Brown algal phlorotannins: Improving and applying chemical methods

Phlorotannins are the least studied group of tannins and are found only in brown algae. Hitherto the roles of phlorotannins, e.g. in plant-herbivore interactions, have been studied by quantifying the total contents of the soluble phlorotannins with a variety of methods. Little attention has been given to either quantitative variation in cell-wall-bound and exuded phlorotannins or to qualitative variation in individual compounds. A quantification procedure was developed to measure the amount of cell-wall-bound phlorotannins. The quantification of soluble phlorotannins was adjusted for both large- and small-scale samples and used to estimate the amounts of exuded phlorotannins using bladder wrack (Fucus vesiculosus) as a model species. In addition, separation of individual soluble phlorotannins to produce a phlorotannin profile from the phenolic crude extract was achieved by high-performance liquid chromatography (HPLC). Along with these methodological studies, attention was focused on the factors in the procedure which generated variation in the yield of phlorotannins. The objective was to enhance the efficiency of the sample preparation procedure. To resolve the problem of rapid oxidation of phlorotannins in HPLC analyses, ascorbic acid was added to the extractant. The widely used colourimetric method was found to produce a variation in the yield that was dependent upon the pH and concentration of the sample. Using these developed, adjusted and modified methods, the phenotypic plasticity of phlorotannins was studied with respect to nutrient availability and herbivory. An increase in nutrients decreased the total amount of soluble phlorotannins but did not affect the cell-wall-bound phlorotannins, the exudation of phlorotannins or the phlorotannin profile achieved with HPLC. The presence of the snail Thedoxus fluviatilis on the thallus induced production of soluble phlorotannins, and grazing by the herbivorous isopod Idotea baltica increased the exudation of phlorotannins. To study whether the among-population variations in phlorotannin contents arise from the genetic divergence or from the plastic response of algae, or both, algae from separate populations were reared in a common garden. Genetic variation among local populations was found in both the phlorotannin profile and the content of total phlorotannins. Phlorotannins were also genetically variable within populations. This suggests that local algal populations have diverged in their contents of phlorotannins, and that they may respond to natural selection and evolve both quantitatively and qualitatively.

Enhancing performance of paper coatings by tailor-made plastic pigments

The paper industry is constantly looking for new ideas for improving paper products while competition and raw material prices are increasing. Many paper products are pigment coated. Coating layer is the top layer of paper, thus by modifying coating pigment also the paper itself can be altered and value added to the final product. In this thesis, synthesis of new plastic and hybrid pigments and their performance in paper and paperboard coating is reported. Two types of plastic pigments were studied: core-shell latexes and solid beads of maleimide copolymers. Core-shell latexes with partially crosslinked hydrophilic polymer core of poly(n-butyl acrylate-co-methacrylic acid) and a hard hydrophobic polystyrene shell were prepared to improve the optical properties of coated paper. In addition, the effect of different crosslinkers was analyzed and the best overall performance was achieved by the use of ethylene glycol dimethacrylate (EGDMA). Furthermore, the possibility to modify core-shell latex was investigated by introducing a new polymerizable optical brightening agent, 1-[(4-vinylphenoxy)methyl]-4-(2-henylethylenyl)benzene which gave promising results. The prepared core-shell latex pigments performed smoothly also in pilot coating and printing trials. The results demonstrated that by optimizing polymer composition, the optical and surface properties of coated paper can be significantly enhanced. The optimal reaction conditions were established for thermal imidization of poly(styrene-co-maleimide) (SMI) and poly(octadecene-co-maleimide) (OMI) from respective maleic anhydride copolymer precursors and ammonia in a solvent free process. The obtained aqueous dispersions of nanoparticle copolymers exhibited glass transition temperatures (Tg) between 140-170ºC and particle sizes from 50-230 nm. Furthermore, the maleimide copolymers were evaluated in paperboard coating as additional pigments. The maleimide copolymer nanoparticles were partly imbedded into the porous coating structure and therefore the full potential of optical property enhancement for paperboard was not achieved by this method. The possibility to modify maleimide copolymers was also studied. Modifications were carried out via N-substitution by replacing part of the ammonia in the imidization reaction with amines, such as triacetonediamine (TAD), aspartic acid (ASP) and fluorinated amines (2,2,2- trifluoroethylamine, TFEA and 2,2,3,3,4,4,4-heptafluorobuthylamine, HFBA). The obtained functional nanoparticles varied in size between 50-217 nm and their Tg from 150-180ºC. During the coating process the produced plastic pigments exhibited good runnability. No significant improvements were achieved in light stability with TAD modified copolymers whereas nanoparticles modified with aspartic acid and those containing fluorinated groups showed the desired changes in surface properties of the coated paperboard. Finally, reports on preliminary studies with organic-inorganic hybrids are presented. The hybrids prepared by an in situ polymerization reaction consisted of 30 wt% poly(styrene- co-maleimide) (SMI) and high levels of 70 wt% inorganic components of kaolin and/or alumina trihydrate. Scanning Electron Microscopy (SEM) images and characterization by Fourier Transform Infrared Spcetroscopy (FTIR) and X-Ray Diffraction (XRD) revealed that the hybrids had conventional composite structure and inorganic components were covered with precipitated SMI nanoparticles attached to the surface via hydrogen bonding. In paper coating, the hybrids had a beneficial effect on increasing gloss levels.

Effects of algal turbidity on foraging and antipredator behaviour of the three-spined stickleback (Gasterosteus aculeatus)

The aim of this thesis was to examine how aquatic organisms, such as fish, behave in an altered environmental condition. Many species of fish use vision as their primary tool to gain information about their surrounding environment. The visual conditions of aquatic habitats are often altered as a result of anthropogenic disturbance, such as eutrophication that initiates algal turbidity. In general, turbidity reduces the visibility and can be hypothesized to have an influence on the behaviour of fish. I used the three-spined stickleback (Gasterosteus aculeatus) as a model species and conducted four studies in the laboratory to test how algal turbidity affects its behaviour. In this thesis, two major behavioural aspects are discussed. The first is antipredator behaviour. In study I, the combined effects of turbidity and shoot density on habitat choice (shelter vs open) behaviour was tested on a group of sticklebacks (20 fish) in the presence and absence of piscivorous perch (Perca fluviatilis). In study II, I examined the behavioural responses of feeding sticklebacks when they were exposed to the sudden appearance of an avian predator (the silhouette of a common tern, Sterna hirundo). The study was done in turbid and clear water using three different groups sizes (1, 3 and 6 fish). The second aspect is foraging behaviour. Study III & IV focused on the effects of algal turbidity on the foraging performance of sticklebacks. In study III, I conducted two separate experiments to examine the effects of turbidity on prey consumption and prey choice of sticklebacks. In this experiment turbidity levels and the proportion of large and small prey (Daphnia spp.) were manipulated. In study IV, I studied whether a group of six sticklebacks can distribute themselves according to food input at two feeding stations in a way that provided each fish with the same amount of food in clear and turbid water. I also observed whether the fish can follow changes in resource distribution between the foraging patches. My results indicate an overall influence of algal turbidity on the antipredator and foraging behaviour of sticklebacks. In the presence of a potential predator, the use of the sheltered habitat was more pronounced at higher turbidity. Besides this, sticklebacks reduced their activity levels with predator presence at higher turbidity and shoot density levels, suggesting a possible antipredator adaptation to avoid a predator. When exposed to a sudden appearance of an avian predator, sticklebacks showed a weaker antipredator response in turbid water, which suggests that turbidity degrades the risk assessment capabilities of sticklebacks. I found an effect of group size but not turbidity in the proportion of sticklebacks that fled to the shelter area, which indicates that sticklebacks are able to communicate among group members at the experimental turbidity levels. I found an overall negative effect of turbidity on food intake. Both turbidity and changes in the proportion of prey sizes played a significant role in a stickleback’s prey selection. At lower turbidity levels (clear <1 and 5 NTU) sticklebacks showed preferences for large prey, whereas in more turbid conditions and when the proportion of large to small prey increased sticklebacks became increasingly random in their prey selection. Finally, my results showed that groups of sticklebacks disperse themselves between feeding stations according to the reward ratios following the predictions of the ideal free distribution theory. However, they took a significantly longer time to reach the equilibrium distribution in turbid water than in clear water. In addition, they showed a slower response to changes in resource distribution in a turbid environment. These findings suggest that turbidity interferes with the information transfer among group foragers. It is important to understand that aquatic animals are often exposed to a degraded environment. The findings of this thesis suggest that algal turbidity negatively affects their behavioural performance. The results also shed light on the underlying behavioural strategies of sticklebacks in turbid conditions that might help them adapt to an altered environmental situation and increase their survival. In conclusion, I hold that although algal turbidity has detrimental effects on the antipredator and foraging behaviour of sticklebacks, their behavioural adjustment might help them adapt to a changing environment.