Michele S. Stanley

Effects of temperature and nutrient regimes on biomass and lipid production by six oleaginous microalgae in batch culture employing a two-phase cultivation strategy

Commercial success of algal-based biofuels depends on growth characteristics and lipid metabolism of the production species. The oleaginous microalgae, Thalassiosira pseudonana, Odontella aurita, Nannochloropsis oculata, Isochrysis galbana, Chromulina ochromonoides, and Dunaliella tertiolecta, were cultivated under a matrix of two temperatures (10 and 20 °C) and two nutrient regimes (deplete and replete). For all species, a strong negative correlation between growth rate and lipid content was observed. Multiple stressors have no additive effect on lipid accumulation. Total oil content (fatty acid methyl esters, FAMEs, pg cell(-1)) was increased more by nutrient limitation than by temperature stress. In response to nutrient stress, N. oculata emerged as the most robust species with an increase in lipid accumulation of up to three to four-fold compared to the accumulation under nutrient sufficient conditions. Although stress conditions led to reduced fatty acid unsaturation in most taxa due to increased triacylglycerol (TAG) production, a high proportion of eicosapentaenoic acid (EPA) was maintained in O. aurita.

Biogas from Macroalgae: is it time to revisit the idea?

The economic and environmental viability of dedicated terrestrial energy crops is in doubt. The production of large scale biomass (macroalgae) for biofuels in the marine environment was first tested in the late 1960’s. The culture attempts failed due to the engineering challenges of farming offshore. However the energy conversion via anaerobic digestion was successful as the biochemical composition of macroalgae makes it an ideal feedstock. The technology for the mass production of macroalgae has developed principally in China and Asia over the last 50 years to such a degree that it is now the single largest product of aquaculture. There has also been significant technology transfer and macroalgal cultivation is now well tried and tested in Europe and America. The inherent advantage of production of biofuel feedstock in the marine environment is that it does not compete with food production for land or fresh water. Here we revisit the idea of the large scale cultivation of macroalgae at sea for subsequent anaerobic digestion to produce biogas as a source of renewable energy, using a European case study as an example.

Overcoming biological constraints to enable the exploitation of microalgae for biofuels

Microalgae have significant potential to form the basis of the next biofuel revolution. They have high growth and solar energy conversion rates. Furthermore, their osmotolerance, metabolic diversity and capacity to produce large amounts of lipids have attracted considerable interest. Although there are a handful of commercially successful examples of the photoautotrophic mass-culture of algae, these have focused on the production of higher value products (pigments, health-foods etc.). The technical and commercial challenges to develop an economically viable process for biofuels are considerable and it will require much further R&D. In this paper the biological constraints, with a particular focus on strain selection are discussed.

A rapid and general method for measurement of protein in micro-algal biomass

A convenient small-scale extraction method for lyophilized micro-algae is described that dispenses with labor-intensive homogenization and is widely applicable to algae from different phyla. The procedure employs an optimized sequential extraction in trichloroacetic acid (TCA) and NaOH to achieve chemical lysis. Conditions were tested using several micro-algal strains to develop a method that was generally applicable. Incubation of lyophilized material in 24% (w/v) TCA at 95 °C followed by a hot alkaline treatment was found to be effective for strains that are resistant to conventional extraction approaches, such as the Chlorella and the Eustigmatophycean species. The single-tube extraction procedure can be complete in 4h and is conveniently followed by the Lowry assay, requiring a further 30 min. Overall, this method proved to be generally applicable and ideal either for single samples or for high-throughput screening of multiple algal strains for protein content.

Inhibition of fungal spore adhesion by zosteric Acid as the basis for a novel, nontoxic crop protection technology.

ABSTRACT To explore the potential for nontoxic crop protection technologies based on the inhibition of fungal spore adhesion, we have tested the effect of synthetic zosteric acid (p-(sulfo-oxy) cinnamic acid), a naturally occurring phenolic acid in eelgrass (Zostera marina L.) plants, on spore adhesion and infection in two pathosystems: rice blast caused by Magnaporthe grisea and bean anthracnose caused by Colletotrichum lindemuthianum. We have shown that zosteric acid inhibits spore adhesion to model and host leaf surfaces and that any attached spores fail to develop appressoria, and consequently do not infect leaf cells. Low concentrations of zosteric acid that are effective in inhibiting adhesion are not toxic to either fungus or to the host. The inhibition of spore adhesion in the rice blast pathogen is fully reversible. On plants, zosteric acid reduced (rice) or delayed (bean) lesion development. These results suggest that there is potential for novel and environmentally benign crop protection technologies based on manipulating adhesion.

Cellular and molecular approaches to understanding primary adhesion in Enteromorpha: an overview

The attachment of motile spores of the green alga Enteromorpha to the substratum is an active process involving an irreversible commitment to adhesion and the secretion of an adhesive. This paper provides an overview of the spore adhesion processes and outlines the results of an experimental approach towards the molecular characterisation of the adhesive, based on the use of monoclonal antibody (mAb) technology. Hybridomas were produced to settled spores displaying secreted adhesive. Candidates producing mAbs to putative adhesive were selected using a range of criteria based on cellular localisation, time of secretion and functional inhibition of adhesion. MAb Ent 6 immunolabelled fibrillar material which was secreted during the early stages of adhesion and low (nM) concentrations of this mAb, or its F(ab)2 fragments, strongly inhibited the attachment of zoospores. A related antibody (Ent 1) also labelled the spore adhesive apparatus, but the antigen appeared to be secreted later during the adhesion process and was predominantly associated with the developing cell wall. Ent 1 also inhibited settlement in spore adhesion assays but the effect was most pronounced at later time points which suggests that this antigen does not have a role in the earliest stages of adhesion. Immunolocalisation showed that both antigens were absent from the cytoplasm or organelles of vegetative tissue but labelled the vegetative cell wall, suggesting a relationship between cell wall components and materials involved in primary adhesion. Both mAbs labelled the Golgi region of settled spores, suggesting continued synthesis of both antigens after adhesion. Both mAbs recognised a 110 kDa N‐linked polydisperse and heterogeneous glycoprotein in extracts of swimming spores under denaturing conditions. In native form the antigens behaved as high molecular weight aggregates (Mr>1.3 × 106). The antigens became progressively insoluble after zoospore attachment. Taken together, the data suggest that the two antibodies recognise closely related, polydisperse, self‐aggregating cell wall glycoproteins in which there is some structural variation to suit alternative roles in primary adhesion and cell wall formation. The two mAbs Ent 1 and Ent 6 partially discriminate between these structural and functional variants. A model for zoospore adhesion is discussed in which adhesion is viewed as an extension of cell wall synthesis, with cross‐links between glycoproteins and other cell wall matrix components providing a strong physical continuum between the cell and the adhesive at the substratum interface.

Unlocking nature’s treasure-chest: screening for oleaginous algae

Micro-algae synthesize high levels of lipids, carbohydrates and proteins photoautotrophically, thus attracting considerable interest for the biotechnological production of fuels, environmental remediation, functional foods and nutraceuticals. Currently, only a few micro-algae species are grown commercially at large-scale, primarily for “health-foods” and pigments. For a range of potential products (fuel to pharma), high lipid productivity strains are required to mitigate the economic costs of mass culture. Here we present a screen concentrating on marine micro-algal strains, which if suitable for scale-up would minimise competition with agriculture for water. Mass-Spectrophotometric analysis (MS) of nitrogen (N) and carbon (C) was subsequently validated by measurement of total fatty acids (TFA) by Gas-Chromatography (GC). This identified a rapid and accurate screening strategy based on elemental analysis. The screen identified Nannochloropsis oceanica CCAP 849/10 and a marine isolate of Chlorella vulgaris CCAP 211/21A as the best lipid producers. Analysis of C, N, protein, carbohydrate and Fatty Acid (FA) composition identified a suite of strains for further biotechnological applications e.g. Dunaliella polymorpha CCAP 19/14, significantly the most productive for carbohydrates, and Cyclotella cryptica CCAP 1070/2, with utility for EPA production and N-assimilation.

Comparison of screening methods for high-throughput determination of oil yields in micro-algal biofuel strains.

The phenotypic and phylogenetic diversity of micro-algae capable of accumulating triacylglycerols provides a challenge for the accurate determination of biotechnological potential. High-yielding strains are needed to improve economic viability and their compositional information is required for optimizing biodiesel properties. To facilitate a high-throughput screening programme, a very rapid direct-derivatization procedure capable of extracting lyophilized material for GC analysis was compared with a scaled-down Folch-based method. This was carried out on ten micro-algal strains from 6 phyla where the more rapid direct-derivatization approach was found to provide a more reliable measure of yield. The modified Folch-based procedure was found to substantially underestimate oil yield in one Chlorella species (P < 0.01). In terms of fatty acid composition however, the Folch procedure proved to be slightly better in recovering polyunsaturated fatty acids, in six out of the ten strains. Therefore, direct-derivatization is recommended for rapid determination of yields in screening approaches but can provide slightly less compositional accuracy than solvent-based extraction methods.

Biofouling community composition across a range of environmental conditions and geographical locations suitable for floating marine renewable energy generation

Abstract Knowledge of biofouling typical of marine structures is essential for engineers to define appropriate loading criteria in addition to informing other stakeholders about the ecological implications of creating novel artificial environments. There is a lack of information regarding biofouling community composition (including weight and density characteristics) on floating structures associated with future marine renewable energy generation technologies. A network of navigation buoys were identified across a range of geographical areas, environmental conditions (tidal flow speed, temperature and salinity), and deployment durations suitable for future developments. Despite the perceived importance of environmental and temporal factors, geographical location explained the greatest proportion of the observed variation in community composition, emphasising the importance of considering geography when assessing the impact of biofouling on device functioning and associated ecology. The principal taxa associated with variation in biofouling community composition were mussels (Mytilus edulis), which were also important when determining loading criteria.

Assessment of saccharification and fermentation of brown seaweeds to identify the seasonal effect on bioethanol production

Brown seaweeds such as the kelps are attractive sources of biomass for bioethanol production, but fully optimised saccharification and fermentation conditions have yet to be established. To address this, various saccharification methods using dilute and concentrated acid and enzymes were trialled on three kelp species, Laminaria digitata, Laminaria hyperborea and Saccharina latissima, collected through a full seasonal cycle. Enzymatic hydrolysates were then fermented using Saccharomyces cerevisiae and Pichia angophorae to identify seasonal variations in ethanol yields. Highest glucose yields were achieved using concentrated acid, followed by enzymatic and dilute acid saccharification, respectively. The effect of seasonality showed that the highest glucose and ethanol yields were from kelps harvested during the autumn months and lowest during winter and spring months. However, the season at which biomass was collected did not have any measurable impact on the method of saccharification. Differences in ethanol yields between seaweed species were found with P. angophorae producing more ethanol from L. digitata and L. hyperborea hydrolysates, whilst S. cerevisiae was better for fermentation of S. latissima hydrolysates. It was observed that ethanol conversion yields with S. cerevisiae were higher than the theoretical maximum based on the yield of glucose identified, suggesting that other sugars in addition to glucose were co-fermented. For glucose liberation from seaweeds, terrestrial-derived cellulose and hemicellulose enzyme blends were suitable, but for liberation of all sugar monomers from seaweed polymers, other hydrolytic enzymes need to be investigated. In addition, fermentative microorganisms that are more tolerant of salinity and polyphenols are still required and ideally, be strains that can be engineered to ferment all carbohydrate sources present in kelps.