The total synthesis of capsaicin analogues was performed in one pot, starting from compounds that can be derived from lignin. Heterogeneous palladium nanoparticles were used to oxidise alcohols to aldehydes, which were further converted to amines by an enzyme cascade system, including an amine transaminase. It was shown that the palladium catalyst and the enzyme cascade system could be successfully combined in the same pot for conversion of alcohols to amines without any purification of intermediates. The intermediate vanillyl-amine, prepared with the enzyme cascade system, could be further converted to capsaicin analogues without any purification using either fatty acids and a lipase, or Schotten-Baumann conditions, in the same pot. An aldol compound (a simple lignin model) could also be used as starting material for the synthesis of capsaicin analogues. Using l-alanine as organocatalyst, vanillin could be obtained by a retro-aldol reaction. This could be combined with the enzyme cascade system to convert the aldol compound to vanillylamine in a one-step one-pot reaction.

Screening of expression constructs suitable for protein pharmaceuticals is often done in batch cultivations. But the production of the recombinant protein is made during fed-batch cultivations. The two types of cultivations are different and therefore may good expression constructs that grow poorly in batch cultivations but good in fed-batch cultivations be rejected. Therefore would it be desirable to develop a fed-batch method that can be used in shake flasks. Biosilta has developed a method where starch is broken down into glucose by an enzyme creating fed-batch conditions. This method has been tried out and analyzed during this project. It is shown that the cells grown under these conditions can be glucose limited. However, at a later stage of the cultivation the cells produce a large amount of acetate and pH is not stable. The system builds on a booster tablet which content is unknown. If the booster is not added to the cultivations the cells stop growing, this indicates that there is some other limitation than just glucose. It is also seen that the amount of protein that is produced during this fed-batch mimic cultivation is much lower than that is produced during normal batch cultivations. I would therefore not recommend EnBase as a screening method.

Microalgae are considered as a potential feedstock for many promising applications. Some active substances in microalgae have plant biostimulation effects potentially useful in agriculture. However, to produce such a microalgal biomass, specific microalgae cultivation and post-treatment processes must be designed to preserve active substances. A particular focus is provided on cultivation (tubular photobioreactor) and different plausible post-treatment scenarios for microalgae separation (flocculation and centrifugation) and preservation (sterilization and drying). For each step, yield and energy consumption are modeled using data taken from literature or lab and pilot scale experiments. Industrial equipment for scale-up process is also studied by comparing existing systems. These models enable to make an economic evaluation of the whole process and to study its profitability for each scenario. The breakeven price is calculated as a function of the production rate. Several parameters are suggested to improve system efficiency and profitability at the end of this study. However, a better microalgae characterization and more experiments on potential post-treatment systems are required to improve the accuracy of the model.

Material degradation and a decrease of fuel quality are common phenomena when storing biomass. A magnitude of 7.8% has been reported to degrade over five months when storing spruce wood chips in the winter in Central Europe. This thesis presents a theoretical study of biomass storage. It includes investigations of bio-chemical, chemical and physical processes that occur during storage of chipped woody biomass. These processes lead to degradation caused by micro-activity, chemical oxidation reactions and physical transformation of water. Micro-activity was modeled with Monod kinetics which are Michaelis-Menten type of expressions. The rate expressions were complemented with dependency functions describing the impact of oxygen, moisture and temperature. The woody biomass was divided into three fractions. These fractions represent how hard different components of the wood are to degrade by microorganisms. Chemical oxidation was modeled as a first order rate expression with respect to the active components of the wood. Two different cases have been simulated during the project. Firstly, an isolated system with an initial oxygen concentration of air was considered. This case displayed a temperature increase of approximately 2˚C and a material degradation less than 1%. The second case considered an isolated system with an endless depot of oxygen. This case resulted in degradation losses around 0.45-0.95% in the temperature range between 65-80˚C during approximately 300 days of storage. The temperature increased slowly due to chemical oxidation.

Pharmaceutical residues are found in the environment due to extensive use in human and veterinary medicine. The active pharmaceutical ingredients (APIs) have a potential impact in non-target organisms. Municipal wastewater treatment plants (WWTPs) are not designed to remove APIs.

In this thesis, two related matters are addressed 1) evaluation of advanced treatment to remove APIs from municipal wastewater and 2) the prevalence and degradation of APIs in the Baltic Sea.

A stationary pilot plant with nanofiltration (NF) and a mobile pilot plant with activated carbon and ozonation were designed to study the removal of APIs at four WWTPs. By NF, removal reached 90%, but the retentate needed further treatment. A predictive model of the rejection of APIs by NF was developed based on the variables: polarizability, globularity, ratio hydrophobic to polar water accessible surface and charge. The pilot plants with granular and powdered activated carbon (GAC) and (PAC) removed more than 95% of the APIs. Screening of activated carbon products was essential, because of a broad variation in adsorption capacity. Recirculation of PAC or longer contact time, increased the removal of APIs. Ozonation with 5-7 g/m³ ozone resulted in 87-95% removal of APIs. Elevated activity and transcription of biomarkers indicated presence of xenobiotics in regular effluent. Chemical analysis of APIs, together with analysis of biomarkers, were valuable and showed that GAC-filtration and ozonation can be implemented to remove APIs in WWTPs, with decreased biomarker responses.

Sampling of the Baltic Sea showed presence of APIs in 41 out of 43 locations. A developed grey box model predicted concentration and half-life of carbamazepine in the Baltic Sea to be 1.8 ng/L and 1300 d respectively.

In conclusion, APIs were removed to 95% by GAC or PAC treatment. The additional treatment resulted in lower biomarker responses than today and some APIs were shown to be widespread in the aquatic environment.

Användning av etanol som drivmedel och en efterfråga på gröna kemikalier driver utvecklingen av bioetanol framåt. Etanolpiloten, SEKAB, i Örnsköldsvik är en av få anläggningar i världen med kompetens och kunskap att producera bioetanol baserat på lignocellulosa. På senare tid har det dock uppstått problem vid etanolframställningen på grund av att en del jästodlingar blivit kontaminerade av bakterier vilket lett till ett sämre utbyte av biomassa och etanol. Det huvudsakliga syftet med detta examensarbete var att ta reda på orsaken till dessa misslyckade jästodlingar.

Examensarbetet delades upp i två huvudsakliga problemområden. Förutom orsaken till de kontaminerade odlingarna studerades även funktionen hos en ny jäststam, Saccaromyces cerevisiae torrjäst, i syfte att undersöka om det finns bättre alternativ till den jäststam som används i etanopiloten i nuläget.

En specialstudie av rengöringen av odlingstankar och ledningar i etanolpiloten utfördes i syfte att kartlägga var i utrustningen som infektionsrisken är som störst. Försöken påvisade att det huvudsakliga problemet kan lokaliseras till den största jästodlingstanken. Där befinner sig jästen under en längre tid i en miljö som är gynnsam för tillväxt av både jäst och bakterier. En annan orsak till de infekterade odlingarna är att rengöringen av utrustningen inte har skett på rätt sätt, samt att temperaturen hos tvättkemikalierna har varit för låg. En viktig slutsats är därför att bättre rutiner vid hanteringen av jästodlingsutrustningen samt att större noggrannhet i samband med rengöringen bör eftersträvas.

En bidragande orsak till de infekterade odlingarna kan också härröra från uppodlingsprocessen av ympjäst som i dagens läge sker på laboratorium. Genom att använda en stam av S. cerevisiae som köps in i frystorkad form kan flera steg i jästodlingsprocessen elimineras. Det både förkortar odlingsprocessen och minskar infektionsrisken. S. cerevisiae torrjäst undersöktes både i laboratorium och i etanolpiloten. Tre olika odlingsskalor användes, skakflaskor (250 ml), labfermentorer (3 l) och pilotskala (10m³). Försöken påvisar höga utbyten av både biomassa och etanol. För att kunna hålla nere produktionskostnaderna för etanolframställningen är det viktigt att jästen som används går att odla på det hydrolysat som produceras vid förbehandlingen av råvaran. Försök i pilotskala visar på lovande resultat vid uppodling av S. cerevisiae torrjäst när hela 70 % av sockerkällan kommer från hydrolysat. Ytterligare utvärdering och optimering av odlingsprocessen samt en ekonomisk jämförelse mellan de tillgängliga jäststammarna krävs dock innan S. cerevisiae torrjäst eventuellt kan användas kontinuerligt i pilotskala. 

High capacity magnetic protein A agarose beads, LOABeads PrtA, were used in the development

of a new process for affinity purification of monoclonal antibodies (mAbs) from non-clarified

CHO cell broth using a pilot-scale magnetic separator. The LOABeads had a maximum binding

capacity of 65 mg/mL and an adsorption capacity of 25–42 mg IgG/mL bead in suspension for an

IgG concentration of 1 to 8 g/L. Pilot-scale separation was initially tested in a mAb capture step

from 26 L clarified harvest. Small-scale experiments showed that similar mAb adsorptions were

obtained in cell broth containing 40 Å~ 106 cells/mL as in clarified supernatant. Two pilot-scale

purification runs were then performed on non-clarified cell broth from fed-batch runs of 16 L,

where a rapid mAb adsorption ≥96.6% was observed after 1 h. This process using 1 L of magnetic beads had an overall mAb yield of 86% and 16 times concentration factor. After this single protein

A capture step, the mAb purity was similar to the one obtained by column chromatography, while

the host cell protein content was very low, <10 ppm. Our results showed that this magnetic bead

mAb purification process, using a dedicated pilot-scale separation device, was a highly efficient

single step, which directly connected the culture to the downstream process without cell clarification.

Purification of mAb directly from non-clarified cell broth without cell separation can provide

significant savings in terms of resources, operation time, and equipment, compared to legacy procedure of cell separation followed by column chromatography step.

The interest for perfusion is increasing nowadays. This new focus has emerged from a synergy of a demand for disposable equipment and the availability of robust cell separation device, as well as the need for higher flexibility and lower investment cost. The cell separation devices mostly used today are based on filtration, i.e. alternating flow filtration, tangential flow filtration, spin-filter, or acceleration/gravity, i.e. inclined settler, centrifuge, acoustic settler. This paper gives an introduction to the basic concepts of perfusion and its practical implementation. It reviews the actual cell separation devices and describes the approaches used in the field to develop and optimize the perfusion processes.

An inflatable bioreactor bag for cell cultivation, which comprising a top and a bottom sheet of flexible material, joined together to form two end edges and two side edges, wherein one baffle or a plurality of baffles extend from the bottom sheet in a region where the shortest distance to any one of the two end edges is higher than about one fourth of the shortest distance between the two end edges.

A method has been developed to tune the DO and pH controller PID parameters for pilot / large scale mammalian cultivation. Our approach is to identify a model of the variable to be controlled (e.g. DO, pH) and to design several possible PID controllers based on this model. The controllers were first tested in computer simulations, followed by wet simulation and finally the best controller was tested on the real process. The approach is developed for the tuning of the DO controller of a 50 L bioreactor using microbubble continuous oxygen flow. The method, called lag control here, is based on a lead lag control design using Bode analysis where the prediction part is omitted. Experiments show that the approach results in a highly satisfactory DO control. The oxygen microbubbles were almost completely consumed before reaching the liquid surface so the oxygen flow used to maintain the DO gave an excellent indication of the cellular oxygen consumption. The control system was robust against all the perturbations, i.e. cell growth, cell bleed, addition of air-saturated fresh medium, DO set point change and a second gas sparger used to strip out the carbon dioxide. This approach was also successfully used for the tuning of a 400 L bioreactor DO controller and pH controller.

Surface expression has attracted much recent interest, and it has been suggested for a variety of applications. Two such applications are whole-cell biocatalysis and the creation of live vaccines. For successful implementation of these applications there is a need for flexible surface expression systems that can yield a high level of expression with a variety of recombinant fusion proteins. The aim of this work was thus to create a surface expression system that would fulfil these requirements.

A novel surface expression system based on the AIDA-I autotransporter was created with the key qualities being are good, protein-independent detection of the expression through the presence of two epitope tags flanking the recombinant protein, and full modularity of the different components of the expression cassette. To evaluate the flexibility of this construct, 8 different model proteins with potential use as live-vaccines or biocatalysts were expressed and their surface expression levels were analysed.

Positive signals were detected for all of the studied proteins using antibody labelling followed by flow cytometric analysis, showing the functionality of the expression system. The ratio of the signal from the two epitope tags indicated that several of the studied proteins were present mainly in proteolytically degraded forms, which was confirmed by Western blot analysis of the outer membrane protein fraction. This proteolysis was suggested to be due to protein-dependent stalling of translocation intermediates in the periplasm, with indications that larger size and higher cysteine content had a negative impact on expression levels. Process design with reduced cultivation pH and temperature was used to increase total surface expression yield of one of the model proteins by 400 %, with a simultaneous reduction of proteolysis by a third. While not sufficient to completely remove proteolysis, this shows that process design can be used to greatly increase surface expression. Thus, it is recommended that future work combine this with engineering of the bacterial strain or the expression system in order to overcome the observed proteolysis and maximise the yield of surface expressed protein.

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.

Background: Bacterial surface display is of interest in many applications, including live vaccine development, screening of protein libraries and the development of whole cell biocatalysts. The goal of this work was to understand which parameters result in production of large quantities of cells that at the same time express desired levels of the chosen protein on the cell surface. For this purpose, staphylococcal protein Z was expressed using the AIDA autotransporter in Escherichia coli.

Results: The use of an OmpT-negative E. coli mutant resulted in successful expression of the protein on the surface, while a clear degradation pattern was found in the wild type. The expression in the mutant resulted also in a more narrow distribution of the surface anchored protein within the population. Medium optimisation showed that minimal medium with glucose gave more than four times as high expression as LB-medium. Glucose limited fed-batch was used to increase the cell productivity and the highest protein levels were found at the highest feed rates. A maintained high surface expression up to cell dry weights of 18 g l(-1) could also be achieved by repeated glucose additions in batch cultivation where production was eventually reduced by low oxygen levels. In spite of this, the distribution in the bacterial population of the surface protein was narrower using the batch technique.

Conclusions: A number of parameters in recombinant protein production were seen to influence the surface expression of the model protein with respect both to the productivity and to the display on the individual cell. The choice of medium and the cell design to remove proteolytic cleavage were however the most important. Both fed-batch and batch processing can be successfully used, but prolonged batch processing is probably only possible if the chosen strain has a low acetic acid production.

Today, it is considered state-of-the-art to engineer living organisms for various biotechnology applications. Even though this has led to numerous scientific breakthroughs, the enclosed interior of bacterial cells still restricts interactions with enzymes, pathways and products due to the mass-transfer barrier formed by the cell envelope. To promote accessibility, we propose engineering of biocatalytic reactions and subsequent product deposition directly on the bacterial surface. As a proof-of-concept, we used the AIDA autotransporter vehicle for Escherichia coli surface expression of tyrosinase and fully oxidized externally added tyrosine to the biopolymer melanin. This resulted in a color change and creation of a black cell exterior. The capture of ninety percent of a pharmaceutical wastewater pollutant followed by regeneration of the cell bound melanin matrix through a simple pH change, shows the superior function and facilitated processing provided by the surface methodology. The broad adsorption spectrum of melanin could also allow removal of other micropollutants.

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.

Introduction: Hitherto the production of the biopolymer cyanophycin (CGP) using recombinant Escherichia coli strains and cheap mineral salts medium yielded only trace amounts of CGP (<0.5%, w/w) of the cell dry matter (CDM). This was probably due to the instability of the plasmids encoding the cyanophycin synthetase. Material and methods: In this study, we developed an anabolism-based media-dependent plasmid addiction system (PAS) to enhance plasmid stability, and we established a process based on a modified mineral salts medium yielding a CGP content of 42% (w/w) at the maximum without the addition of amino acids to the medium for the first time. This PAS is based on different lysine biosynthesis pathways and consists of two components: (1) a knockout of the chromosomal dapE disrupts the native succinylase pathway in E. coli and (2) the complementation by the plasmid-encoded artificial aminotransferase pathway mediated by the dapL gene from Synechocystis sp. PCC 6308, which allows the synthesis of the essential lysine precursor L,L-2,6-diaminopimelate. In addition, this plasmid also harbors cphAC595S, an engineered cyanophycin synthetase gene responsible for CGP production. Results: Cultivation experiments in Erlenmeyer flask and also in bioreactors in mineral salts medium without antibiotics revealed an at least 4.5-fold enhanced production of CGP in comparison to control cultivations without PAS. Discussion: Fermentation experiments with culture volume of up to 400 l yielded a maximum of 18% CGP (w/w) and a final cell density of 15.2 g CDM/l. Lactose was used constantly as an effective inducer and carbon source. Thus, we present a convenient option to produce CGP with E. coli at a technical scale without the need to add antibiotics or amino acids using the mineral salts medium designed in this study.

Background: Glycerol, whose formation contributes to cellular redox balancing and osmoregulation in Saccharomyces cerevisiae, is an important by-product of yeast-based bioethanol production. Replacing the glycerol pathway by an engineered pathway for NAD(+)-dependent acetate reduction has been shown to improve ethanol yields and contribute to detoxification of acetate-containing media. However, the osmosensitivity of glycerol non-producing strains limits their applicability in high-osmolarity industrial processes. This study explores engineering strategies for minimizing glycerol production by acetate-reducing strains, while retaining osmotolerance. Results: GPD2 encodes one of two S. cerevisiae isoenzymes of NAD(+)-dependent glycerol-3-phosphate dehydrogenase (G3PDH). Its deletion in an acetate-reducing strain yielded a fourfold lower glycerol production in anaerobic, low-osmolarity cultures but hardly affected glycerol production at high osmolarity. Replacement of both native G3PDHs by an archaeal NADP(+)-preferring enzyme, combined with deletion of ALD6, yielded an acetate-reducing strain the phenotype of which resembled that of a glycerol-negative gpd1 Delta gpd2 Delta strain in low-osmolarity cultures. This strain grew anaerobically at high osmolarity (1 mol L-1 glucose), while consuming acetate and producing virtually no extracellular glycerol. Its ethanol yield in high-osmolarity cultures was 13% higher than that of an acetate-reducing strain expressing the native glycerol pathway. Conclusions: Deletion of GPD2 provides an attractive strategy for improving product yields of acetate-reducing S. cerevisiae strains in low, but not in high-osmolarity media. Replacement of the native yeast G3PDHs by a heterologous NADP(+)-preferring enzyme, combined with deletion of ALD6, virtually eliminated glycerol production in high-osmolarity cultures while enabling efficient reduction of acetate to ethanol. After further optimization of growth kinetics, this strategy for uncoupling the roles of glycerol formation in redox homeostasis and osmotolerance can be applicable for improving performance of industrial strains in high-gravity acetate-containing processes.

The potential of using droplet microfluidics for screening mammalian cell factories has been limited by the difficulty in achieving continuous cell division during cultivation in droplets. Here, we report the influence of droplet size on mammalian cell division and viability during cultivation in droplets. Chinese Hamster Ovary (CHO) cells, the most widely used mammalian host cells for biopharmaceuticals production were encapsulated and cultivated in 33, 180 and 320 pL droplets for 3 days. Periodic monitoring of the droplets during incubation showed that the cell divisions in 33 pL droplets stopped after 24 h, whereas continuous cell division was observed in 180 and 320 pL droplets for 72 h. The viability of the cells cultivated in the 33 pL droplets also dropped to about 50% in 72 h. In contrast, the viability of the cells in the larger droplets was above 90% even after 72 h of cultivation, making them a more suitable droplet size for 72-h cultivation. This study shows a direct correlation of microfluidic droplet size to the division and viability of mammalian cells. This highlights the importance of selecting suitable droplet size for mammalian cell factory screening assays.

Perfusion operation mode is currently under fast expansion in mammalian cell based manufacturing of biopharmaceuticals, not only for labile drug protein but also for stable proteins such as monoclonal antibodies (mAbs). Perfusion mode can advantageously offer a stable cell environment, long-term production with high productivity and consistent product quality. Intensified high cell density culture (HCDC) is certainly one of the most attractive features of a perfusion process due to the high volumetric productivity in a small footprint that it can provide. Advancements in single-use technology have alleviated the intrinsic complexity of perfusion processes while the maturing in cell retention devices has improved process robustness. The knowledge for perfusion process has been gradually built and the “continuous” concept is getting more and more acceptance in the field.

This thesis presents the development of robust perfusion process at very high cell densities in various culture systems. Four HCDC perfusion systems were developed with industrial collaborators with three different mAb producing Chinese Hamster Ovary (CHO) cell lines: 1-2) WAVE Bioreactor™ Cellbag prototype equipped with cell separation by hollow fiber filter utilizing Alternating Tangential Flow (ATF) and Tangential Flow Filtration (TFF) techniques; 3) Fiber matrix based CellTank™ prototype; 4) Glass stirred tank bioreactor equipped with ATF. In all the systems, extremely high viable cell densities above 130 million viable cells per milliliter (MVC/mL) up to 214 MVC/mL were achieved. Steady states were maintained and studied at 20-30 MVC/mL and 100-130 MVC/mL for process development. Perfusion rate selection based on cell specific perfusion rate (CSPR) was systematically investigated and exometabolome study was performed to explore the metabolic footprint of HCDC perfusion process.