Extraction of microalgae derived lipids with supercritical carbon dioxide in an industrial relevant pilot plant

Microalgae are capable of producing up to 70% w/w triglycerides with respect to their dry cell weight. Since microalgae utilize the greenhouse gas CO₂, they can be cultivated on marginal lands and grow up to ten times faster than terrestrial plants, the generation of algae oils is a promising option for the development of sustainable bioprocesses, that are of interest for the chemical lubricant, cosmetic and food industry. For the first time we have carried out the optimization of supercritical carbon dioxide (SCCO₂) mediated lipid extraction from biomass of the microalgae Scenedesmus obliquus and Scenedesmus obtusiusculus under industrrially relevant conditions. All experiments were carried out in an industrial pilot plant setting, according to current ATEX directives, with batch sizes up to 1.3 kg. Different combinations of pressure (7–80 MPa), temperature (20–200 °C) and CO₂ to biomass ratio (20–200) have been tested on the dried biomass. The most efficient conditions were found to be 12 MPa pressure, a temperature of 20 °C and a CO₂ to biomass ratio of 100, resulting in a high extraction efficiency of up to 92%. Since the optimized CO₂ extraction still yields a crude triglyceride product that contains various algae derived contaminants, such as chlorophyll and carotenoids, a very effective and scalable purification procedure, based on cost efficient bentonite based adsorbers, was devised. In addition to the sequential extraction and purification procedure, we present a consolidated online-bleaching procedure for algae derived oils that is realized within the supercritical CO₂ extraction plant.

     

A QTL that confers resistance to Colorado potato beetle (Leptinotarsa decemlineata [Say]) in tetraploid potato populations segregating for leptine

Genetic resistance to Colorado potato beetle (Leptinotarsa decemlineata [Say]) from Solanum chacoense has been incorporated in the tetraploid potato selection, ND4382-19, which is highly resistant and contains moderate level of foliar leptines. We recently reported using ND4382-19 progeny, population ND5873 (ND4382-19 × Chipeta), to map two genes that segregated as complementary epistatic genes that allow accumulation of leptinidine (Lep) and acetyl-leptinidine (AL) on chromosomes 2 and 8, respectively. We describe here the characterization of a second half-sib population NDG116 (ND4382-19 × N142-72). In this population, solasodine from parent N142-72, which has Solanum berthaultii in its background, was predominant over solanidine-based alkaloids. Concentrations of solanidine, leptinidine, and acetyl-leptinidine were 15-, 5-, and 14-fold lower than in the ND5873 population. Nevertheless, Lep and AL mapped to the same locations on chromosomes 2 and 8 of parent ND4382-19, respectively. The two populations were evaluated for resistance to Leptinotarsa in field assays, and by detached leaf assay for population NDG116. In both families, QTL analysis identified a major QTL from ND4382-19 on the distal end of chromosome 2, close to the Lep locus. The contribution of this QTL to resistance ranged from 11 to 34% for ND5873 at four field sites. Contribution to resistance from the linkage group that contains the gene AL for the accumulation of leptine was not detected. In family NDG116, the same chromosome 2 QTL was detected for field and detached leaf assays, explaining 26 and 12% of the variance for defoliation and larval development, respectively. These data may indicate another resistance mechanism besides leptine in the Leptinotarsa resistance observed in these populations.     

The polysialic acid-specific O-acetyltransferase OatC from Neisseria meningitidis serogroup C evolved apart from other bacterial sialate O-acetyltransferases

Neisseria meningitidis serogroup C is a major cause of bacterial meningitis and septicaemia. This human pathogen is protected by a capsule composed of alpha2,9-linked polysialic acid that represents an important virulence factor. In the majority of strains, the capsular polysaccharide is modified by O-acetylation at C-7 or C-8 of the sialic acid residues. The gene encoding the capsule modifying O-acetyltransferase is part of the capsule gene complex and shares no sequence similarities with other proteins. Here, we describe the purification and biochemical characterization of recombinant OatC. The enzyme was found as a homodimer, with the first 34 amino acids forming an efficient oligomerization domain that worked even in a different protein context. Using acetyl-CoA as donor substrate, OatC transferred acetyl groups exclusively onto polysialic acid joined by alpha2,9-linkages and did not act on free or CMP-activated sialic acid. Motif scanning revealed a nucleophile elbow motif (GXS286XGG), which is a hallmark of alpha/beta-hydrolase fold enzymes. In a comprehensive site-directed mutagenesis study, we identified a catalytic triad composed of Ser-286, Asp-376, and His-399. Consistent with a double-displacement mechanism common to alpha/beta-hydrolase fold enzymes, a covalent acetylenzyme intermediate was found. Together with secondary structure prediction highlighting an alpha/beta-hydrolase fold topology, our data provide strong evidence that OatC belongs to the alpha/beta-hydrolase fold family. This clearly distinguishes OatC from all other bacterial sialate O-acetyltransferases known so far because these are members of the hexapeptide repeat family, a class of acyltransferases that adopt a left-handed beta-helix fold and assemble into catalytic trimers.     

Biphasic ethylene production during the hypersensitive response in Arabidopsis: A window into defense priming mechanisms?

The hypersensitive response (HR) is a cell death phenomenon associated with localized resistance to pathogens. Biphasic patterns in the generation of H₂O₂, salicylic acid and ethylene have been observed in tobacco during the early stages of the HR. These biphasic models reflect an initial elicitation by pathogen-associated molecular patterns followed by a second phase, induced by pathogen-encoded avirulence gene products. The first phase has been proposed to potentiate the second, to increase the efficacy of plant resistance to disease. This potentiation is comparable to the “priming” of plant defenses which is seen when plants display systemic resistance to disease. The events regulating the generation of the biphasic wave, or priming, remains obscure, however recently we demonstrated a key role for nitric oxide in this process in a HR occurring in tobacco. Here we use laser photoacoustic detection to demonstrate that biphasic ethylene production also occurs during a HR occurring in Arabidopsis. We suggest that ethylene emanation during the HR represents a ready means of visualising biphasic events during the HR and that exploiting the genomic resources offered by this model species will facilitate the development of a mechanistic understanding of potentiating/priming processes.Key words: hypersensitive response, biphasic patterns, potentiation, defense priming, ethylene, ArabidopsisThe Hypersensitive Response (HR) is a cell death process which occurs at the site of attempted pathogen attack and which has been associated with host resistance.¹ Much work on the regulation of the HR has indicated the importance of H₂O₂,² and NO.³ A feature of H₂O₂ generation during the HR is its biphasic pattern (Fig. 1A). The first rise reflects elicitation by pathogen-associated molecular patterns (PAMPs)⁴ and the second reflects the interaction between a pathogen-encoded avirulence (avr) gene product with a plant resistance (R) gene. A key aspect of the first rise is the initiation of salicylic acid (SA) synthesis which potentiates the second rise and hence the potency of plant defense and the HR.⁵Open in a separate windowFigure 1Patterns of defense signal generation during the Pseudomonas syringae pv. phaseolicola elicited-hypersensitive response in tobacco (Nicotiana tabacum). Generation of (A) H₂O₂ (●, Mur¹⁸); (B) nitric oxide (◇; Mur¹² (C) salicylic acid (SA, ■¹⁹) and (D) ethylene (○ Mur⁹) during a HR elicited by Pseudomonas syringae pv. phaseolicola (Psph) in tobacco cv. Samsun NN. In (A) a phase where SA acts to augment the second rise in H₂O₂—the potentiation phase—is highlighted. The potentiation phase is likely to be similar to defense “priming”.⁶ Methodological details are contained within the appropriate references. (E) A possible model for biphasic defense signal regulation during the Psph-elicited HR in tobacco. During an initial phase NO and H₂O₂ act to initiate SA biosynthesis, where SA and NO act to initiate a “H₂O₂ biphasic switch”. This could initially suppress both SA and the H₂O₂ generation but subsequently acts to potentiate a second phase of H₂O₂ generation. This in turn increases SA biosynthesis which could act with NO to initiate the “C₂H₄ biphasic switch” to potentiate ethylene production. These (and other) signals contribute to initiation of the HR and SAR.This potentiation mechanism appears to be similar to defense priming; when whole plants display systemic resistance to disease as opposed to a localized resistance against pathogens. Priming can be initiated (the “primary stimulus”) following attack with a necrotizing pathogen (leading to “systemic acquired resistance”, SAR) or non-pathogenic rhizosphere bacteria (to confer “induced systemic resistance”, ISR). In the primed state a plant stimulates a range of plant defense genes, produces anti-microbial phytoalexins and deposits cell wall strengthening molecules, but only on imposition of a “secondary stimulus”.⁶ Such secondary stimuli include SA³ or PAMPs⁷ and is likely to be mechanistically similar to the potentiation step in the biphasic pattern of H₂O₂ generation (shaded in Fig. 1A). Accordingly, the two phases in the biphasic wave represent primary and secondary stimuli in priming.Highlighting a similarity between local HR-based events and priming, adds further impetus to efforts aiming to describe the underlying mechanism(s), however both phenomena remain poorly understood. Besides SA, both jasmonates and abscisic acid (ABA) have been shown to prime defenses as have a range of non-plant chemicals, with β-aminobutyric acid (BABA) being perhaps most widely used.^6,8 Mutants which fail to exhibit BABA-mediated potentiation were defective in either a cyclin-dependent kinase-like protein, a polyphosphoinositide phosphatase or an ABA biosynthetic enzyme.⁸We have recently investigated biphasic ethylene production during the HR in tobacco elicited by the nonhost HR-eliciting bacterial pathogen Pseudomonas syringae pv. phaseolicola.⁹ As with H₂O₂ generation, this pattern reflected PAMP-and AVR-dependent elicitation events and included a SA-mediated potentiation stage. Crucially, we also showed that NO was a vital component in the SA-potentiation mechanism. When this finding is integrated with our other measurements of defense signal generation in the same host-pathogen system the complexity in the signaling network is revealed (Fig. 1). NO generation (Fig. 1B) appeared to be coincident with the first rise in H₂O₂ (Fig. 1A) which initiated SA biosynthesis^10,11 and together would contribute to the first small, but transient, rise in that hormone (Fig. 1C). In line with established models⁵ this momentary rise in SA coincides with the potentiation phase (shaded in Fig. 1A) required to augment the second rise in ROS. However, ethylene production seems to be correlated poorly with the patterns of NO, H₂O₂ and SA (Fig. 1D). Nevertheless, biphasic ethylene production was found to reflect PAMP and AVR-dependent recognition and included a SA-mediated potentiation step.⁹ Hence, ethylene production could be used as a post-hoc indicator of the potentiation mechanism. Therefore, our discovery that the second wave of ethylene production—a “biphasic switch”—is influenced by NO acting with SA could also be relevant to the H₂O₂ generation. Significantly, the second phases in both H₂O₂ and ethylene production occur exactly where SA and NO production coincides; in the case of H₂O₂ generation 2–4 h post challenge and with ethylene 6 h onwards (Fig. 1E).Thus, ethylene production represents a readily assayable marker to indicate perturbations in the underlying biphasic and possible priming mechanisms. As we have demonstrated, laser photoacoustic detection (LAPD) is a powerful on-line approach to determine in planta ethylene production in tobacco^9,12 but any mechanistic investigations would be greatly facilitated if the genetic resources offered by the model species Arabidopsis could be exploited.To address this, Arabidopsis Col-0 rosettes were vacuum infiltrated with either Pseudomonas syringae pv. tomato (Pst) avrRpm1 (HR-eliciting), the virulent Pst strain and the non-HR eliciting and non-virulent Pst hrpA strain. Ethylene production was monitored by LAPD (Fig. 2A). Significantly, Pst avrRpm1 initiated a biphasic pattern of ethylene production whose kinetics were very similar to that seen in tobacco (compare Figs. 2A with ​with1D).1D). Inoculations with Pst and Pst hrpA only displayed the first PAMP-dependent rise in ethylene production. Thus, these data establish that Arabidopsis can be used to investigate biphasic switch mechanism(s) in ethylene production during the HR and possibly defense priming. When considering such mechanisms, it is relevant to highlight the work of Foschi et al.¹³ who observed that biphasic activation of a monomeric G protein to cause phase-specific activation of different kinase cascades. Interestingly, ethylene has been noted to initiate biphasic activation of G proteins and kinases in Arabidopsis, although differing in kinetics to the phases seen during the HR.¹⁴ Further, plant defense priming has been associated with the increased accumulation of MAP kinase protein.⁶Open in a separate windowFigure 2Ethylene in the Pseudomonas syringae pv. tomato elicited-hypersensitive response in Arabidopsis thaliana. (A) Ethylene production from 5 week old short day (8 h light 100 µmol.m².sec⁻¹) grown Arabidopsis rosette leaves which were vacuum infiltrated with bacterial suspensions (2 × 10⁶ colony forming units.ml⁻¹) of Pseudomonas syringae pv. tomato (Pst) strains detected using laser photoacoustic detection (LAPD). Experimental details of the ethylene detection by LAPD are detailed in Mur et al.⁹ The intercellular spaces in leaves were infiltrated with the HR-eliciting strain Pst avrRpm1, (■), the virulent strain Pst (△) or the non-virulent and non-HR eliciting derivative, Pst hrpA (◇). (B) The appearance of Arabidopsis Col-0 and etr1-1 leaves at various h following injection with 2 × 10⁶ c.f.u.mL⁻¹ with of Pst avrRpm1. (C) Explants (1 cm diameter discs) from Arabidopsis leaf areas infiltrated with suspensions of Pst avrRpm1 were placed in a 1.5 cm diameter well, bathed in 1 mL de-ionized H₂O. Changes in the conductivity of the bathing solution, as an indicator of electrolyte leakage from either wild type Col-0 (◆), mutants which were compromised in ethylene signaling; etr1-1 (□), ein2-2 (▲) or which overproduced ethylene; eto2-1 (●) were measured using a conductivity meter. Methodological details are set out in Mur et al.⁹A further point requires consideration; the role of ethylene as a direct contributor to plant defense.¹⁵ The contribution of ethylene to the HR has been disputed,¹⁶ but in tobacco we have observed that altered ethylene production influenced the formation of a P. syringae pv. phaseolicola elicited HR.⁹ In Arabidopsis, cell death in the ethylene receptor mutant etr1-1 following inoculation with Pst avrRpm1 is delayed compared to wild type (Fig. 2B). When electrolyte leakage was used to quantify Pst avrRpm1 cell death, both etr1-1 and the ethylene insensitive signaling mutant ein2-1 exhibited slower death than wild-type but in the ethylene overproducing mutant eto2, cell death was augmented (Fig. 2C). These data indicate that ethylene influences the kinetics of the HR.Taking these data together we suggest that the complexity of signal interaction during the HR or in SAR/ISR could be further dissected by combining the genetic resources of Arabidopsis with measurements of ethylene production using such sensitive approaches as LAPD.     

Multimerisation of A disintegrin and metalloprotease protein-17 (ADAM17) is mediated by its EGF-like domain

A disintegrin and metalloprotease protein 17 (ADAM17) is a transmembrane zinc dependent metalloprotease. The catalytic activity of the enzyme results in the shedding of a broad range of membrane proteins. The release of the corresponding ectodomains induces a switch in various physiological and pathophysiological processes. So far there is not much information about the molecular mechanism of ADAM17 activation available. As for other transmembrane proteases, multimerisation may play a critical role in the activation and function of ADAM17. The present work demonstrates that ADAM17 indeed exists as a multimer in the cell membrane and that this multimerisation is mediated by its EGF-like domain.     

Changing Realities—Perspectives on Balinese Rice Cultivation

This paper discusses issues of agrarian change in south-central Bali. The proximity to urban areas, especially the tourist centers along the southern coast, provides many off-farm employment opportunities for small-scale farming households. Although rice farming continues, for many households it has become a side business. The flexible nature of rice farming in terms of labor input and available casual off-farm work allows farming households to allocate their available labor to a variety of on-farm and off-farm income generating activities. The subak which unites farmers in the irrigation and cultivation of the rice crop plays an important role in supporting this flexibility. Still, the future of rice farming and the organization behind it looks rather dim with a younger generation unwilling to work in the “mud” and little appreciation of the many benefits the subak provides not only to the farming but to the wider community.     

Links between plant and fungal communities across a deforestation chronosequence in the Amazon rainforest

Understanding the interactions among microbial communities, plant communities and soil properties following deforestation could provide insights into the long-term effects of land-use change on ecosystem functions, and may help identify approaches that promote the recovery of degraded sites. We combined high-throughput sequencing of fungal rDNA and molecular barcoding of plant roots to estimate fungal and plant community composition in soil sampled across a chronosequence of deforestation. We found significant effects of land-use change on fungal community composition, which was more closely correlated to plant community composition than to changes in soil properties or geographic distance, providing evidence for strong links between above- and below-ground communities in tropical forests.     

Temporal stability in forest productivity increases with tree diversity due to asynchrony in species dynamics

Theory predicts a positive relationship between biodiversity and stability in ecosystem properties, while diversity is expected to have a negative impact on stability at the species level. We used virtual experiments based on a dynamic simulation model to test for the diversity–stability relationship and its underlying mechanisms in Central European forests. First our results show that variability in productivity between stands differing in species composition decreases as species richness and functional diversity increase. Second we show temporal stability increases with increasing diversity due to compensatory dynamics across species, supporting the biodiversity insurance hypothesis. We demonstrate that this pattern is mainly driven by the asynchrony of species responses to small disturbances rather than to environmental fluctuations, and is only weakly affected by the net biodiversity effect on productivity. Furthermore, our results suggest that compensatory dynamics between species may enhance ecosystem stability through an optimisation of canopy occupancy by coexisting species.