Kjetill Østgaard

INDUCTION OF CYTOKINE PRODUCTION FROM HUMAN MONOCYTES STIMULATED WITH ALGINATE

Summary Alginates are polysaccharides with gel-forming properties composed of 1,4-linked β-D-mannuronic acid (M), α-L-guluronic acid (G), and alternating (MG) blocks. Alginate can be used as a matrix for implanted cells in vivo. In this study, we have examined the ability of alginates and their components to stimulate human monocytes to produce tumor necrosis factor-α, interleukin-6, and interleukin-1. Alginates stimulated the monocytes to produce high levels of all three cytokines. Low G alginates were approximately 10 times more potent in inducing cytokine production compared with high G alginates. The M-blocks and the MG-blocks, but not the G-blocks, stimulated the cytokine production. The results demonstrate that the mannuronic acid residues are the active cytokine inducers in alginates.

Ethanol production from seaweed extract

Extracts from Laminaria hyperborea could possibly be fermented to ethanol commercially. In particular, seaweed harvested in the autumn contains high levels of easily extractable laminaran and mannitol. Four microorganisms were tested to carry out this fermentation, one bacterium and three yeasts. Only Pichia angophorae was able to utilise both laminaran and mannitol for ethanol production, and its substrate preferences were investigated in batch and continuous cultures. Laminaran and mannitol were consumed simultaneously, but with different relative rates. In batch fermentations, mannitol was the preferred substrate. Its share of the total laminaran and mannitol consumption rate increased with oxygen transfer rate (OTR) and pH. In continuous fermentations, laminaran was the preferred substrate at low OTR, whereas at higher OTR, laminaran and mannitol were consumed at similar rates. Optimisation of ethanol yield required a low OTR, and the best yield of 0.43 g ethanol (g substrate)−1 was achieved in batch culture at pH 4.5 and 5.8 mmol O2 l−1 h−1. However, industrial production of ethanol from seaweed would require an optimisation of the extraction process to yield a higher ethanol concentration. Journal of Industrial Microbiology & Biotechnology (2000) 25, 249–254.

Homogeneous alginate gels: A technical approach

Abstract Homogeneous alginate gels can be made by internal liberation of calcium ions. Non-toxic gels at neutral pH were achieved by mixing particles of CaCO 3 with the slowly hydrolysing proton donor d -glucono-δ-lactone into the alginate solutions. This paper summarizes the essential factors controlling the properties of the resulting gel, such as pH, optical clarity, gel homogeneity, gel strength and syneresis.

Production of ethanol from mannitol by Zymobacter palmae

Extracts from brown seaweeds could possibly be fermented to ethanol, particularly seaweeds harvested in the autumn, which contain high levels of easily extractable laminaran and mannitol. Few microorganisms are able to utilise mannitol as a substrate for ethanol production and Zymobacter palmae was tested for this purpose. Bacterial growth as well as ethanol yield depended on the amount of oxygen present. Strictly anaerobic growth on mannitol was not observed. At excessive aeration, a change in the fermentation pattern was observed with high production of acetate and propionate. Under oxygen-limiting conditions, the bacteria grew and produced ethanol in a synthetic mannitol medium with a yield of 0.38 g ethanol (g mannitol)−1. Z. palmae was also successfully applied for fermentation of mannitol from Laminaria hyperborea extracts. Journal of Industrial Microbiology & Biotechnology (2000) 24, 51–57.

Quantitative determination of chitosans by ninhydrin

Abstract A simple method for quantitative analysis of chitosan based on the ninhydrin reaction is described. Only the 2-amino-2-deoxy-β- d -glucopyranose (GlcN) units of chitosans were found to form coloured products with ninhydrin. This reaction of chitosans with ninhydrin was sensitive and reproducible. Linear calibration curves were obtained in the concentration range of 10–120 mg/l, depending on the chemical composition of the chitosans. The amount of colour produced per GlcN unit decreased with decreasing fraction of acetylated units ( F A ). This imperfect stoichiometry was studied in more detail by comparing a series of β-(1→4)-linked GlcN oligomers. Dimer, trimer and tetramer produced 82, 67 and 61% of the colour relative to that of the monomer, respectively. The yield of almost fully deacetylated chitosan ( F A =0.01) with different molecular weights reached a constant value at 40% when the degree of polymerization increased to more than 10. Differences in reaction rates were also observed. While monomer, dimer and chitosan with F A =0.6 reacted rapidly, other chitosans did not fully complete the reaction within 30 min. Due to this behaviour, the reaction of chitosans with ninhydrin might be used for quantitative analysis only when a reliable calibration against a reference of a similar F A is available.

Interactions between chitosans and bacterial suspensions : adsorption and flocculation

Abstract Chitosans with different chemical composition were applied to flocculate Escherichia coli suspensions. The adsorption of chitosans was followed both by applying fluorescence labeled chitosans and monitoring changes in zeta potential values of the bacterial cells. The effects of chitosan molecular weight and composition, identified by the fraction of acetylated units ( F A ), were examined, as well as environmental conditions such as pH and ionic strength. The adsorption of chitosans to E. coli cells increased strongly with pH, the adsorbed amounts increased approximately 40% if pH was increased from pH 5 to 6.5. Despite their low charge density, the chitosans with higher F A adsorbed in higher amounts and reversed the cell surface charge most effectively. The chitosans with low molecular weights adsorbed most. Ionic strength did not affect the adsorption of highly acetylated chitosan with F A 0.49, whereas for chitosan with F A 0.01, adsorption increased with ionic strength. The combination of flocculation and adsorption data clearly showed that charge neutralization was not the main flocculation mechanism. Several results pointed to bridging as one dominating mechanism for flocculation.

Efficiency of chitosans applied for flocculation of different bacteria.

Three types of well-characterized chitosans of different composition were applied to flocculate 8 different bacterial species. The aim of this study was to relate chitosan structure and flocculation characteristic to general bacterial characteristics such as the cell surface charge and hydrophobicity. Large differences in the flocculation efficiency of chitosan were found between different bacterial suspensions, both regarding the effective chitosan concentrations and the optimal type of chitosan. However, no correlation was observed between general surface characteristics of bacteria and flocculation by chitosan of different composition. It may be concluded that purely electrostatic interactions did not play a dominant role in flocculation of Gram-negative bacteria in this study. The presence of GlcNAc residues had clearly beneficial effects on flocculation in such cases.

Alginate-based solid media for plant tissue culture

SummaryA new method for solid medium plant tissue culture based on in situ gelation of alginate is proposed as an alternative to agar-based media. In situ gelation by the use of dispersed CaCO3 and the slowly hydrolysing acid glucono-δ-lactone (GDL) was the basis for the use of alginate as a gelling agent. Inexpensive alginate-based media can be made in a wide range of pH values. Biological tests of these gels, concerning sterile seed growth and microcalli plating of Brassica napus (cv. Topas) and biomass production of Panax ginseng callus, showed results equal to those achieved with agar-based gels.

Regeneration, Cultivation and Differentiation of Plant Protoplasts Immobilized in Ca-alginate Beads

Summary A method for growing plant protoplasts immobilized in Ca-alginate is presented in detail. Two types of protoplasts, isolated from hypocotyls of Brassica napus cv. Niklas and leaf mesophyll tissue of Nicotiana sanderae cv. Crimson Bedder, have been used as examples to illustrate the general applicability of the method. The major advantage of the method is improved cellular protection during the first critical weeks after isolation. This is a protection both against mechanical strain and strong gradients in environmental conditions. Higher plating efficiency may thus be achieved and handling simplified, particularly when changing nutrient medium. Both regeneration and differentiation have repeatedly been achieved.

Alginate degradation during anaerobic digestion of Laminaria hyperborea stipes

Polyphenols and divalent metal ions present in the tissue may seriously affect the degradation of alginate during anaerobic digestion of brown seaweeds. Laminaria hyperborea stipes, harvested at 59 °N off the Norwegian coast in the autumn, were degraded at different concentrations of polyphenols in anaerobic batch reactors at 35 °C and pH 7. This was done by removing or adding the mechanically peeled outer phenolic layer of the algae, and using methanogenic and alginate degrading inocula already adapted to L. hyperborea degradation. Initial alginate released from the algal particles was affected by NaOH titrations because the Ca/Na-ratio was reduced. After a rapid consumption of the mannitol, alginate lyases were induced, and guluronate lyases showed the highest extracellular activity. Then the microbes digested 0.12–0.23 g Na-alginate L−1 h−1. Later the degradation rate of alginates declined almost to zero, and 13–50% of the alginate remained insoluble. The total solubilisation of alginates was apparently limited by both Ca-crosslinked guluronate residues and complexation with compounds such as polyphenols. The methane production had a lag phase that increased at higher amounts of soluble polyphenols, and the total fermentation probably also became product inhibited if soluble compounds such as acetate, ethanol and butyrate were accumulated.