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- Post3D Bioprinting Meniscus for Future Tissue Replacement in Osteoarthritis Affected Knee Joints(2020) Pettersson, Ida; Olsson Widjaja, Amanda; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Gatenholm, Paul; Simonsson, StinaProgressive cartilage defects in human knee joints are a worldwide health issue that exposes a great burden on society as well as on single individuals. Sudden traumas to the knees can induce tears in the structures of the joints such as the articular cartilage and the meniscus. Lesions in the cartilage tissue of the knee joints have a high probability to eventuate in osteoarthritis (OA). The emerging technique of 3D bioprinting tissue is a novel approach for cartilage repair and regeneration. The choice of bioink and cell type are important factors that considerably impact the resulting cartilage repair potential after the process of 3D bioprinting. Induced pluripotent stem cells possess the ability to differentiate into almost any cell type and their potential in future regenerative medicine is of great interest. The present study has investigated the possibility of 3D bioprinting chondrocytederived human induced pluripotent stem (iPS) cells into meniscus structures with initiated cartilage regeneration. A combination of nanofibrillated cellulose (NFC) and alginate (A) was used as bioink. At first, the bioink was subjected to optimization to augment printing properties and cell viability during and after bioprinting. The composition of nanofibrillated cellulose and alginate originated from a ratio of 60/40 NFC/A (% w/w) that was compared with one ratio of 48/52 NFC/A (% w/w) and one ratio of 80/20 NFC/A (% w/w). The optimization of bioink involved measurements of printability, rheology, and cell viability. The formation of microtissues enabled differentiation of iPS cells toward extracellular matrix (ECM) producing cells prior to 3D bioprinting. 3D bioprinted menisci, as well as cultured microtissues, were analyzed with LIVE/DEAD assay, immunohistochemical analysis, and quantitative reverse transcription polymerase chain reaction (qRT PCR). The work also comprised an in vivo assay of 3D bioprinted constructs transplanted subcutaneously in mice to enable the evaluation of tissue formation in a realistic milieu. Induced pluripotent stem cells further modified to express green fluorescence protein under the aggrecan promotor have been subjected to a screening of a library of small Food and Drug Administration (FDA) approved molecules. Changes in intensity indicated induction or inhibition of the synthesis of aggrecan. Moreover, the screening highlighted molecules included in the induction or inhibition of aggrecan production. These molecules will be subjected to further investigations to evaluate their possible contribution in future OA-treatments. The prevailing work demonstrates the feasibility of utilizing microtissues formed by iPS cells for the 3D bioprinting of cartilage tissue. The culturing of iPS cells as microtissues has proven to induce differentiation and synthesis of components included in the ECM, both in vitro and in vivo. Simultaneously, the study has included a comparison of three bioinks that resulted in an optimized protocol for the production of the bioink composed of 60/40 NFC/A (% w/w). This optimized bioink fulfilled the requirements of being printable while supporting cell viability.
- PostA CRISPR approach to manipulating NK cell receptor ligand expression(2018) Kristensson, Linnea; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological EngineeringNatural killer (NK) cells are important innate lymphocytes in the immune system and have been considered to be vital in fighting cancer, virus infections as well as regulating immune responses. The activity of NK cells is regulated via a complex signaling between inhibitory and activating receptors. Ligands for most of the ac-tivating receptors have been discovered but no endogenous ligand for one of the important natural cytotoxicity receptors, NKp46 has been identified. Therefore, the long term aim of this thesis project was to define ligand candidates for the acti-vating receptor NKp46 with intermediate aims to manipulate expression of NK cell activating receptor ligands in the K562 cell line using a CRISPR approach. The result from the performed cytotoxicity assays demonstrated the importance of the activating receptors NKp30 and DNAM-1 in elimination of K562 cells and the expression of the complementary ligands NCR3LG1, PVR and NECTIN2 was confirmed using RT-qPCR. The successful creation of a Cas9-expressing K562 cell line was demonstrated, including verification of Cas9 protein expression using both flow cytometry and western blot. Following that, a constructed plasmid with the gRNA for the B7-H6 gene, NCR3LG1, was generated and shown to create a cleavage and mutation within the gene. The engineered cell line was ultimately shown to have a reduced expression of the B7-H6 protein, indicating the successful knockout of the NCR3LG1 gene.
- 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.
- 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.
- PostA Rolling Circle Amplification-Based Methodology for Making Long, SequenceRepeating, DNA Duplexes(2018) Dekoning, Nora; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological EngineeringIn vitro studies of sequence-specific DNA-protein interactions using techniques where long DNA molecules is needed are currently limited by the size of synthesized DNA molecules, or is restricted to the commercially available DNA. For single molecule DNA imaging techniques using for instance nanochannels confinement, the small sized molecules make the interactions difficult to detect. The commercially available DNA, on the other hand, does not allow any freedom in choosing or changing the DNA sequence. Therefore, a method for producing long DNA molecules containing a sequence of choice would alleviate these limitations and greatly improve the possibility to study DNA-protein interactions. The general concept of this paper was to create long, double-stranded DNA molecules with a sequence that is specifically designed to interact with the protein internal host factor. To make such molecules, three experimental procedures were developed, building on the single-stranded DNA products from a rolling circle amplification (RCA) reaction. The first experimental procedure was based on the presumed perfect annealing of smaller complementary strands to the single-stranded RCA product. The second experiment assumed that single-stranded gaps would be present in the duplex after annealing, hence the addition of a polymerisation reaction. In the third experiment, the RCA was run for 24 hours, to allow double-stranded product to be formed in the RCA. The DNA strands were visualized using fluorescence microscopy, with the goal of studying them and their protein interactions in nanochannels. To be able to use this detection method, an external fluorescent dye, YOYO, is used in the main aim of the project. However, as these types of dyes change the native structure of DNA, an extra aim was to use the fluorescent base analogue tC incorporated into one of the duplex strands, leaving the native structure intact. All three experimental procedures were shown to be capable of producing apparently double-stranded DNA molecules, that were larger than 100 kilo-base pairs albeit with a broad size distribution. This shows that the main aim in terms of procuing DNA molecules has been completed. The tC-containing DNA molecules were not visible under the microscope with the settings used, but appears to be promising.
- PostA Step Toward Personalized Cancer Treatment Simultaneous Detection of Multiple Types of Chemotherapyinduced DNA Damage Using Single Molecule Imaging(2022) Foss, Ebba; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological Engineering; Westerlund, Fredrik; Akwasi Aning, ObedChemotherapy is commonly used to treat cancer today, either alone or more commonly as part of combination therapy. Response to a certain chemotherapeutic agent is highly individual, both in terms of treatment efficacy and the extent to which healthy cells are affected. For several drugs, induced DNA damage provides the main cytotoxic effect, and a method for evaluating this damage could therefore prove a powerful tool in treatment planning. In this thesis, a single molecule imaging approach is used to assess chemotherapy-induced DNA damage, allowing visualisation of damage sites on individual DNA strands. While previous studies have focused on one damage type, or collective damage without distinction between types, a novel modification to pre-existing techniques that allows for this distinction has recently been demonstrated. In this thesis, the alkylating agent temozolomide was used to illustrate how different damage types can be distinguished with a single molecule imaging approach. This is done using repair enzymes associated with different DNA repair pathways. The repair enzymes sequentially incorporate spectrally distinct fluorescent nucleotides at the damage site which are then visualized as fluorescent spots of two different colours on individual DNA molecules. This distinction could be shown with high repeatability in terms of colour ratio. While both enzymes used separately clearly repaired the treated DNA, there appeared to be an overlap when applying them sequentially. This could suggest a problem with enzyme specificity. Further exploration of this issue is needed to verify the feasibility of single molecule imaging for the purpose of simultaneous detection of chemotherapy-induced DNA damage types.
- PostAccelerated shelf life tests of wheat tortillas A study of microbial and textural deterioration in wheat tortilla(2018) Nilsson, Fredrik; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological EngineeringThis study tried to shorten the time required for shelf life studies in product development of wheat tortillas, by developing accelerated shelf life test (ASLT) models for microbial and textural shelf life. ASLTs speed up product degradation by exposure to extreme environmental conditions and in this case were temperature and relative humidity (RH) used. The samples were divided into four storage groups: 3°C and 70% RH, 20°C and 30% RH, 27°C and 80% RH as well as 40°C and 80% RH. Texture was determined with a fold/roll-method, an instrument and by a sensory panel. Microbial concentrations were determined by a laboratory company. Gas composition inside the bags, tortilla pH, water content and water activity were measured to determine possible links to the shelf life. It was not possible to calculate an ASLT model for the microbial growth because of unknown starting number of microbes. The microbial tests however showed that a high temperature and humidity caused the number of bacteria to rapidly increase above what is considered acceptable. Microbial ASLTs thus seem possible, but more tests with a lower detection limit are needed to create a model. To avoid bad taste from nonpathogenic bacterial growth, consumers are recommended to avoid storing the tortillas in high temperature and/or humidity. Textural ASLTs also seem possible in regards of rollability and foldability, but more tests are needed to precisely determine the accelerating factor. Stickiness and translucency seems to deteriorate much slower than rollability and foldability under normal storage conditions. It is therefore suggested to be enough to check that freshly baked tortillas meet the quality requirements of stickiness and translucency. Tortillas seemed to lose textural quality at the same rate, or slower, in refrigerator compared to room conditions. Longer shelf life studies are therefore suggested, to verify if this is the case. If so, storage in a refrigerator could possibly prolong shelf life of wheat tortillas. Changes in the modified atmosphere inside the bags seems to correlate with textural degradation. A study with atmospheric air instead would therefore be interesting as an attempt to determine if it retards textural degradation in wheat tortillas.
- PostAdaptive evolution of Yarrowia lipolytica(2017) Hellgren, John; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological EngineeringWe need renewable resources to allow sustainable production of fuels. By using lipid accu-mulating yeast, biodiesel can be produced in a sustainable way from resources that were previously not used, for example lignocellulose-based sources such as agricultural waste. However, for this process to be proﬁtable, the tolerance of the yeast needs to be improved. This project aims to improve the tolerance of the oleaginous yeast Yarrowia lipolytica to-wards osmotic and saline stress by using the method adaptive laboratory evolution. This method has previously been shown to be eﬃcient in constructing strains to tolerate new conditions without the need of prior knowledge. After evolving Y. lipolytica for 220 gen-eration in minimal medium containing 1.4 M NaCl, an improved performance in the same medium was observed, along with evolved cross-tolerance towards low pH. This indicates that this adaptive evolution of Y. lipolytica resulted in improved ionic tolerance rather than pure osmotic tolerance. The evolved strains were sent for whole genomic sequencing to ﬁnd out which mutations that caused this phenotype. During this project, two previous CRISPR/Cas9 strategies were combined and adapted for eﬃcient markerless reverse en-gineering. When genome data arrives, this strategy will be used for reconstruction of can-didate mutations to ﬁnd out which mutations are important for the observed phenotype. The gained knowledge from this evolution experiment can later be used for constructing a robust industrial strain that eﬃciently converts lignocellulose-based material to biodiesel, allowing sustainable production of fuels.
- PostAdaptive laboratory evolution of Saccharomyces cerevisiae for increased tolerance towards compounds of industrial interest(2016) Malina, Carl; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological EngineeringOver the last decades, microbial production of fuels and chemicals has become an increas-ingly attractive alternative to petroleum-based production. This has created a demand cell factories able to produce a wide range of compounds. The yeast Saccharomyces cere-visiae is widely used in biotechnology with successful applications in the production of both bulk and fine chemicals. However, in order to reach the full potential of yeast as a cell factory challanges still remain. These include creating strains tolerant to stress conditions, such as inhibition at the high product titers required in industrial production. Traits conferring these properties are often complex and encoded by several genes. In order to obtain strains with improved tolerance, adaptive laboratory evolution (ALE) is often used. This thesis was part of an ongoing project of ALE for increased tolerance towards compounds with potential industrial applications. The work in this thesis can be divided into two main parts. The first part was screening for tolerance of S. cerevisiae towards four diols and two diamines by microplate cultivation. For both the diols and diamines, clear trends of decreasing fitness as compound concentration was increased was seen. For the diols tested, it seems increasing toxicity correlates with increasing chain length and branching. The second part was characterization of pimelic acid tolerant strains from ALE, by shake flask cultivation and HPLC analysis of metabolites. In addi-tion, the genomes of 21 strains were resequenced. Results from the shake flask cultures showed that, in presence of pimelic acid, most strains have an impaired growth on non-fermentable carbon sources. Furthermore, HPLC analysis of metabolites revealed that glycerol and acetate accumulated during cultivation while ethanol was slowly consumed, implicating a defective respiratory system. Through genome resequencing, in total 47 genes were found to be mutated across all of the evolved strains.
- PostAggregation av Parkinsonproteinet -synuklein (vildtyp och sjukdomsvarianterna A30P och A53T) samt interaktion med liposomer(2017) Lorentzon, Emma; Olsson, Anna; Sundvall, Nathalie; Tingvall Gustafsson, Johanna; Torell Björkman, Agnes; Vånder, Frida; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological EngineeringParkinsons sjukdom ¨ar en vanlig ˚aldersrelaterad neurodegenerativ sjukdom, som leder till b˚ade motoriska och kognitiva problem. Sjukdomen karakt¨ariseras av ab-normal ansamling av proteinaggregat, kallade Lewykroppar, inneh˚allande amyloida ﬁbrer. Dessa ﬁbrer best˚ar till stor del av proteinet α-synuklein, som fr¨amst ˚aterﬁnns i de presynaptiska nervterminalerna, d¨ar det interagerar med bland annat fosfolipider och andra molekyler. Syftet med projektet ¨ar att studera hur tre varianter av α-synuklein, vildtyp samt tv˚a sjukdomsalstrande mutationer, A30P och A53T, aggregerar samt interage-rar med liposomer. Liposomerna best˚ar av DOPC, DOPS, DOPE och kolesterol f¨or att efterlikna synaptiska vesiklar i hj¨arnan. Proteinstruktur samt aggregationskinetik in¨arvaro av liposomer unders¨oktes m.h.a. cirkul¨ar dikroism och ﬂuorescenspektro-skopi. Studien visar att samtliga α-synukleinvarianter interagerar med liposomerna. Vildtypen och A53T-mutanten uppvisar en likartad strukturell f¨or¨andring, i form av en ¨okning av α-helixstruktur, i n¨arvaro av liposomer, medan A30P-mutanten uppvisade l¨agre grad av strukturf¨or¨andring. Aggregationskinetiken f¨or samtliga proteinvarianter inhiberas av de syntetiska liposomerna, vilket observeras m.h.a. tioﬂavin T ﬂuorescens. Prov inneha˚llande enbart protein aggregerar snabbare ¨an i n¨arvaro av liposomer. Slutsatsen ¨ar att samtliga varianter av α-synuklein interagerar med membran-modellen, som inducerar en f¨or¨andring av deras sekund¨arstruktur. Aggregationen f¨or samtliga proteinvarianter inhiberas i n¨arvaro av membranmodellen.
- PostAltered immune phenotype in patients with cirrhosis results in impaired vaccine-induced immunity following COVID-19 vaccination(2022) Blick, Elin; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Nygård, Yvonne; Martner, Anna
- PostAnaerobic Digestion using Mini Reactors. Validation and optimization of analytical instruments for anaerobic bioprocesses(2020) Larsson, Yrsa; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Franzén, Carl-Johan; Habagil, MosheI vattenreningsverk används anaerob nedbrytning för biologisk stabilisering av slamvattnet. I den anaeroba nedbrytningen skapas metan och koldioxid med andra ord biogas. Biogasen tas tillvara och kan användas som grön energikälla, men produktionen är inte alltid pålitlig i vilka mängder biogas som produceras. För att optimera och utveckla biogasproduktionen används småskaliga biogasreaktorer i laboratoriemiljöer. Fram tills idag så är det främst semikontinuerliga reaktorer som kommer till användning som behöver manuell matning och dataavläsning varje dag. VIVAB i Varberg har investerat i automatiska reaktorer som ska förenkla laborativa undersökningar med mindre spill, automatisk avläsning och ta bort det vardagliga manuella momentet. Målet med arbetet under den här rapporten, har varit att möjliggöra en ersättning av de semikontinuerliga reaktorerna med de optimerade automatiska reaktorerna. Vid uppstart av de automatiska reaktorerna uppkom flera problem, som upptäcktes via höga värden på de organiska fettsyrorna och låg biogasproduktion. Optimering av reaktorutformningen skedde genom följande korrigeringar. Identifiering och eliminering av gasläckor, utbyte av delar av utrustningen och utformning av ett rör som möjliggör utflöde från reaktorn. Reaktorerna var sammankopplade med en PLC controller som via en skärm tillät enkla förändringar av reaktorerna vad gäller matning, temperatur, mixning och kalibrering av gasmätare och flödespump. Resultat från reaktorerna sparades i realtid i PLC controller som registrerade mätdata från reaktorerna varje gång gasmätaren läste av gasproduktion. Gasmätaren uppvisade i några fall fel resultat då gasmätningsutrustningen fastnat och inte läste av biogasproduktionen. För att undvika utelämning av resultat sammankopplades ytterligare en gasmätare, μ-Flow som kunde påvisa uteblivna data och bidra till en pålitlig kalibrering av gasmätaren. De automatiska reaktorerna fungerade efter optimeringen mer eller mindre självgående med undantag för matning av slam var 4–5 dag. Detta innebär en stor minskning i arbetsbelastning vid laborationer och analyser vid optimering och utveckling av biogasproduktion som i efterhand kan appliceras på storskalig biogasproduktion.
- PostAnalysis of avenanthramides in oats with Ultra High Performance Liquid Chromatography(2016) Astner, Martin; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological EngineeringAvenanthramides have been shown to have beneficial properties such as strong antioxidant activity and anti-inflammatory effects in humans, and are a key defence molecule for oats. The goal of this project was to create and validate a method for quantification of avenanthramides in oats that would take less than 20 minutes. A new extraction method and new liquid chromatography method were tested, as well as the introduction of an internal standard to improve quantification. We found that the internal standard selected was not stable during extraction and that it was necessary to use an external standard curve. We found that the liquid chromatography method was reproducible for standard compounds and that we could get separation of the three main avenathramides in oats for a total run time of 15 minutes. The extraction component of the method requires further work to improve recovery and stability before this method can be used for quantification of avenanthramides in oats.
- PostAntimicrobial silk(2016) Floderus Savonen, Lotta; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological EngineeringSpider silk has shown potential for use as a biomaterial. If fused to antimicrobial peptides (AMPs), recombinant spider silk could be used for medical applications and reduce the need to use conventional antibiotics to battle infections. Recombinant spider silk 4RepCT was fused to the AMPs Magainin I, Lactoferricin and a synthetically derived AMP referred to as WGR. Polystyrene disks were coated with the AMP-silk fusion proteins and the disks were incubated with cultures of Staphylococcus aureus and Escherichia coli, to test the AMP-silks antimicrobial activity. All AMP-silk fusion proteins significantly decreased bacterial adhesion of S. aureus to the disks after 48 hours of incubation compared to uncoated disks. The Mag- and WGR-silks were e↵ective already after 24 hours. The recombinant silk itself seemed to have an antimicrobial e↵ect by reducing bacterial adhesion of both bacterial strains to the polystyrene disks. Results indicated that addition of the AMPs improved this e↵ect on S. aurues, but not on E.coli.
- 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.
- PostArtbestämning och karaktärisering av laktosväxande vildjäst från Nigeria(2022) Good, Johanna; Bellot Avendaño, Gelmy; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Geijer, Cecilia; Persson, KarlDuring cheese production, whey is a residual product, containing disaccharide lactose and other nutrients and minerals. Large quantities of whey must be disposed of yearly, which becomes costly since it requires further processing. The possibility of using microorganisms to convert this cheap and available resource into valuable bioproducts is an attractive approach. However, few known yeasts can perform this task without extensive genomic engineering. The discovery of yeast species that naturally produce valuable biochemicals could increase the value of whey in the industry. Therefore, from a large collection of wild Nigerian yeast isolates, our research sought to identify new yeast species that can grow on lactose. This was done by identifying species with DNA sequence data from a collection of ~6000 wild Nigerian yeast strains, were ~230 was found to grow on lactose. One representative isolate from 15 selected species were chosen for cultivating, HPLC-analysis, and literature review. The species were selected based on findings in literature such as usage in industry, bioproduct formation and biohazard level. The selected species include Apiotrichum mycotoxinovorans, Candida orthopsilosis, Candida pseudointermedia, Clavispora lusitaniae, Cutaneotrichosporon curvatum, Cyberlindnera fabianii, Kodamaea ohmeri, Meyerozyma caribbica, Meyerozyma carpophila, Moesziomyces antarcticus, Papiliotrema flavescens, Rhodotorula mucilaginosa, Trichosporon insectorum, Vishniacozyma taibaiensis and Yarrowia lipolytica. The literature research of this study revealed that all yeast species may have industrial potential, but more research is needed, especially for C. pseudointermedia, M. carpophila and V. taibaiensis, for which information was lacking. Species that produced fatty lipids deemed of special interest, due to the potential for future use in biotechnology. The result from the cultivation showed that half of the species grew well on lactose medium, which was backed up by the HPLC analysis. M. antarcticus was the only species which had greater growth in lactose medium than in glucose medium, and which also, shown trough the HPLC analysis, produced products that differed from other species. Although a few species showed good potential, further research is needed to determine if the species is suitable future industrial usage.
- PostAssessment of fermentability of steam-pretreated spruce tips, needles and branches for bioethanol applications(2018) Asp, Tobias; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological EngineeringThe production of bioethanol from fermentation-based bioprocesses utilizing ligno-celluosic feedstocks is an option for replacing fossil based fuels. By using genetically modified yeasts that co-consume glucose and xylose, it is possible to ferment lingo-cellulosic materials such as spruce for bioethanol production. In this project the fermentability of spruce tips, needles and branches, pretreated by acid-catalyzed steam explosion according to a design of experiments plan, was evaluated in terms of ethanol titer, rate and yield as well as cell viability. In order to ferment the spruce tips, needles and branches, suÿcient cell concentrations are needed. A preculture method was developed where enough cells were produced and harvested in the same physiological state in di˙erent batches for simultaneous saccharification and co-fermentation. A anaerobic shake flask system was used to ferment the spruce tips, needles and branches. Final ethanol titers of up to 10 g l-1, average volumetric ethanol production rates of up to 0.35 g l-1 h-1 and final yields of ethanol on available glucose and on available glucose together with xylose of up to 0.19 gEthanol gGlucose-1 and 0.16 gEthanol gGlucose+Xylose-1 respectively were observed. The final cell concentrations, colony forming units and growth rates observed were all fairly low values of up to 3.00 x 105 cells ml-1, 1.19 x 107 CFU ml-1 and 2.36 x 10-2 h-1 respectively. Even though there may be some indications on preferable pretreatment conditions in terms of fermentability, more testing and experiments are required to make statisti-cally significant recommendations.However, trends observed in this project points to either high temperature and short time or low temperature and long time as prefer-able pretreatment process conditions. Materials pretreated under these conditions showed the highest final titers and yields.
- PostAttempted Isolations of Cholesterol-to-Coprostanol Reducing Bacteria in the Human Gut(2023) Antonsson, Selma; Chalmers tekniska högskola / Institutionen för life sciences; Chalmers University of Technology / Department of Life Sciences; Landberg, Rikard; Tremaroli, ValentinaAtherosclerotic cardiovascular disease (ASCVD) is the leading cause of death globally. In early development of atherosclerosis, retention of cholesterol in artery walls is a key step. Cholesterol both endogenously produced and absorbed from the diet ends up in the gut, where bacteria may reduce it to coprostanol. Unlike cholesterol, coprostanol cannot be reabsorbed into circulation from the intestines, leading to long-standing hypotheses that high cholesterol-to-coprostanol conversion may lower blood cholesterol and thus the risk of ASCVD. Metagenomic evidence for both conversion being health-associated and ASCVD being a microbiota-modulated disease is mounting, emphasising the potential importance of microbial cholesterol metabolism in the gut. This project thus aimed to isolate cholesterol-converting bacteria from the human gut to further characterise them. Conversion was initially studied in a pure Eubacterium coprostanoligenes culture as a positive control. However, the type strain was quickly outcompeted by a contaminant whereupon culturing of faecal samples from two healthy donors was initiated. Over four sample series, several media compositions and cultivation approaches were investigated. Coprostanol was not observed in any culture. Pathway intermediates could not be analysed and as such it is possible, although improbable, that partial conversion took place. There was also no way of detecting the potential presence coprostanoligenic but non-converting bacteria. Altogether, these results reiterate on the previously established fastidiousness of coprostanoligenic bacteria. Further attempts would benefit from more research determining the conditionssupporting cholesterol conversion, as a better understanding of those conditions is a first step in characterising the bacteria performing conversion. Eventually, this might enable research on supporting their growth using prebiotics or the feasibility of their application as next-generation probiotics for improved cardiovascular health.
- PostB cells maturation in infancy and its affect in early allergy(2019) Karlsson, Emma; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological EngineeringThe last decades, the presence of allergy has increased in countries belonging to the Western world and over 25 % of Swedish young people are aﬀected of allergic symptoms. One reasonable explanation is the hygiene hypothesis, mentioned by David Strachan 1989, that associate the increase in allergy development with a cleaner lifestyle. Previous studies showed that children who grew up on a farm with animals had a more maturated immune system and therefore developed less allergy. With samples received from the study named Nutritional impact on Immunological maturation during Childhood in relation to the Environment(NICE),I have studied the B cells maturation during early time in life. By studying subpopulations of B cells, speciﬁc surface markers are stained with ﬂuorochromes to be analysed with a ﬂow cytometer. Thus, the development of B cell subpopulations from birth up to 4 months can be charted. Several surface markers can indicate the same subpopulation, i.e. CD27+ and CD24hiCD38lo that are expressed by memory B cells. However, the percentage diﬀer from each other and the most liable reason is that the surface markers have diﬀerent functions in the B cells, and are therefore not only expressed during one phase of the development of B cells. My results shows that the development of percentage of subpopulations of B cells diﬀer from each other from birth up to 4 months. Correlation tests indicate that CD27+ and CD24hiCD38lo could both be used to detect memory B cells. Correlation tests at number of subpopulations had higher correlation coeﬃcient and were strongly signiﬁcant at birth. Therefore, it would be interesting to continue the analysis of numbers of B cells populations at 48h, 1, 4 and 12 months of life. In the NICE study, so far 452 children have been examined for allergy and 6.2 % suﬀered from eczema, 2.4 % of food allergy and 6.2 % of the children had developed asthmaat1yearofage. Inthisthesis, there was no signiﬁcant associations between B cell maturity and the development of allergy.
- PostBakteriella polysackarider - enzymatisk syntes och jakt på nya nedbrytande enzymer(2019) Bergentall, Martina; Chau, Christina; Engström, Enya; Johansson, Sofia; Lövgren, Sandra; Nordanger, Emma; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological EngineeringA common problem in society today is tooth-related diseases caused by dental plaque. Dental plaque is a microbial biofilm which works like a protecting shield for organisms to live within, and consists of mostly water and polysaccharides. Dental plaque can eventually lead to diseases such as caries or tooth decay, hence treating dental plaque at an early stage is of importance. Despite preventional methods in use today, dental plaque is still a widespread global problem suggesting that these methods are insufficient. There are a limited number of enzymes known today which can break down mutan or alternan, which are two polysaccharides that can be found in the dental plaque matrix. This project aimed to isolate and categorise enzymes from microorganisms with the ability to break down mutan and alternan. A long-term goal with finding new enzymes able to break down the polysaccharides is to incorporate these enzymes in dental hygiene products. Since mutan and alternan are not commercially available they were produced from the enzymes GtfJ and GtfL, and then purified by centrifugation and filtration. To confirm the purity, the polysaccharides were tested by reducing sugar analysis and thereafter freeze-dried before use. The polysaccharides were then used as a carbon source in cultivation plates and in liquid media when cultivating microorganisms. Field studies were carried out for collection of samples in two green belts around the Gothenburg area. The sampling included water and soil samples from different biotopes, which were introduced to cultivation plates made up of agar and alternan or mutan. A cultivation plate only containing agar was used as control. Growth was studied for a couple of weeks, and some colonies which showed growth with a clearing zone were restreaked on additional cultivation plates. Thereafter, samples were inoculated into liquid media for faster growth and to generate a sufficient amount of DNA for sequencing. Samples which showed growth after inoculation, and had a clearing zone when restreaked, were analysed by 16S and ITS sequencing. Results from the sequencing showed the possible presence of a known fungal species, Brettanomyces bruxellensis, and also the probable presence of five fungal and bacterial species belonging to the genera Cladosporium and Paenibacillus. Out of the six species discovered, five of them were able to break down mutan and one of them was able to break down alternan. Due to lack of time the purpose of isolating and characterising enzymes from said microorganisms could not be achieved, and hence it is a possible subject for further studies. These studies may involve enzyme efficiency and function in vivo, along with comparisons between several di erent enzymes in order to obtain optimal enzyme characteristics.