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BioEthanol : fuel of the future?


Microbial fermentations are potential producers of sustainable energy carriers. In this
study, 68 samples were used for the isolation of ethanol and hydrogen producing
bacteria from various carbon substrates from geothermal springs in Iceland. 16S rRNA
analysis revealed that most of low temperature (50°C) enrichments indicated the
presence of bacteria belonging to Thermoanaerobacterium, Caloramator and
Clostridium. At higher temperatures (60°C) Thermoanaerobacterium and
Thermoanaerobacter became more dominant and at 70 and 75°C only members of the
genera Thermoanaerobacter and Caldicellulosiruptor were present. Low temperature
enrichments (50°C) produced mainly ethanol, acetate and butyrate from monosugar
fermentation and two enrichments produced more than 1.3 mol-EtOH/mol-glucose and
two enrichments produced more than 1.0 mol-EtOH/mol xylose. Most of the
enrichments that directed their carbon flow to ethanol belong to Caloramator,
Paenibacillus and Thermoanaerobacterium. At higher temperatures, several
enrichments produced more than 1.0 mol-EtOH/mol-glucose and xylose, especially at
60°C, mostly belonging to Thermoanaerobacter and Thermoanaerobacterium. Butyrate
was only detected in one of the 18 enrichments at 60°C and not at higher temperatures.
Only one enrichment of a total of 16 at 70 and 75°C produced more than 1.0 mol-
EtOH/mol glucose but the carbon flow was directed more towards acetate and hydrogen
formation. Seven cultures were investigated further with respect to phylogeny and
physiology. Six of the strains belonged to Thermoanaerobacterium and one to
Paenibacillus. Basic characteristics concerning Topt and pHopt were performed on all
seven strains, indicating that the Thermoanaerobacterium strain grow best at around
60°C and at low pH between 5 and 6. The Paenibacillus strain was a moderate
thermophile (Topt; 50°C) and had a pHopt at 8.0. Paenibacillus was the fastest growing
bacterium with the shortest generation time of 0.39 h (pHopt) and 0.71 h (Topt). The
Thermoanaerobacterium strains showed generation times between 0.81h to 1.99h. All
strains were highly saccharolytic degrading broad spectrum of pentoses, hexoses and
disaccharides as well as xylan and pectin. All strains produced more than 1 mol-
EtOH/mol-glucose and xylose and could grow in the presence of 3.2% ethanol (v/v) but
not at 6.4%. Different initial substrate loadings (glucose) indicated that at glucose
concentrations between 50 and 100 mM, a clear inhibition occurred leading to
inefficient glucose degradation and lower end product formation. Growth of two strains AK66 and AK17 was also investigated on hydrolysates (0.75% w/v) made from cellulose
(Whatman paper), newspaper, hemp leaves and hemp fibers (Cannabis sativa), barley
straw (Hordeum vulgare) and grass (Phleum pratense). End product formation was
analyzed from the cultures after fermentation. Strain AK66 produced most ethanol (20 to
24 mM) from cellulose but weak acid and base pretreatment did not enhance ethanol
yields. On other biomass types, lower ethanol yield were observed (lowest on barley
straw; 4 mM, highest on hemp stem, 12 mM) but were increased significically by
pretreatments. Yields of ethanol by strain AK17 were considerable higher, ranging from
5 mM on hemp leaves to 50 mM on pure cellulose. Acidic pretreatment enhanced
ethanol yields mostly on hemp leaves (5 mM to 18 mM) and base pretreatment on grass
(20 mM to 28 mM). Other products from hydrolysate fermentation for both strains were
acetate, carbon dioxide and hydrogen, but in lower amounts.


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