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The production of biofuels and biobased products from low-cost abundant renewable non-food lignocellulosic biomass will be vital to sustainable development because it will bring benefits to the environment, the economy, and the national security. The largest technical and economic challenge for emerging biorefineries is cost-effective release of fermentable sugars from recalcitrant structure of lignocellulosic biomass. Cellulose- and organic-solvent-based lignocelluloses fractionation (COSLIF) technology was employed to overcome biomass recalcitrance. Surface response methodology (SRM) showed that optimal COSLIF pretreatment conditions were 85% (w/v) H₃PO₄ and ~50 °C, regardless of moisture contents in biomass from 5-15% (w/w) for common reed. Under these conditions, the pretreated biomass was hydrolyzed fast with high glucan digestibilities at low enzyme loadings (i.e., one FPU of cellulase per gram of glucan). Crystallinity index (CrI) measurements by X-ray diffraction (XRD) and cross polarization/magic angle spinning (CP/MAS) ¹³C nuclear magnetic resonance (NMR), and cellulose accessibility to cellulase (CAC) determinations of COSLIF-pretreated biomass confirmed that highly ordered hydrogen-bonding networks in cellulose fibers of biomass were disrupted through cellulose dissolution in a cellulose solvent. This disruption of hydrogen bonding networks among cellulose chains resulted in a drastic increase in CAC values. Fourier transform infrared (FTIR) analyses on COSLIF-pretreated biomass revealed conformational changes in specific hydrogen bonding among cellulose chains due to COSLIF. While CrI is believed to be a key substrate characteristic that impacts enzymatic cellulose hydrolysis, studies in this thesis showed CrI values varied greatly depending on measurement techniques, calculation approaches, and sample preparation conditions. A correlation between CAC values and glucan digestibility of pretreated biomass showed that substrate accessibility is a key substrate characteristic impacting enzymatic cellulose hydrolysis. In summary, COSLIF can effectively overcome biomass recalcitrance. The resulting pretreated biomass has high CAC values, resulting in fast hydrolysis rates and high enzymatic glucan digestibilities of COSLIF-pretreated biomass at low enzyme usage. / Ph. D.

Excessive use of fossil fuels to meet everyday energy demands has led to adverse environmental impacts like global warming and high dependence on foreign oil. Development of cellulosic feedstocks provides energy security and also reduces the burden on food crops like corn and sugarcane used for ethanol production. This thesis uses cost-benefit analysis to ascertain the profitability of producing cellulosic ethanol from Switchgrass and Sweet Sorghum Bagasse. First, breakeven price of producing Switchgrass and Sweet Sorghum is calculated to obtain a raw material (feedstock) cost for ethanol production. Next, net present value (NPV) and minimum ethanol selling price (MESP) for Switchgrass and Sweet sorghum are calculated. Lastly, risk analysis is performed and its impacts on NPV are calculated for two farmer categories. The results show that ethanol production from Switchgrass and Sweet Sorghum is commercially feasible and generates a Net Present Value (NPV) of $39.54 million for Switchgrass and $96.76 million for Sweet Sorghum at an ethanol selling price of $2.17 per gallon. At NPV zero the MESP for Switchgrass and Sweet Sorghum is estimated to be $2.10 and $1.96 per gallon respectively. The risk analysis results revealed that there is a 9.5 percent probability that the NPV for a risk-averse Switchgrass farmer will be less than zero. On the other hand, the probability of the NPV being less than zero is 67.4 percent. The overall analysis indicates that ethanol production from Switchgrass and Sweet Sorghum is a promising option. Reduction in feedstock prices, optimization of the conversion process and additional revenues from by-products can make cellulosic ethanol more competitive with current gasoline prices. / Master of Science

Main goal of the dissertation is pointing out the different aspects of biofuels market, focus on forms of regulation, their use and analysis of their pros and cons.

Biofuels in civil aviation is actively studied in recent years to identify potential alternative jet fuels to meet stringent environmental regulations imposed to tackle degraded air quality caused by fossil fuel combustion. In this context, the aviation industry prefers to develop ‘drop-in’ fuels which may not require substantial modifications in existing jet engine technologies. The thesis aims at evaluating the atomization characteristics of camelina- and jatropha-derived drop-in biofuels discharging from simplex swirl atomizer used in aircraft gas turbine engines. The test fuels are characterized in detail and all fuels meet current ASTM D7566 specifications. The experiments are conducted by discharging fuel spray into quiescent atmospheric air in a fuel spray booth to obtain spray characteristics such as fuel discharge behaviour, spray cone angle, breakup behaviour of swirling fuel sheet and spray drop size distribution. The characteristics of sheet breakup are deduced from the captured images of biofuel sprays and the measurements of spray droplet size distribution are obtained using Spraytec (laser-diffraction instrument). A systematic comparison is made between the biofuel sprays and the 100% Jet A-1 (conventional aviation kerosene) sprays to evaluate the drop-in feature of the biofuels. All the measured spray characteristics of the biofuel sprays follow the Jet A-1 both in qualitative and quantitative terms which ensure the drop-in nature of the tested biofuels. The minor differences observed in the comparison of the quantitative spray measurements are attributed to the variation in the fuel properties. This claim is supported using the predictions obtained from the liquid film breakup model and the empirical correlation reported in the literature for the determination of sheet breakup characteristics and mean drop size for sprays discharging from simplex swirl atomizers.

The transport sector is seen as particularly problematic when concerns about climate change and dependency on fossil energy are discussed. Because of this, bioenergy is strongly promoted for use in the transport sector, both on a European level and nationally in Sweden. Even though bioenergy is considered one of the key solutions, it is generally agreed that both supply- and demand-side measures will be needed to achieve a change to a more sustainable transport system. One of the reasons for this is the limited availability of biomass, especially agricultural feedstocks competing with food or feed production. Woody biomass, however more abundant, is also exposed to tough competition from other sectors. In this thesis, the role of biogas as a vehicle fuel in a future sustainable transport system is discussed together with the prerequisites needed to realise such a transport system. Biogas is a biofuel that could be produced in several different ways: by anaerobic digestion, which is a first-generation production route, by gasification, which is a second-generation process, and by catalytic reduction of carbon dioxide, a third-generation technology. The main focus in this thesis is on biogas produced by anaerobic digestion and the results show that there is a significant potential for an increase compared to today’s production. Biogas from anaerobic digestion, however, will only be able to cover a minor part of the demand in the Swedish transport sector. Considering biogas of the second and third generations, the potential for production is more uncertain in a mid-term future, mainly due to competition for feedstock, the possibility to produce other fuels by these processes, and the present immaturity of the technology. The limited potential for replacing fossil vehicle fuels, either by biogas or other renewable fuels, clearly shows the need for demand-side measures in the transport system as well. This thesis shows the importance of technical and non-technical means to decrease the demand for transport and to make the transport as efficient as possible. The results show that both energy-efficient vehicles and behavioural and infrastructural changes will be required. Policies and economic incentives set by governments and decision-making bodies have a prominent role to play, in order to bring about a shift to a more sustainable transport system, however, measures taken on individual level will also have a great impact to contribute to a more sustainable transport system. / <p>QC 20121116</p>

The work was concerned with experimental study of the turbulent flame development process of alcohol fuels, namely ethanol and butanol, in an optically accessed spark ignition research engine. The fuels were evaluated in a single cylinder engine equipped with full-bore overhead optical access operated at typical stoichiometric part-load conditions with images captured using high-speed natural light imaging techniques (or chemiluminescence). The differences in flame development between the fuels was analysed to understand better the impact of high and low alcohol content fuels on combustion. Advanced image analysis, in conjunction with Ricardo WAVE simulation, allowed for the conclusion that the faster burning exhibited by ethanol was the result of the marginally higher laminar burning velocity providing a faster laminar burn phase and accelerating the flame into the turbulent spectrum thus reducing bulk flame distortion and better in-cylinder pressure development. Such physical reactions are often over-looked in the face of chemical differences between fuels. A further study into the variation of maximum in-cylinder pressure values was conducted focussing on iso-octane and ethanol. This study identified two phenomena, namely “saw-toothing” and “creep” in which cluster of cycles feed into one another. From this it became clear that the presence of high pressure during the exhaust process had a large influence on the following cycles. This is another often overlooked phenomenon of direct cycle-to-cycle variation whereby incylinder pressures during blowdown can dictate the duration, load or stability output of the following cycle. Finally the work investigated the impact on flame development of alcohol fuels when the overlap duration was altered. While the engine produced counterintuitive figures of residual gas, ethanol was confirmed as having greater synergy with EGR by displaying less impacted combustion durations c.f. iso-octane. Care should be taken however when analysing these results due to the unique valve configuration of the engine.

Microalgae synthesize and store large volumes of lipids (potentially over 25% of dry weight) which could provide a renewable source of biodiesel. Traditional extraction techniques often produce poor lipid yields particularly from microalgae with robust cell walls. This project investigated the role of power ultrasound as a cell disruption step in lipid extraction from four microalgal species. Nile Red staining was used to assess the time when ultrasound induced increased membrane permeability in each species and lipids were extracted using an ultrasound assisted Bligh and Dyer extraction method. A 20 kHz probe system (40% amplitude, 0.086 W/cm3) caused increased lipid recovery from dry biomass in all cases; D. salina (no cell wall) from 15 to 22.5% of dry biomass after 1 minute (26% when stressed with 35 g/L NaCl). C. concordia (thin cell wall) from 7.5 to 10.5% of dry biomass after 2 minutes (27% with 25% nitrogen reduction in growth media). N. oculata (thick cell wall) from 6.5 to 10% of dry biomass after 16 minutes (31.5% when stressed with 30 g/L NaCl). The stressed cultures yield could be improved to 35% when ultrasound was combined with S070 beating beads. Chlorella sp. (thick cell wall) from 6.3 to 8.7% of dry biomass, after 16 minutes (44% was achieved when harvested at day 9 instead of 15). A Dual Frequency Reactor (16 and 20 kHz, 0.01 W/cm3) flow system with S070 beads demonstrated that high lipid extraction yields could be achieved on a larger level with N. oculata. After 4:48 minutes sonication 24% lipid recovery was achieved. This system could theoretically increase daily microalgal oil production from 3.96 to 5.76 L per day when compared to conventional techniques, at an extra production cost of only 2.9 p/litre (1.5% increase). D. salina, N. oculata and C. concordia resumed normal growth following sonication at 20 kHz after 1-20 days (8 minutes treatment for D. salina, 60 minutes treatment for N. oculata and 16 minutes treatment for C. concordia). It was found that the supernatant of sonicated D. salina and C. concordia when added to established cultures were able to boost their growth.

The development of biofuels has received much attention due to the global warming and limited resources associated with fossil fuels. Butanol has been identified as a potential option due to its advantages over ethanol, for example higher energy density, compatibility with current infrastructure and its possibility to be blended with gasoline at any ratio. Yeast Saccharomyces cerevisiae can be used as a producer of butanol. However, butanol toxicity to the host limits the yield produced. In this study, four strains of yeast isolated from the habitats of lager beer, ale, wine and baker ́s yeast were grown in YPD media containing isobutanol concentrations of 1.5 %, 2 %, 3 % and 4 %. Growth was measured to determine the most tolerant strain. Gene expression for the genes RPN4, RTG1 and ILV2 was also measured, to determine its involvement in butanol stress. The genes have in previous studies seen to be involved in butanol tolerance or production, and the hypothesis was that they all should be upregulated in response to butanol exposure. It was found that the yeast strain isolated from lager beer was most tolerant to isobutanol concentrations of 2 % and 3 %. It was also found that the gene RPN4 was upregulated in response to isobutanol stress. There was no upregulation of RTG1 or ILV2, which was unexpected. The yeast strain isolated from lager beer and the gene RPN4 is proposed to be investigated further, to be able to engineer a suitable producer of the biofuel butanol.

The development of sustainable replacements for fossil fuels has been spurred by concerns over global warming effects. Biofuels are typically produced through fermentation of edible crops, or forest or agricultural residues requiring cost-intensive pretreatment. An alternative is to use photosynthetic cyanobacteria to directly convert CO2 and sunlight into fuel. In this thesis, the cyanobacterium Synechocystis sp. PCC 6803 was genetically engineered to produce the biofuel n­-butanol. Several metabolic engineering strategies were explored with the aim to increase butanol titers and tolerance. In papers I-II, different driving forces for n-butanol production were evaluated. Expression of a phosphoketolase increased acetyl-CoA levels and subsequently butanol titers. Attempts to increase the NADH pool further improved titers to 100 mg/L in four days. In paper III, enzymes were co-localized onto a scaffold to aid intermediate channeling. The scaffold was tested on a farnesene and polyhydroxybutyrate (PHB) pathway in yeast and in E. coli, respectively, and could be extended to cyanobacteria. Enzyme co-localization increased farnesene titers by 120%. Additionally, fusion of scaffold-recognizing proteins to the enzymes improved farnesene and PHB production by 20% and 300%, respectively, even in the absence of scaffold. In paper IV, the gene repression technology CRISPRi was implemented in Synechocystis to enable parallel repression of multiple genes. CRISPRi allowed 50-95% repression of four genes simultaneously. The method will be valuable for repression of competing pathways to butanol synthesis. Butanol becomes toxic at high concentrations, impeding growth and thus limiting titers. In papers V-VI, butanol tolerance was increased by overexpressing a heat shock protein or a stress-related sigma factor. Taken together, this thesis demonstrates several strategies to improve butanol production from cyanobacteria. The strategies could ultimately be combined to increase titers further.