The effect of power ultrasound on algae blooms (Microcystis aeruginosa) over a 30 minute period was assessed using 200 and 400 mL suspensions of optical density of 2.0 at 680 nm. The frequencies employed were 20, 40, 580 (40%, 80%, and maximum intensity), 864 (40%, 80% and maximum intensity) and 1146 kHz (40%, 80% and maximum intensity). Ultrasound can induce two different effects on algal cells; inactivation at high power (≥ 0.0022 Wcm-3) and de-agglomeration at low power (≤ 0.0042 Wcm-3). Ultrasonic effects were observed using haemocytometer, optical density, UV-visible spectrometer, fluorospectrometer and flow cytometry. Using a 40 kHz bath (0.0214 Wcm-3) led to de-agglomeration resulting in an overall increase in algae of -0.28% by haemocytometer and -4.20% by optical density. The highest inactivation achieved was 91.54% (haemocytometer) and 44.63% (optical density) using 1146 kHz (maximum intensity, 0.0248 Wcm-3) and 200 mL suspension. In terms of efficiency to achieve inactivation (i.e. inactivation % / power) the best result was observed at 864 kHz (40% power setting, 0.0042 Wcm-3) with 200 mL suspension giving 8226.19 by haemocytometer and 5011.90 by optical density. This initial part of the study allowed a comparison to be made of the ultrasonic parameters that would lead to optimum algae removal in terms of acoustic energy input. The haemocytometer results for cells number were generally higher than those indicated by optical density which is probably due to the fact that the former records only cell numbers remaining whereas the latter is an overall measure of algae concentration (ruptured cells will still register, because their contents remain in suspension). Studies on de-agglomeration and inactivation were also undertaken using small or medium-scale ultrasonic equipment that were models for industrial scale systems. The following volumes of algae suspension and equipment were employed: Sonolator (Sonic Corporation, 5L flow), 16 kHz and 20 kHz Dual Frequency Reactor (DFR, Advanced Sonics LLC, 1L static and 3.5 L flow), 20 kHz Vibrating Tray (Advanced Sonics LLC, 1.5L static) and 20 kHz ultrasonic probe (made at Southeast University, 4L static). The most effective inactivation effects were obtained with the DFR reactor in static mode and 60% power setting for 10 minutes which achieved reductions calculated at 79.25% using haemocytometry and 60.44% by optical density. The third part of this study was to gain a greater understanding of the basic mechanisms of the action of ultrasound on algae and to interpret this in terms of its potential for algal cell removal and control. Algal cell activity was assessed by three methods: using a UV-visible spectrometer (Shimazu, 2450PC), a fluorometer (Shimazu, RF5301) and a flow cytometer (BD FACS Calibur). Ultrasonic damage to Chlorophyll A was revealed through observation of the loss in UV-Vis spectrophotometer peaks around 600 nm together with the decrease in fluorometer results for peaks around 500 and 680 nm. Flow cytometer results were able to identify the number of both intact cells and damaged/ruptured cells thus giving greater insight into the mechanism of ultrasonic inactivation. The direct rupture of cells by power ultrasound was prevalent at low frequencies ≤ 40 kHz due to the mechanical effects of cavitation collapse and inactivation of algal cells by free radicals occurred at high frequencies ≥ 100 kHz and medium powers where mechanical effects are much reduced. In conclusion, this work has shown that power ultrasound can provide a suitable method to control algal growth in small and medium laboratory scales. Scale-up beyond this point is the subject of further research but the results herein clearly demonstrate the importance of choosing the correct ultrasonic parameters in terms of frequency, power and exposure time.

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.

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Chemistry / Master / Master of Philosophy

<p>Two decades ago the recruitment of YOY perch (<em>Perca fluviatilis</em>) started to decline along the Swedish east cost of the Baltic Sea. Factors that influence recruitment are e.g. eutrophication that causes habitat losses and overfishing of cod (<em>Gadus morhua</em>) which causes cascading effects in the food web. Filamentous cyanobacterial blooms are often toxic and has increased in the Baltic Sea and its coastal waters. The aim of this field study was to evaluate the effects of salinity and cyanobacterial exposure on fitness related parameters of young of the year (YOY) perch (<em>Perca Fluviatilis</em>) in a natural environment. Our study was performed in the Curonian Lagoon (Lithuania) in August 2009. The lagoon offers a temporary salinity gradient (wind induced influxes from the Baltic Sea) ranging from 7 psu in the north to 0 psu in the south. Submerged enclosures containing YOY perch were set up at three different locations along the salinity gradient in the Lagoon (referred to as North, Middle, South). The duration of the experiment was 21 or 27 days, depending on treatment. Measurements of perch condition were specific growth rate, somatic condition index (SCI) and whole fish lipid and protein content. Average chl <em>a</em> values for the three stations during the experimental time were: north 180 ± 70 µg/l chl <em>a</em>, middle 133 ± 36 µg/l chl <em>a</em> and south 180 ± 52 µg/l chl <em>a</em>. The North and the Middle stations experienced two different salinity influxes reaching a maximum salinity of 6.5 psu at the northern station. The duration of each saline influx was approximately 4-6 days. The saline water did not reach the Southern station at any time. Results show that perch from the southern station were in best condition in terms of specific growth rate and contents of total lipids. Compared to the South the perch condition declined to the Middle station and was lowest at the Northern station which experienced the highest degree of fluctuation in terms of salinity and cyanobacterial exposure. Examination of the abundance of the main food resource at the different stations revealed no statistical differences, which suggest that availability of food was not a factor in explaining the differences in growth.  The results possibly indicate that a changing environment with the potential synergistic negative effects of salinity and cyanobacteria has a higher negative impact on YOY perch condition compared to constantly high concentrations of cyanobacteria.</p>

This research was an attempt to characterize previously constructed mutants with a specifically altered psbO gene which encodes a 33 kDa protein active in photosynthesis. This polypeptide was believed to function in stabilization of manganese ions during photolysis of water at the photosystem II. The initial phase of this work was concerned with determining the manganese content of the genetically manipulated PS II particles of the photosynthetically active cyanobacteria.We found however, that the results of the isolation procedure for PS II particles of photosynthetically active cyanobacteria as described by Burnap et al. was not reproducible in our research organism. This prevented the chemical characterization of function of these particles as had been planned.In the second phase of the research sequencing of the mutated gene was to be performed for several clones in order to determine the kinds of specific alterations that had been made. The mutated genes had been cloned into both pUC1 20 and pPGV5 vectors which were transformed into Escherichia OR (EQQJi) and the cyanobacterium Synechococcus PCC 7942, respectively.Several attempts were mad o isolate plasmid DNA from both the transformed E QQJI and cyanobacterium. Isolation of pUC120 DNA was not achieved due to the toxicity of the 33 kDa protein product of the psbO gene in sgJj. The pPGV5 plasmid isolation was successful and PCR-sequencing was performed. However, the sequencing did not result in a readable sequence. Instead, banding patterns showed more than one nucleotide per lane. Since pPGV5 contains a strong constitutive promoter, a large amount of mutant protein was being produced. Our findings suggested that transformed cyanobacteria may have been under pressure to revert the altered gene to wild-type. Thus, upon growth of a single colony to a larger volume, a heterogeneous population of cells with different sizes of plasmids may have resulted. Restriction analysis of isolated plasmid DNA confirmed the presence of multiple-sized plasmid molecules. Therefore, this research has shown that the previously constructed mutants are not stable enough to characterize for alterations in manganese binding. / Department of Biology

The 33 kDa manganese stabilizing protein (MSP) has been proposed to provide ligands to stabilize Mn ions in the water lysis reaction of photosystem II of photosynthesis. In previous research site-directed mutagenesis had been performed on regions of the psbO gene encoding two aspartic acid residues of MSP which were thought to have the potential to form carboxyl bridges with Mn ions. The purpose of this research was to analyze these mutants. Plasmids pUC120-33 (#1,3,5,7,9,11,15) containing mutant psbO genes could not be isolated from E.coli because the expressed MSP was toxic to the cells. However, a psbO mutant gene carried in pPGV5-33 (#7) was isolated from E.coli and transformed into cyanobacterium Svnechococcus PCC 7942. Cyanobacterial cells carrying the MSP mutant showed a susceptibility to intensive light (100 footcandles) with a decrease of 30% in the growth rate within the first 100 hours after inoculation. This result suggested a possible function of the MSP in protecting the oxygen evolving complex from intensive light exposure. However, the mutant appeared to revert after this time probably due to homologous gene recombination with the wild type gene. In order to further analyze the function of mutants without recombination occurring, the construction of an MSP deletion was attempted using insertion of a kanamycin cartridge into the middle of the psbO gene. The inactivated psbO gene was transformed into E.coli and transformants were selected by kanamycin resistance. However, plasmid DNA carrying the interrupted genes could not be isolated, probably due to toxicity of the expression product in E.coli cells. Thus, future studies should be directed to reconstruction of a deletion mutant by direct transformation into cyanobacterial cells. Once a deletion mutant has been constructed analyses of the site-directed mutations could be performed in cyanobacteria. / Department of Biology

No description available.

The tidal-fresh James River experiences recurring blooms of toxin-producing cyanobacteria, including Microcystis aeruginosa. However, cyanobacteria cell densities in the James are relatively low. Our purpose was to identify key factors suppressing cyanobacteria growth in the face of eutrophication. A mesocosm experiment was designed to test the effects of light, mixing and grazing on cyanobacteria abundance, with nutrients held constant. We predicted that toxic cyanobacteria would be most abundant under stagnant conditions, with enhanced light, with no bivalve grazers present. Abundances of indicator gene copies and phytoplankton counts supported this hypothesis. However, chlorophyll-a, phycocyanin, and the toxin microcystin were all found to be most abundant under mixed conditions with ambient light. Statistically, light and mixing were important in controlling toxic cyanobacteria abundance, with little to no effect observed for bivalve grazers. Our findings suggest that toxin production may be regulated by factors independent from those driving algal growth and cyanobacteria abundance.

The goal of this research is to identify and characterize enzymes involved in bilin attachment to the phycobiliprotein allophycocyanin in the cyanobacterium Synechococcus sp. PCC 7002. Candidates for lyases responsible for attachment of phycocyanobilin to allophycocyanin are two cpeS-like genes termed cpcS and cpcU, and one cpeT-like gene termed cpcT. In vitro bilin attachment reactions were conducted in the presence of the recombinant substrate apo-allophycocyanin (HT-ApcAB). Size exclusion HPLC showed that CpcS and HT-CpcU form a 1:1 heterodimeric complex and that HT-ApcAB is present as a monomer (áâ). Absorbance and fluorescence spectroscopy illustrated that both CpcS and HT-CpcU were required to get holo-allophycocyanin with phycocyanobilin attached to the cysteine-81 residue. Absorbance of the product at 615 nm was consistent with holo-monomeric allophycocyanin. Experiments were performed with HT-ApcD ApcB and HT-ApcF ApcA, but size exclusion HPLC showed they were in aggregated form.