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- PostStudy of the Bioaugmentation of Grease Separators Using the GOR BioSystem™(2014) De Santa Izabel Alves, Aline; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological EngineeringGrease separators (GS) are the first preventive strategy to reduce the entry of fats, oils and grease (FOG) into the sewage system. Since its patent in 1883, very little has been developed in order to improve the function and maintenance of these devices. One of the strategies to upgrade the GS performance is through the use of bioaugmentative systems. However,limited scientific-based knowledge of the performance and bioaugmentation of GS is available to promote sound regulations regarding the use, maintenance and improvement of GSs in Sweden. Thus, an important consideration of this study was to evaluate the function of accepted GSs and the effects of bioaugmentative systems in its performance. Grease separators from three different food-producing establishments (FSEs) (one restaurant from an elementary school and two full service restaurants) located in Danderyd and Stockholm municipalities were evaluated. The GS of one of the full service restaurants was treated with a bioaugmentative system (GOR BioSystem™) and monitored for changes in the effluent quality and FOG accumulation compared to an untreated control period. This evaluation consisted of two cycles, lasting approximately one month (five weeks) each, which was in accordance to the regulation for the intervals between the pump outs in the Stockholm region. The GS from an elementary school in Danderyd municipality was treated and monitored for ten weeks with the same bioaugmentative system (GOR BioSystem™) while one untreated standard GS from a full service restaurant in Stockholm was monitored for five weeks as a control. Regular analysis including FOG (either ether soluble, emulsified and separable fat), biochemical oxygen demand, chemical oxygen demand, total suspended solids, pH,temperature, conductivity, grease cap thickness and bottom solids accumulation and odor was performed. In general, the chemical and physical parameters were similar in both treated and untreated cycles in the full service restaurant analyzed with and without the treatment with GOR BioSystemTM. However, lower levels of grease cap development and solids accumulation and odor were achieved in the treated cycle. No grease accumulation was detected in the GS bioaugmented for ten weeks while a considerable amount of both grease and solids accumulated in the control untreated GS after 25 activity days. There were no indications that the bioaugmentation promoted by GOR BioSystem™ led to a deterioration of the effluent quality or released additional amounts of oil and greases in the collection system. Furthermore, it is expected that the use of GOR BioSystem™ can improve the rationalization of the handling of organic waste with the reduction of waste transports.
- PostA platform for Acetyl-CoA synthesis using xylose as feedstock in Saccharomyces cerevisiae(2015) Englund Örn, Oliver; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological EngineeringGlobal rise in temperature and diminishing oil reserves has stimulated a market of alternative replacements to traditional petroleum based products. An alternative is the use of a bio refinery capable of converting biomass to the normally petroleum based products. The baker yeast Saccharomyces cerevisiae is an attractive cell factory as its existing large-scale infrastructures for bioethanol production. However, it cannot utilize xylose, an otherwise unusable part of the plant biomass, which represents of utmost importance in the bio-refinery development. To also have a strain capable to produce a wide range of products, it could be used as a platform to base a bio refinery upon. Therefore, the aim of this study is to generate platform strains capable of forming acetyl-CoA, an intermediate metabolite in many of the cells metabolic reactions and also for many other industrially relevant bio-chemicals. With this goal in mind, the metabolism of S. cerevisiae was engineered. The genes encoding an isomerase-based xylose assimilation pathway (RTG, XI, XKS), and a phosphoketolase pathway (XPK, PTA), were cloned into the yeast strain CEN.PK113-5D to enable the yeast to take up and convert xylose into acetyl-CoA. The functionality of this synthetic pathway were evaluated for the production of 3-hydroxypropionic acid via introduction of ACC1** and MCR genes into the engineered strains. By characterisation of all the engineered strains on glucose growth we found increase of acetate production in strains with the phosphoketolase pathway expressed, indicating the in vivo activity of this pathway. However, expression of the xylose assimilation pathway through genome integration did not render the strains able to grow on xylose, suggesting the low efficiency of the assembled xylose assimilation pathway. To overcome this adaptive laboratory evolution is recommended.
- PostEvaluation of new methods to determine antimicrobial efficiency(2015) Lancon, Oceane; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological EngineeringThe work for this master's thesis, which was performed at Medibiome, was a part of a Vinnova-funded national project “Innovation mot Infektion” for the establishment and evaluation of a draft standard FprEN 16615 in order to evaluate different disinfection and analytical methods. Healthcare associated infections are a global issue and the need for new methods to reduce contamination and evaluate efficiency of disinfection is increasingly recognized in scientific, clinical and political communities. In order to evaluate the antibacterial efficiency of cleaning and disinfecting products, test floorings were inoculated with bacterial suspensions and an interfering substance, and wiped with a product. A unitary weight was used to standardize the wiping. The bacteria load on the surface was swabbed after wiping and the remaining bacterial load was evaluated after plate cultivation.The ATP bioluminescence analysis was used during a similar procedure to replace the plate cultivation method. The enzyme luciferase provided light emission while degrading the ATP contained into bacteria. The light emission is measured to determine the bacterial load on the surface. The compatibility of wiping materials and cleaning agents was also tested with a test suspension of S. aureus. The recovery of S.aureus and P. aeruginosa after different drying times on surfaces was studied. The results show that three disinfectants meet the requirements of the standard FprEN 1661: DES45, Rely+On™ Virkon® and Wet Wipe. Input was transmitted to the standard committee, especially regarding the need to sterilize the wipes, to standardize and improve the release of bacteria from the swab, and to decrease the weight of the granite block for more clinical relevance. ATP analysis could be performed without background by using an interfering substance modified with Triton X-100 at 0.2%. It seems to be a promising technique, as it is both fast and cost-efficient but further study needs to be done in order to solve technical issues and standardize the method
- PostMethod Development for Measuring Cleaning Agent Efficiency at a Laboratory Scale. Cleaning agent efficiency in dissolution of UHT fouling at different concentrations and temperatures.(2015) Hulting, Fredrik; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological EngineeringIn the milk industry, the production must be stopped daily in order to clean the process equipment from deposits that are formed at the inside surface when the products are heat treated. This leads reduced production time since no products can be treated when the equipment are being cleaned. The heat treatment of milk products is necessary to ensure that the product is safe to consume. When heat treating the milk, certain compounds in the milk become unstable and form a deposit called fouling inside the heating equipment. This fouling layer reduces the heat transfer from the heat equipment to the product that is supposed to be heat treated. In order to remove the fouling deposit cleaning with first alkaline and then acidic detergent are used. It can take up to a few hours before the equipment is cleaned depending on the degree of fouling. The aim of this master’s thesis is to develop a laboratory method for measuring cleaning efficiency of UHT fouling by using cleaning detergents at different temperatures and concentrations. The method was created by looking on how a Cleaning-In-Place process is performed and imitates these steps at a laboratory level, by performing experiments on storage, drying, size and separation of fouling. The cleaning method was evaluated according to repeatability and ability to distinguish cleaning effects of the different cleaning liquids. A cleaning method for dissolution of UHT fouling was achieved where it was possible to detect trends of degree of dissolved fouling when different temperature and concentration of cleaning liquids were used in the sodium hydroxide step. The repeatability of the cleaning method was sufficient to be able to detect the cleaning trends. The cleaning effect trials showed that higher temperature and concentration leads to more fouling dissolved. It was also seen that with higher concentration of alkaline detergent a lower temperature could be used to achieve a similar cleaning compared to lower concentration of alkaline detergent.
- PostCharacterizing plasmids from a nosocomial outbreak using optical DNA mapping(2015) Müller, Vilhelm; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological Engineering
- PostDevelopment and evaluation of an immunoassay for Keratin 5/19 complex in lung cancer(2015) Andersson, Sofia; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological Engineering14 antibody combinations were evaluated as candidates for a keratin 5/19 enzymeimmunoassay. By enzyme immune assay and western blot, 1A12+Ks19.1 was determined as the superior combination. The most successful test assay was the two-step enzyme immunoassay with 2 hours incubation time for the biotinylated antibody and 1 hour incubation for the HRPconjugated antibody. The experiments used to evaluate antibody combinations also indicates a structural difference between keratin 5/19 in pleura liquid compared to serum and that K5/19 complex containing the N-terminal is likely to be present in serum in higher extent than complex with intact C-terminal. The western blot also revealed cross-reaction of G-2 antibody with keratin 8, although G-2 is stated specific for keratin 5 by its manufacturer.  A randomized lung cancer study including 110 lung cancer plasma samples, 52 serum samples of benign lung disease and 52 plasma samples from healthy blood donors was performed. The study was carried out by analyzing the samples in the constructed 1A12+Ks19.1 test and CYFRA21-1. The constructed test was not able to discriminate between histological classes of lung cancer in the lung cancer study. The ability of the test to detect early stage lung cancer varied among the different histological classes. Adenocarcinoma and large cell carcinoma samples detected by the 1A12+Ks19.1 test were progressed cancer, stage III or stage IV. For small cell lung cancer 3 of the 13 early stage lung cancer were detected, indicating the test is thus not suitable for detecting early stages of these histological classes. For Squamous cell carcinoma in lung early stages are detected in larger extent than stage III and IV for K5/19 (1A12+Ks19.1) as a separate test, but primarily in combination with CYFRA21-1 where 9 of 13 early stage samples were detected. From this study one can recommend to perform further studies on early stage SCC in lung to determine the relevance of CYFRA21-1 in combination with 1A12 as diagnostic tool.
- PostReplace Synthetic Flavorings with Natural Flavorings - Product Development and Method for Accelerated Shelf Life Studies Development(2015) Klangefjäll, Josefine; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological EngineeringFlavorings are concentrated products consisting of the flavor molecule together with carriers. Flavorings are divided into two major groups, synthetic flavorings and natural flavorings. The consumers are more and more demanding products without synthetic flavorings since they do not want to have “chemicals” in the food. This project was part of the replacement process at the food company Santa Maria AB. The aim was to replace the synthetic flavorings in the Tortilla cheese chips, and Cheese dip mix with natural flavorings or other natural components. Complementary to the product development work a method to do accelerate shelf life studies of seasoned tortilla chips was developed. From the study it was obtained that natural flavors are generally not as complete and clean in taste as the synthetic ones, but together with other taste boosting components it is possible to achieve the same or better taste profile as previously without a too high cost. Also an accelerated shelf life method based on high temperature stress, possible to use when doing product development work of seasoned tortilla chips, was developed. The shelf life of the Tortilla cheese chips with the synthetic cheese flavors replaced was measured with the method to be seven months
- PostEffekten av Kemiras processhjälpmedel BDP866 i Hulesjöns avloppsreningsverks rötgas kammare för matavfall(2015) Andersson, Amanda; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological EngineeringBiogas tillverkas i stor utsträckning med mikrobiell nedbrytning i rötkammare. För att ha en stabil gasproduktion krävs det bland annat att bakteriekulturen i rötkammaren är välmående och har en rik mångfald. För att uppnå detta kan det ibland vara nödvändigt att tillsätta extra tillskott av olika ämnen. I rötkammaren för matavfall har Hulesjöns reningsverk valt att dosera Kemiras processhjälpmedel BDP688 för att optimera processen. BDP688 innehåller bland annat spårämnen som kobolt, nickel och selen, samt järnklorid. Denna kombination av spårämnen skall vara bra för bakterierna i det metanbildande steget och därmed reducera höga halter av organiska flyktiga syror. Järnklorid sänker pH-värdet och binder svavel. Detta processhjälpmedel har reningsverket blivit rekommenderade att dosera till rötkammaren för matavfall då den har producerat lite gas och haft höga värden av flyktiga organiska syror. Syftet med arbetet är att utforska hur flyktiga organiska syror, övriga processparametrar samt gasproduktionen påverkas av tillsatsen av BDP866. Detta har studerats genom att utföra kontinuerlig provtagning under en 10 veckors period som startade 4 veckor efter att processhjälpmedlet hade börjat doserats till rötkammaren. Resultatet av undersökningen var att mängden flyktiga organiska syror minskade, de flesta processparametrarna förbättrades och nådde stabilitet samt att gasproduktionen ökade. Detta tyder på att tillsatsen av processhjälpmedlet har resulterat i en stabil process som kan kompensera för störningar.
- PostshRNA silencing of receptor NC007 in human mesangial cells(2015) Lassén, Emelie; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological Engineering
- PostNetwork coupled astrocytes as a model system to study inflammatory properties(2015) Werner, Tony; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological EngineeringBefore antibiotics and vaccines were discovered, microbes were the primary cause of mortality rates. However, with the drastically altered life style that comes with economic development, diseases that are non-infectious, chronic and recurrent have replaced microbial diseases as the biggest challenge to human health. Cancer, cardiovascular- and neurodegenerative diseases are some examples with potentially debilitating outcomes, and the common denominator among these have been recognized to be the pathways of chronic low-grade inflammation. However, inflammation is not well understood and research must thus be conducted to better understand the condition and elucidate potential therapeutic solutions. Because of this, scientists have employed both in vivo and in vitro systems to study inflammation but because of the lack of knowledge, especially regarding neuroinflammation, in vitro assays have produced many contradictory results. A reliable in vitro system is an attractive option to in vivo systems, as it is relatively inexpensive, quick, less complex and eliminates the ethical concern for animals. The aim of this project was to develop a model cell system consisting of network coupled astrocytes and analyze changes in biomarkers when exposed to the pro-inflammatory mediators, lipopolysaccharide (LPS) and interleukin-1 beta (IL-1), for 24 hours, 3, 6 and 9 days. Analyses were done by enzyme-linked immunosorbent assay, immunofluorescence, and Western blot. The astrocyte cell cultures were investigated under different cultivation parameters, of which two different seras were tested; one of which has been shown to promote microglial proliferation. The cultures with the serum that has been shown to be beneficial to microglial proliferation had drastic effects on some of the investigated biomarkers, with a stronger induction of the toll-like receptor 4, reduced the Na+/K+-ATPase instead of an increase and an induced transcription of excitatory amino acid transporter 2. However, both seras reorganized astrocytic actin filaments similarly, caused a polymerization of globular actin, neither released the pro-inflammatory cytokines IL-1 or tumor necrosis factor alpha and in both serum groups the alteration to the biomarkers returned to control levels during longer stimulations than 24 hours. These results suggest that the astrocytes and microglia are not capable of initiating an inflammation by themselves, but microglia do appear to be at least partly responsible for observed alterations to known biomarkers. Furthermore, as previous studies have produced inflammatory reactive astrocytes by LPS or IL-1, the results imply that additional unknown factors are missing, possibly an unrecognized contaminating cell type.
- PostInvestigations of Bi-phasic Drug Depletion in Liver Microsomes and Hepatocytes in Metabolic Stability Studies(2015) Engsevi, Madeleine; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological EngineeringDevelopment of a new drug is a long and costly process and to avoid attritions due to inadequate metabolic and pharmacokinetic data it is important to determine these early by the use of in vitro systems. The intrinsic clearance (CLint) is calculated from the disappearance of test compound in the incubation plate over time and for most compounds this disappearance is linear, however, some compounds show a bi-phasic depletion profile which can cause under predictions of in vivo clearance when using the same calculation model. To improve the quality of the assay for these compounds the reasons behind the bi-phasic depletion profile were investigated. A set of 26 carboxylic acids were incubated with rat hepatocytes and human liver microsomes (HLM) on robot and the disappearance was determined by LC-MSMS on HSS T3, CSH and BEH C18 columns. The incubations with HLM suggest that conjugative metabolism dominated for this set of compounds. Viability and matrix matched zero time point had no impact on the depletion curve. Mass/chromatographic interferences from co-elution of metabolites were observed for two of the compounds which likely had an impact on the response but this could not explain the bi-phasic profile. The identities of the hydroxy and acyl glucuronide metabolites were confirmed on quadrupole time-of-flight (Q-TOF). A correlation was found between high LogD7.4 and a more bi-phasic appearance at higher cell concentrations. The depletion was modelled with a bi-exponential model that produced unbiased CLint values that were less prone to cause under prediction of in vivo clearance
- PostThe Implication of the JAK-STAT Pathway in Myxoid Liposarcoma(2015) Bäcksten, Karin; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological Engineering
- PostP2Y2 RECEPTOR PEPDUCINS HIJACK AND ACTIVATE THE FORMYL PEPTIDE RECEPTOR 2 IN HUMAN NEUTROPHILS(2015) Holdfeldt, André; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological EngineeringNeutrophils are the most abundant phagocytic cell type and have a critical role in the innate immune system. They are attracted to the site of infection/inflammation through a number of surface expressing chemoattractant G-protein coupled receptors (GPCRs). Activation of neutrophil GPCRs mediates not only directional migration, but also triggers the release of reactive oxygen species and granule stored enzymes. The formyl peptide receptor 1 (FPR1), displaying high binding affinity for the “danger signal” formyl peptides derived from bacteria or damaged mitochondria, was the first chemoattractant GPCRs cloned and has therefore served as a model receptor for our understanding of neutrophil physiology. In addition to FPR1, neutrophils express also the closely related formyl peptide receptor 2 (FPR2) and the danger signaling ATP recognition receptor (P2Y2-R). All GPCRs share a similar seven transmembrane helical structure in which the extracellular domains are involved in ligand binding whereas the intracellular parts are engaged in G-protein coupling and signaling transduction. Recent research has proposed a group of cell penetrating molecules, so called pepducins that can modulate GPCR signaling from the inside. Pepducins are lipopeptides derived from one of the intracellular lopes of a GPCR, they are supposed to allosterically activate or inhibit the receptor from it is derived. In this study, we investigated the ATP receptor signaling by applying pepducins from the P2Y2 receptor. Our data show that these pepducins do not target their cognate receptor as the currently “pepducin model” proposed and instead activate FPR2. This “model” has also been challenged by the recent findings from pepducins from FPR1 and β2-adrenergic receptor
- PostEnhancing the prebiotic properties of malted barley(2015) Persson, Jenny; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological EngineeringPrebiotics can improve health indirectly by promoting probiotic bacteria in the gut and production of beneficial metabolites by the gut microbiota. In this project the possibility to enhance the potential prebiotic properties of malted barley was explored. The aim was to find yeast strains with high potential to modify the polysaccharide composition, in particular the β-glucan profile, in malted barley and investigate if fermentation of barley malt materials by these yeast strains can promote growth of probiotic bifidobacteria and the production of organic acids. Firstly, the β-glucan content in Brewers’ Spent Grain was determined. Thereafter, yeasts were screened for production of β-glucanase enzyme. The growth of strains showing this ability was studied in the malt materials in order to select optimal strains to be used in further experiments with bifidobacteria. Pichia butronii TY01 and Pichia kudriavzevii TY3 were found to be most suitable for fermentation of the malt materials based on β-glucanase activity and highest increase in cell numbers during growth in malt materials. The potential impact of the fermented malt materials on gut health was evaluated by analysing growth of Bifidobacterium bifidum and Bifidobacterium breve in malt-based media and the subsequent production of organic acids, in particular lactic acid. Indications of increased growth of Bifidobacterium in fermented malt was observed. Furthermore, a higher concentrations of lactic- and acetic acid was found in fermented malt compared to unfermented samples. Brewers’ Spent Grain generated lower organic acid concentrations compared to malted barley, possibly due to differences in the β-glucan content. In conclusion, this study showed increased prebiotic properties of barley malt materials fermented by β-glucanase producing yeast strains compared to unfermented materials.
- PostBioprospecting for novel laminarin-degrading enzymes in marine microorganisms - A step towards the use of macroalgae in bioprocesses(2015) Olsson, Joakim; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological EngineeringCurrently, efforts are being made to find alternative biomasses for biofuel production, which are not competing with food production. Several kelp species have potential for large-scale cultivation, harvest and biorefinery processes. On the Swedish west coast, Laminaria digitata and Saccharina latissima are two such species. These kelps can contain up to 33% of the storage carbohydrate laminarin (dry weight), which can be hydrolysed to glucose and subsequently converted into bioethanol by for instance yeast. Enzymes able to digest the β-1,3 or β-1,6 bonds of the laminarin, to release glucose, are in large unknown and this project has been about finding microorganisms expressing such enzymes, known as laminarases. Samples from partly decomposed L. digitata and S. latissima specimens were streaked on nutrient agar plates for proliferation and isolation of surface microorganisms. Isolated organisms were subsequently screened for growth on laminarin and promising strains were identified through 16s/18s rRNA sequencing. After growth experiments on liquid medium with different carbon sources, two bacterial strains, Pseudoalteromonas ssp., were selected for further characterisation. The two strains were grown in algae extract and the sugar composition was monitored over time. In the extract, mainly mannitol and laminarin were present but glucose was formed during the cultivation, indicating the presence of hydrolytic enzymes, after which the hydrolytic activity on pure laminarin was investigated. The supernatants of one of the strains showed in vitro activity, further indicating the presence of extracellular hydrolytic enzymes. More investigations are needed on whether the enzymes can be used in any type of process. This thesis work has been successful in isolating marine organisms with laminarin-degrading activity.
- PostP27 deficiency accelerates the development of PTEN-deficiency-induced myeloproliferative disease(2015) Shao, Jingchen; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological EngineeringPTEN acts as a phosphatase for PIP3 and negatively regulates the PI3K/AKT pathway, and CDKN1B (P27KIP1) is a cyclin-dependent kinase inhibitor that regulates G0 to S phase transitions by binding to and regulating the activity of cyclin-dependent kinases. Genetic alternations of Pten or Cdkn1b are common in hematological malignancies. Combined loss of PTEN and P27KIP1 expression is associated with tumor cell proliferation and poor prognosis in prostate cancer. However, it is not so clear how two mutations would cooperate in leukemogenesis. Here, we show that combined inactivation of PTEN or P27KIP1 in the hematopoietic compartment in mice results in a more severe myeloproliferative disease phenotype with shorter lifespan, lower hemoglobin and more enlarged spleen, lever comparted inaction of Pten or p27KIP1 alone.
- PostA kinetically-constrained FBA-model of the synthesis of aromatic amino acid-derived products in Saccharomyces cerevisiae(2015) Janasch, Markus; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological EngineeringDuring the last decades the research interest for creating a more sustainable and environmentallyfriendly society and industry has increased dramatically. The employment of icroorganisms to create valuable compounds such as fuels and chemicals from renewable resources has gained high popularity. With directed modifications in the metabolism of these microorganisms, metabolic engineering seeks to optimize the properties of these organisms for an efficient bioprocess to produce valuable compounds. One characteristic of metabolic engineering is the extensive use of computational models to predict the behavior of the metabolism and thereby identifying suitable targets for genetic interventions in an in silico to in vivo progress. Flux Balance Analysis (FBA) is a popular analysis method for metabolic models, as it only requires the underlying stoichiometric network of the metabolism modelled. With FBA the optimal flux distribution, given a certain objective to be maximized, can be calculated with linear programming algorithms. As FBA only takes the stoichiometry and reaction directionality into account, further physiological constraints have to be included in the framework to increase the predictive strength of the simulations. In this thesis, an expanded form of FBA, that includes kinetic enzyme parameters as additional constraints on the system, was used to analyze a metabolic model of the popular industrially-used organisms Saccharomyces cerevisiae, that included, additionally to the native metabolism, also recombinant enzymatic steps to form the plant secondary metabolites resveratrol and naringenin from the aromatic amino acids phenylalanine and tyrosine. Resveratrol and naringenin have been found to be beneficial to human health by offering anti-inflammatory, anti-carcinogenesis and anti-oxidant properties. The kinetically-constrained model was successfully used to estimate the impact of introducing recombinant pathways on the protein pool in the cell as well as to identify the enzymatic steps offering the highest control over the flux towards these products. This might be used in metabolic engineering to estimate the efficiency of metabolic pathways in the context of the whole metabolism form a protein cost point of view.
- PostAnvändning av svenska makroalger som substrat för bioetanolproduktion(2015) Davidsson, Christoffer; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological EngineeringThis project investigates Saccharina latissima seaweeds, harvested in Sweden, as substrate for bioethanol fermentation. Saccharina latissima is also called sugar kelp and contains large amounts of the polysaccharide laminarin which was the target of interest in this project. The seaweed was milled before mixed with strong base followed by acid to extract the laminarin into a solution. After extraction, three methods for hydrolysing the laminarin into glucose were evaluated: sulphuric acid and autoclaving, commercial laminarinase enzyme, and enzymes from the naturally occuring bacterium Pseudoalteromonas sp. After hydrolysis fermentation was performed in small batches with a commercial strain of brewers' yeast. The results show that both sulphuric acid and enzymes from Pseudoalteromonas sp. did release glucose, which was successfully fermented into ethanol.
- PostIncreasing production of 3-hydroxypropionic acid by modulating the activity of acetyl-CoA carboxylase 1 in Saccharomyces cerevisiae(2015) Koendjbiharie, Jeroen Girwar; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological Engineering3-Hydroxypropionic acid (3-HP) is an attractive chemical that can be produced from biomass, because it can be used as a precursor for a lot of commercially interesting products and thus contribute to a more sustainable, bio-based economy. The synthesis of 3-HP has already previously been shown to be possible in Saccharomyces cerevisiae via the heterologous expression of malonyl-CoA reductase (MCRCa). 3-HP production was further improved via the overexpression of an acetyl-CoA carboxylase 1 double mutant that is no longer phosphorylated by Snf1 (Acc1**). However, malonyl-CoA is also a precursor for fatty acids, and the increased fatty acid production associated with the overexpression of Acc1** is undesirable when aiming to produce 3-HP, and could, at least partly, originate from the presumed association of Acc1 with the cytosolic side of the ER membrane. It was hypothesized that by bringing the two enzymes, Acc1** and MCRCa, together with a scaffold, the 3-HP production could be increased by channeling the acetyl-CoA directly towards 3-HP and minimizing the leakage towards fatty acid synthesis. To investigate the use of a scaffold, the following two research questions were proposed: 1) Is it possible to abolish the association of Acc1 with the ER membrane by fusing Acc1 to a docking domain? 2) Is the 3-HP production improved when Acc1 is linked to MCRCa via a protein scaffold? Due to the limited time of the project it was not possible to adequately address the proposed questions. However, it was clear that it was not possible to effectively abolish the presumed association of Acc1** with the ER membrane by fusing dockerin a (Da) from C. thermocellum endoglucanase to the C-terminus of Acc1**, as it rendered to enzyme non-functional, most likely as the result of incorrect folding. The non-functionality of Acc1**-Da was not an inherent feature of Acc1** when fusing a protein domain to the C-terminus, as Acc1** was still functional with an enhanced green fluorescent protein (eGFP) fused to the C-terminus, as well as to the N-terminus, making the use of a scaffold still a viable strategy to improve the production of 3-HP and other malonyl-CoA derived products. In parallel, the extent in which Acc1** activity is regulated by the efficiency of its biotinylation by Bpl1 was also investigated. Cultivating S. cerevisiae with Acc1** integrated in the genome in the presence of different biotin concentrations did not lead to an increased production of 3-HP, both with and without overexpressing BPL1 via a multiple copy plasmid. This suggested that Acc1** activity is not significantly regulated by its biotinylation. However, to be conclusive about the ACCase activity, it should be directly measured, rather than indirectly, as the 3-HP production is affected by more than just the activity of Acc1**.
- PostMicrostructure of Instant Coffee Foam. Confocal Microscopy Method Development and Production Related Parameters Affecting Foam Kinetics(2015) Nilsson, Gustav; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological EngineeringCoffee foam is despite being an important parameter for the sensory impressions a cup of coffee provides perhaps the least studied part of this enormous commercial product consumed worldwide. In terms of microstructure and rheology the field is considerably lacking in data. This project aimed to develop a method to study coffee foam from instant coffee, by confocal laser scanning microscopy (CLSM) with the intention of providing data that may be used for production purposes. The instrument setup and operation of the method were defined, efficient dyes identified and common artefacts explained. The average bubble area was measured as a function of time by image analysis, the distribution and movement of particles in the lamellae could be observed and in particular surface active lipids and proteins could be stained and detected. A significant advantage of the method compared to e.g. rheological measurements in a rheometer turned out to be the small sample volumes needed. In addition, a simple method for measuring drainage using a USB-microscope was developed and applied to the foam. The analyzed parameters were filtering (pore sizes of 0.20-0.80 μm), pH adjustment (pH 4.0-6.3), hydrophobic particle addition (~1 μm) as well as the combined effects of filtering and particle addition to the coffee pre-foaming. Growth rate of average bubble area in coffee foam was shown to be virtually linear for at least the first 25 minutes, as oppose to what was described in literature for dynamic liquid foams in general, where growth was described as logarithmic. Extrapolating the growth of average bubble area to minute zero as a measure of foamability proved viable. Previously reported data of increased foamability in the pH range of 5.7-6.3 was confirmed. The results showed that higher pH values (>pH 5.0) had a negative impact on foam stability, and a lower pH increased foam stability. Filtering and particle addition showed that the interaction between various particles in the coffee play an important role for both foamability and foam stability