Skye R. Thomas-Hall

Improved Photobiological H2 Production in Engineered Green Algal Cells

Oxygenic photosynthetic organisms use solar energy to split water (H2O) into protons (H+), electrons (e-), and oxygen. A select group of photosynthetic microorganisms, including the green alga Chlamydomonas reinhardtii, has evolved the additional ability to redirect the derived H+ and e- to drive hydrogen (H2) production via the chloroplast hydrogenases HydA1 and A2 (H2 ase). This process occurs under anaerobic conditions and provides a biological basis for solar-driven H2 production. However, its relatively poor yield is a major limitation for the economic viability of this process. To improve H2 production in Chlamydomonas, we have developed a new approach to increase H+ and e- supply to the hydrogenases. In a first step, mutants blocked in the state 1 transition were selected. These mutants are inhibited in cyclic e- transfer around photosystem I, eliminating possible competition for e- with H2ase. Selected strains were further screened for increased H2 production rates, leading to the isolation of Stm6. This strain has a modified respiratory metabolism, providing it with two additional important properties as follows: large starch reserves (i.e. enhanced substrate availability), and a low dissolved O2 concentration (40% of the wild type (WT)), resulting in reduced inhibition of H2ase activation. The H2 production rates of Stm6 were 5-13 times that of the control WT strain over a range of conditions (light intensity, culture time, ± uncoupler). Typically, ∼540 ml of H2 liter-1 culture (up to 98% pure) were produced over a 10-14-day period at a maximal rate of 4 ml h-1 (efficiency = ∼5 times the WT). Stm6 therefore represents an important step toward the development of future solar-powered H2 production systems.

The Metabolome of Chlamydomonas reinhardtii following Induction of Anaerobic H2 Production by Sulfur Depletion

The metabolome of the model species Chlamydomonas reinhardtii has been analyzed during 120 h of sulfur depletion to induce anaerobic hydrogen (H2) production, using NMR spectroscopy, gas chromatography coupled to mass spectrometry, and TLC. The results indicate that these unicellular green algae consume freshly supplied acetate in the medium to accumulate energy reserves during the first 24 h of sulfur depletion. In addition to the previously reported accumulation of starch, large amounts of triacylglycerides were deposited in the cells. During the early 24- to 72-h time period fermentative energy metabolism lowered the pH, H2 was produced, and amino acid levels generally increased. In the final phase from 72 to 120 h, metabolism slowed down leading to a stabilization of pH, even though some starch and most triacylglycerides remained. We conclude that H2 production does not slow down due to depletion of energy reserves but rather due to loss of essential functions resulting from sulfur depletion or due to a build-up of the toxic fermentative products formate and ethanol.

Isolation and evaluation of oil-producing microalgae from subtropical coastal and brackish waters.

Microalgae have been widely reported as a promising source of biofuels, mainly based on their high areal productivity of biomass and lipids as triacylglycerides and the possibility for cultivation on non-arable land. The isolation and selection of suitable strains that are robust and display high growth and lipid accumulation rates is an important prerequisite for their successful cultivation as a bioenergy source, a process that can be compared to the initial selection and domestication of agricultural crops. We developed standard protocols for the isolation and cultivation for a range of marine and brackish microalgae. By comparing growth rates and lipid productivity, we assessed the potential of subtropical coastal and brackish microalgae for the production of biodiesel and other oil-based bioproducts. This study identified Nannochloropsis sp., Dunaniella salina and new isolates of Chlorella sp. and Tetraselmis sp. as suitable candidates for a multiple-product algae crop. We conclude that subtropical coastal microalgae display a variety of fatty acid profiles that offer a wide scope for several oil-based bioproducts, including biodiesel and omega-3 fatty acids. A biorefinery approach for microalgae would make economical production more feasible but challenges remain for efficient harvesting and extraction processes for some species.

Transcriptome for Photobiological Hydrogen Production Induced by Sulfur Deprivation in the Green Alga Chlamydomonas reinhardtii

ABSTRACT Photobiological hydrogen production using microalgae is being developed into a promising clean fuel stream for the future. In this study, microarray analyses were used to obtain global expression profiles of mRNA abundance in the green alga Chlamydomonas reinhardtii at different time points before the onset and during the course of sulfur-depleted hydrogen production. These studies were followed by real-time quantitative reverse transcription-PCR and protein analyses. The present work provides new insights into photosynthesis, sulfur acquisition strategies, and carbon metabolism-related gene expression during sulfur-induced hydrogen production. A general trend toward repression of transcripts encoding photosynthetic genes was observed. In contrast to all other LHCBM genes, the abundance of the LHCBM9 transcript (encoding a major light-harvesting polypeptide) and its protein was strongly elevated throughout the experiment. This suggests a major remodeling of the photosystem II light-harvesting complex as well as an important function of LHCBM9 under sulfur starvation and photobiological hydrogen production. This paper presents the first global transcriptional analysis of C. reinhardtii before, during, and after photobiological hydrogen production under sulfur deprivation.

Towards sustainable sources for omega-3 fatty acids production

Omega-3 fatty acids eicosapentaenoic acid (EPA) and docohexaenoic acid (DHA), provide significant health benefits for brain function/development and cardiovascular conditions. However, most EPA and DHA for human consumption is sourced from small fatty fish caught in coastal waters and, with depleting global fish stocks, recent research has been directed towards more sustainable sources. These include aquaculture with plant-based feeds, krill, marine microalgae, microalgae-like protists and genetically-modified plants. To meet the increasing demand for EPA and DHA, further developments are needed towards land-based sources. In particular large-scale cultivation of microalgae and plants is likely to become a reality with expected reductions in production costs, yield increasese and the adequate addressing of genetically modified food acceptance issues.

Cold-adapted yeasts from Antarctica and the Italian Alps—description of three novel species: Mrakia robertii sp. nov., Mrakia blollopis sp. nov. and Mrakiella niccombsii sp. nov.

Worldwide glaciers are annually retreating due to global overheating and this phenomenon determines the potential lost of microbial diversity represented by psychrophilic microbial population sharing these peculiar habitats. In this context, yeast strains, all unable to grow above 20°C, consisting of 42 strains from Antarctic soil and 14 strains isolated from Alpine Glacier, were isolated and grouped together based on similar morphological and physiological characteristics. Sequences of the D1/D2 and ITS regions of the ribosomal DNA confirmed the previous analyses and demonstrated that the strains belong to unknown species. Three new species are proposed: Mrakia robertii sp. nov. (type strain CBS 8912), Mrakia blollopis sp. nov. (type strain CBS 8921) and a related anamorphic species Mrakiella niccombsii sp. nov. (type strain CBS 8917). Phylogenetic analysis of the ITS region revealed that the new proposed species were closely related to each other within the Mrakia clade in the order Cystofilobasidiales, class Tremellomycetes. The Mrakia clade now contains 8 sub-clades. Teliospores were observed in all strains except CBS 8918 and for the Mrakiella niccombsii strains.

Ethylene response factor 6 is a regulator of reactive oxygen species signaling in Arabidopsis.

Reactive oxygen species (ROS) are produced in plant cells in response to diverse biotic and abiotic stresses as well as during normal growth and development. Although a large number of transcription factor (TF) genes are up- or down-regulated by ROS, currently very little is known about the functions of these TFs during oxidative stress. In this work, we examined the role of ERF6 (ETHYLENE RESPONSE FACTOR6), an AP2/ERF domain-containing TF, during oxidative stress responses in Arabidopsis. Mutant analyses showed that NADPH oxidase (RbohD) and calcium signaling are required for ROS-responsive expression of ERF6. erf6 insertion mutant plants showed reduced growth and increased H2O2 and anthocyanin levels. Expression analyses of selected ROS-responsive genes during oxidative stress identified several differentially expressed genes in the erf6 mutant. In particular, a number of ROS responsive genes, such as ZAT12, HSFs, WRKYs, MAPKs, RBOHs, DHAR1, APX4, and CAT1 were more strongly induced by H2O2 in erf6 plants than in wild-type. In contrast, MDAR3, CAT3, VTC2 and EX1 showed reduced expression levels in the erf6 mutant. Taken together, our results indicate that ERF6 plays an important role as a positive antioxidant regulator during plant growth and in response to biotic and abiotic stresses.

Phylogenetic and molecular analysis of hydrogen-producing green algae

A select set of microalgae are reported to be able to catalyse photobiological H2 production from water. Based on the model organism Chlamydomonas reinhardtii, a method was developed for the screening of naturally occurring H2-producing microalgae. By purging algal cultures with N2 in the dark and subsequent illumination, it is possible to rapidly induce photobiological H2 evolution. Using NMR spectroscopy for metabolic profiling in C. reinhardtii, acetate, formate, and ethanol were found to be key compounds contributing to metabolic variance during the assay. This procedure can be used to test algal species existing as axenic or mixed cultures for their ability to produce H2. Using this system, five algal isolates capable of H2 production were identified in various aquatic systems. A phylogenetic tree was constructed using ribosomal sequence data of green unicellular algae to determine if there were taxonomic patterns of H2 production. H2-producing algal species were seen to be dispersed amongst most clades, indicating an H2-producing capacity preceded evolution of the phylum Chlorophyta.

Cryptococcus statzelliae sp. nov. and three novel strains of Cryptococcus victoriae, yeasts isolated from Antarctic soils

A morphological and physiological characterization of yeast strains CBS 8908, CBS 8915, CBS 8920, CBS 8925(T) and CBS 8926, isolated from Antarctic soils, was performed. Phylogenetic analyses of the sequences of the D1/D2 regions and the adjacent internal transcribed spacer (ITS) regions of the large-subunit rDNA of these strains placed them into the Tremellales clade of the Hymenomycetes. The sequence data identified strains CBS 8908, CBS 8915 and CBS 8920 as belonging to the species Cryptococcus victoriae. Strains CBS 8925(T) and CBS 8926 were found to represent an unique clade within the Hymenomycetes, with Dioszegia crocea CBS 6714(T) being their closest phylogenetic relative. Fatty acid composition and proteome fingerprint data for these novel strains were also obtained. No sexual state was observed. A novel basidiomycetous species, Cryptococcus statzelliae, is proposed for strains CBS 8925(T) and CBS 8926.

Cryptococcus nyarrowii sp nov., a basidiomycetous yeast from Antarctica

In December 1997, 196 soil and snow samples were collected from Vestvold Hills, Davis Base, Antarctica. Two isolates, CBS 8804T (pink colonies) and CBS 8805 (yellow colonies), were shown by proteome analysis and DNA sequencing to represent the same species. Results from the sequencing of the D1/D2 region of the large rDNA subunit placed this species in the hymenomycetous tree in a unique sister clade to the Trichosporonales and the Tremellales. The clade consists of Holtermannia corniformis CBS 6979 and CBS strains 8804T, 8805, 8016, 7712, 7713 and 7743. Morphological and physiological characteristics placed this species in the genus Cryptococcus, with characteristics including the assimilation of D-glucuronate and myo-inositol, no fermentation, positive Diazonium blue B and urease reactions, absence of sexual reproduction and production of starch-like compounds. Fatty acid analysis identified large proportions of polyunsaturated lipids, mainly linoleic (C18:2) and, to a lesser extent, linolenic (C18:3) acids. On the basis of the physiological and phylogenetic data, isolates CBS 8804T and CBS 8805 are described as Cryptococcus nyarrowii sp. nov.