In this case study the authors have tried to fill the gap of technology transferliterature focused on the biopharmaceutical industry. The technology transferliterature displays a clear industry-specific gap, mostly focused on heavy- andpharmaceutical industries. The authors have tried to find the key aspects of asuccessful technology transfer from the literature on the subject from alldifferent industries. The authors have then used these aspects to create atheoretical framework of the aspects that are possibly applicable in thebiopharmaceutical industry. A case study has been conducted at The Company which has a long pedigreeas one of the most innovative companies within the biopharmaceuticalindustry. The Company both develops and manufactures diagnostic tests forantibodies in animals, and their products are today widely known within theindustry. The authors have conducted a series of interviews, a non-participantobservation and also reviewed documentation of previous productsdevelopment processes. These qualitative methods have provided bothempirical evidence of similarities between the technology transfer literatureand a biopharmaceutical technology transfer process, as well as evidence ofwhat aspects are of importance in the biopharmaceutical industry. Using thisabductive research strategy, the authors have determined the key aspects thatare conceivably applicable in the biopharmaceutical industry. These are Goalcombability, Communication and documentation, Transfer plan andInterdepartmental collaboration. These aspects have not been implementedand therefore not been tested at The Company.

A significant by-product of bioethanol plants is whole stillage, commonly used to produce animal feed due to its nutritious value, has a potential to be used to produce various value-added products while eliminating a costly process step is an alternative approach. In this study, production and separation of additional ethanol, fungal biomass and enzyme were successfully achieved with the cultivation in membrane bioreactors in batch process condition. Process optimization studies regarding fermentation and filtration conditions were carried out. Up to 10.4 g/l ethanol per litre of used whole stillage can be produced in simultaneous saccharification and fermentation (SSF) condition without any pH adjustment and additional pretreatment step. Also, 50% diluted whole stillage provided 87% higher ethanol production comparing to non-diluted medium. Moreover, 71 % higher biomass production was obtained with the filtrate of 50% diluted whole stillage comparing to 25% diluted one. Considering the achieved results, a two-stage cultivation using SHF (Separate Hydrolysis and Fermentation) strategy in membrane bioreactors for separation of ethanol, lignin-rich stream, protein-rich fungal biomass and enzymes was proposed. The present thesis showed that the integration of filamentous fungi with membrane bioreactors can increase the range of products that can be produced from whole stillage.

Oomycetes have long been considered as a separate class within the kingdom Fungi, but they are in fact closer to brown algae. They are currently classified in the Stramenopile eukaryotic kingdom, which includes heterokont algae and water molds. The major cell wall polysaccharides in Oomycetes are b-(1à3) and b-(1à6)-glucans, as well as cellulose, which has never been reported in any fungal species. Chitin - the major cell wall polysaccharide in fungi - occurs in minor amounts in the walls of some Oomycetes. Some Oomycete species are pathogens of great economical importance. For example, species of the genus Phytophthora are well studied plant pathogens that cause considerable economical losses in agriculture. Saprolegniosis, a fish disease caused by species from the genus Saprolegnia, is a major problem in the aquaculture industry and represents a threat to populations of salmonids in natural habitats. Currently, there are no chemicals available that are at the same time efficient Oomycete inhibitors, environmentally friendly and safe for human consumption of treated fishes. The biosynthesis of cellulose in Oomycetes is poorly understood, even though this biochemical pathway represents a potential target for new Oomycete inhibitors. In this work, cellulose biosynthesis was investigated in two selected Oomycetes, the plant pathogen Phytophthora infestans and the fish pathogen Saprolegnia monoica. A new Oomycete CesA gene family was identified. It contains four homologues designated as CesA1, CesA2, CesA3 and CesA4. The gene products of CesA1, 2 and 4 contain Pleckstrin Homology domains located at the N-terminus. This represents a novel feature, unique to the Oomycete CesA genes. CesA3 is the dominantly expressed CesA homologue in the mycelium of both S. monoica and P. infestans, while CesA1 and CesA2 are up-regulated in virulent life stages of P. infestans. CesA4 was expressed only in minute amounts in all investigated types of cells. Gene silencing by RNA interference of the whole CesA gene family in P. infestans lead to decreased amounts of cellulose in the cell wall. The inhibitors of cellulose synthesis DCB and Congo Red had an up-regulating effect on SmCesA gene expression, which was accompanied by an increased b-glucan synthase activity in vitro. In addition, these inhibitors slowed down the growth of the mycelium from S. monoica. Zoospores from P. infestans treated with DCB were unable to infect potato leaves and showed aberrant cell wall morphologies similar to those obtained by silencing the CesA gene family. Altogether these results show that at least some of the CesA1-4 genes are involved in cellulose biosynthesis and that the synthesis of cellulose is crucial for infection of potato by P. infestans. / QC 20101110

DNA detection has an increasing importance in our everyday lives, with applications ranging from microbial diagnostics to forensic analysis. Currently, as the associated costs decrease, DNA diagnostic techniques are routinely used not only in research laboratories, but also in clinical and forensic practice. The present thesis aims to unravel the potential of cellulose filter paper to be a viable candidate for DNA array support. There are two papers in this study. In Paper I, we studied the method of functionalizing the surface of filter paper and the possibility to detect DNA on acitve paper using fluorescence. In Paper II, we investigated visualization and throughput of DNA detection with magnetic beads on active filter papers, an assay which requires no instrumentation (scanner). The findings in Paper I show that XG-NH2 and PDITC can functionalize the cellulose filter paper and that the activated filter papers can covalently bind oligonucleotides modified with amino groups to detect DNA. The detection limit of the assay is approximately 0.2 pmol. In Paper II, visualization of DNA detection on active paper is achieved without instrumentation, based on the natural color of magnetic beads. Furthermore, successful multiplex detection supports the potential to increase the throughput of DNA detection on active papers. In summary, these studies show that active cellulose filter paper is a good DNA array support candidate as it provides a user-friendly and cost-efficient DNA detection assay. The methods described in Paper I and II are possible sources of development to a point-of-care device for on-site analysis of DNA contents in a sample. / <p>QC 20131111</p>

Banana peels account for about 61 million tons of waste each year globally. The aim of this project was to investigate the possibility of using banana peels as a substrate to cultivate edible filamentous fungi. The peels were subjected to physical and thermal pretreatments while variables such as changes in the medium pH, biomass concentration, fungal strain dependence, and protein content of the fungal biomass were analyzed. The experiments were carried out in three phases. The purpose of phase I was to identify which of the four fungal strains among Neurospora intermedia, Aspergillus oryzae, Rhizopus oryzae, and Rhizopus oligosporus could grow in a medium containing ball-milled banana peel powder (BPP) only. In phase II, the best performing strains from phase I in terms of biomass concentration, i.e., A. oryzae and R. oryzae, were cultivated using banana peel broth (BPB) obtained from thermal pretreatment of BPP. During this phase, the impact of medium supplementation with yeast extract was also assessed. The biomass yield for A. oryzae and R. oryzae 2.9 g/L and 1.6 g/L, respectively, yeast supplementation compared to 2.7 g/L and 0.7 g/L, respectively, without supplementation. In phase III, the experiments performed in phase II without yeast extract supplementation were scaled up, after which protein analysis was performed. A crude protein content of 8.82% was determined for A. oryzae, while in R. oryzae, a higher value of 21.1% was obtained. The protein content from both fungal strains was much higher than that present in the BPP, which was 4.8 g/L. The results showed the potential of using banana peel as a substrate to produce edible fungal biomass with higher protein content and thus has potential applications as animal feed or human food. Further studies are needed to optimize the process in order to raise the fungal biomass yield as well as increase the protein content of the biomass. In addition, comprehensive characterization of the fungal biomass would reveal other important components, such as the amino acid profile.

Anaerob jäsning antas vara en bra och effektiv metod för behandling av organiskt avfall. Avloppsvatten från denna process är mer utmanande beroende främst på dess höga innehåll av bland annat flyktiga fettsyror (VFA). Mikroalger har en stor potential för hållbart avlägsnande av näringsämnen från vatten samtidigt som algbiomassa kan användas för produktion av bio-gödsel, biobränsle och bioplaster. Därför var syftet med denna studie att undersöka möjligheten att odla två svenska stammar av mikroalger, anpassade till det nordiska klimatet, på olika syntetiska kulturer. Kulturerna innehöll två bestämda totalkoncentrationer av ättiksyra, propionsyra och smörsyra med tre olika förhållande till varandra. Stammarna som studeras i detta experiment var Chlorella vulgaris (13–1) och Chlorococcum sp. (MC-1). Stammarna visade olika förmågor att tolerera VFA som kolkälla. Chlorococcum sp. kunde ge betydligt högre biomassakoncentrationer i närvaro av VFA än C. vulgaris. Vid totalkoncentrationen 2 g/L VFA och den största halten av ättiksyra erhölls den högsta biomassakoncentrationen. C. vulgaris i kulturerna med VFA visade samma beteende som i referensodlingen utan VFA. De slutliga biomassakoncentrationerna i närvaro av VFA liknade den biomassakoncentrationen från referensodlingen. Att Chlorococcum sp. kan växa på VFA kan minska miljöpåverkan av industriella utflöden från anaerob jäsning i Sverige och andra nordiska länder. / Anaerobic fermentation is believed to be a good and effective method for treating organic waste. Wastewater from this process is more challenging mainly due to its high content of for instance volatile fatty acids (VFA). Microalgae have shown great potential for the sustainable removal of nutrients from water sources. At the same time, algal biomass can be used to produce bio-fertilizer, biofuel, and bioplastics. Therefore, the purpose of this study was to investigate the possibility of growing two Swedish microalgae strains, adapted to the Nordic climate, on different synthetic cultures. The cultures contain two determined total concentrations of acetic acid, propionic acid, and butyric acid with three different ratios to each other. The strains studied in this experiment were Chlorella vulgaris (13–1) and Chlorococcum sp. (MC-1). The strains showed different abilities to tolerate VFA as a carbon source under the same biotic and abiotic conditions. Chlorococcum sp. was able to produce significantly higher biomass concentrations in the presence of VFA than in the reference culture without VFA. The culture with the total concentration of 2 g/L of VFA and the ratio that had the greatest content of acetic acid gave the highest biomass concentration. C. vulgaris was not affected by VFA and the algal cells in the cultures with VFA show the same behaviour as in the reference culture. Moreover, the final biomass concentrations in the presence of VFA were similar to the biomass concentration from the optimal culture. Since Chlorococcum sp. could grow on VFA it can reduce the environmental impact of industrial effluents from anaerobic fermentation in Sweden and other Nordic countries.

Antibodies have become indispensable tools in diagnostics, research and as therapeutics. There are several strategies to generate monoclonal antibodies (mAbs) in order to avoid the drawbacks of polyclonal antibodies (pAbs) for therapeutic use. Moreover, the growing interest in precision medicine requires a well-characterized target and antibody to predict the responsiveness of a treatment. This thesis describes the use of epitope information and display technologies to generate and characterize antibodies. In Paper I, we evaluated if the epitope information of a well-characterized pAb could be used to generate mAbs with retained binding characteristics. In Paper II, the epitope on the complement protein C5 towards Eculizumab was mapped with surface display, the results of which explained the non-responsiveness of Eculizumab treatment among a patient group due to a mutated C5 gene. With this in mind, we showed efficacy in treatment of the mutated C5 variants using a drug binding to another site on C5, suggesting that our approach can be used to guide treatment in precision medicine. In Paper III, a Gram-positive bacterial display platform was evaluated to complement existing platforms for selection of human scFv libraries. When combined with phage display, a thorough library screening and isolation of nano-molar binders was possible. In Paper IV, a solid phase method for directed mutagenesis was developed to generate functional affinity maturation libraries by simultaneous targeting of all six CDRs. The method was also used to create numerous individual mutants to map the paratope of the parent scFv. The paratope information was used to create directed libraries and deep sequencing of the affinity maturation libraries confirmed the viability of the combination approach. Taken together, precise epitope/paratope information together with display technologies have the potential to generate attractive therapeutic antibodies and direct treatment in precision medicine. / <p>QC 20170418</p>

Food waste and fashion pollution are two of the significant global environmental issues throughout the recent past. In this research, it was investigated the feasibility of making a leather-like material from bread waste using biotechnology as the bridging mechanism. The waste bread collected from the supermarkets were used as the substrate to grow filamentous fungi species Rhizopus Delemar and Fusarium Venenatum. Tanning of fungal protein fibres was successfully performed using vegetable tanning, confirmed using FTIR and SEM images. Furthermore, glycerol and a biobased binder treatment was performed for the wet-laid fungal microfibre sheets produced. Overall, three potential materials were able to produce with tensile strengths ranging from 7.74 ± 0.55 MPa to 6.92 ± 0.51 MPa and the elongation% from 16.81 ± 1.61 to 4.82 ± 0.36. The binder treatment enhanced the hydrophobicity even after the glycerol treatment, an added functional advantage for retaining flexibility even after contact with moisture. The fungal functional material produced with bread waste can be tailored successfully into leather substitutes using an environmentally benign procedure.

Consumption of citrus fruits and citrus juice production creates wastes, which could be valorized by using it for cultivating fungi. Before cultivation, the medium needs to be sterilized though autoclavation. Larger volumes used when autoclaving requires longer heating cycles and therefore runs the risk of degrading the medium to a greater extent. This research examines the effects of the volume lemon waste medium used while sterilizing. The aim is to find the largest volume still providing good growth for the filamentous fungus used, Rhizopus Delemar. Lemon waste was provided by Herrljunga Musteri AB and was pre-treated at 45°C for 2h. The liquid was strained and autoclaved in different volumetric series ranging from 200 – 10 000 mL, that was then used in 200 mL shake flask cultivations.  A scale up in two 3,5 L bubble column reactors was also performed from the 10 000 mL autoclaved medium, after not observing severe impacts on growth. Testing was done by weighing biomass and HPLC analysis of sugars. The yield of the biomass in the shake flasks ranged from 0,11 – 0,14 g/g sugars and the biomass concentration ranged between 2,4 - 3,0 g/L. Overall, the volume of autoclavation seems to not too be of great concern when cultivating R. Delemar on lemon waste medium in the analyzed ranges.

Xyloglucans are polysaccharides found as storage polymers in seeds and tubers, and as cross-linking glycans in the cell wall of plants. Their structure is complex with intricate branching patterns, which contribute to the physical properties of the polysaccharide including its binding to and interaction with other glycans such as cellulose. Xyloglucan is widely used in bulk quantities in the food, textile and paper making industries. With an increasing interest in technically more advanced applications of xyloglucan, such as novel biocomposites, there is a need to understand and control the properties and interactions of xyloglucan with other compounds, to decipher the relationship between xyloglucan structure and function, and in particular the effect of different branching patterns. However, due to the structural heterogeneity of the polysaccharide as obtained from natural sources, relevant studies have not been possible to perform in practise. This fact has stimulated an interest in synthetic methods to obtain xyloglucan mimics and analogs with well-defined structure and decoration patterns. Glycosynthases are hydrolytically inactive mutant glycosidases that catalyse the formation of glycosidic linkages between glycosyl fluoride donors and glycoside acceptors. Since its first conception in 1998, the technology is emerging as a useful tool in the synthesis of large, complex polysaccharides. This thesis presents the generation and characterisation of glycosynthases based on xyloglucanase scaffolds for the synthesis of well-defined homogenous xyloglucan oligo- and polysaccharides with regular substitution patterns.