Fed-batch process for the psychrotolerant marine bacterium Pseudoalteromonas haloplanktis

<p>Abstract</p> <p>Background</p> <p><it>Pseudoalteromonas haloplanktis </it>is a cold-adapted γ-proteobacterium isolated from Antarctic sea ice. It is characterized by remarkably high growth rates at low temperatures. <it>P. haloplanktis </it>is one of the model organisms of cold-adapted bacteria and has been suggested as an alternative host for the soluble overproduction of heterologous proteins which tend to form inclusion bodies in established expression hosts. Despite the progress in establishing <it>P. haloplanktis </it>as an alternative expression host the cell densities obtained with this organism, which is unable to use glucose as a carbon source, are still low. Here we present the first fed-batch cultivation strategy for this auspicious alternative expression host.</p> <p>Results</p> <p>The key for the fed-batch cultivation of <it>P. haloplanktis </it>was the replacement of peptone by casamino acids, which have a much higher solubility and allow a better growth control. In contrast to the peptone medium, on which <it>P. haloplanktis </it>showed different growth phases, on a casamino acids-containing, phosphate-buffered medium <it>P. haloplanktis </it>grew exponentially with a constant growth rate until the stationary phase. A fed-batch process was established by feeding of casamino acids with a constant rate resulting in a cell dry weight of about 11 g l^-1(OD₅₄₀= 28) which is a twofold increase of the highest densities which have been obtained with <it>P. haloplanktis </it>so far and an eightfold increase of the density obtained in standard shake flask cultures.</p> <p>The cell density was limited in the fed-batch cultivation by the relatively low solubility of casamino acids (about 100 g l^-1), which was proven by pulse addition of casamino acid powder which increased the cell density to about 20 g l^-1(OD₅₄₀= 55).</p> <p>Conclusion</p> <p>The growth of <it>P. haloplanktis </it>to higher cell densities on complex medium is possible. A first fed-batch fermentation strategy could be established which is feasible to be used in lab-scale or for industrial purposes. The substrate concentration of the feeding solution was found to influence the maximal biomass yield considerably. The bottleneck for growing <it>P. haloplanktis </it>to high cell densities still remains the availability of a highly concentrated substrate and the reduction of the substrate complexity. However, our results indicate glutamic acid as a major carbon source, which provides a good basis for further improvement of the fed-batch process.</p

SISTEM PENDUKUNG KEPUTUSAN POLA OLAHRAGA BERDASARKAN HASIL YANG INGIN DICAPAI MENGGUNAKAN FUZZY DATABASE MODEL TAHANI

Pada saat ini, perkembangan di dunia kesehatan telah berkembang secara cepat sehingga mendorong para ahli untuk merancang sebuah teknologi yang dapat mengambil keputusan didalam bidang kesehatan. Kesehatan merupakan hal sangat mahal dan sangat penting bagi keberlangsungan hidup manusia. Untuk mendapatkan tubuh yang sehat tentunya di butuhkan olahraga yang teratur. Olahraga dilakukan juga harus dengan porsi yang dibutuhkan oleh tubuh. Ketidaktahuan akan porsi olahraga yang dibutuhkan oleh tubuh manusia ini yang menjadi masalah bagi kebanyakan orang. Penelitian ini menggunakan metode studi literature dalam pengumpulan data serta fuzzy database model tahani. Pengembangan sistemnya menggunakan metode waterfall. Pemodelan analisis dan desain menggunakan bahasa pemograman PHP dan database server MySQL. Metode pengujian menggunakan pengujian white box.  Hasil penelitian ini adalah sebuah sistem pendukung keputusan pola olahraga berbasis website yang dapat memudahkan pengguna dalam menentukan pola olahraga yang cocok dilakukan sesuai data kriteria yaitu umur, berat badan dan tinggi badan

Proteomanalyse und Bioprozessentwicklung des psychrophilen, marinen Bakteriums Pseudoalteromonas haloplanktis TAC125

Das aus antarktischem Meereis stammende und auch bei geringen Temperaturen schnell wachsende γ Proteobakterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125) ist ein Modellorganismus für kälteangepasste Bakterien und Enzyme. Zusätzlich ist es ein alternativer Expressionswirt für die lösliche Überproduktion von heterologen Proteinen, die in etablierten Expressionswirten zur Bildung von inclusion bodies neigen. Bisher sind bei PhTAC125 im Rahmen der Erforschung von Kälteanpassungsmechanismen bzw. der Optimierung des kälteangepassten Expressionssystems nur Teilaspekte der Physiologie, des Stoffwechsels und der Bioprozessoptimierung untersucht worden. Bis zum Beginn dieser Dissertation gab es kaum Experimente, die sich mit der dynamischen und nahezu ganzheitlichen Betrachtung der Veränderung zellulärer Zustände und des Stoffwechsels von PhTAC125 beschäftigt haben. Darüber hinaus sind trotz der Fortschritte bei der Etablierung von PhTAC125 als alternativer Expressionswirt die bisher erzielten Biomassekonzentrationen gering. Aus diesem Grund wurden in dieser Dissertation zur Untersuchung des Stoffwechsels die exponentielle Wachstumsphase sowie vergleichende Untersuchungen verschiedener Wachstumsphasen und zellulärer Kompartimente auf Basis der Proteomanalytik durchgeführt. Zusätzlich konnte mit Hilfe der Fed-Batch-Kultivierungstechnik die Biomassekonzentration im Vergleich zu den herkömmlichen Methoden deutlich gesteigert werden. Zur Untersuchung der Physiologie und des Stoffwechsels von PhTAC125 während des exponentiellen Wachstums wurde das Proteom analysiert. Neben den typischen stark exprimierten Kategorien der exponentiellen Wachstumsphase wie Protein- u. Nukleotidbiosynthese, Aminosäure- u. Kohlenstoffmetabolismus, stellten sich vor allem die Proteine des TonB abhängigen Transportsystems (TBDT), sowie der Kategorien Entgiftung und Coenzyme für PhTAC125 als wichtig heraus. Das TBDT ist wegen seiner hohen Abundanz im Proteom und seiner potentiellen Beteiligung am Transport von Proteinabbauprodukten für PhTAC125 ähnlich bedeutend wie die anderen Kategorien mit einer hohen Anzahl stark exprimierter Proteine. Auch die Proteine zum Schutz vor ROS (reactive oxygen species) und die der Biosynthesewege der Coenzyme sind besondere und bedeutende Merkmale von PhTAC125. Der ROS Schutz ist bei kälteangepassten Bakterien während des Wachstums bei geringen Temperaturen (≤ 20°C) mit erhöhter Sauerstofflöslichkeit und der damit verbundenen verstärkten ROS-Bildung essentiell. Die Verfügbarkeit der meisten Biosynthesewege der Coenzyme im Proteom von PhTAC125 ist ein besonderes Charakteristikum gegenüber vielen anderen Wasser- und Bodenmikroorganismen und kennzeichnet einen potentiellen Wachstumsvorteil. Zur vergleichenden Untersuchung der unterschiedlichen zellulären Kompartimente von PhTAC125 wurden 2D-Gelbilder des Cyto- und Periplasmas erstellt und gegenübergestellt. Das periplasmatische Kompartiment war wesentlich durch Signalpeptid-haltige Proteine des TBDT, Porine und periplasmatische Peptidasen und Chaperone charakterisiert. Die Untersuchung der Proteomsignaturen unter Nährstofflimitationsbedingungen basierte auf dem Vergleich der späten exponentiellen und stationären Wachstumsphase mit der exponentiellen Wachstumsphase. Beide Wachstumsphasen waren durch Kategorien mit hoher Anzahl an gering exprimierten Proteinen dominiert. Dabei handelte es sich vor allem um die Kategorien der Nukleotid-, Protein- und RNS-Biosynthese. Diese potentiell reprimierten Kategorien der späten exponentiellen und stationären Wachstumsphase waren in Verbindung mit der Limitation der meisten Aminosäuren ein deutlicher Hinweis auf die stringent response. In diesem Zusammenhang schienen die stärker exprimierten Proteine (TBDT, Porine, Peptidasen/Protease und PilQ) positiv durch die stringent response eguliert zu sein, um das Überleben unter Nährstofflimitationbedingungen zu garantieren. Bei der Bioprozessoptimierung zur Steigerung der Biomassekonzentration von PhTAC125 wurden zwei verschiedene FB-Strategien durchgeführt. Bei der ersten Strategie wurde eine komplexe Aminosäurequelle (Casamino Acids) als Substrat eingesetzt und über konstante oder exponentielle Substratzufütterungsprofile eine optische Dichte (OD) von 30 erreicht. Im Vergleich zu den bisherigen in der Literatur beschriebenen Bioprozessen von PhTAC125 wurde die finale Biomassekonzentration 3 fach erhöht. Bei der zweiten FB-Strategie wurde ein „definierteres“ Substrat bestehend aus Glycerol und Glutamat für die Fütterungslösung eingesetzt. Mit einer anfänglichen exponentiellen gefolgt von einer konstanten Fütterungsrate konnte die Biomassekonzentration (OD = 86) gegenüber den veröffentlichen Ergebnissen 8 fach gesteigert werden. Zusammenfassend konnten erste proteombasierte Aussagen zur Physiologie und zum Stoffwechsel von PhTAC125 getroffen und erste Bioprozessstrategien zur gezielten Biomassesteigerung entwickelt werden.Pseudoalteromonas haloplanktis TAC125 (PhTAC125) is a cold-adapted γ-proteobacterium isolated from Antarctic sea ice. It is characterised by remarkably high growth rates at low temperatures and is one of the model organisms for cold-adapted bacteria and enzymes. In addition P. haloplanktis has been suggested as an alternative host for the soluble overproduction of heterologous proteins which tend to form inclusion bodies in established expression hosts. Until now only partial aspects of its physiology and metabolism have been investigated. For this reason one focus of this dissertation was the detailed analysis of proteomic signatures under exponential growth and nutrient starvation conditions. Despite the progress in establishing P. haloplanktis as an alternative expression host, the cell densities obtained with this organism are still low. Therefore the second focus of this work was the development of fed-batch cultivation strategies for this auspicious alternative expression host to reach high biomass concentrations. The analysis of PhTAC125s physiology and metabolism of the exponential growth phase was based on the investigation of the exponential growth on a soy-peptone supplemented artificial seawater medium at 16°C. The proteome of the exponential growth phase was characterised by a high number and abdundance of proteins of typical exponential growth dependent types like amino acid and carbohydrate metabolism as well as protein and nucleotide biosynthesis. The proteins of the TonB dependent transport system (TBDT) and of the categories detoxification and coenzymes seemed to be special metabolic features of the marine microbe during exponential growth. The high number and high abundance of the TBDT proteins in the proteome and their potential role in the transport of protein degradation products emphasised their great importance for PhTAC125. Despite a lower abundance of detoxifying enzymes, they had similar prominence. These enzymes were mainly involved in the protection against reactive oxygen species (ROS) of which number is drastically increased due to higher oxygen solubility at low temperatures. The enzymes for the coenzyme biosynthesis were additionally a special characteristic of the psychrotolerant marine bacterium. These biosynthesis pathways distinguished PhTAC125s growth advantage over many other water and soil microorganisms. During the exponential growth another focus was the proteome analysis of the differences between the cyto- and periplasmatic compartments. The periplasmatic compartment was dominated by a high number of signal peptide-sequence-containing proteins mainly by TBDT, porins and periplasmatic peptidases and chaperones. The most prominent categories were the TBDT and the peptidases. They play an important role in the import of nutrients into the periplasm and are necessary for their continuative transport into the cytoplasm. Therefore these proteins seemed to be essential for P. haloplanktis. For the analysis of proteomic signatures under nutrient starvation conditions the late exponential and stationary growth phase was compared with the exponential growth phase. Both growth phases were dominated by categories with high numbers of low expressed proteins. These categories particularly included nucleotides/nucleic acids biosynthesis, protein and RNA biosynthesis. These potential repressed categories of the late exponential and stationary phase, combined with the limitation of the most amino acids, were a distinct allusion to stringent response. In this connection the stronger expressed proteins (TBDT, porins, peptidases/proteases and PilQ) seemed to be positively regulated by sringent response to guarantee the survival under nutrient limitations. The content of the last part of the dissertation were experiments to increase the biomass concentration of P. haloplanktis. Two different fed batch strategies were tested to reach higher biomass yields. The first strategy was based on continuous feeding of a complex medium containing a complex amino acid source (casamino acids). This strategy resulted in a 3-fold increase of the optical density (OD540 = 30) compared to the biomass concentrations described in the literature. The second strategy was based on a more defined feeding solution with glycerol and glutamic acid as carbohydrate and nitrogen source. The initial exponential feeding was followed by a continuous one which finally resulted in an up to 8 fold increase of the optical density (OD540 = 86) compared to literature. In summary first proteome-based conclusions of the physiology and metabolism of P. haloplanktis during exponential growth and nutrient limitation conditions could be made. Furthermore two fed batch strategies for increasing biomass yields of PhTAC125 could be established

CFK/Titan, ein Hybridwerkstoff zur verbesserten Kopplung von Faserverbundstrukturen

Bei der Konstruktion von Bauteilen aus Kohlen-stofffaserverbundkunststoff (CFK) steht jeder Inge-nieur vor der Problematik der Kopplung der ver-schiedenen Baugruppen. In der Luft- und Raum-fahrt werden zu diesem Zweck in der Vielzahl Bol-zenverbindungen eingesetzt. Diese Methode ist aus der Metalltechnik bekannt und es liegen für diese Technik entsprechende Erfahrungen vor. Zudem ist diese Form der Verbindung fast überall anwendbar und erfordert nur einen geringen Zusatzaufwand. Allerdings stehen diesen Vorzügen Defizite in der Festigkeit der Laminate aufgrund der Kerbwirkung der Bohrungen gegenüber. Das Einbringen von Bohrungen und die Kraftübertragung über die Boh-rungsränder gilt als nicht fasergerecht. Folglich sind meist Verstärkungen bzw. Aufdickungen des Mate-rials lokal im Bereich der Verbindung erforderlich. Alternativ müsste die Festigkeit der Bolzenverbin-dung als Auslegungskriterium für das Bauteil he-rangezogen werden

CFRP/Titanium Hybrid Material Improving Composite Structure Coupling

One of the most important challenges during the development of composite structures is found in the design of structural interconnections. Although composite technology offers the advantage of reducing structural coupling by means of integral design and manufacturing techniques, joining parts to subassemblies remains unavoidable due to inspection, repair, material limitation, production methods and transportation requirements. One of the main methods currently used for joining components is mechanical fastening, which has the advantage of no special surface preparation requirements, easy disassembly and inspection and represents a reliable wellestablished and well-known method from its origin in the design of metallic structures. To realize the great potential of composites in lightweight aircraft structures, it is absolutely necessary to ensure that the joints do not reduce structural efficiency. This challenge is much more severe with composite materials than metals due to the notch sensitivity of composite materials, their brittleness and their reduced bearing strength and shear strength at highly anisotropic behaviour. That means, material advantages of high directional stiffness and strength cannot be exploited without disadvantages in weight: stress concentrations at holes require a local buildup thicker laminate which excites further local stresses due to eccentricities. This aspect is aggravated by the fact that joint efficiency decreases with increasing directional structure performance. Several design approaches have been developed in the past in view of optimising mechanical fastening of composite structures. One method of increasing mechanical joint efficiency consists in reinforcing the joining area with thin metal laminates, which provide a higher bearing strength and shear strength of the joint in comparison to pure composite material. Thus, both material performance and joint efficiency can be kept high. The suitability of Titanium as reinforcing material was researched at the Institute of structural mechanics of the German Aerospace Center (DLR). Extensive research work dealt with the compatibility of titanium and carbon composite material, some plies being replaced by thin titanium sheets, and was mainly focused in the investigation of the strength increase of bolted joints. Experimental results showed a significant gain of bearing strength and bolted joint strength, whereas interlaminar shear strength featured only a minimal decrease. A higher specific strength could also be achieved by relatively low titanium rates. Furthermore, an efficient design of the transition from pure composite to hybrid titanium composite material was developed, which enables the local use of hybrid titanium composite material in structure interconnections and force transmission points of composite structures

Hybrid Titanium Composite Material Improving Composite Structure Coupling

One of the most important challenges during the development of composite structures is found in the design of structural interconnections. Although composite technology offers the advantage of reducing structural coupling by means of integral design and manufacturing techniques, joining parts to subassemblies remains unavoidable due to inspection, repair, material limitation, production methods and transportation requirements. One of the main methods currently used for joining components is mechanical fastening, which has the advantage of no special surface preparation requirements, easy disassembly and inspection and represents a reliable wellestablished and well-known method from its origin in the design of metallic structures. To realize the great potential of composites in lightweight aircraft structures, it is absolutely necessary to ensure that the joints do not reduce structural efficiency. This challenge is much more severe with composite materials than metals due to the notch sensitivity of composite materials, their brittleness and their reduced bearing strength and shear strength at highly anisotropic behaviour. That means, material advantages of high directional stiffness and strength cannot be exploited without disadvantages in weight: stress concentrations at holes require a local buildup thicker laminate which excites further local stresses due to eccentricities. This aspect is aggravated by the fact that joint efficiency decreases with increasing directional structure performance. Several design approaches have been developed in the past in view of optimising mechanical fastening of composite structures. One method of increasing mechanical joint efficiency consists in reinforcing the joining area with thin metal laminates, which provide a higher bearing strength and shear strength of the joint in comparison to pure composite material. Thus, both material performance and joint efficiency can be kept high. The suitability of Titanium as reinforcing material was researched at the Institute of structural mechanics of the German Aerospace Center (DLR). Extensive research work dealt with the compatibility of titanium and carbon composite material, some plies being replaced by thin titanium sheets, and was mainly focused in the investigation of the strength increase of bolted joints. Experimental results showed a significant gain of bearing strength and bolted joint strength, whereas interlaminar shear strength featured only a minimal decrease. A higher specific strength could also be achieved by relatively low titanium rates. Furthermore, an efficient design of the transition from pure composite to hybrid titanium composite material was developed, which enables the local use of hybrid titanium composite material in structure interconnections and force transmission points of composite structures

Flow optimised design of a novel point-of-care diagnostic device for the detection of disease specific biomarkers

For the development of a novel, user-friendly and low cost point-of-care diagnostic device for the detection of disease specific biomarkers a flow optimised design of the test system is investigated. For this, computational fluid dynamics and rapid prototyping methods are used. The result is a functional design, which can be used for further experimental investigations

Design and Technology Development for a Composite Standard Body Fuselage

The goal of the DLR’s “Black Fuselage” project is to reduce the weight of the standard fuselage of a passenger aircraft by 30% and to cut its costs by 40% by using CFRP. In particular, higher safety standards in case of impact and crash are to be attained with the new structure. A CFRP design concept for a fuselage that takes into consideration the above-mentioned goals is discussed and a corresponding manufacturing method is presented here. Some of these construction ideas were displayed by a full-scale demonstrator that was presented at the International Aerospace Exhibition in Berlin in 2002 (ILA 2002)

New Design Concepts for an CFRP Fuselage

The goal for the next generation of aircraft in comparison to todays structures is to reduce the weight of the fuselage by 30% and manufacturing costs by 40%. This objective makes an advancement in technology necessary, and this is expected to happen with the switch from aluminium alloys to carbon fibre reinforced plastics (CFRP). Within the framework of an HGF (Helmholz-Gesellschaft) project that is being sponsored by BMBF (Federal Ministry of Education and Research), intensive work on a black fuselage is being carried out at the German Aerospace Center (DLR) and the Forschungszentrum Karlsruhe (FZK). One goal of this project was the realization of the full-scale demonstration structure of a CFRP fuselage with a new design concept that has been adapted to the material by means of more economical manufacturing technologies. The manufactured section of a typical fuselage structure therefore reflects importantinsights and results of this project