Environmental and nutritional parameters influencing the growth of Skeletonema cultures and optimization of the large-scale production in photobioreactors

Microalgae have a large variety of applications and their biomass can be produced in a sustainable way. The diatom genus Skeletonema comprises a unique biochemical composition and is already widely used in the cultivation of shrimps and molluscs. The biomass of Skeletonema strains is rich in essential biomolecules, including fucoxanthin and polyunsaturated fatty acids and hold a high market demand. However, the large-scale production of this species is still challenging and needs to be optimised. In this context, different trials were conducted from lab- to industrial-scale and the growth of Skeletonema sp. cultures was observed and evaluated. In the laboratory, using Algem® photobioreactors (PBRs), obtained results revealed that autumn conditions are favourable over winter conditions, and cultures with lower inoculum concentrations show a higher specific growth rate. In addition, an initial silicate concentration of at least 0.11 mM favours the growth of the culture and the species growth is tolerant to pH values within 7.0 to 8.5. After the lab-scale optimization, cultures were successfully scaled-up under outdoor conditions, using flat panel PBRs of 100 - 1000 L and later to tubular PBRs up to 19000 L. In the production PBRs, it was found that nutrient starvation impairs the physiological state of cultures and diluted cultures suffer from photoinhibition by radiation maxima of 1500 μmol m-² s-1. A maximal daily growth rate of 0.331 d-1 was observed in a tubular PBR, exposed to an average radiation and temperature of 521 μmol m-² s-1 and 15.24 °C, respectively. The average macronutrient composition of Skeletonema sp. biomass as percentage of dry weight was 20.88 % carbohydrates, 31.52 % proteins and 18.14 % lipids. Overall, the lab-scale optimization and large-scale production of Skeletonema was successfully achieved and the biochemical composition of the biomass seems favourable for the nutrition of aquaculture organisms.Microalgas são os produtores primários e essenciais nos ecossistemas aquáticos. São organismos fotossintéticos eficientes. A sua biomassa tem uma variedade de aplicações e pode ser produzida de maneira sustentável. Dentro das microalgas existe o grupo único das diatomáceas, caracterizado pelo frústula de sílica. A espécie de diatomácea Skeletonema tem uma composição bioquímica única e é bastante usada na nutrição dos moluscos em cativeiro (por exemplo ostras), crustáceos e Artémia, que por sua vez são usados como fonte de alimento na aquacultura. A biomassa da estirpe Skeletonema é rica em biomoléculas valiosas como fucoxantina e ácidos gordos não saturados, de importância nutricional. A biomassa é importante comercialmente para a indústria cosmética e na área da medicina para o tratamento do cancro e doenças neurológicas. Algumas espécies de microalgas já são produzidas a grande escala, mas a produção da Skeletonema ainda demostra dificuldades. Dentro deste tópico, foram realizados diferentes ensaios, de escala laboratorial e outros de escala industrial, com o objetivo de otimizar a produção da biomassa da Skeletonema sp. O crescimento das culturas de Skeletonema sp. foi observado e avaliado por diferentes parâmetros, nomeadamente densidade ótica, peso seco e concentração celular. A medida de densidade ótica com uso de um espectrofotómetro é um método com protocolo bem estabelecido e rápido. Mas com algumas imprecisões, que se pode dever á pouca homogeneização da cultura ou as modificações fisiológicas da célula. O peso seco tem interesse industrial, pois representa a concentração da biomassa na cultura. Obtém-se através da filtração e secagem de um volume da cultura. A obtenção da concentração celular através da densidade ótica é uma medida mais precisa, pois só a espécie de cultivo é contada. Este método também pode medir sem precisão o peso seco quando o tamanho celular é variável. O estado fisiológico das células foi monitorizado através da observação ao microscópio e por fluorometria. A microscopia revelou que as células demonstravam estar em condição saudável, também foi observado de maneira direta a mudança na aparência. A habilidade fotossintética duma célula é avaliada pela fluorometria. O método é baseado na determinação do movimento dos eletrões durante os processos de oxidação e redução no fotossistema da célula. Após o recobro no escuro as células são expostas a uma intensidade de luz e pulso definido que ativam os aparatos fotossintéticos das células. Os parâmetros determinados foram a fluorescência mínima F0, que representa as células fotossintéticas ativas e a eficiência do fotossistema II Fv/Fm, um indicador de stress da cultura. A observação por fluorometria mostra o estado fisiológico de uma célula de maneira rápida e quantitativa. No laboratório, diferentes parâmetros de cultura foram testados com o uso de sistema de Algem®, para inferir sobre o crescimento em condições especificas da estirpe Skeletonema sp. Foram testados duas estripes específicas com as condições de temperatura e radiação da estação de outono e inverno. Num ensaio foi testado diferentes concentrações iniciais de inóculo valores de 2.105, 4.105, 6.105 e 1.106 células mL-1. Concentrações de silicatos no meio testadas foram desde 0.05, 0.11, 0.22 a 0.43 mM. Os valores fisiológicos de pH de interesse foram entre o 7.0 e 8.5. Os resultados demonstraram que diferentes estirpes têm diferentes padrões de crescimento, e que a condição mais favorável para o crescimento foi o outono. Uma cultura com um valor de inóculo mais baixo mostrou um valor de crescimento específico maior. O valor Fv/Fm numa cultura com 0.05 mM de silicatos no meio, foi significativamente mais baixo, indicando falta deste elemento criando stress na cultura de células, visto que a sílica é essencial para o crescimento das diatomáceas. Uma concentração inicial de pelo menos 0.11 mM favoreceu o crescimento das culturas. A estirpe demonstrou tolerância a valores de pH entre 7.0 e 8.5. Apos uma otimização de escala laboratorial, as culturas de Skeletonema sp. foram testadas com sucesso numa grande escala em condições ao ar livre, em fotobioreatores de painéis lisos de 100-1000 L. O processo de aumento para grande escala incluiu vários destes painéis, este processo demorou 28 a 73 dias. A biomassa foi produzida num fotobioreactor tubular de 10000L a 19000 L e foram armazenados em renovações semi-contínuas de meio de cultura. As culturas cresceram nos fotobioreatores tubulares até ao máximo 33 dias. O crescimento da Skeletonema sp. em fotobioreatores ao ar livre foi medida através da densidade ótica e do peso seco. A observação fluorométrica deu uma avaliação exata das culturas, mostrando enfraquecimento na função de aparatos fotossintéticos das células, equivalente ao stress da cultura. No aumento da escala e na produção em fotobioreatores, o valor Fv/Fm foi reduzido na falta de nutrientes ou quando culturas mais diluídas foram expostas a valores máximos de radiação de 1500 μmol m-² s-1. Culturas, que cresceram durante alguns dias em fotobioreatores ao ar livre não mostraram uma redução no valor Fv/Fm quando expostas a radiação, porém as culturas transferidas de laboratório há pouco tempo tendem a sofrer mais pela radiação súbita. A observação indicou a habilidade da Skeletonema sp. possui para se adaptar às condições de radiação. Um valor máximo de crescimento específico de 0.331 d-1, observado num fotobioreator tubular, durante uma radiação de 521 μmol m-² s-1 e temperatura de 15.24 °C. Este valor máximo foi similar aos valores médios de crescimento específico observado no sistema Algem®. A produtividade foi avaliada da seguinte forma: a biomassa recolhida é dividida pelo total por o tempo da produção e o volume do reator ou a sua área da produção. A média da produtividade volumétrica foi 0.034 g L.1 d.1 e a da produtividade por área é de 1.447 g m2 d-1. Os valores foram parecidos aos valores de um relatório sobre a cultivação da Skeletonema sp. na escala piloto ao ar livre. Valores da produtividade máxima foram calculados baseados na determinação diário do crescimento. A análise revelou uma grande variabilidade no próprio fotobioreator e entre os fotobioreatores conduzidos durante a época examinada. Em geral, as condições climáticas no local da produção suportaram o crescimento da Skeletonema sp. Em dias de radiação extremamente alta, as culturas devem ser sombreadas e arrefecidos com o objetivo de reduzir o efeito da fotoinibição. Por outro lado, o crescimento foi reduzido nos dias de baixa radiação. A produção de Skeletonema sp. tem de ser gerida da melhor forma para otimizar a produção da sua biomassa. A composição média dos macronutrientes da biomassa de Skeletonema sp., dado em percentagem do peso seco, foi 20.88 % carboidratos, 31.52 % proteínas e 18.14 % lípidos. A espécie pode ser considerada como um alimento rico na aquacultura. Seguido a literatura, Skeletonema sp. contem pigmentos e ácidos gordos com aplicações importantes. A microalga tem atividade anticancerígena e neuro-protetiva. Estes factos mostraram o potencial de aplicações de alto valor. Em suma, a otimização em ensaios laboratoriais e a produção em grande escala de Skeletonema sp. foi realizada com sucesso e a composição bioquímica da biomassa mostrou benefícios na alimentação e para a saúde de organismos aquáticos

Protein Activity Sensing in Bacteria in Regulating Metabolism and Motility

Bacteria have evolved complex sensing and signaling systems to react to their changing environments, most of which are present in all domains of life. Canonical bacterial sensing and signaling modules, such as membrane-bound ligand-binding receptors and kinases, are very well described. However, there are distinct sensing mechanisms in bacteria that are less studied. For instance, the sensing of internal or external cues can also be mediated by changes in protein conformation, which can either be implicated in enzymatic reactions, transport channel formation or other important cellular functions. These activities can then feed into pathways of characterized kinases, which translocate the information to the DNA or other response units. This type of bacterial sensory activity has previously been termed protein activity sensing. In this review, we highlight the recent findings about this non-canonical sensory mechanism, as well as its involvement in metabolic functions and bacterial motility. Additionally, we explore some of the specific proteins and protein-protein interactions that mediate protein activity sensing and their downstream effects. The complex sensory activities covered in this review are important for bacterial navigation and gene regulation in their dynamic environment, be it host-associated, in microbial communities or free-living

Seismo-Stratigraphy and Sedimentology of Holocene Sediments off Grande Rivière de la Baleine, Southeastern Hudson Bay, Québec

The regional distribution of Holocene sediments of eastern Hudson Bay off the Grande Rivière de la Baleine mouth was mapped using a grid of reflection seismic lines (approximately 300 km long and covering an area of approximately 800 km2) and data from 7 piston cores. Based on the seismic records and piston cores, 4 stratigraphic units overlying the Proterozoic bedrock (unit 1) were defined and interpreted: (unit 2) glacial till deposited by a westward flowing ice sheet, (unit 3) rhythmically bedded clays and silts presumably deposited in glacial Lake Ojibway, (unit 4) postglacial marine muds deposited in the Tyrrell Sea overlain by undifferentiated modern marine muds, and (unit 5) distal fluviodeltaic sediments from Grande Rivière de la Baleine. Similar stratigraphie units have been described onshore. Textural and geochemical analyses suggest that unit 3 rhythmites are true varves; dark "summer" laminae were deposited mainly by underflows during the open water season, and light "winter" laminae were deposited by overflows-interflows along thermal stratifications under a seasonal ice cover. Unit 5 covers approximately 400 km2 and occurs as a deltaic constructional wedge protruding as far as 11 km offshore of the Grande Rivière de la Baleine entrance with thicknesses reaching 30 m along the coast. It was deposited between 3500 BP and the present from remobilization of glacial sediments farther upstream due to river downcutting during emergence.La répartition régionale des sédiments holocènes de l'est de la baie d'Hudson, face à l'embouchure de la Grande rivière de la Baleine, a été cartographiée à l'aide d'un grille de profils de sismique réflexion (d'environ 300 km de long et d'une superficie de 800 km2) et de données provenant de sept forages par carottier à piston. Quatre unités stratigraphiques reposant sur le socle protérozoïque (unité 1) ont été définies et interprétées : un till (unité 2) déposé par un glacier progressant vers l'ouest, des argiles et silts stratifiés (unité 3) probablement déposés dans le Lac glaciaire Ojibway, des boues marines postglaciaires (unité 4) déposés dans la Mer de Tyrrell auxquelles se superposent des boues marines récentes indifférenciées et des sédiments fluvio-deltaiques distaux (unité 5) provenant de la Grande rivière de la Baleine. Les mêmes unités stratigraphiques ont été décrites sur la côte. Les analyses granulométriques et géochimiques laissent croire que les rythmites de l'unité 3 sont des varves; les couches foncées d'été ont surtout été déposées par des courants de densité s'écoulant sur le fond pendant la période libre de glace et les couches pâles d'hiver ont été transportés en suspension le long de stratifications thermiques sous un couvert de glace saisonnier. L'unité 5 couvre environ 400 km2 et forme un prisme s'étendant jusqu'à 11 km de l'embouchure de la Grande rivière de la Baleine avec des épaisseurs atteignant 30 m le long de la côte. La mise en place de l'unité 5 s'est faite entre 3500 BP et aujourd'hui et résulte de l'érosion et de l'apport de sédiments d'origine glaciaire provenant de l'amont durant l'émergence du chenal fluvial.Man hat die régionale Verteilung von Holozàn-Sedimenten der ôstlichen Hudson Bay in offener See gegenùber der Mùndung von Grande Rivière de la Baleine kartographiert, mit Hilfe eines Rasters der seismischen Reflexionslinien (etwa 300 km lang und ein Gebiet von etwa 800 km2 bedeckend) und der Daten aus 7 Kernen von Kolbenbohrungen. Vier ùber dem anstehenden Gestein des Protero-zoikums (Einheit 1) liegende stratigraphische Einheiten wurden bestimmt und interprétiez: (Einheit 2) glaziales Till, das von einer westwàrts flieBenden Eisdecke abge-lagert wurde, (Einheit 3) rhythmisch geschichteter Lehm und Schlamm, der wohl in dem glazialen Ojibwaysee abgelagert worden war, (Einheit 4) postglazialer Meeresschlamm, der in der Tyrrell-See abgelagert wurde und uber dem undifferenzierter neuzeitlicher Meeresschlamm liegt, und (Einheit 5) distale FluB-Deltasedimente von Grande Rivière de la Baleine. Àhnliche stratigraphische Einheiten sind am Strand beschrieben worden. Die Analysen von Textur und Geochemie lassen vermuten, daB Rhythmite der Einheit 3 echte Warwen sind; die dun-klen "Sommer"-Schichten wurden hauptsâchlich durch Unterstrômungen wàhrend der Saison offenen Wassers abgelagert, und die hellen "Winter"-Schichten wurden durch Ùberlauf-Zwischenlauf entlang der therma-len Schichtungen unter der saisonbedingten Eisdecke abgelagert. Einheit 5 bedeckt etwa 400 km2 und hat die Form eines deltaartigen Keils, der bis zu 11 km in die Mundung von Grande Rivière de la Baleine hineinragt und eine Dicke bis zu 30 m entlang der Kûste erreicht

Functional expression of TLR5 of different vertebrate species and diversification in intestinal pathogen recognition

Toll-like receptor 5 (TLR5) is activated by bacterial flagellins and plays a crucial role in the first-line defence against pathogenic bacteria and in immune homeostasis, and is highly conserved in vertebrate species. However, little comparative information is available on TLR5 functionality. In this study, we compared TLR5 activation using full-length and chimeric TLR5 of various vertebrate species (human, chicken, mouse, pig, cattle). Chimeric TLR5 receptors, consisting of human transmembrane and intracellular domains, linked to extracellular domains of animal origin, were generated and expressed. The comparison of chimeric TLR5s and their full-length counterparts revealed significant functional disparities. While porcine and chicken full-length TLR5s showed a strongly reduced functionality in human cells, all chimeric receptors were functional when challenged with TLR5 ligand Salmonella FliC. Using chimeric receptors as a tool allowed for the identification of ectodomain-dependent activation potential and partially host species-specific differences in response to various enteric bacterial strains and their purified flagellins. We conclude that both the extra- and intracellular determinants of TLR5 receptors are crucial for compatibility with the species expression background and hence for proper receptor functionality. TLR5 receptors with a common intracellular domain provide a useful system to investigate bacteria- and host-specific differences in receptor activation

Systematic site-directed mutagenesis of the Helicobacter pylori CagL protein of the Cag type IV secretion system identifies novel functional domains

The Cag Type IV secretion system, which contributes to inflammation and cancerogenesis during chronic infection, is one of the major virulence factors of the bacterial gastric pathogen Helicobacter pylori. We have generated and characterized a series of non-marked site-directed chromosomal mutants in H. pylori to define domains of unknown function of the essential tip protein CagL of the Cag secretion system. Characterizing the CagL mutants, we determined that their function to activate cells and transport the effector CagA was reduced to different extents. We identified three novel regions of the CagL protein, involved in its structural integrity, its possible interaction with the CagPAIT4SS pilus protein CagI, and in its binding to integrins and other host cell ligands. In particular two novel variable CagL motifs were involved in integrin binding, TSPSA, and TASLI, which is located opposite of its integrin binding motif RGD. We thereby defined functionally important subdomains within the CagL structure, which can be used to clarify CagL contributions in the context of other CagPAI proteins or for inhibition of the CagT4SS. This structure-function correlation of CagL domains can also be instructive for the functional characterization of other potential VirB5 orthologs whose structure is not yet known

Systematic site-directed mutagenesis of the Helicobacter pylori CagL protein of the Cag type IV secretion system identifies novel functional domains

The Cag Type IV secretion system, which contributes to inflammation and cancerogenesis during chronic infection, is one of the major virulence factors of the bacterial gastric pathogen Helicobacter pylori. We have generated and characterized a series of non-marked site-directed chromosomal mutants in H. pylori to define domains of unknown function of the essential tip protein CagL of the Cag secretion system. Characterizing the CagL mutants, we determined that their function to activate cells and transport the effector CagA was reduced to different extents. We identified three novel regions of the CagL protein, involved in its structural integrity, its possible interaction with the CagPAIT4SS pilus protein CagI, and in its binding to integrins and other host cell ligands. In particular two novel variable CagL motifs were involved in integrin binding, TSPSA, and TASLI, which is located opposite of its integrin binding motif RGD. We thereby defined functionally important subdomains within the CagL structure, which can be used to clarify CagL contributions in the context of other CagPAI proteins or for inhibition of the CagT4SS. This structure-function correlation of CagL domains can also be instructive for the functional characterization of other potential VirB5 orthologs whose structure is not yet known

PLoS Genet.

The Helicobacter pylori cag pathogenicity island (cagPAI) encodes a type IV secretion system. Humans infected with cagPAI-carrying H. pylori are at increased risk for sequelae such as gastric cancer. Housekeeping genes in H. pylori show considerable genetic diversity; but the diversity of virulence factors such as the cagPAI, which transports the bacterial oncogene CagA into host cells, has not been systematically investigated. Here we compared the complete cagPAI sequences for 38 representative isolates from all known H. pylori biogeographic populations. Their gene content and gene order were highly conserved. The phylogeny of most cagPAI genes was similar to that of housekeeping genes, indicating that the cagPAI was probably acquired only once by H. pylori, and its genetic diversity reflects the isolation by distance that has shaped this bacterial species since modern humans migrated out of Africa. Most isolates induced IL-8 release in gastric epithelial cells, indicating that the function of the Cag secretion system has been conserved despite some genetic rearrangements. More than one third of cagPAI genes, in particular those encoding cell-surface exposed proteins, showed signatures of diversifying (Darwinian) selection at more than 5% of codons. Several unknown gene products predicted to be under Darwinian selection are also likely to be secreted proteins (e.g. HP0522, HP0535). One of these, HP0535, is predicted to code for either a new secreted candidate effector protein or a protein which interacts with CagA because it contains two genetic lineages, similar to cagA. Our study provides a resource that can guide future research on the biological roles and host interactions of cagPAI proteins, including several whose function is still unknown

The SASSCAL contribution to climate observation, climate data management and data rescue in Southern Africa

A major task of the newly established "Southern African Science Service Centre for Climate Change and Adaptive Land Management" (SASSCAL; www.sasscal.org) and its partners is to provide science-based environmental information and knowledge which includes the provision of consistent and reliable climate data for Southern Africa. Hence, SASSCAL, in close cooperation with the national weather authorities of Angola, Botswana, Germany and Zambia as well as partner institutions in Namibia and South Africa, supports the extension of the regional meteorological observation network and the improvement of the climate archives at national level. With the ongoing rehabilitation of existing weather stations and the new installation of fully automated weather stations (AWS), altogether 105 AWS currently provide a set of climate variables at 15, 30 and 60 min intervals respectively. These records are made available through the SASSCAL WeatherNet, an online platform providing near-real time data as well as various statistics and graphics, all in open access. This effort is complemented by the harmonization and improvement of climate data management concepts at the national weather authorities, capacity building activities and an extension of the data bases with historical climate data which are still available from different sources. These activities are performed through cooperation between regional and German institutions and will provide important information for climate service related activities

ADP-heptose enables Helicobacter pylori to exploit macrophages as a survival niche by suppressing antigen-presenting HLA-II expression

The persistence of Helicobacter pylori in the human gastric mucosa implies that the immune response fails to clear the infection. We found that H. pylori compromises the antigen presentation ability of macrophages, because of the decline of the presenting molecules HLA-II. Here, we reveal that the main bacterial factor responsible for this effect is ADP-heptose, an intermediate metabolite in the biosynthetic pathway of lipopolysaccharide (LPS) that elicits a pro-inflammatory response in gastric epithelial cells. In macrophages, it upregulates the expression of miR146b which, in turn, would downmodulate CIITA, the master regulator for HLA-II genes. Hence, H. pylori, utilizing ADP-heptose, exploits a specific arm of macrophage response to establish its survival niche in the face of the immune defense elicited in the gastric mucosa

Novel Immunomodulatory Flagellin-Like Protein FlaC in Campylobacter jejuni and Other Campylobacterales

The human diarrheal pathogens Campylobacter jejuni and Campylobacter coli interfere with host innate immune signaling by different means, and their flagellins, FlaA and FlaB, have a low intrinsic property to activate the innate immune receptor Toll-like receptor 5 (TLR5). We have investigated here the hypothesis that the unusual secreted, flagellin-like molecule FlaC present in C. jejuni, C. coli, and other Campylobacterales might activate cells via TLR5 and interact with TLR5. FlaC shows striking sequence identity in its D1 domains to TLR5-activating flagellins of other bacteria, such as Salmonella, but not to nonstimulating Campylobacter flagellins. We overexpressed and purified FlaC and tested its immunostimulatory properties on cells of human and chicken origin. Treatment of cells with highly purified FlaC resulted in p38 activation. FlaC directly interacted with TLR5. Preincubation with FlaC decreased the responsiveness of chicken and human macrophage-like cells toward the bacterial TLR4 agonist lipopolysaccharide (LPS), suggesting that FlaC mediates cross-tolerance. C. jejuni flaC mutants induced an increase of cell responses in comparison to those of the wild type, which was suppressed by genetic complementation. Supplementing excess purified FlaC likewise reduced the cellular response to C. jejuni. In vivo, the administration of ultrapure FlaC led to a decrease in cecal interleukin 1β (IL-1β) expression and a significant change of the cecal microbiota in chickens. We propose that Campylobacter spp. have evolved a novel type of secreted immunostimulatory flagellin-like effector in order to specifically modulate host responses, for example toward other pattern recognition receptor (PRR) ligands, such as LPS. IMPORTANCE Flagellins not only are important for bacterial motility but are major bacterial proteins that can modulate host responses via Toll-like receptor 5 (TLR5) or other pattern recognition receptors. Campylobacterales colonizing the intestinal tracts of different host species harbor a gene coding for an unusual flagellin, FlaC, that is not involved in motility but is secreted and possesses a chimeric amino acid sequence composed of TLR5-activating and non-TLR5-activating flagellin sequences. Campylobacter jejuni FlaC activates cells to increase in cytokine expression in chicken and human cells, promotes cross-tolerance to TLR4 ligands, and alters chicken cecal microbiota. We propose that FlaC is a secreted effector flagellin that has specifically evolved to modulate the immune response in the intestinal tract in the presence of the resident microbiota and may contribute to bacterial persistence. The results also strengthen the role of the flagellar type III apparatus as a functional secretion system for bacterial effector proteins