To increase profitability for biogas production, new innovative substrates and condition of operations needs to be implemented. At the current state, fats, oils and greases (FOGs) represent a promising substrate even though it brings operational challenges to the anaerobic digestion process. By utilizing hydrodynamic cavitation (HC) as a pre-treatment of the FOGs, the efficiency of FOGs’ co-digestion with wastewater sludge can be significantly improved. Preliminary experiments conducted on oil and water demonstrates that the HC pre-treatment improves the oil solubilisation as well as forms stable oil and water emulsion that last for several hours. The pre-treatment also improved the soluble chemical oxygen demand (COD) of biosludge (BiSl) by up to 115% and the initial degradation rate by up to 35%. In a semi-continues system, this allowed a significant increment in the specific methane yield depending on the organic loading rate (OLR) applied1. With sufficient process optimization, the HC-pre-treatment may prove to be an energy efficient and effective pre-treatment of FOGs.

The thesis deals with bioinformatics analysis, the aim of which was to find a suitable producer of PHA for new generation industrial biotechnologies from the collection of found thermophilic bacteria. Part of experiments was the finding of several thermophilic bacteria based on the similarity of the protein sequence of the phaC gene of the bacterium Cupriavidus necator. The next part of thesis was a literature search of the abilities of these thermophilic bacteria focused on culture conditions and the spectrum of usable substrates. Subsequently, five bacteria were selected for use in NGBI based on the information obtained. Freely available databases were used during the experimental work, and evolutionary analysis were performed in MEGA X and Operon-mapper. Rubrobacter xylanophilus with collection number DSM 9941 was selected from the collection of bacterial strains as the most promising PHA producer for NGIB. The high culture temperature of up to 70 ° C and a large amount of utilized carbohydrate substrates were considered decisive. An interesting result of the analysis was to find the gene sequences of two classes of PHA synthase – I. and III. class, as for a single bacterial strain from the entire collection. Additional genes linked to PHA metabolism were found in genome analysis.

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.

The world is currently faced with the enormous challenge of slowing down human triggered global warming. As the global energy demand increases, there is an urgent need for renewable and carbon-neutral fuel-sources. Isoprene and isobutene are crude-oil derived, short, volatile and reactive hydrocarbons that can be polymerised into longer chains to be used as jet fuel. Isoprene has previously been produced from the cyanobacterial strain Synechocystis sp. PCC 6803 but there has been no reported isobutene synthesis from any photosynthetic organism. This work aimed to synthesise isobutene in Synechocystis using a cytochrome P450 from Cystobasidium minutum with reported isobutene production capability. Substrate availability was to be provided through the insertion of two heterologous enzymes, IpdC from Salmonella typhimurium and PadA from Escherichia coli. Both IpdC and PadA were successfully expressed in Synechocystis but the functional activities of IpdC, PadA and the cytochrome P450 in Synechocystis remains undetermined. This project also had the aim to design and construct a photo-bioreactor and gas collection system capable of producing and harvesting isoprene directly from an engineered Synechocystis strain. Herein lies a description of a closed system photobioreactor connected to a cold-trap that was able to concentrate isoprene produced from Synechocystis to measurable amounts.

Energy balance of large-scale and small-scale scenarios of macroalgae harvesting for biogas production was assessed from the energy balance perspective. Evaluation was based on primary energy Input Output (IO) ratio where all primary energy inputs into the stages of the process life-cycle were summarized and divided by the final energy output from the system. Estimations were made for three cases of possible methane yield from macroalgae as well as for different scenarios of macroalgae co-digestion with other feedstock. Anaerobic digestion of macroalgae as a single substrate both on a small- and large-scale is energy efficient only in case when their methane potential is at the average or high level with the IO ratio of 0.47 and 0.32 correspondently. In general co-digestion with other substrates is more preferable with respect to process condition and energy balance. Large-scale scenario is more stable and efficient than small-scale with the lowest IO ratio for co-digestion with crops. This is explained by the fact that biogas plant operation is among the most energy demanding stages which on the small-scale requires about 65 % of the input energy when this number for large-scale plant does not exceed 28 %. Energy inputs into digestate handling, feedstock pre-treatment and biogas upgrading, that are next most energy consuming stages, is greatly affected by the assumptions made about amount of substrate, produced biogas and transportation distance. When considering the maximal distance between macroalgae harvesting point and biogas production site and to which at which the energy balance remains positive then digestate handling becomes the most energy demanding process stage.

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

The first method for DNA analysis in forensics was presented in 1985. Since then, the introduction of the polymerase chain reaction (PCR) has rendered possible the analysis of small amounts of DNA and automated sequencing and fragment analysis techniques have facilitated the analyses. In most cases short tandemly repeated regions (STRs) of nuclear DNA are analysed in forensic investigations, but all samples cannot be successfully analysed using this method. For samples containing minute amounts of DNA or degraded DNA, such as shed hairs, analysis of mitochondrial DNA (mtDNA) is generally more successful due to the presence of thousands of copies of mtDNA molecules per cell. In Sweden, ~40 % of all households have cats or dogs. With ~9 million humans shedding ~100 scalp hairs per day, and ~1.6 million cats and ~1 million dogs shedding hairs it is not surprising that shed hairs are one of the most common biological evidence found at crime scenes. However, the match probability for domestic dog mtDNA analysis has only been investigated in a few minor studies. Furthermore, although breed –sequence correlations of the noncoding mtDNA control region (CR) have been analysed in a few studies, showing limited correlations, no largescale studies have been performed previously. Thus, there have not been any comprehensive studies of forensic informativity of dog mtDNA. In the two papers presented in this thesis we have tried to lay a foundation for forensic use of analysis of domestic dog mtDNA. In the first paper, CR sequences were analysed and the exclusion capacity was investigated for a number of different populations. This is also the first comprehensive study of the correlation between mtDNA CR type and breed, type, and geographic origin of domestic dogs. Since the exclusion capacity for analysis of domestic dog CR sequences is relatively low, it was investigated in the second paper to what extent the discrimination power is improved by analysis of coding sequence. The exclusion capacity improved considerably when 3,000 base pairs of coding sequences where analysed in addition to CR sequences. This study will hopefully work as a basis for future development of analysis of dog mtDNA for forensic purposes. / QC 20101123

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>

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