The 8.6 kb kalilo linear mitochondrial plasmid of Neurospora intermedia was found to give rise to multiple transcripts of 8.6, 4.4, 4.0, 1.3, 1.2, and 0.9 kb. A transcription map has been generated which shows similarities to other linear plasmids. These transcripts are all transcribed from a single, unique promoter sequence reiterated near the ends of the terminal inverted repeats of the linear plasmid. The transcripts are not processed, but instead utilize optional transcription stop sites. An analysis of sub-cellular RNA fractions has confirmed the mitochondrial location of kalilo transcription. The strong association of kalilo-specific RNA with rRNA to yield RNA artifacts is reported. Kalilo-specific RNA appears to be selectively unstable in affected strains of N. intermedia; this may be a general consequence of linear plasmid RNA. The 5' RNA start site was determined by primer extension and RNA sequencing. The sequence in this region does not show homology to any known mitochondrial, plasmid, nor nuclear promoter, and may constitute a novel element. The transcription start site shares homology with the terminus of the linear plasmid, and marks the end of a long series of direct repeats; therefore, the plasmid RNA polymerase may be bifunctional, it may recognize sequences at the ends of the plasmid as well as at the promoter. The analysis of the insertional behaviour of the linear mitochondrial plasmid was studied in parallel subculture series of the organism. It was determined that insertion, per se is not the event required to kill the organism. Generation of inserts of kalilo in the mtDNA is necessary, but not sufficient, for death to occur in all cases. An analysis of insertion sites has found one new site and good agreement with previously published locations. Insertion does not always appear to be random, so cultures may inherit undetectable amounts of mtlS-kalDNA. The analysis of insertion sites in one strain has suggested a novel possible structure for the mtDNA. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

A survey of natural isolates of N. intermedia revealed five variants with respect to growth phenotype. These variants showed stop-start behavior in growth tubes, sometimes never attaining the full tube length. These strains are mainly.female sterile. The stop-start phenotype was not transmitted via the male parent in any cross. In two crosses, maternal transmission was demonstrated. One strain was investigated for the presence of virus-like particles, but none were found. Cytochrome spectra show a deficiency of cytochromes a. and/or b_ relative to the amount of c. In respiration studies, the one strain tested proved to be cyanide resistant and salicyl hydroxamic acid sensitive. These characteristics are also found in extranuclear mutants of N. ovassa. However, the analogy no longer holds at the mitochondrial ribosome level. An analysis of four of the strains disclosed that three are large subunit deficient. Only small subunit deficient strains have been found among the cytoplasmic mutants of N. ovassa. Restriction enzyme analysis was also carried out on the variants. It was found that they possess additional mitochondrial DNA compared to normal N. intermedia. One Eco RI fragment of M.W. 2.4 x 10 was found to be common to all variants tested, but is not found in the normal strains. There were other DNA differences which were unique to each abnormal strain. These findings suggest that the basis for the abnormal phenotypes .is some heritable factor associated with the mitochondria. / Science, Faculty of / Botany, Department of / Graduate

Senescence, the progressive loss of growth potential culminating in death, is common among Kauaian strains of Neurospora intermedia. Senescence is initiated by the insertion of kalilo DNA into the mitochondrial DNA. Mitochondrial DNA molecules carrying the insert accumulate and death occurs when the insert is equimolar with the mitochondrial DNA. The inserted form of kalilo DNA is referred to as mtlS-kalDNA. Studies on the somatic transmission of mtlS-kalDNA in ascospore series have revealed that kalilo DNA is capable of assuming new locations within the mitochondrial DNA. It is proposed that these novel insertions originate from intramitochondrial movement and an autonomous form of kalilo DNA, mtFF-kalDNA, is predicted to be an intermediate in movement. Novel insertion of kalilo DNA appears to depend on the form of mtlS-kalDNA transmitted sexually. If a mutagenic insert is transmitted, senescence is initiated at the onset of vegetative growth of the ascospores and no novel insertions are detected. The lifespans of these ascospores are quite short, death occurring in 10 subcultures or less. Transmission of a nonmutagenic insert delays the onset of senescence until either a novel insertion or a rearrangement of the transmitted insert occurs. The lifespans of these ascospores usually exceed 10 subcultures and are variable. Information obtained from tetrad analysis has revealed that novel insertion of kalilo DNA may also be under the influence of the host genome. A senescent Kauaian strain was identified which shows some but not all characteristics of kalilo senescence. In this strain and its derivatives, the behaviour of mtlS-kalDNA is erratic and in, some cultures the characteristic mitochondrial biochemical deficiencies, normally accompanying kalilo senescence, are not observed. It is suspected that kalDNA is not responsible for senescence in this strain and its derivatives but rather some other unknown factor is affecting the normal growth patterns of these cultures. Kauaian strains were surveyed for the presence of dsRNA to determine whether kalDNA has a viral origin. Only one senescent strain contains detectable amounts of dsRNA which was not homologous with a kalDNA probe. The survey identified six nonKauaian strains which contain dsRNA and seven dsRNA species were delineated. Although the presence of dsRNA is not relevant to kalilo senescence, analysis of dsRNA in a genetically-well defined organism like Neurospora may give insight into the significance of dsRNA in fungi in general. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

Jordens befolkning förväntas ha överstigit 9 miljarder till 2050, vilket leder till en ökning av matbehov med 70%. Ungefär en tredjedel av all producerad mat slängs idag, speciellt grönsaker, bröd och annan mat med kort hållbarhetsdatum. Det är väldigt viktigt att hitta sätt att minska och återanvända avfallet om miljöpåverkan ska kunna minskas. Brödrester är en stor del av Sveriges totala matavfall och använt spannmål (BSG) står för den största delen av biprodukter från bryggningsindustrin. De kan tillsammans användas som substrat för att fermentera en svampburgare som är rik på näring och protein. Denna studien syftar till att bestämma vilken filamentös svamp, N. intermedia eller R. oryzae, som ger de bästa resultaten med avseende på konsistens, proteininnehåll, smak och utseende. Detta gjordes genom textur- och proteinanalys på svampburgarna. Ingen av svamparna utmärkte sig med märkvärt bättre resultat gällande konsistensen, men resultaten för studien hade en del osäkerheter i resultatet på grund av otillräcklig tillväxt. Med avseende på smak var N. intermedia att föredra, då den hade en sötare och mer behaglig smak. Vad gäller utseende så ser båda burgarna väldigt aptitliga ut, och de anses ha stor potential som köttsubstitut, men vidare forskning inom området krävs. / Earth’s population is expected to have exceeded 9 billion people by 2050, leading to a 70% increase in food demand. Today, approximately one quarter to one third of all food produced goes to waste, especially vegetables, bread and other foods with short shelf life. Finding ways to reduce and reuse the waste is very important if the impact on the environment is to be decreased. Stale bread accounts for a large part of Sweden’s total food waste and brewers spent grain (BSG) is the brewing industry’s major by-product. Combined, they can be used as a substrate to produce a fungal fermented burger, rich in nutrients and protein. This study aims to determine which fungus, N. intermedia and R. oryzae, provides the best results regarding texture, protein content, taste and appearance. This was done by performing texture analysis and protein analysis on the fermented burgers. None of the fungi showed significantly better results than the other regarding texture. However, the results from this study have a few uncertainties due to lack of growth. The fungus that was preferred when considering taste was N. intermedia, which had a sweeter and more pleasant taste in general. Regarding the appearance of the fermented burgers, they are very different but they both are equally as appealing to the eye. The fermented burgers show great potential as meat substitute, but extended research is required.

Det pågår ständigt forskning på att göra bioetanolen till ett mer konkurrenskraftigt bränsle gentemot fossila bränslen. Flera försök till att optimera processen både ekonomiskt och energimässigt görs i flera länder världen över. I detta examensarbete har den filamentösa svampen Neurospora intermedia odlats i tunndrank som är en restprodukt från bioetanolsproduktionen. Olika utspädningar på tunndranken har använts för att se vilken som producerar mest bioetanol. Enligt detta examensarbete produceras det mest bioetanol när N. intermedia odlas i 50 % utspädd tunndrank. När den optimala utspädningen hade hittats prövades det vilken tid under odlingen som N. intermedia producerade den högsta koncentrationen bioetanol. Efter 39 timmar hade det producerats 4,34 g/l bioetanol och det var även vid denna tidpunkt som biomassan hade de bästa protein- och RNA-innehållen för att kunna torka biomassan och producera djurfoder till lantbruket.

Mycelial pellet formation by filamentous fungi is one of the most researched topics in fungal biotechnology research. Pellets are generally formed as a result of a complex interaction process through the influence of many cultivation factors such as inoculum size, pH, dissolved oxygen level, agitation system, nucleating agents, additives, trace metals, CO2, temperature, reactor types, carbon substrate, rheology, culture modes, fermenter geometry, nitrogen and phosphate levels etc. Each factor has varying effects on the growth morphologies of different fungal species. Fungal growths in the form of pellets have several advantages and pose a potential solution to overcome the problems associated with the filamentous fungal growth in large scale industrial bioreactors. The aim of the present work was to study pellet formation of edible filamentous fungus Neurospora intermedia, focusing on the molecular aspects of the fungal pellets with special interest to investigate the role of cell signaling second messenger cyclic 3', 5’-adenosine mono- phosphate (cAMP). It was found that Neurospora intermedia stimulate cAMP in the pellet form than filamentous form. The industrial applications of fungal pellets for generating value added products were also studied and observed fermentation in individual and co fermented first and second-generation ethanol substrate, showed an ethanol yield maximum of 0.25 ± 0.05 g/g dry substrate. The growth of fungal pellets in presence of inhibitors (such as acetic acid, HMF and furfural) resulted in about 11% to 45% increase in ethanol production as compared to filamentous forms, at similar growth conditions in the liquid straw hydrolysate.

Establishing a commercial, lignocellulose-based, second-generation ethanol process has received several decades of attention by both researchers and industry. However, a fully economically viable process still remains a long-term goal. The main bottleneck to this achievement is the recalcitrance of lignocellulosic feedstocks, although there are several other factors, such as the huge investment required for second-generation ethanol facilities. An intelligent alternative solution discussed in this thesis is an integrated approach using firstgeneration ethanol plants for second-generation processes. Wheat is the major feedstock for first-generation ethanol in Europe; therefore, wheat-based lignocellulose waste, such as wheat straw, bran, and whole stillage fiber (a waste stream from first-generation wheat-based ethanol plants) was the primary focus of the integration model in this thesis. Since the major share of first-generation ethanol plant economics focuses on the animal feed DDGS (Distillers’ dried gains with solubles), the integration of lignocellulose should be designed in order to maintain DDGS quality. An ethanol-producing edible filamentous fungus, Neurospora intermedia, a potential protein source in DDGS, was considered for use as the fermenting microbe. The morphological and physiological aspects of this fungus were studied in the thesis, leading to the first report of fungal pellet development. An alternative approach of using dilute phosphoric acid to pretreat lignocellulose, as it does not negatively affect fungal growth or DDGS quality, was demonstrated in both the laboratory and on a 1m3 pilot scale. Furthermore, the process of hydrolysis of pretreated lignocelluloses and subsequent N. intermedia fermentation on lignocellulose hydrolysate was also optimized in the laboratory and scaled up to 1 m3 using an in-house pilot-scale airlift bioreactor. Fungal fermentation on acid-pretreated and enzyme-hydrolyzed wheat bran, straw and whole stillage fiber resulted in a final ethanol yield of 95%, 94% and 91% of the theoretical maximum based on the glucan content of the substrate, respectively. Integrating the first- and second-generation processes using thin stillage (a waste stream from first-generation wheat-based ethanol plants) enhanced the fungal growth on straw hydrolysate, avoiding the need for supplementing with extra nutrients. Based on the results obtained from this thesis work, a new model for integrated first- and second-generation ethanol using edible filamentous fungi processes that also adds value to animal feed (DDGS) was developed.

Vegetable oil is more difficult to degrade by microorganisms in comparison to carbohydrates and protein. Thus, it creates serious environmental and health concerns if oil-derived waste streams produced by restaurants and industries remain untreated. In this study, a strategy has been developed to grow filamentous fungi in pure olive oil so that it can be used as a bench-mark for growth in olive oil mill sidestreams. The growth of different strains (Aspergillus oryzae, Neurospora intermedia and Rhizopus oryzae) was tested in pure olive oil. A pre-germination stage using glucose as carbon and energy source, or the addition of yeast extract, were found necessary for successful fungal growth in olive oil. Here, A. oryzae showed a superior performance in comparison to N. intermedia and R. oryzae. Medium pH did not impact A. oryzae growth in olive oil, whereas a concentration higher than 40 g/L of the latter impaired the growth of the ascomycete. Obtained biomasses from A. oryzae and N. intermedia cultivations in olive oil were analyzed and compared for protein, fat, ash, and alkali-insoluble material (cell wall content), where the presence of olive oil had a steering effect. The fungal biomass of A. oryzae, obtained from cultivation in the absence of olive oil, contained 0.33% fat and 48% protein, whereas the respective values in the presence of olive oil were 31% and 14%. Similar trends on fat and protein contents were observed for the biomass of N. intermedia. Sudan black staining was also performed on fresh biomass which clearly indicated the presence of oil globules inside the fungal cells. This research can be a fundamental step towards treatment of oil-based waste streams, which entails high-energy and costs if treated, or environmental impacts during informal discharges. Moreover, the fact that the composition of fungal biomass can be steered through addition of olive oil increases the versatility of the originated biomass for various applications, namely in feed, food and biofuel production.