Giuliana d’Ippolito

Potential of lipid metabolism in marine diatoms for biofuel production

BackgroundDiatoms are an ecologically relevant group of microalgae that are not commonly considered for bio-oil production even if they are responsible for massive blooms at sea. Seventeen diatom species were screened for their capacity to produce biomass and lipids, in relation to their growth rate. Triglyceride levels were also assessed as a preferential source of biofuels.ResultsUsing statistical analysis, two centric diatoms, Thalassiosira weissflogii and Cyclotella cryptica, were selected as good candidates for oil production. Lipid levels significantly increased when the two diatoms were cultivated in a two-stage process under nitrogen limitation. The effect was less pronounced in cultures where silicon was reduced to 20% of the standard supply. Nitrogen limitation did not affect growth rates but led to lipid remodeling and de novo synthesis of triacylglycerols.ConclusionsTriacylglycerols in T. weissflogii and C. cryptica can account for up to 82% and 88% of total glycerolipids, thereby suggesting that the two species are promising candidates for large-scale experimentation for biofuel production.

15S-Lipoxygenase metabolism in the marine diatom Pseudo-nitzschia delicatissima

In recent years, oxylipins (lipoxygenase-derived oxygenated fatty acid products) have been reported in several bloom-forming marine diatoms. Despite increasing attention on the ecophysiological role of these molecules in marine environments, their biosynthesis is largely unknown in these microalgae. Biochemical methods, including tandem mass spectrometry, nuclear magnetic resonance and radioactive probes were used to identify structures, enzymatic activities and growth-dependent modulation of oxylipin biosynthesis in the pennate diatom Pseudo-nitzschia delicatissima. Three major compounds, 15S-hydroxy-(5Z,8Z,11Z,13E,17Z)-eicosapentaenoic acid (15S-HEPE), 15-oxo-5Z,9E,11E,13E-pentadecatetraenoic acid and 13,14-threo-13R-hydroxy-14S,15S-trans-epoxyeicosa-5Z,8Z,11Z,17Z-tetraenoic acid (13,14-HEpETE), were produced by three putative biochemical pathways triggered by eicosapentaenoic acid-dependent 15S lipoxygenase. Oxylipin production increases along the growth curve, with remarkable changes that precede the demise of the culture. At least one of the compounds, namely 15-oxoacid, is formed only in the stationary phase immediately before the collapse of the culture. Synthesis and regulation of phyco-oxylipins seem to correspond to a signaling mechanism that governs adaptation of diatoms along the growth curve until bloom termination. Factors triggering the process are unknown but synthesis of 15-oxoacid, constrained within a time-window of a few days just before the collapse of the culture, implies the involvement of a physiological control not directly dependent on distress or death of diatom cells.

LIPOXYGENASE PRODUCTS IN MARINE DIATOMS: A CONCISE ANALYTICAL METHOD TO EXPLORE THE FUNCTIONAL POTENTIAL OF OXYLIPINS1

Oxylipins are oxygenated derivatives of polyunsaturated fatty acids (PUFAs) that act as chemical mediators in many ecological and physiological processes in marine and freshwater diatoms. The occurrence and distribution of these molecules are relatively widespread within the lineage with considerable species‐specific differences due to the variability of both the fatty acids recognized as substrates and the enzymatic transformations. The present review provides a general introduction to recent studies on diatom oxylipins and describes an analytical method for the detection and assessment of these elusive molecules in laboratory and field samples. This methodology is based on selective enrichment of the oxylipin fraction by solvent extraction, followed by parallel acquisition of full‐scan UV and tandem mass spectra on reverse phase liquid chromatography (LC) peaks. The analytical procedure enables identification of potential genetic differences, enzymatic regulation, and ecophysiological conditions that result in different oxylipin signatures, thus providing an effective tool for probing the functional relevance of this class of lipids in plankton communities. Examples of oxylipin measurements in field samples are also provided as a demonstration of the analytical potential of the methodology.

PLASTICITY OF OXYLIPIN METABOLISM AMONG CLONES OF THE MARINE DIATOM SKELETONEMA MARINOI (BACILLARIOPHYCEAE)1

Diatom oxylipins have been observed to deleteriously impact copepod reproductive success. However, field studies have revealed very variable and case‐dependent results. Therefore, the plasticity of diatom oxylipin metabolism was studied among four clones of the marine diatom Skeletonema marinoi Sarno et Zingone. Diatom oxylipin metabolism was studied by two lipoxygenase (LOX) activity assays carried out at different pH values and by oxylipin quantification. The four clones showed no major metabolic differences in terms of protein content or growth rate. However, two of the clones produced significantly higher levels of oxylipins than the other two. LOX activity measurements also indicated clonal variability in fatty acid oxidative metabolism. The presence of clone‐specific differences in oxylipin metabolism may play a role in shaping diatom population dynamics by conferring selective advantages to certain clones.

Specificity of Lipoxygenase Pathways Supports Species Delineation in the Marine Diatom Genus Pseudo-nitzschia

Oxylipins are low-molecular weight secondary metabolites derived from the incorporation of oxygen into the carbon chains of polyunsaturated fatty acids (PUFAs). Oxylipins are produced in many prokaryotic and eukaryotic lineages where they are involved in a broad spectrum of actions spanning from stress and defense responses, regulation of growth and development, signaling, and innate immunity. We explored the diversity in oxylipin patterns in the marine planktonic diatom Pseudo-nitzschia. This genus includes several species only distinguishable with the aid of molecular markers. Oxylipin profiles of cultured strains were obtained by reverse phase column on a liquid chromatograph equipped with UV photodiode detector and q-ToF mass spectrometer. Lipoxygenase compounds were mapped on phylogenies of the genus Pseudo-nitzschia inferred from the nuclear encoded hyper-variable region of the LSU rDNA and the plastid encoded rbcL. Results showed that the genus Pseudo-nitzschia exhibits a rich and varied lipoxygenase metabolism of eicosapentaenoic acid (EPA), with a high level of specificity for oxylipin markers that generally corroborated the genotypic delineation, even among genetically closely related cryptic species. These results suggest that oxylipin profiles constitute additional identification tools for Pseudo-nitzschia species providing a functional support to species delineation obtained with molecular markers and morphological traits. The exploration of the diversity, patterns and plasticity of oxylipin production across diatom species and genera will also provide insights on the ecological functions of these secondary metabolites and on the selective pressures driving their diversification.

Establishment of Genetic Transformation in the Sexually Reproducing Diatoms Pseudo-nitzschia multistriata and Pseudo-nitzschia arenysensis and Inheritance of the Transgene

We report the genetic transformation of the planktonic diatoms Pseudo-nitzschia arenysensis and Pseudo-nitzschia multistriata, members of the widely distributed and ecologically important genus Pseudo-nitzschia. P. arenysensis and P. multistriata present the classical size reduction/restitution life cycle and can reproduce sexually. Genetic transformation was achieved with the biolistic method, using the H4 gene promoter from P. multistriata to drive expression of exogenous genes. The transformation was first optimized introducing the Sh ble gene to confer resistance to the antibiotic zeocin. Integration of the transgene was confirmed by PCR and Southern blot analyses. Subsequently, we simultaneously transformed in P. arenysensis two plasmids, one encoding the β-glucuronidase (GUS) gene together with the plasmid carrying the Sh ble resistance gene, demonstrating the possibility of co-transformation. By transforming a gene encoding a fusion between the histone H4 and the green fluorescent protein (GFP), we demonstrated that fluorescent tagging is possible and that studies for protein localization are feasible. Importantly, we crossed P. arenysensis- and P. multistriata-transformed strains with a wild-type strain of opposite mating type and demonstrated that the transgene can be inherited in the F1 generation. The possibility to transform two diatom species for which genetic crosses are possible opens the way to a number of new approaches, including classical loss of function screens and the possibility to obtain different combinations of double transformants.

Autoinhibitory sterol sulfates mediate programmed cell death in a bloom-forming marine diatom

Cell mortality is a key mechanism that shapes phytoplankton blooms and species dynamics in aquatic environments. Here we show that sterol sulfates (StS) are regulatory molecules of a cell death program in Skeletonema marinoi, a marine diatom-blooming species in temperate coastal waters. The molecules trigger an oxidative burst and production of nitric oxide in a dose-dependent manner. The intracellular level of StS increases with cell ageing and ultimately leads to a mechanism of apoptosis-like death. Disrupting StS biosynthesis by inhibition of the sulfonation step significantly delays the onset of this fatal process and maintains steady growth in algal cells for several days. The autoinhibitory activity of StS demonstrates the functional significance of small metabolites in diatoms. The StS pathway provides another view on cell regulation during bloom dynamics in marine habitats and opens new opportunities for the biochemical control of mass-cultivation of microalgae.Phytoplankton blooms are shaped by a period of rapid growth followed by massive cell death. Here the authors show that sterol sulfates accumulate in aging cells of a bloom-forming marine diatom and trigger an oxidative burst that leads to a mechanism of apoptosis-like death.

Toxigenic effects of two benthic diatoms upon grazing activity of the sea urchin: morphological, metabolomic and de novo transcriptomic analysis

Diatoms are unicellular algae playing a key role as photosynthetic organisms in the world’s ocean food webs. The chemical ecology of planktonic diatoms is well documented, but few studies have reported on the effects of benthic diatoms on their consumers, also due to difficulties in the collection, quantification and massive culturing of benthic species. Here for the first time we investigate the effects of feeding on two abundantly occurring benthic diatoms, Nanofrustulum shiloi and Cylindrotheca closterium, isolated from the leaves of the seagrass Posidonia oceanica, on the sea urchin Paracentrotus lividus. Adult P. lividus were fed for one month on diets of either one of the two diatoms and on the green alga Ulva rigida, used as a feeding control. By combining morphological, metabolomic and de novo transcriptomic approaches, we demonstrate toxigenic effect on embryos generated by females fed with these benthic diatoms. Furthermore, chemical analysis reveal the presence of polyunsaturated aldehydes only for N. shiloi, and a high production of other oxylipins (cytotoxic compounds on their grazers and on cancer cell lines) for both diatoms, including some additional peaks not correlated to the canonic oxylipins commonly observed in planktonic diatoms. These findings open new perspectives in the study of diatom secondary metabolites influencing their grazers.

Potential of Hydrogen Fermentative Pathways in Marine Thermophilic Bacteria: Dark Fermentation and Capnophilic Lactic Fermentation in Thermotoga and Pseudothermotoga Species

Hydrogen is a clean energy vector that could help to face the current environmental issues of greenhouse gas emissions and, over a longer time scale, to replace the depleting nonrenewable fuels. Biological production by fermentation of waste and residues has the potential to surrogate the current technologies of production of this gas. In this chapter we report a summary of the fermentative pathways related to hydrogen production in the thermophilic microorganisms of the genera Thermotoga and Pseudothermotoga that embrace several marine species with the highest hydrogen yields among eubacteria. The contribution includes a brief review of dark fermentation (DF) and capnophilic lactic fermentation (CLF), the two processes related to hydrogen synthesis in these organisms, together with a discussion of new data concerning the distribution of CLF in these bacteria. The data show a varied scenario with different metabolic capabilities spread across the two genera. Under standard conditions, CLF is active only in few species of Thermotoga genus. The study underlines the great potential of these microbes in the valorization of agro-food waste and production of fuel and chemicals. In particular, the metabolic and biochemical diversity of Thermotoga and Pseudothermotoga species, together with their resilience to different environmental conditions, suggests the possibility to overtake many of the bottlenecks related to operational factors such as substrates, temperature, pH, hydraulic retention time, and hydrogen partial pressure.