mesophilic enzymes. Here, we report on the development of a platform to produce four different thermostable cell wall degrading enzymes in the chloroplast of Chlamydomonas reinhardtii. The enzyme blend was composed of the cellobiohydrolase CBM3GH5 from C. saccharolyticus, the β-glucosidase celB from P. furiosus, the endoglucanase B and the endoxylanase XynA from T. neapolitana. In addition, transplastomic microalgae were engineered for the expression of phosphite dehydrogenase D from Pseudomonas stutzeri, allowing for growth in non-axenic media by selective phosphite nutrition. The cellulolytic blend composed of the glycoside hydrolase (GH) domain GH12/GH5/GH1 allowed the conversion of alkaline-treated lignocellulose into glucose with efficiencies ranging from 14% to 17% upon 48h of reaction and an enzyme loading of 0.05% (w/w). Hydrolysates from treated cellulosic materials with extracts of transgenic microalgae boosted both the biogas production by methanogenic bacteria and the mixotrophic growth of the oleaginous microalga Chlorella vulgaris. Notably, microalgal treatment suppressed the detrimental effect of inhibitory by-products released from the alkaline treatment of biomass, thus allowing for efficient assimilation of lignocellulose-derived sugars by C. vulgaris under mixotrophic growth

Caratterizzazione fisiologica e molecolare del trasporto ad alta affinit\ue0 dell\u2019ammonio in radici di mais

Nella maggior parte dei suoli, ammonio (NH4+) e nitrato (NO3-) sono le forme di azoto prevalentemente disponibili per la nutrizione delle piante. Bench\ue9 la concentrazione di NH4+ nel terreno sia sensibilmente inferiore a quella di NO3- (da 10 a 1000 volte minore), molte specie vegetali mostrano una preferenza verso l\u2019assorbimento di NH4+, in quanto il processo di assimilazione non richiede una reazione di riduzione come nel caso del NO3-. D\u2019altro canto una nutrizione azotata basata esclusivamente su NH4+ ha effetti deleteri sulla crescita vegetativa (riduzione della biomassa ipogea ed epigea) e sull\u2019assorbimento di cationi. Si ritiene che per la maggior parte delle colture le condizioni ottimali di nutrizione azotata siano quelle che vedono la presenza contemporanea dei due nutrienti. Gli studi relativi alla caratterizzazione dei meccanismi di assorbimento dell\u2019 NH4+ sono meno presenti in letteratura rispetto a quelli che riguardano il NO3-. In precedenti lavori condotti con riso e Arabidopsis \ue8 stato dimostrato che la velocit\ue0 di assorbimento di NH4+ in funzione della concentrazione esterna di substrato segue un andamento bifasico, mostrando una cinetica di tipo Michaelis-Menten a basse concentrazioni (< 1 mM) e un andamento lineare a concentrazioni pi\uf9 elevate, rivelando l\u2019esistenza di un sistema di trasporto ad alta affinit\ue0 e uno a bassa affinit\ue0. Il trasporto di NH4+ a livello della membrana plasmatica avviene a carico di carriers, appartenenti alla famiglia ammonium transporter/methylamine permease/rhesus (AMT/MEP/Rh). In Arabidopsis thaliana sono stati identificati 5 membri della famiglia AMT1. I geni AtAMT1;1, AtAMT1;3 e AtAMT1;5 sono prevalentemente espressi nella radice di piante carenti di azoto e la loro attivit\ue0 contribuisce in maniera predominante al trasporto ad alta affinit\ue0 del NH4+. Lo scopo di questo lavoro \ue8 la caratterizzazione biochimica e molecolare del trasporto di NH4+ in radici di plantule della linea T250 di mais, allevate in coltura idroponica. Gli esperimenti di uptake condotti con diversa concentrazione esterna di 15NH4+ e diversi pH hanno consentito di calcolare le velocit\ue0 nette di assorbimento, di determinare gli intervalli di concentrazione in cui si osservano i trasporti ad alta e a bassa affinit\ue0 e le rispettive costanti cinetiche, e la dipendenza da pH del processo di trasporto. Attraverso analisi di omologia di sequenza sono stati identificati nel genoma di mais (http://www.maizesequence.org) 5 putativi geni codificanti AMT1. Le informazioni ottenute permetteranno di analizzare le variazione di espressione di questi geni nelle radici di piante di mais in risposta alle fluttuazioni della concentrazione di NH4+

Development of the “Highly Sensitive Dog” questionnaire to evaluate the personality dimension “Sensory Processing Sensitivity” in dogs

<div><p>In humans, the personality dimension ‘sensory processing sensitivity (SPS)’, also referred to as “high sensitivity”, involves deeper processing of sensory information, which can be associated with physiological and behavioral overarousal. However, it has not been studied up to now whether this dimension also exists in other species. SPS can influence how people perceive the environment and how this affects them, thus a similar dimension in animals would be highly relevant with respect to animal welfare. We therefore explored whether SPS translates to dogs, one of the primary model species in personality research. A 32-item questionnaire to assess the “highly sensitive dog score” (HSD-s) was developed based on the “highly sensitive person” (HSP) questionnaire. A large-scale, international online survey was conducted, including the HSD questionnaire, as well as questions on fearfulness, neuroticism, “demographic” (e.g. dog sex, age, weight; age at adoption, etc.) and “human” factors (e.g. owner age, sex, profession, communication style, etc.), and the HSP questionnaire. Data were analyzed using linear mixed effect models with forward stepwise selection to test prediction of HSD-s by the above-mentioned factors, with country of residence and dog breed treated as random effects. A total of 3647 questionnaires were fully completed. HSD-, fearfulness, neuroticism and HSP-scores showed good internal consistencies, and HSD-s only moderately correlated with fearfulness and neuroticism scores, paralleling previous findings in humans. Intra- (N = 447) and inter-rater (N = 120) reliabilities were good. Demographic and human factors, including HSP score, explained only a small amount of the variance of HSD-s. A PCA analysis identified three subtraits of SPS, comparable to human findings. Overall, the measured personality dimension in dogs showed good internal consistency, partial independence from fearfulness and neuroticism, and good intra- and inter-rater reliability, indicating good construct validity of the HSD questionnaire. Human and demographic factors only marginally affected the HSD-s suggesting that, as hypothesized for human SPS, a genetic basis may underlie this dimension within the dog species.</p></div