No / Violacein is a bisindole pigment occurring as a biosynthetic product of Chromobacterium violaceum and Janthinobacterium lividum. It has some structural similarities to the cyanobacterial UV-protective pigment scytonemin, which has been the subject of comprehensive spectroscopic and structural studies. A detailed experimental Raman spectroscopic study with visible and near-infrared excitation of violacein produced by C. violaceum has been undertaken and supported using theoretical DFT calculations. Raman spectra with 514 and 785 nm excitation of cultivated cells as well as extracts and Gaussian (B3LYP/6-311++G(d,p)) calculations with proposed molecular vibrational assignments are reported here.

<p dir="ltr">Phenylalanine (Phe) is an essential amino acid that has uses in the feed, food and pharmaceutical industries. There is a large and growing market for Phe as a precursor to the production of artificial sweetener. Industrially, Phe is produced by feeding glucose to genetically modified strains of heterotrophic organisms such as <i>E. coli </i>or Corynebacterium in a stainless-steel fermenter. Cyanobacteria are photosynthetic microalgae which could replace heterotrophic production thereby reducing land use for crops required for glucose production. SYN-PHE, a strain of <i>Synechococcus</i> elongatus sp. PCC 11801 which was previously developed in the Morgan lab at Purdue University, produces Phe at 1 g/L in 3 days in shake flask cultures. In this thesis, a techno-economic analysis of Phe production by <i>E. coli </i>and SYN-PHE were compared. Results indicate that Phe produced by SYN-PHE is a promising competitor of <i>E. coli </i>produced Phe at an industrial scale. Further strain engineering to improve the titer of Phe is needed to be economically competitive. Additionally, efforts are needed for low capital cost photobioreactors that can enable both high biomass concentrations and high Phe titers.</p>

published_or_final_version / Ecology and Biodiversity / Doctoral / Doctor of Philosophy

The overall aim of the study was to provide better insights to the ecological role and impact of cyanobacteria in Baltic Sea (BS) bay, coastal and open sea areas. Biomass and heterocyst development of diazotrophic, heterocystous cyanobacteria were monitored over several years simultaneously as physical parameters such as nutrients and temperature. Nitrogen fixation was estimated as well as its transfer in the BS food web. Even after decades of debate there is still controversy whether eutrophication of lakes and estuaries/coastal areas should be managed by reducing phosphorus only or also nitrogen. Central to this debate is whether nitrogen fixation by cyanobacteria can replace shortages of combined nitrogen quickly enough to make phosphorus the limiting nutrient and nitrogen removal pointless or even harmful. Also, it is not clear if available combined nitrogen inhibits heterocystous cyanobacterial nitrogen fixation and if it is used for their growth in situ. A large ecosystem-wide experiment started in Himmerfjärden bay in year 1997, where the N-loadings and release depth from a modern sewage treatment plant (STP), located in the inner part of the bay, were modulated. The STP creates a steep gradient of nutrients and stable nitrogen isotopes, which can be used to study uptake of combined nitrogen, as well as biomass development and primary productivity. A 35-year long data series was used to achieve good insights into phytoplankton development and primary productivity in the Baltic Sea over the last couple of decades. These in vivo long time series, based on monitoring data, in combination with shorter series (2-3 seasons, including measurements of colony stoichiometry and stable isotopes), have resulted in a unique meta-dataset, allowing for high-resolution observations into the role of the cyanobacteria in the Baltic Sea ecosystem. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.</p>

Synthetic biology views a complex biological system as an ensemble in the hierarchy of parts, devices, systems, and networks. The practice of using engineering rules such as decoupling and standardization to understand, predict, and re-build novel biological functions from model-driven designed genetic circuits is emphasized. It is one of the top ten technologies that could help solving the current and potential risks in human society. Cyanobacteria have been considered as a promising biological system in conducting oxygenic photosynthesis to convert solar energy into reducing power, which drives biochemical reactions to assimilate and generate chemicals for a specific purpose such as CO2 fixation, N2 fixation, bioremediation, or fuels production. The promoter is a key biological part to construct feedback loops in genetic circuits for a desired biological function. In this thesis, promoters that don't work in the cyanobacterium Synechocystis PCC 6803 in terms of promoter strength, and dynamic range of gene regulation are identified. Biological parts, such as ribosome binding sites, and reporter genes with and without protease tags were also characterized with the home-built broad-host-range BioBrick shuttle vector pPMQAK1. The strong L03 promoter, which can be tightly regulated in a wide dynamic range by the foreign Tet repressor, was created through an iterative promoter engineering cycle. The iteration cycle of DNA breathing dynamic simulations and quantification of a reporting signal at a single-cell level should guide through the engineering process of making promoters with intended regulatory properties. This thesis is an essential step in creating functional promoters and it could be applied to create more diverse promoters to realize the emphasized practices of synthetic biology to build synthetic cyanobacteria for direct fuel production and CO2 assimilation.

As cyanobacteria (also known as blue green algae) produce a range of cyclic peptides which are highly toxic, capillary electrophoresis and associated techniques have been investigated to assess their applicability for toxin monitoring in the water bodies of kwaZulu Natal, South Africa. Capillary electrophoresis (CE) is a technique in which charged molecules can be efficiently separated in a buffer solution within a capillary tube under the influence of a strong electric field. Two CE modes, namely capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MECC) were initially evaluated using a laboratory-built CE instrument. The former mode lacked selectivity due to the similar charge to size ratio of the algal toxins. However, with the latter mode, incorporation of a surfactant (sodium dodecyl sulphate) into the buffer, produced sufficient resolution between components. Parameters including surfactant concentration, buffer ionic strength, buffer pH and operating voltage were systematically optimized to separate the four algal toxins under investigation (microcystin YR, microcystin LR, microcystin RR and nodularin). The optimum separation conditions were: 30 mM borax, 9 mM sodium dodecyl sulphate, pH 9.18, 30 kV applied voltage, 10 s hydrodynamic injection, 70 cm x 50 Ilm Ld. bare fused silica capillary (LEFF 40 cm) and UV detection at 238 nm. Under these conditions, typical detection limits were in the low ng/IlL range (14.13 ng/IlL for microcystin LR to 29.85 ng/ILL for nodularin). The MECC method was evaluated in terms of migration time precision, efficiency and resolution, peak area and normalised peak area precision. Standard deviation values for retention times acquired using replicate electrokinetic injections ranged from 0.018 to 0.054 and 0.069 to 0.148 for hydrodynamic injections. Normalised peak area precision for replicate hydrodynamic injections were in the range 84 to 97 % RSD, while improved % RSD values of 11.5 to 18.7 were achieved for electrokinetic injections. Due to poor precision resulting from the lack of automation on the laboratory built CE system, poor correlation between increasing concentration and a corresponding change in normalised peak areas were achieved. The MECC method developed was applied to the analysis of an algal scum extract to illustrate the technique. A general problem with CE is that it suffers from poor detection sensitivity. Hence in this study, alternative injection modes, sample concentration strategies and alternative detection techniques were investigated in an attempt to improve detection limits for algal toxins. Using optimized electrokinetic injection conditions, detection limits were five to ten times better than those obtained with hydrodynamic injections. On-line sample concentration methods were partially successful. Field amplified back and forth MECC in which analyte injected in the entire column volume and subsequently focused in a narrow band by manipulating the electric field, resulted in an enormous sensitivity enhancement that ranged from 197 times for microcystin RR to 777 times for microcystin YR when compared to hydrodynamic injections. Field amplified sample stacking (FASI) was ineffective for toxin preconcentration, while electro-extraction produced detection limits ranging from 0.27 ng/J.tL for microcystin YR to 1.08 ng/J.tL for microcystin RR. Solid phase extraction, in which analytes are first trapped and concentrated on HPLC material in a cartridge and then eluted in a more concentrated form for injection, was found to be practical only in the offline mode. A concentration detection limit of less than 0.002 ng/J.tL was obtained. Attempts with on-line solid phase extraction failed due to problems associated with coupling the cartridge with the separation capillary. Finally, laser induced fluorescence (LIF) detection was investigated as an alternative to UV detection. Unfortunately, the algal toxins were not amenable to LIF detection because tagging with the fluorescent moiety, fluorescein isothiocyanate (FITC), was prevented by the stereochemistry of these cyclic peptides. A comparative study between HPLC and MECC revealed that the former displayed poor efficiency peaks and long analysis times for toxin analysis. However HPLC was superior in terms of retention time precision (0.12 to 0.64 % RSD) and area precision (1.78 to 2.86 % RSD). Mass detection limits for MECC (0.0142 to 0.0603 ng) were far superior to those achieved by HPLC (0.55 to 1.025 ng). In addition to HPLC and MECC, a preliminary investigation of micro-high performance liquid chromatography (J.tHPLC) and capillary electrochromatography (CEC) for the analysis of algal toxins was made using 50 J.tm Ld. capillary columns packed in-house, with reverse phase HPLC packing material. / Thesis (M.Sc.)-University of Natal, 1997.

The detection and control of microorganisms such as bacteria is important in a wide range of industries and clinical settings. Detection, binding and removal of such pathogenic contaminants can be achieved through judicious consideration of the targets which are available at or in the bacterial cell. Polymers have the ability to present a number of binding ligands for cell targeting on one macromolecule and so avidity of interaction can be greatly increased. The goal of the project was to test whether polymers generated with bacteria in situ would have their composition significantly altered to determine if a templating process was occurring. It was also anticipated that the templated polymers would have better re-binding properties than those produced in the absence of bacteria. A series of chemical functionalities were analysed for their binding properties to bacteria. The functionalities were chosen with consideration to the cell surface characteristics. Further to identification of the most binding and least binding functionalities the polymers were tested for their cytotoxicity against bacteria and human epithelial cells. Concentration ranges were determined which could facilitate bacterial binding and templating yet minimise the lethality of the processes. Templated polymers of the bacteria were generated using a novel method of atom transfer radical polymerisation (ATRP) which we have termed bacterial activated atom transfer radical polymerisation (b-ATRP). This polymerisation method has maximised the potential for templating processes to occur during the polymerisation. Templated polymers differed in both their composition and their binding behaviour to non-templated polymers. The bacterial organic reduction process has also been demonstrated to have greater scope for use within the organic chemistry field as demonstrated by the use of this system to enable in "click-chemistry" via the reduction of copper.

Cyanobacteria are widely distributed in marine, freshwater and terrestrial habitats. Some cyanobacterial genera can convert di-nitrogen gas (N2) to bioavailable ammonium, i.e. perform nitrogen (N) fixation, and are therefore of profound significance for N cycling. N fixation by summer blooms of cyanobacteria is one of the largest sources of new N for the Baltic Sea. This thesis investigated N fixation by cyanobacteria in the Baltic Sea and explored the fate of fixed N at different spatial and temporal scales. In Paper I, we measured cell-specific N fixation by Aphanizomenon sp. at 10 ºC, early in the season. Fixation rates were high and comparable to those in late summer, indicating that Aphanizomenon sp. is an important contributor to N fixation already in its early growth season. In Paper II, we studied fixation and release of N by Aphanizomenon sp. and found that about half of the fixed N was rapidly released and transferred to other species, including autotrophic and heterotrophic bacteria, diatoms and copepods. In Paper III, we followed the development of a cyanobacterial bloom and related changes in dissolved and particulate N pools in the upper mixed surface layer. The bloom-associated total N (TN) increase was mainly due to higher particulate organic N (PON) concentrations, but also to increases in dissolved organic nitrogen (DON). About half the PON-increase could be explained by the sum of N-fixing cyanobacteria, other phytoplankton (&gt;2µm) and zooplankton, indicating that production was stimulated by the N fixation. In Paper IV, we used a growth model based on measured photosynthesis–irradiance relationships to explore the production potential of Aphanizomenon sp. The model included data on irradiance, biomass, temperature and light attenuation (1999–2013). Until the bloom peak, the modelled production matched the measured biomass, indicating low production losses. Over the whole season, the modelled production could explain a substantial part of the summer TN increase, assuming that plausible losses (such as grazing or cell lysis) are retained within the upper mixed layer. Complementing the other data, we also investigated the nutrient content (Paper I) and varying cell width (Paper IV) of Aphanizomenon sp. By a combination of approaches, this thesis has contributed new information on cyanobacterial N fixation rates, the transfer of fixed N to other organisms in the food web and shown the potential for fixed N to stimulate summer primary and secondary production in the Baltic Sea. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>

No description available.

This study was conducted to evaluate the relative roles of trophic dynamics and nutrient concentrations in the development of cyanobacteria blooms. The motivation for this research was to gain insights into how food webs respond to ecosystem-scale changes, using Lake Champlain as a case study. I sought to link field-based observations with experimentally derived data on mechanisms to better understand the processes that drive cyanobacteria blooms. My research addressed three specific topics: (1) associations among phytoplankton and nutrient concentration trends over time, (2) the impacts of planktivory by invasive fish on the ambient zooplankton community, and (3) the role of herbivore zooplankton grazers in determining the composition of the phytoplankton community. I found little evidence of a strong association between nutrient concentrations and phytoplankton community composition during summer months in shallow bays of Lake Champlain prone to annual cyanobacteria blooms. Fish diet analysis indicated that invasive white perch (Morone americana) and alewife (Alosa pseudoharengus) selectively graze on large zooplankton, which has likely led to substantial declines in zooplankton biomass. I used these results to inform the design of a mesocosm study, which tested the effects of zooplankton grazing on phytoplankton and provided support for the theory that large zooplankton grazing pressure changes the size structure, abundance and composition of phytoplankton. High nutrient concentrations support increased levels of ecosystem productivity, but cascading trophic dynamics are additional forces that are likely contributing to the determination of phytoplankton community composition. Collectively, my research suggests that in shallow bays of Lake Champlain, selective grazing by invasive planktivorous fish is shifting the size structure of the zooplankton grazer community and has likely contributed to conditions that favor dominance by cyanobacteria in summer.