Filamentösa svampar används inom många biotekniska områden. Svamparnas biomassa kan användas till föda eller djurfoder, medan biprodukter som svampar producerar kan användas inom områden som industri samt medicin. Kultiveringen av Neurospora intermedia och Aspergillius oryzae genomförs på smör, raps och frityrolja i detta arbete för att få en större förståelse för hur svamparna kan växa på olika lipider samt skillnaden som kan uppstå mellan olika arter av filamentösa svampar. Under arbetes studeras biomassakoncentration, proteinhalt samt pH. Även problem som att svamparna kan lagra lipider i cellerna och att det kan påverka resultaten genom en ökning av biomassakoncentrationen diskuteras.  Kultiveringarna genomfördes i Erlenmeyerflaskor med 20 g/L lipid samt saltblandning för att bestå med nödvändiga näringsämnen. För A.oryzae genomförs även kultivering i en airliftbioreaktor för att förstå hur svampen beter sig i större skala. Högst koncentrationen torrbiomassa som erhålls ifrån Erlenmeyerflaskorna är 18,49±1,90 g/L för A.oryzae samt17,31±1,14 g/L för N.intermedia med en proteinhalt för torr biomassa upp till ca 14%.Utbytetmellan torr biomassa och lipid som tillsatts i kultiveringen hamnade på 94,00%±0,06%(rapsolja) för A.oryzae samt 81,69%±0,007% (frityrolja) för N.intermedia. De höga utbytena kan förklaras med att svamparna kan lagra lipider i cellerna vilket även ger de höga koncentrationen av torr biomassa. För att förstå morfologin, lipidhalt i biomassan samt pH beteende behövs mer arbete utföras med ändring av parametrar som pH justeringar, temperatur, luftflöde samt analys av biomassan för att utreda hur mycket lipider som lagras. / Filamentous fungi are used in many different biotechnological fields. The fungal biomass can be used for food or feed, while by-products that the fungi produce can be used in different industries or medical applications. The cultivations of Neurospora intermedia and Aspergillius oryzae implemented on butter, rapeseed and frying oil that is used in this work is for the larger understanding of how the fungi can grow on different lipids and the difference between species of fungi. Biomass concentration, protein content and pH are the main focus during this work, but problems like lipids storage in the biomass that can affect the results by increase of the total biomass concentration is discussed. The cultivations were implemented in Erlenmeyerflasks with 20 g/L lipid with added saltsolution with necessary nutrients. For A.oryzae cultivation in an airlift bioreactor was carried out for the understanding of larger scale cultivation. Highest concentration of dry biomass obtained from Erlenmeyerflasks are 18,49±1,90 g/L for A.oryzae and 17,31±1,14 g/L for N.intermedia with protein content for dry biomass up to 14%.The yield for dry biomass perlipid added during the cultivation ended at 94,00%±0,06% (rapeseed oil) for A.oryzae and 381,69%±0,007% (frying oil) for N.intermedia. During cultivation the cells can store lipids and that explains the high yields and biomass concentrations. For the understanding of morphology, the amount of lipids in the biomass and pH behaviour needs more work that includes changes of parameters like pH adjustment, temperature, airflow and analysis of the biomass for lipid content.

Chromatography units, used in the production of pharmaceuticals, degrade with use and need to be changed or repackaged. This study investigates the effectiveness of two statistical methods, principal component analysis and simple and one-point multiparameter technique, for determining degradation in the Fibro chromatography unit. The methods have been shown to be effective on resin chromatography columns but not before tested on the relatively new Fibro chromatography unit. The statistical methods are implemented in an unreleased version of the monitoring and control software Unicorn. This implementation aims to be a proof of concept for including more complex methods for monitoring runs directly in the software, easing the workflow of operators by removing the need to export measurements to a third-party program. The methods were tested on measurements of absorbance, conductivity, and pressure from two series of chromatograms performed on two Fibro chromatography units. One of the units was defective and broke down halfway through the series. Principle component analysis could clearly visualize a difference between early and late runs on the defective unit. The same could only be achieved for the non-defective unit by excluding measurements of pressure. Simple and one-point multiparameter technique visualized trends from early to late in the series which were much clearer for the defective unit. Both methods showed signs of predicting degradation in a Fibro chromatograpy unit but require validation on chromatogram series with more direct measurements of performance and a wider range of failure causes.

Fish has for a long time been a very important source of protein for human kind and with the world population at an all-time high, 7.5 billion and rapidly growing, the demand for fish as a food source is also at an all-time high and rapidly increasing. This has in turn led to overexploitation of many of the fish stocks of the world ocean’s and in many cases to depletion of fish stocks. The demand for sustainable food sources and sustainable usage of the world ocean’s fish stocks is therefore a subject with great deal of interest today. Much of the fish caught today are used for production of fish meal for usage as fish food at fish farms, which also increases the depletion of fish stocks around the globe. One way of dealing with this problem is to replace the fish meal as protein source in fish feed with protein from agricultural crops which in many cases are done today by usage of soy bean protein. This however poses another problem as the agricultural crops take up vast amount of land, in many cases obtained by diminishing the rainforests in the area. Another usage for the soybean would be as a direct human food source. Agricultural products are also dependent on environmental conditions to ensure reasonable production. The problems related to production of fish meal and soy has sparked the idea of using microorganisms for production of Single cell protein for usage as protein source in fish feed. Single cell protein can be produced in closed fermentation vessels and can be produced at a controlled rate and under controlled manners, while taking up negligible land space. During this thesis, the production of single cell protein from spent sulfite liquor using the filamentous fungi Paecilomyces variotii was examined. The aim of the project was to examine the effect of cultivation parameters (i.e., pH, temperature and nutrients) on the production of biomass as well as the protein content of the biomass. The correlation of the biomass growth and protein content have also been examined. The project was carried out by performing several experiment cultivations using spent sulfite liquor provided by Domsjö Fabriker in Örnsköldsvik. This process enables the utilization of a residual stream from the pulp industry which gives this process a huge environmental upside compared to similar processes as for example the commercial production of Quorn (a Single cell based food product) which utilizes pure glucose. The results showed that the protein content will steadily decrease as the biomass production increases hence it is desirable to keep the cultivation time at a minimum while maximizing biomass production during that time frame. It also points towards that the highest protein content is present in the young cell mass. The key conclusion from this thesis is however that it is possible to lower the pH of the cultivation from pH 6 down to pH 4.5 while still maintaining the biomass production and increasing the protein content. The highest obtained protein content was 62.7% at pH 4.5. The high protein content might be due to a slightly longer lag phase in the beginning of the cultivation which yields a higher number of younger cells in the final broth thus increasing protein content. Running the process at a lower pH is a huge advantage for industrial implementation as this on large scale means significant lower amounts of chemicals needed for pH adjusting of the spent sulfite liquor which renders the process much more economical. This is because pH adjustment today is one of the most costly process steps in the production of bioethanol from spent sulfite liquor.

Polyhydroxyalkanoater (PHA) är en grupp av polymerer som är biobaserade, biologiskt nedbrytbara och som kan uppvisa varierande fysiokemiska egenskaper. Således har PHA potential att ersätta konventionell och fossilbaserad plast så som polypropylen (PP) och polyetentereftalat (PET).  PHA framställas genom mikrobiologisk fermentering av organiskt material och innesluter sig i bakteriernas celler som sfäriska lagringsdepåer när bakterier matas med en kolkälla. PHAt kan sedan extraheras ut från bakterierna med hjälp av lösningsmedel, en metod som ger höga utvinningsnivåer men som kräver stora mängder lösningsmedel. Ska metoden vara hållbar och ha industriell relevans är det nödvändigt att lösningsmedelsåtervinning tillämpas.   Industriell framställning av PHA sker idag med rena bakteriekulturer som matas med odlade sockerrika grödor. Detta leder till att konkurrens om markanvändning samt vatten- och energiresurser för främst livsmedelsproduktion uppstår. Samtidigt skapas restströmmar från industri och samhälle innehållande organiskt material och blandade bakteriekulturer vilka kan utnyttjas för att producera PHA.   I detta arbete har PHA-processen modellerats i processimuleringsprogrammet CHEMCAD där data på restströmmar från skogsindustrin utnyttjas för PHA-produktion. I modellen har uppströmsprocesserna fermentering, anrikning, PHA-ackumulering och avvattning samt nedströmsprocesserna lösningsmedelsextraktion och separation inkluderats. Nedströmsprocesserna har modellerats för två fall. Ett fall där extraktion gjorts med 100 % torr biomassa (extraktionssystem 1) samt ett fall där extraktion gjorts med våt biomassa (extraktionssystem 2). Därtill implementerades intern värmeväxling vilket optimerades genom införandet av pinchanalys.   Målet med projektet var att upprätta en energianalys av PHA processen, identifiera energibehovet för de enskilda PHA-processerna, analysera om lösningsmedelsextraktion görs mest energieffektivt med våt eller torr biomassa samt att utvärdera hur intern värmeväxling kan bidra till att energieffektivisera PHA-processen.  Resultaten som erhölls visade att behovet av extern värme kunde minska från 49 till 13 MJ/ kg PHA och kylbehovet från 43 till 11 MJ/ kg PHA efter införandet av intern värmeväxling för extraktionssystem 1. När extraktion utfördes enligt extraktionssystem 2 minskade det externa kylbehovet från 98 till 48 MJ/kg PHA och det externa värmebehovet från 60,9 till 8,1 MJ/ kg PHA. I jämförelse med det totala specifika värme- och kylbehovet var det totala specifika elbehovet för PHA-processen lågt vilket kan ses som positivt då el är högvärdig och dyr energi. När extraktion utfördes enligt extraktionssystem 1 var det totala specifika elbehovet 3,55 MJ/ kg PHA och för extraktionssystem 2 var motsvarande siffra 4,1 MJ/ kg PHA. Störst elbehov uppstod i processen för luftning av PHA-ackumuleringstanken vilket krävde 2,8 MJ/kg PHA. / Polyhydroxyalkanoates (PHAs) is a group of biobased and biodegradable polymers which can exhibit flexible physicochemical properties. Thus, PHA has the potential to replace conventional and fossil-based plastic such as polypropylene (PP) and polyethylene terephthalate (PET).  PHA is produced through microbiological fermentation by organic matter and is stored in the cells of bacteria as spherical storage depots when the bacteria are feed by a carbon source. The PHA can then be extracted from the bacteria using solvent, a method resulting in high yields of PHA but on the other hand large quantities of solvent is required and if the method will be sustainable and have industrial relevance it is necessary to apply solvent recycling in the system.  Today industrial production of PHA takes place using pure bacterial cultures and cultivated sugar-rich crops. This contributes to a competition of land use and water and energy resources, mainly for food production. At the same time, waste streams are generated from industry and society containing organic material and mixed bacterial cultures which can be used to produced PHA.  In this work the process for PHA production has been modelled in the process simulation program CHEMCAD using data from waste streams produced from forest industry. In the model the following upstream processes has been included; acidogenic fermentation, enrichment, PHA-accumulation, dewatering and the downstream processes solvent extraction and separation. Moreover, the downstream processes have been modelled for two cases; one case where extraction takes place using 100 % dry biomass (extraction system 1) and one case where extraction takes place using wet biomass (extraction system 2). In addition, internal heat exchanging was implemented which was optimized through the introduction of pinch analysis.   The aim of the project was to establish an energy analysis of the PHA process, identify the energy needs of the individual PHA processes, analyse whether solvent extraction is made most energy efficient using wet or dry biomass and to evaluate how internal heat exchanging can contribute to make to the PHA process more energy efficient.   The results obtained showed that the need for external heating and cooling could decrease from 49 to 13 MJ/ kg PHA and 43 till 11 MJ/ kg PHA after the implementation of internal heat exchanging for extraction system 1. When extraction was performed according to extraction system 2 the external cooling demand was reduced from 98 to 48 MJ/kg PHA and the externa heating demand was reduced from 60,9 to 8,1 MJ/ kg PHA. In comparison with the total specific heating and cooling demand, the total specific electricity demand for the PHA process was low, which can be seen as positive as electricity is high-value and expensive energy. When extraction was performed according to extraction system 1, the total specific electricity requirement was 3,55 MJ/ kg PHA and for extraction system 2 the corresponding value was 4,1 MJ/ kg PHA. The greatest electricity demand arose in the process of aerating the PHA accumulation reactor with 2,8 MJ/kg PHA.

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.

No description available.

The area of surface expression has gathered a lot of interest from research groups all over the world and much work is performed in the area. Autotransporters have been used for surface expression in Gram-negative bacteria. One of the more commonly used autotransporters is the Adhesin Involved in Diffuse Adherence (AIDA) of pathogenic Escherichia coli. The surface expression of enzymes and vaccine epitopes offer several advantages. Surface expressed enzymes gain similar properties to immobilised enzymes, mainly simplified handling and separation using centrifugation. Surface expressed vaccine epitopes can have longer half-lives inside the animal that is to be immunized and surface groups on the host cell can act as adjuvants, increasing the immune response and leading to a better immunisation.    However, while much basic research is directed towards mechanisms of surface expression using autotransporters there are few reports regarding production of surface expressed protein. Thus the aim of this work was the optimisation of the yield and productivity of surface expressed protein. Protein Z, an IgG-binding domain of Staphylococcal protein A, was used as a model protein for the investigation of which cultivation parameters influenced surface expression. The choice of cultivation medium gave the largest impact on expression, which was attributed to effects based on the induction of the native promoter of AIDA. The AIDA system was then used for the expression of two Salmonella surface proteins, SefA and H:gm, with potential for use as vaccine epitopes. SefA was verified located on the cell surface, and H:gm was found in the outer membrane of the host cell, though only in proteolytically truncated forms lacking the His6-tag used for detection. This proteolysis persisted in E. coli strains deficient for the outer membrane protease OmpT and was concluded to be dependent on other proteases. The removal of proteolysis and further optimisation of the yield of surface-expressed protein are important goals of further work. / QC 20111123 / Vinnova: BIO-AMINES / SIDA Vietnam: Production of viral proteins for vaccine development

Producing pharmaceuticals in Escherichia coli inevitably comes with an extensive purification process. This is because many of the native proteins of E. coli are immunogenic to humans, especially the heat and pH resistant endotoxins located in the membrane of E. coli. These native proteins drive up the cost of the purification, which led to a request from the biopharmaceutical company Affibody AB. They want a review on the possibilities of producing their unique Affibody®-molecules in a new, less problematic host cell. Based on a previous bachelor project, Affibody AB chose Pichia pastoris as the candidate. P. pastoris is a methylotrophic yeast that is increasing in use when it comes to producing pharmaceuticals. In this review, multiple ways of utilizing P. pastoris are presented. The process proposals are based on 4 different promoters, pAOX1, pAOX2, pFLD1 and the pGAP. The AOX1- and AOX2-promoters and the FLD1-promoter are inducible promoters that require an inducer-molecule. An inducible promoter presents the best control of the process. The GAP-promoter is a constitutive promoter, meaning that the gene is expressed continuously. A constitutive promoter provides a process which requires fewer steps and ingredients. If the Affibody®-molecules were to be produced with P. pastoris as the host cell, the products would contain less immunogenic substances. Further, P. pastoris is also a very effective option when it comes to producing protein extracellularly. This would ultimately lead to a purification process that requires less resources.

Surface expression of recombinant proteins has attracted a lot of attention due to its potential in applications such as enzyme production, vaccine delivery and bioremediation. Autotransporters have been used for surface expression of a variety of proteins, but the expression systems reported in literature have typically been inflexible and incapable of detecting proteolysis, thereby limiting surface expression yield. In this thesis, a modular surface expression system, utilizing dual tag detection, was therefore created. It was based on the adhesin involved in diffuse adherence (AIDA-I) autotransporter, and was here used to express the model proteins SefA and H:gm on the cell surface of Escherichia coli. Due to the dual tag detection system, proteolysed H:gm could be successfully verified on the cell surface. By optimizing cultivation conditions, surface expression yield of SefA was increased by 300 %, and proteolysis reduced by 33 %. While proteolysis could not be eliminated completely, the work presented in this thesis is a major step towards a general system for surface expression of a wide range of proteins in varied applications. / <p>QC 20130506</p>

A big challenge within pulp and paper mills is the large quantities of waste activated sludge (WAS) that is produced during the wastewater treatment. The WAS is made up of biological cells and extra polymeric substances (EPS) and can bind a large amount of water causing difficulties to dewater the WAS. This study aimed to determine how to improve the dewatering properties of the WAS by using hydrolysis. Hydrolysis will cause the cells to disrupt and the bound water in the cells and the water trapped by the EPS can be released. Specifically, this study investigated what impact hydrolysis with heat, alkalis, and acids had on the WAS dewatering properties. In addition to the impact on the dewatering properties, the release of organic material and nutrients from the cells has also been important for biomethane production. In this study, it was specifically NH4-N, PO43- and COD that have been studied. WAS from paper mills have in general poor methane potential so it was of interest to see how the WAS was affected by hydrolysis and how hydrolysis could improve the methane production. To test the hypothesis of whether hydrolysis could affect the WAS and improve the dewatering properties, several experiments were performed. The experiments included thermal hydrolysis at temperatures of 70-90 °C, acidic hydrolysis with acids such as spent acid and acid water, and alkalis such as green liquor sludge and EOP. All acids and alkalis used in the study were chemicals that exist at the paper mills included in this study. To test the dewatering properties, methods such as TS analysis on the accept, CST-analysis, and a belt press were used. Analyses were also performed on the reject to measure the suspended solids and the nutrients NH4 – N, PO43– and COD in the WAS. This study did also determine what effect hydrolysed WAS had on the biomethane potential. In this study were the paper mills BillerudKorsnäs in Skärblacka and SCA in Östrand included. Hence was sludge from the two mills of interest to analyse. This study has shown better dewatering properties with an increase in the total solids (in the accept) after the thermal hydrolysis, the acidic hydrolysis with spent acid, and the alkali hydrolysis with green liquor sludge. Specifically did the acidic hydrolysis with spent acid improve the dewatering properties in terms of an increase in TS in %. The biggest increase in TS in % could be seen after using 10% spent acid ratio. The TS for the WAS from SCA Östrand increased in this experiment by 107 %. The thermal hydrolysis also showed promising results both in terms of dewatering properties and in the release of organic material. The biochemical methane potential test results showed a better and more rapid stabilized production of biomethane after hydrolysis of WAS compared to untreated WAS. The thermal hydrolysis both increased the rate of production and the total amount of methane produced. The thermally hydrolysed WAS from SCA Östrand improved the methane production from 77 Nml methane/g VS to 95 Nml methane/ g VS. The WAS from BillerudKorsnäs improved the methane production from 40 Nml methane/ g VS to 55 Nml methane/ g VS. These results, both from the methane potential tests and the results of the increased dewatering properties, show that the concept with hydrolysing should be evaluated further for improving the dewatering of the WAS.